Patent Application
20130347134
The present invention provides, inter alia, a transgenic non-human animal, such as a transgenic mouse, containing in its genome a polynucleotide encoding a von Willebrand factor (VWF) polypeptide. Nucleic acid sequences and vectors for generating the transgenic non-human animals, and methods using the transgenic non-human animals are provided as well.
This application contains references to amino acids and/or nucleic acid sequences that have been filed concurrently herewith as sequence listing text file 0352923.txt, file size of 137 KB, created on Jun. 20, 2013. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. §1.52(e)(5).
The ability of platelets to rapidly stick to the damaged wall of arterial blood vessels is critical for preventing blood loss (hemorrhage). Inappropriate deposition of these hemostatic cells in arterial blood vessels due to pathological disease processes such as atherosclerosis can result in lack of blood flow to vital organs such as the heart and brain. Thus a delicate balance exists between providing adequate hemostasis without causing blockage of blood vessels by excessive platelet deposition (a.k.a. thrombus formation).
von Willebrand Factor (VWF) is a multidomain, plasma glycoprotein of complex multimeric structure which is synthesized by vascular endothelial cells and megakaryocytes (Jaffe et al., 1973; Nachman et al., 1977; Sporn et al., 1985). Its presence in the blood is vital to maintaining the integrity of the vasculature. To accomplish this task, VWF forms a “bridge” between the injured vessel wall and platelets by virtue of its ability to interact with extracellular matrix components, such as collagen, and receptors expressed on platelets, such as glycoprotein Ib alpha (Sakariassen et al., 1979; Meyer et al., 1983; Cruz et al., 1995; Handa et al., 1986; Murata et al., 1991; Fressinaud et al., 1988). It also binds to and confers stability to factor VIII (Wiss et al., 1977). The importance of this glycoprotein in hemostasis is underscored by the occurrence of clinical bleeding when the plasma VWF levels fall below 50 IU/dL (type I von Willebrand's disease, abbreviated as “VWD”), or when functional defects in the protein occur (Type 2 VWD, which includes 4 subtypes: Type 2A, Type 2B, Type 2M, and Type 2N) (Ewenstein et al., 1997; Sadler et al., 1995).
Upon surface immobilization of VWF at sites of vascular injury, it is the role of the A1 domain of VWF (which includes e.g., residues 1240-1481, such as residues 1260-1480) to initiate the process of platelet deposition at sites of vascular injury and under conditions of high rates of shear flow (>1,000 s−1) (Ruggeri et al., 2006). The critical nature of this interaction is exemplified by the bleeding disorder, termed type 2M VWD, which results from the incorporation of loss-of-function mutations within this domain that perturb interactions with GPIb alpha (Sadler et al., 2006; Rabinowitz et al., 1992; Cruz et al., 2000). In addition, recombinant VWF multimers lacking the A1 domain cannot support platelet adhesion at high rates of flow despite retaining the ability to interact with collagen (Sixma et al., 1991).
The structure of the A1 domain includes the α/β fold with a central β-sheet flanked by α-helices on each side as well as one intra-disulfide bond (Cys1272-Cys 1458), but no MIDAS motif (Emsley et al., 1998). Its overall shape is cuboid, with the top and bottom faces forming the major and minor binding sites, respectively, that interact with the concave surface of GPIb α. The most extensive contact site buries about 1700 Å2 of surface area, interacting with leucine-rich repeat (LRR) five to eight and the C-terminal flank of the GPIb α (Huizing a et al., 2002). For this to occur, the β-switch region of this platelet receptor undergoes a conformation change so that it aligns itself with the central beta sheet of the A1 domain. The smaller site (about 900 Å2) accommodates the binding of the β-finger and the first LRR of GPIb α, an event that appears to require the displacement of the amino-terminal extension of the A1 domain. Based on these findings as well as the preferential localization of mutations in humans within this region, which enhance GPIb α binding, it is speculated that the amino-terminal extension regulates the adhesive properties of this domain. This is also supported by the fact that recombinant A1 proteins lacking this extension have a higher affinity for this platelet receptor (Sugimoto et al., 1993). Despite these observations, the physiological relevance of such structural changes in this receptor-ligand pair remains to be determined as well as the contribution of other domains to this process.
In addition to its role in hemostasis, VWF also contributes to pathological thrombus formation on the arterial side of the circulation. This may be the consequence of injury to the blood vessel wall from inflammatory disease states and/or medical/surgical interventions. Pathological thrombus formation is the leading cause of death in the Western world. Thus, pharmaceutical companies have committed considerable resources towards the research and design of drugs to prevent or treat thrombosis. However, there remains an urgent need to develop new and improved therapies such as those aimed at reducing platelet and/or VWF interactions with the injured arterial wall. One major hurdle hindering drug development in this field is the lack of an appropriate small animal model of thrombosis to test promising therapies. For instance, differences in the structure or isoform of protein receptors or ligands on mouse vs. human platelets that are critical for the activation and/or binding of these cells to the injured vessel wall preclude testing of drugs developed against human platelets in a mouse model of thrombosis. Moreover, this issue cannot be overcome by simply transfusing mice with human platelets because mouse VWF does not support significant interactions with human cells (see below). Thus, the development of “humanized” mouse models of hemostasis and thrombosis would potentially expedite drug discovery and testing.
Biophysical and molecular approaches are essential for understanding the structure-function relationship between a receptor and its ligand. Thus, the ability to study such interactions in an appropriate physiological and/or pathological setting is desirable. To do so, one requires an animal model that is amenable to genetic manipulation and has receptor-ligand interactions that closely resemble those found in humans. Thrombosis models in hamsters and guinea pigs have proven useful in pharmacological studies, but a mouse model would prove to be more beneficial based on the ability to insert or delete genes of interest, accessibility of tissues for study, and cost and ease of handling (Yamamoto, et al., 1998, Azzam et al., 1995). Regarding GPIb α-VWF interactions, two groups have significantly advanced the understanding of the importance of these interactions in mediating thrombosis by generating mice deficient in these proteins (Denis, et al., 1998, Ware, et al., 2000). Yet, no information regarding the role of the biophysical properties of the GPIb α-VWF-A1 in regulating the processes of thrombosis and hemostasis are obtained.
Thus, there is a need for a biological platform for testing of drugs to be used in human beings. The present invention addresses these and other needs.
One embodiment of the present invention is a transgenic non-human animal. This animal comprises in its genome a polynucleotide encoding a von Willebrand factor (VWF) polypeptide, wherein the transgenic non-human animal expresses the VWF and forms a thrombus when in the presence of human platelets.
Another embodiment of the present invention is a transgenic mouse. This mouse comprises in its genome a polynucleotide encoding a von Willebrand factor (VWF) polypeptide, wherein the transgenic mouse expresses the VWF and forms a thrombus when in the presence of human platelets.
A further embodiment of the present invention is a nucleic acid sequence. This nucleic acid sequence comprises SEQ ID NO:13.
An additional embodiment of the present invention is a vector. This vector comprises a nucleic acid sequence comprising SEQ ID NO:13.
A further embodiment of the present invention is a mouse-human chimeric polypeptide sequence. This sequence comprises the amino acid sequence of SEQ ID NO:25.
An additional embodiment of the present invention is a method for identifying a candidate agent that modulates human platelet mediated thrombosis. This method comprises:
Yet another embodiment of the present invention is a method for identifying a candidate agent that modulates human platelet mediated thrombosis. This method comprises:
Another embodiment of the present invention is a method for determining whether platelet function or morphology in a subject is abnormal. This method comprises:
a) affixing a protein comprising a VWF-A1 domain obtained from a transgenic non-human animal disclosed herein to a surface of a flow chamber;
b) perfusing through the flow chamber a volume of blood or plasma from a subject at a shear flow rate of at least about 100 s−1;
c) perfusing a targeted molecular imaging agent into the flow chamber; and
d) comparing the flow rate of the blood or plasma from the subject as compared to a normal flow rate, so as to determine whether the subject's platelet function or morphology is abnormal.
Yet another embodiment of the present invention is a method for producing chimeric von Willebrand Factor A1 protein that specifically binds to human platelets. This method comprises:
(a) providing a non-human animal expressing a chimeric von Willebrand Factor A1 protein, wherein the chimeric protein causes the platelet binding specificity of the non-human animal von Willebrand Factor A1 protein to change to be specific for human platelets; and
(b) harvesting the chimeric von Willebrand Factor A1 from the non-human animal, which specifically binds human platelets.
Another embodiment of the present invention is a method for calibrating an aggregometry device or a device for measuring clot formation or retraction. This method comprises:
a) providing hematologic data obtained from a subject, wherein blood or platelets from the subject is assessed by the device;
b) determining whether or not a thrombotic event occurs in a transgenic non-human animal disclosed herein, wherein the animal is perfused with a sample of blood or platelets from the subject; and
c) correlating data obtained from step (b) with the data obtained in step (a) so as to calibrate the device, wherein a certain data obtained from the device is indicative of the corresponding thrombotic outcome determined in the transgenic non-human animal.
This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
, loxP sites.
One embodiment of the present invention is a transgenic non-human animal. This animal comprises in its genome a polynucleotide encoding a von Willebrand factor (VWF) polypeptide, wherein the transgenic non-human animal expresses the VWF and forms a thrombus when in the presence of human platelets.
Assays for thrombus formation are known in the art (See e.g., Harrison's Principle of Internal Medicine, 15th ed. (Chapter 116) 2001, McGraw Hill, Columbus, Ohio) and include the in vivo (the tail bleeding assay, for example) and in vitro (ex vivo platelet adhesion study, for example) assays disclosed in the Example section. More details with respect to methods for assessing thrombotic events in vivo are set forth below.
In one aspect of this embodiment, the VWF polypeptide comprises the A1 domain of human VWF polypeptide, preferably amino acids 1240P through 1481G of the full length human VWF polypeptide, the amino acid sequence of which is depicted in SEQ ID NO:6. The literature appears to loosely define the bounds of the A1 domain of human VWF to include amino acids 1240-1481, 1260-1480, and other ranges (see, e.g., Emsley et al., 1998 (residues 1238-1472); Bonnefoy et al., 2006 (residues 1262-1492); Huang et al., 2009 (residues 1260-1468)).
In another aspect of this embodiment, the VWF polypeptide is at least 85%, preferably at least 90%, identical to the amino acid sequence depicted in SEQ ID NO:25, which is a chimeric VWF protein in which the human VWF A1 domain replaces the non-human, e.g., mouse, VWF A1 domain. For example, the VWF polypeptide is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% identical to the amino acid sequence depicted in SEQ ID NO:25.
In an additional aspect of this embodiment, the polynucleotide encodes a VWF to which AvW3 specifically binds. AvW3 is a monoclonal antibody that specifically recognizes the human VWF-A1 domain and blocks VWF's interaction with GPIb. (See e.g., Mancuso et al., 1996; Kroner et al., 1992; Rathore et al., 2003). AvW3 is available from commercial vendors such as Linscott's USA, catalog #GTI-V3A, Mill Valley, Calif.).
In yet another aspect of this embodiment, the animal may be any useful non-human laboratory or agricultural animal. For example, the animal may be selected from the group consisting of mouse, rat, hamster, guinea pig, rabbit, dog, goat, horse, and monkey. Preferably, the animal is a mouse.
Another embodiment of the present invention is a transgenic mouse. This mouse comprises in its genome a polynucleotide encoding a von Willebrand factor (VWF) polypeptide, wherein the transgenic mouse expresses the VWF and forms a thrombus when in the presence of human platelets.
In one aspect of this embodiment, the VWF polypeptide comprises amino acids 1240P through 1481G of SEQ ID NO:6.
In another aspect of this embodiment, the VWF polypeptide is at least 85%, preferably at least 90%, identical to the amino acid sequence depicted in SEQ ID NO:25. For example, the VWF polypeptide is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% identical to the amino acid sequence depicted in SEQ ID NO:25.
In an additional aspect of this embodiment, the polynucleotide encodes a VWF to which the monoclonal antibody AvW3 specifically binds.
A further embodiment of the present invention is a nucleic acid sequence. This nucleic acid sequence comprises, consists essentially of, or consists of, SEQ ID NO:13, which depicts the nucleic acid sequence encoding the human VWF A1 domain. We note that SEQ ID NO:13 is not a naturally occurring nucleotide sequence, because while greater than 85% of the human A1 domain sequence was substituted for its murine counterpart, a small portion of the murine A1 domain sequence still remains.
An additional embodiment of the present invention is a vector. This vector comprises, consists essentially of, or consists of, the nucleic acid sequence depicted in SEQ ID NO:13. For example, the nucleic acid sequence of one such vector is depicted in SEQ ID NO:11.
A further embodiment of the present invention is a mouse-human chimeric polypeptide sequence. This sequence comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO:25.
In another aspect of this embodiment, the VWF polypeptide is at least 85%, preferably at least 90%, identical to the amino acid sequence depicted in SEQ ID NO:25. For example, the VWF polypeptide is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% identical to the amino acid sequence depicted in SEQ ID NO:25.
An additional embodiment of the present invention is a method for identifying a candidate agent that modulates human platelet mediated thrombosis. This method comprises:
In one aspect of this embodiment, the VWF polypeptide comprises amino acids 1240P through 1481G of SEQ ID NO:6.
In another aspect of this embodiment, the VWF polypeptide is at least 85%, preferably at least 90%, identical to the amino acid sequence depicted in SEQ ID NO:25. For example, the VWF polypeptide is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% identical to the amino acid sequence depicted in SEQ ID NO:25.
In an additional aspect of this embodiment, the polynucleotide encodes a VWF to which the monoclonal antibody AvW3 specifically binds.
In a further aspect of this embodiment, the animal is as defined above. The animal may be selected from the group consisting of mouse, rat, hamster, guinea pig, rabbit, dog, goat, horse, and monkey. Preferably, the animal is a mouse.
Yet another embodiment of the present invention is a method for identifying a candidate agent that modulates human platelet mediated thrombosis. This method comprises:
In one aspect of this embodiment, the VWF polypeptide comprises amino acids 1240P through 1481G of SEQ ID NO:6.
In another aspect of this embodiment, the VWF polypeptide is at least 85%, preferably at least 90%, identical to the amino acid sequence depicted in SEQ ID NO:25. For example, the VWF polypeptide is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% identical to the amino acid sequence depicted in SEQ ID NO:25.
In an additional aspect of this embodiment, the polynucleotide encodes a VWF to which the monoclonal antibody AvW3 specifically binds.
In a further aspect of this embodiment, the evaluating step (i.e., step (d)) comprises the use of a diagnostic assay for determining GPIb-alpha-VWF-A1 protein interaction. As used herein, “GPIb-alpha-VWF-A1 protein interaction” means the binding or other association between the platelet GPIb-alpha and the A1 domain of a chimeric VWF protein according to this embodiment.
In another aspect of this embodiment, the diagnostic assay comprises perfusing platelets into a flow chamber at a shear flow rate of at least 100s−1, wherein the VWF protein is immobilized on a bottom surface of the chamber. In the present invention, other shear flow rates may be used, particularly those set forth in more detail in the Examples below, or as may be determined by one skilled in this art. In one aspect of this embodiment, the administration of the candidate agent and the perfusion of the platelets occur sequentially. For example, the perfusion of platelets may occur prior to administration of the candidate agent. Perfusion of platelets may be followed by perfusion of a labeled agent, as defined in more detail below. The labeled agent may target a platelet receptor, a VWF protein, or a portion thereof. The platelets used in this embodiment preferably are not murine platelets; preferably, they are human platelets.
In another preferred embodiment, the diagnostic assay comprises perfusing platelets into the transgenic mouse. In this embodiment, the administration of the candidate agent and the perfusion of the platelets may occur sequentially. For example, the perfusion of platelets may occur prior to administration of the candidate agent. Perfusion of platelets may be followed by perfusion of a labeled agent. In the present invention, the labeled agent may be any agent capable of providing a detectable signal and that does not substantially interfere with the evaluating step. For example, the labeled agent may comprise one or more of a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand. The labeled agent may target a platelet receptor, a VWF protein, or a portion thereof. The platelets used in this embodiment preferably are not murine platelets; preferably, they are human platelets.
In an additional aspect of this embodiment, the evaluating step comprises detecting an increase or decrease in the dissociation rate between the VWF produced by the transgenic mouse and GPIb-alpha protein by at least two-fold.
Certain candidate agents may slow the on-rate, and/or increase the off-rate (koff) binding kinetics, and/or reduce bond strength of the interaction between VWF-A1, e.g., the VWF produced by a non-human transgenic animal, preferably a transgenic mouse according to the present invention, and GPIb-alpha by at least two-fold, thus resulting in a decreased lifetime of the bond(s). Such candidate agents could reduce thrombosis formation. Other candidate agents may abbreviate off-rate (koff) binding kinetics between VWF-A1, e.g., the VWF produced by a non-human transgenic animal, preferably a transgenic mouse according to the present invention, and GPIb-alpha by at least two-fold, thus resulting in a prolongation in the lifetime of the bond(s). Such candidate agents could promote platelet adhesion due to the compound stabilizing an interaction between VWF-A1, e.g., the VWF produced by a non-human transgenic animal, preferably a transgenic mouse according to the present invention, and GPIb-alpha. To assess binding efficiency between VWF-A1, e.g., the VWF produced by a non-human transgenic animal, preferably a transgenic mouse according to the present invention, and GPIb-alpha, binding kinetics can be determined by measuring translocation velocity, tethering frequency, and bond strength (Fukuda, K., et al., (2005) Nat. Struct. Mol. Biol. 12:152-159; Doggett, et al., (2003) Blood 102(10): 152-60; Doggett, T. A. et al. (2002) Biophys. J. 83, 194-205; Schmidtke and Diamond (2000) J Cell Bio 149(3): 719-29; Mody et al., (2005) Biophys. J. 88: 1432-43, all of which are incorporated by reference in their entirety).
The candidate agents, including compounds identified and tested using the methods described above can be anti-platelet drugs. In one embodiment, the anti-platelet drug can be a cyclooxygenase inhibitor, a phosphodiesterase inhibitor, an adenosine diphosphate receptor inhibitor, a PI3K inhibitor, an adenosine reuptake inhibitors, thrombin receptor inhibitor or inhibitor of any intracellular signaling pathway in platelets, an alphaIIb beta3 inhibitor, an alpha2 beta1 inhibitor, a glycoprotein V inhibitor, a glycoprotein VI inhibitor, a PECAM-1 inhibitor or any adhesion molecule and/or activation pathway critical for human platelet function.
In a further aspect of this embodiment, the evaluating step comprises detecting an increase or decrease of platelet adhesion to a surface expressing VWF produced by the transgenic mouse of the present invention.
In another aspect of this embodiment, the evaluating step comprises detecting an increase or decrease in a stabilization of an interaction between VWF produced by the transgenic mouse of the present invention and GPIb-alpha protein.
In an additional aspect of this embodiment, the evaluating step comprises detecting thrombosis formation.
In yet another aspect of this embodiment, the evaluating step comprises identifying an occurrence of an abnormal thrombotic event in the transgenic mouse. Non-limiting examples of an abnormal thrombotic event may comprise abnormal bleeding, abnormal clotting, death, or a combination thereof. As used herein, “abnormal” refers to clinical abnormality, which is readily determined by a physician.
In a further aspect of this embodiment, the evaluating step comprises any suitable method or assay such as, e.g., dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination thereof. These assays are known in the art. For example, Merkel et al. (1999) discloses using dynamic force microscopy to detect and measure receptor-ligand bonds. These assays are disclosed below in the Examples section.
Another embodiment of the present invention is a method for determining whether platelet function or morphology in a subject is abnormal. This method comprises:
a) affixing a protein comprising a VWF-A1 domain, preferably including amino acids 1240P-1481G of SEQ ID NO:6, obtained from a transgenic non-human animal disclosed herein to a surface of a flow chamber;
b) perfusing through the flow chamber a volume of blood or plasma from a subject at a shear flow rate of at least about 100 s−1;
c) perfusing a targeted molecular imaging agent into the flow chamber; and
d) comparing the flow rate of the blood or plasma from the subject as compared to a normal flow rate, so as to determine whether the subject's platelet function or morphology is abnormal. In this assay method, the lack of platelet binding may suggest functional defects in the subject's platelets.
As used herein, a “subject” is a mammal, preferably, a human. In addition to humans, categories of mammals within the scope of the present invention include, for example, agricultural animals, domestic animals, laboratory animals, etc. Some examples of agricultural animals include bovines, porcines, equines, goats, etc. Some examples of domestic animals include canines, felines, etc. Some examples of laboratory animals include murines, rabbits, guinea pigs, hamsters, etc. In one aspect of this embodiment, the subject is selected from the group consisting of a human, a canine, a feline, a murine, a porcine, an equine, or a bovine.
In another aspect of this embodiment, the affixing comprises:
(i) coating a surface of the chamber with an antibody that specifically binds VWF-A1 domain, preferably including amino acids 1240P-1481G of SEQ ID NO:6 and
(ii) perfusing the VWF-A1 protein, preferably including amino acids 1240P-1481G of SEQ ID NO:6 produced by the transgenic mouse in the flow chamber at a shear flow rate of at least 100 s−1.
In yet another aspect of this embodiment, the targeted molecular imaging agent includes any agent capable of providing a detectable signal and that does not substantially interfere with the method. Preferably, the imaging agent comprises a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination thereof.
In a further aspect of this embodiment, the targeted molecular imaging agent binds to a platelet receptor, a platelet ligand, or any region of a VWF protein or a portion thereof.
In an additional aspect of this embodiment, the targeted molecular imaging agent comprises horseradish peroxidase (HRP) coupled to an antibody that specifically binds to VWF-A1 or a fragment thereof, preferably amino acids 1240P-1481G of SEQ ID NO:6. Following binding, a reaction with diaminobenzidine (DAB) can be performed where DAB is reduced by HRP to produce a brown precipitate at the site of binding. This technique allows for enzymatic, calorimetric detection of binding that can be visualized by transmitted light microscopy. For example, if the antibody is directed at a platelet receptor, and calorimetric detection represents whether the antibody bound to the platelet-VWF-A1 complex, the absence of color would denote the lack of a complex formation, thus suggesting that platelets were unable to bind to VWF-A1.
In another aspect of this embodiment, the comparing step comprises a platelet adhesion assay, fluorescence imaging, a chromogenic indicator assay, a microscopy morphology analysis, or any combination thereof.
In a further aspect of this embodiment, platelets bound to VWF-A1 are less than about 500 cells/mm2.
In yet another aspect of this embodiment, the platelets are substantially spherical.
The normal platelet morphology is discoid with some spherical shaping, but some are substantially spherical in shape. To further analyze platelet morphology, gross platelet histology can be assessed via light microscopy or electron microscopy. In another embodiment, platelets having an abnormal morphology are greater than about 2 μm in diameter. (Ross M H, Histology: A text and atlas 3rd edition, Williams and Wilkins, 1995: Chapter 9). Various assays can be used to assess whether platelet function is normal, such as a platelet adhesion assay, fluorescence imaging, a chromogenic indication, microscopy morphology analysis, or those listed in Harrison's Principle of Internal Medicine, 15th ed. ((Chapter 116) 2001, McGraw Hill, Columbus, Ohio), which are hereby incorporated by reference.
In an additional aspect of this embodiment, the protein comprising the VWF-A1 is affixed to the chamber with any appropriate agent. Representative, non-limiting examples of such an agent include an antibody, a peptide, and a Fab fragment that specifically binds to a VWF polypeptide or a portion thereof.
Yet another embodiment of the present invention is a method for producing chimeric von Willebrand Factor A1 protein that specifically binds to human platelets. This method comprises:
(a) providing a non-human animal expressing a chimeric von Willebrand Factor A1 protein, wherein the chimeric protein causes the platelet binding specificity of the non-human animal von Willebrand Factor A1 protein to change to be specific for human platelets; and
(b) harvesting the chimeric von Willebrand Factor A1 from the non-human animal, which specifically binds human platelets.
In one aspect of this embodiment, the chimeric von Willebrand Factor A1 protein comprises amino acids 1240P through 1481G of SEQ ID NO:6.
In another aspect of this embodiment, the chimeric von Willebrand Factor A1 protein is at least 85%, preferably at least 90%, identical to the amino acid sequence depicted in SEQ ID NO:25. For example, the chimeric von Willebrand Factor A1 protein is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% identical to the amino acid sequence depicted in SEQ ID NO:25.
In an additional aspect of this embodiment, monoclonal antibody AvW3 specifically binds to the chimeric von Willebrand Factor A1 protein.
In yet another aspect of this embodiment, the animal is selected from the group consisting of mouse, rat, hamster, guinea pig, rabbit, dog, goat, horse, and monkey. Preferably, the animal is a mouse.
Another embodiment of the present invention is a method for calibrating an aggregometry device or a device for measuring clot formation or retraction. This method comprises:
a) providing hematologic data obtained from a subject, wherein blood or platelets from the subject is assessed by the device;
b) determining whether or not a thrombotic event occurs in a transgenic non-human animal disclosed herein, wherein the animal is perfused with a sample of blood or platelets from the subject; and
c) correlating data obtained from step (b) with the data obtained in step (a) so as to calibrate the device, wherein a certain data obtained from the device is indicative of the corresponding thrombotic outcome determined in the transgenic non-human animal.
Suitable subjects are as disclosed above.
In one aspect of this embodiment, the thrombotic event comprises blood clotting, abnormal bleeding, abnormal clotting, death, or a combination thereof.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
For recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitly contemplated.
The transgenic non-human animals of the current invention are produced by experimental manipulation of the genome of the germline of the non-human animal. These genetically engineered non-human animals may be produced by several methods well known in the art which include the introduction of a “transgene” that comprises a nucleic acid (for example, DNA such as the A1 domain of VWF) integrated into a chromosome of the somatic and/or germ line cells of a non-human animal via methods known to one skilled in the art or into an embryonal target cell. As used herein, a “transgenic animal” is an animal whose genome has been altered by the introduction of a transgene.
The term “transgene” as used herein refers to a foreign gene that is placed into an organism by introducing the foreign gene into newly fertilized eggs, embryonic stem (ES) cells, or early embryos. The term “foreign gene” refers to any nucleic acid (for example, a gene sequence) that is introduced into the genome of an animal by experimental manipulations. These nucleic acids may include gene sequences found in that animal so long as the introduced gene contains some modification (for example, the presence of a selectable marker gene, a point mutation—such as the base pair substitution mutant that contributes to the amino acid change at amino acid residue 1326 of the A1 domain of VWF, the replacement of mouse VWF A1 domain with the human VWF A1 domain (e.g., 1240P-1481G of SEQ ID NO:6), the presence of a loxP site, and the like) relative to the naturally-occurring gene.
The term “loxP site” refers to a short (34 bp) DNA sequence that is recognized by the Cre recombinase of the E. coli bacteriophage P1. In the presence of Cre recombinase, placement of two loxP sites in the same orientation on either side of a DNA segment can result in efficient excision of the intervening DNA segment, leaving behind only a single copy of the loxP site (Sauer et al., 1988).
The embryonic stem (ES) cells suitable for generating transgenic animals are those that harbor introduced expression vectors (constructs), such as plasmids and the like. Such ES cells are known in the art. The expression vector constructs can be introduced via transfection, lipofection, transformation, injection, electroporation, or infection, or other techniques known in the art. The expression vectors can contain coding sequences, or portions thereof, encoding proteins for expression. Such expression vectors can include the required components for the transcription and translation of the inserted coding sequence.
Introducing targeting vectors (as disclosed below in more detail) into ES cells can generate the VWF transgenic animals of the present invention, in particular those encoding amino acids 1240P-1481G of SEQ ID NO:6. ES cells are obtained by culturing pre-implantation embryos in vitro under appropriate conditions (Evans, et al. (1981) Nature 292:154-156; Bradley, et al. (1984) Nature 309:255-258; Gossler, et al. (1986) Proc. Acad. Sci. USA 83:9065-9069; and Robertson, et al. (1986) Nature 322:445-448). Using a variety of methods known to those skilled in the art, transgenes can be efficiently introduced into the ES cells via DNA transfection methods, which include (but are not limited to), protoplast or spheroplast fusion, electroporation, retrovirus-mediated transduction, calcium phosphate co-precipitation, lipofection, microinjection, and DEAE-dextran-mediated transfection. Following the introduction into the blastocoel of a blastocyst-stage embryo, transfected ES cells can thereafter colonize an embryo and contribute to the germ line of the resulting chimeric animal (see Jaenisch, (1988) Science 240:1468-1474). Assuming that the transgene provides a means for selection, the transfected ES cells may be subjected to various selection protocols to enrich for ES cells that have integrated the transgene prior to the introduction of transfected ES cells into the blastocoel. Alternatively, the polymerase chain reaction (PCR) may be used to screen for ES cells that have integrated the transgene and precludes the need for growth of the transfected ES cells under appropriate selective conditions prior to transfer into the blastocoel.
Alternative methods for the generation of transgenic animals (such as transgenic mice) containing an altered A1 domain of the VWF gene encoding, e.g., amino acids 1240P-1481G of SEQ ID NO:6, are established in the art. For example, embryonic cells at various stages of development can be used to introduce transgenes for the production of transgenic animals and different methods are used that depend on the stage of embryonic cell development. For microinjection methods, the zygote is best suited. In the mouse, the male pronucleus reaches the size of approximately 20 microns in diameter, which allows for reproducible injection of 1-2 picoliters (pl) of suspended DNA solution. A major advantage in using zygotes as a gene transfer target is that in most cases, the injected DNA will be incorporated into the host genome before the first cleavage (Brinster, et al. (1985) Proc. Natl. Acad. Sci. USA 82:4438-4442). Thus, all cells of the transgenic non-human animal (such as a mouse) will carry the incorporated transgene (encoding, e.g., amino acids 1240P-1481G of SEQ ID NO:6), which can result in the efficient transmission of the transgene to the offspring of the founder since 50% of the germ cells will harbor the transgene (see e.g., U.S. Pat. No. 4,873,191).
Yet another method known in the art that can be used to introduce transgenes into a non-human animal is retroviral infection. The developing non-human embryo can be cultured in vitro to the blastocyst stage wherein during this time, the blastomeres can be targets for retroviral infection (Janenich (1976) Proc. Natl. Acad. Sci. USA 73:1260-1264). Enzymatic treatment to remove the zona pellucida can increase infection efficiency of the blastomeres (Hogan et al. (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Plainview, N.Y.). The viral vector system used by one skilled in the art in order to introduce the transgene is usually a replication-defective retrovirus that harbors the transgene (Jahner, D. et al. (1985) Proc. Natl. Acad. Sci. USA 82:6927-6931; Van der Putten, et al. (1985) Proc. Natl. Acad. Sci. USA 82:6148-6152). Transfection can be easily and efficiently obtained via culturing blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart, et al. (1987) EMBO J. 6:383-388). Infection can also be performed at a later stage whereby virus or virus-producing cells are injected into the blastocoele (Jahner, D. et al. (1982) Nature 298:623-628). Most of the founder non-human animals will be mosaic for the transgene since incorporation occurs only in a subset of cells that form the transgenic animal and the founder may additionally contain various retroviral insertions of the transgene at different positions in the genome that generally will segregate in the offspring. Additional methods of using retroviruses or retroviral vectors to create transgenic animals known to those skilled in the art involves microinjecting mitomycin C-treated cells or retroviral particles producing retrovirus into the perivitelline space of fertilized eggs or early embryos (see Haskell and Bowen (1995) Mol. Reprod. Dev. 40:386).
As used herein, von Willebrand factor is abbreviated “VWF”. VWF polypeptides are known in the art. For example, pre-pro-human VWF was assigned the Genbank GI accession number of 401413. VWF polypeptide sequences in other animals, such as, e.g., dog (GI accession number 1478046), cat (GI accession number 974579), pig (GI accession number 243984), Norway rat (GI accession number 1256375), and Equus asinus (GI accession number 974573), are also known. (Jenkins et al., 1998).
The VWF sequences from mouse and human have been aligned as shown in Jenkins et al. (1998). The cDNA sequence encoding pre-pro-human VWF (SEQ ID NO: 7) is readily available to those skilled in the art, under Genbank Accession No. X04385. The translated polypeptide sequence of human VWF is listed in SEQ ID NO: 6. The A1 domain of VWF runs from, e.g., amino acid residue number 1240 (proline) to amino acid residue number 1481 (glycine) in both human and mouse VWF. The amino acid sequence of mouse A1 domain is listed as SEQ ID NO:26, and the amino acid sequence of human A1 domain is listed as SEQ ID NO:27. A portion of the human VWF A1 domain, amino acid residue number 1260 to amino acid residue number 1480 of the amino acid sequence of SEQ ID NO:6, is shown in SEQ ID NO: 1. Additionally, a portion of the mouse VWF A1 domain, amino acid residue number 1260 to amino acid residue number 1480 of the of SEQ ID NO:8, is shown in SEQ ID NO: 2.
VWF is one of the key players in arterial thrombosis, which is a pathological consequence of disease states such as atherosclerosis and which remains a major cause of morbidity and mortality in the Western world, with healthcare cost ranging in the billions of dollars in the USA alone (Circulation 2006; 113:e85). Central to this process is the inappropriate deposition and activation of platelets in diseased vessels that can ultimately occlude the lumen, thus impeding blood flow to vital organ such as the heart and brain.
VWF is a large plasma glycoprotein of complex multimeric structure, which under normal physiological conditions prevents excessive bleeding by promoting platelet deposition at sites of vascular injury, thus “sealing off” leaky blood vessels. In order for this event to occur, VWF must form a “bridge” between receptors expressed on circulating platelets and exposed components of the injured vessel wall. This is the function of the A1 and A3 domains of this plasma protein, respectively. Each is folded into a disulfide-bonded loop structure that is critical for optimal biological activity (
It is the A1 domain that contains residues that compose the binding site for its receptor on platelets known as GPIb alpha, an adhesive event essential for the ability of these cells to rapidly attach to the injured vessel wall. The critical nature of this interaction is exemplified by the bleeding disorder, termed type 2M von Willebrand Disease (VWD), which results from the incorporation of loss-of-function point mutations within this domain that reduce the interaction between VWF-A1 and GPIb alpha (Sadler J E et al., (2006) J. Thromb. Haemost. 4: 2103-14). The A3 domain, on the other hand, is believed to be important in anchoring plasma VWF to sites where extracellular matrix components (i.e. collagen) are exposed as a result of disruption of the overlying vasculature endothelium (Wu D. et al. Blood 2002). Once in contact with exposed elements of the damaged vessel wall, platelets become “activated” through various signaling pathways (i.e. GPVI) enabling other adhesion molecules, such as α2β1 (collagen receptor) and aIIbβ3 (fibrinogen and VWF receptor) integrins, to firmly anchor these cells at the site of injury and to each other (
During the past two decades, there has been considerable progress in understanding how VWF mediates platelet adhesion. Both the VWF cDNA and gene have been cloned and the primary structure of the VWF subunit (
With regard to mediating adhesive interactions with platelets, it has become increasingly evident that the VWF-A1 domain plays a crucial role in this process based on molecular genetic studies of individuals with type 2M or 2B VWD (Meyer et al, 1997; Ginsburg et al, 1993; Hillery et al., 1998; Mancuso et al., 1996; Ruggeri et al., 1980; Cooney et al., 1996). VWD is a common hereditary coagulation abnormality that arises from a quantitative or qualitative deficiency of VWF). VWD affects humans, in addition to dogs and cats. There are three types of VWD: type 1, type 2, and type 3. Type 1 VWD is a quantitative defect, wherein decreased levels of VWF are detected but subjects may not have clearly impaired clotting, Type 2 VWD is a qualitative defect, wherein subjects have normal VWF levels but VWF multimers are structurally abnormal, or subgroups of large or small multimers are absent. Four subtypes exist: Type 2A, Type 2B, Type 2M, and Type 2N. Type 3 is rare and the most severe form of VWD (homozygous for the defective gene). (Braunwald et al., Harrison's Principle of Internal Medicine, 15th ed., (Chapter 116) 2001, McGraw Hill, Columbus, Ohio).
In the majority of cases with type 2M or type 2B VWD, patients with these designated genotypes have single point mutations contained within the disulfide loop (between Cys 1272 and Cys 1458) of this domain. With regard to type 2M VWD, afflicted individuals have significant impairments in hemostasis that appears to result from a lack of or reduced adhesive interactions between GPIb alpha and VWF at sites of vascular injury, and not from an alteration in VWF multimer structure. Structural and functional evidence has been provided in that type 2M mutations, such as 1324G>S, are localized within a region of the A1 domain (
In contrast to type 2M VWD, mutations associated with type 2B VWD are known to enhance the interaction between VWF-A1 and GPIb alpha, that is, they mitigate the requirement for exogenous modulators such as ristocetin or botrocetin to induce platelet agglutination (Ruggeri et al., 1980). Moreover, these altered residues are localized in a region remote from the major GPIb alpha binding site that has been identified by mutagenesis (Meyer et al., 1997;
Surface-immobilization of VWF and subsequent exposure to physiologically relevant shear forces appears to be a prerequisite for its ability to support interactions with platelets as this multimeric protein does not bind appreciably to these cells in the circulation. These hydrodynamic conditions are believed to promote structural changes within the A1 domain that in turn increases its affinity for GPIb alpha (Roth et al., 1991; Siedlecki et al., 1996; Ruggeri et al., 1992). Evidence suggested to support the existence of such an alteration in structure includes the ability of non-physiologic modulators such as the antibiotic ristocetin or the snake venom protein botrocetin to promote platelet agglutination in solution-based assays (Howard et al., 1981; Read et al., 1989). Moreover, this “on” and “off” conformation is exemplified by type 2B VWD. For instance, it was initially hypothesized that incorporation of type 2B mutations into the A1 domain shifted the equilibrium between two distinct tertiary conformations, analogous to those seen in crystal structures of the integrin 1 domain in ligand-free and collagen-bound states (Emsley et al., 2000). The location of the type 2B mutants at sites distinct from the GPIb alpha binding site suggest that they disrupt a region responsible for regulation of binding affinity, thus affecting ligand binding allosterically. The crystal structure of a type 2B mutant A1 domain, 1309I>V, was determined and compared to its wild-type counterpart (Celikel et al., 2000). A change was discovered in the structure of a loop, thought to be involved in GPIb alpha binding, lying on the surface distal to the mutation site. A similar finding has been observed for the VWF-A1 crystal structure containing the identical mutation (
This altered conformation represents a high affinity binding state of the A1 domain. However, the pathway of allosteric change proposed previously, involving the burial of a water molecule, cannot be a general feature of type 2B mutants, and the structural rearrangements appear too subtle to explain the altered kinetics. Interestingly, complex formation between botrocetin and VWF-A1 in which the type 2B mutation 1309I>V has been incorporated, and has demonstrated that most of these structural differences are reversed including: (1) loss of the buried water molecule at the mutation site; (2) the peptide plan between Asp 1323 and Gly 1324 flips back to a conformation similar to that in the WT structure; and (3) the side chain of His 1326 remains in the “mutant” position, although there is some evidence from electron density of an alternative conformation similar to WT. However, this reversion in structure does not correlate with a loss in the function-enhancing activity associated with the type 2B mutation. In fact, the addition of botrocetin further augments the interaction between the mutant A1 domain and platelets in flow (Fukuda et al., 2002). Thus, an alternative mechanism must account for function-enhancing nature of type 2B mutations. Moreover, these subtle alterations in structure did not compare to the large conformational changes in homologous integrin I-type domains that occur on ligand binding.
Recent findings provide support that type 2B mutations may stabilize the binding of a region of GPIb alpha known as the β-hairpin to an area near the location of these altered residues, distinct from that identified by site-directed mutagenesis. Type 2B mutations have been suggested to destabilize a network of interactions observed between the bottom face of the A1 domain and its terminal peptides in the wild type A1 structure, thereby making the binding site accessible (
Another disease of interest is Bernard-Soulier Syndrome, which is a rare disorder caused by a deficiency of the surface platelet receptor GPIb alpha. As a result, platelets fail to stick and clump together at the site of the injury. Functional abnormalities have also been observed in some hereditary platelet disorders wherein the platelets are of abnormal size or shape, such as in May-Hegglin Anomaly and Chediak Higashi syndrome. (Braunwald et al., Harrison's Principle of Internal Medicine, 15th ed. (Chapter 116) 2001, McGraw Hill, Columbus, Ohio).
Progress has been made in understanding the structure of VWF and GPIb alpha proteins and potential alterations in conformation that may regulate this protein-protein interaction. This model is non-limiting. However, the model permits determination of the kinetic and biomechanical basis for 1) the regulation of VWF-A1 domain activity in response to hydrodynamic forces, 2) the alterations in bond kinetics that result from incorporation of type 2B mutations into the VWF-A1 domain, and 3) the susceptibility of the kinetics of the GPIb alpha-VWF-A1 bond to an applied force.
Nucleic acids and Vectors
“Nucleic acid” or “oligonucleotide” or “polynucleotide” used herein mean at least two nucleotides covalently linked together.
Nucleic acids may be synthesized chemically or isolated by one of several approaches established in art. The basic strategies for identifying, amplifying, and isolated desired DNA sequences as well as assembling them into larger DNA molecules containing the desired sequence domains in the desired order, are well known to those of ordinary skill in the art. See, e.g., Sambrook, et al., (1989) Nature November 16; 342(6247):224-5; Perbal, B. et al., (1983) J. Virol. March; 45(3):925-40. DNA segments corresponding to all or a portion of the VWF sequence may be isolated individually using the polymerase chain reaction (M. A. Innis, et al., “PCR Protocols: A Guide To Methods and Applications,” Academic Press, 1990). A complete sequence may be assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981), Nature, 292:756; Nambiar, et al. (1984), Science, 223:1299; Jay et al. (1984), J. Biol. Chem., 259:6311. Thus, procedures for construction and expression of mutant proteins of defined sequence are well known in the art.
The assembled nucleotide sequence can be cloned into a suitable vector. As used herein, a “vector” means a vehicle to carry a nucleic acid into a cell. Vectors include, without limitation, cloning vectors, targeting vectors, and expression vectors.
Vectors generally contain a selectable marker. A selectable marker can include a gene which encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed. Selectable markers may be positive. A positive selectable marker is usually a dominant selectable marker wherein the genes encode an enzymatic activity that can be detected in a mammalian cell or a cell line (including ES cells). Some non-limiting examples of dominant selectable markers include the bacterial xanthine-guanine phosphoribosyl transferase gene (also referred to as the gpt gene) which confers the ability to grow in the presence of mycophenolic acid, the bacterial hygromycin G phosphotransferase (hyg) gene which confers resistance to the antibiotic hygromycin, and the bacterial aminoglycoside 3′ phosphotransferase gene (also referred to as the neo gene) which confers resistance to the drug G418 in mammalian cells. Selectable markers may also be negative. Negative selectable markers encode an enzymatic activity whose expression is toxic to the cell when grown in an appropriate selective medium. One non-limiting example of a negative selectable marker is the HSV-tk gene wherein HSV-tk expression in cells grown in the presence of gancyclovir or acyclovir is catatonic. Growth of cells in selective medium containing acyclovir or gancyclovir therefore selects against cells capable of expressing a functional HSV TK enzyme.
Those of ordinary skill in the art are familiar with numerous cloning vectors, and the selection of an appropriate cloning vector is a matter of choice. The construction of vectors containing desired nucleotide sequences linked by appropriate DNA sequences is accomplished by discussed above. These vectors may be constructed to contain additional DNA sequences, such as bacterial origins of replication to make shuttle vectors in order to shuttle between prokaryotic hosts and mammalian hosts.
A suitable targeting vector contains the modified A1 domain of VWF gene sequence, containing, e.g., a VWF gene sequence modified to encode amino acids 1240P-1481G of SEQ ID NO:6, sufficient to permit the homologous recombination of the targeting vector into at least one allele of the A1 domain of the VWF gene resident in the chromosomes of the target or recipient cell (for example, ES cells). The targeting vector will usually harbor 10 to 15 kb of DNA homologous to the A1 domain of the VWF gene, wherein this 10 to 15 kb of DNA will be divided more or less equally on each side of the selectable marker gene. One non-limiting exemplary targeting vector is shown in SEQ ID NO:11.
Targeting vectors can also be of the replacement-type wherein the integration of a replacement-type vector results in the insertion of a selectable marker into the target gene. Replacement-type targeting vectors may be employed to disrupt a gene (such as the VWF gene or the A1 domain of the VWF gene). This can result in the generation of a null allele; for example, an allele not capable of expressing a functional protein wherein the null alleles may be generated by deleting a portion of the coding region, deleting the entire gene, introducing an insertion and/or a frameshift mutation, and the like. Expression vectors containing sequences encoding the produced proteins and polypeptides, as well as the appropriate transcriptional and translational control elements, can be generated using methods well known to and practiced by those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination which are described in J. Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and in F. M. Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. In one embodiment, loxP expressing targeting vectors are used for transfection methods (such as pDNR-1r vector, pACD4K-C vector, and the like). In other embodiments, Cre-recombinase-expressing plasmids are also utilized (for example, crAVE cre recombinase vectors).
An expression vector containing a nucleotide sequence encoding a protein of interest, such as a VWF-A1 molecule, encoding, e.g., amino acids 1240P-1481G of SEQ ID NO:6, is transfected into a host cell, either eukaryotic (for example, yeast, mammalian, or insect cells) or prokaryotic, by conventional techniques well established in the art. Transfection techniques carried out depend on the host cell used. For example, mammalian cell transfection can be accomplished using lipofection, protoplast fusion, DEAE-dextran mediated transfection, CaPO4 co-precipitation, electroporation, direct microinjection, as well as other methods known in the art which can comprise: scraping, direct uptake, osmotic or sucrose shock, lysozyme fusion or erythrocyte fusion, indirect microinjection such as via erythrocyte-mediated techniques, and/or by subjecting host cells to electric currents. Some of the techniques mentioned above are also applicable to unicellular organisms, such as bacteria or yeast. As other techniques for introducing genetic information into host cells will be developed, the above-mentioned list of transfection methods is not considered to be exhaustive. The transfected cells are then cultured by conventional techniques to produce a VWF-A1 molecule harboring at least one of the mutations previously described, particularly a VWF-A1 molecule encoding amino acids 1240P-1481G of SEQ ID NO:6.
One skilled in the art understands that expression of desired protein products in prokaryotes is most often carried out in E. coli with vectors that contain constitutive or inducible promoters. Some non-limiting examples of bacterial cells for transformation include the bacterial cell line E. coli strains DH5a or MC1061/p3 (Invitrogen Corp., San Diego, Calif.), which can be transformed using standard procedures practiced in the art, and colonies can then be screened for the appropriate plasmid expression. Some E. coli expression vectors (also known in the art as fusion-vectors) are designed to add a number of amino acid residues, usually to the N-terminus of the expressed recombinant protein. Such fusion vectors can serve three functions: 1) to increase the solubility of the desired recombinant protein; 2) to increase expression of the recombinant protein of interest; and 3) to aid in recombinant protein purification by acting as a ligand in affinity purification. In some instances, vectors, which direct the expression of high levels of fusion protein products that are readily purified, may also be used. Some non-limiting examples of fusion expression vectors include pGEX, which fuse glutathione S-tranferase to desired protein; pcDNA 3.1V5-His A B & C (Invitrogen Corp, Carlsbad, Calif.) which fuse 6×-His to the recombinant proteins of interest; pMAL (New England Biolabs, MA) which fuse maltose E binding protein to the target recombinant protein; the E. coli expression vector pUR278 (Ruther et al., (1983) EMBO 12:1791), wherein the coding sequence may be ligated individually into the vector in frame with the lac Z coding region in order to generate a fusion protein; and pIN vectors (Inouye et al., (1985) Nucleic Acids Res. 13:3101-3109; Van Heeke et al., (1989) J. Biol. Chem. 24:5503-5509. Fusion proteins generated by the likes of the above-mentioned vectors are generally soluble and can be purified easily from lysed cells via adsorption and binding to matrix glutathione agarose beads subsequently followed by elution in the presence of free glutathione. For example, the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target can be released from the GST moiety.
Other suitable cell lines, in addition to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences for a VWF-A1 molecule described above, such as, e.g., one encoding amino acid 1240P-1481G of SEQ ID NO:6, may alternatively be used to produce the molecule of interest. Non-limiting examples include plant cell systems infected with recombinant virus expression vectors (for example, tobacco mosaic virus, TMV; cauliflower mosaic virus, CaMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing coding sequences for a VWF-A1 molecule described above, such as, e.g., one encoding amino acid 1240P-1481G of SEQ ID NO:6; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing coding sequences for a VWF-A1 molecule described above, such as, e.g., one encoding amino acid 1240P-1481G of SEQ ID NO:6; yeast (for example, Saccharomyces sp., Pichia sp.) transformed with recombinant yeast expression vectors containing coding sequences for a VWF-A1 molecule described above, such as, e.g., one encoding amino acid 1240P-1481G of SEQ ID NO:6; or mammalian cell lines harboring a vector that contains coding sequences for a VWF-A1 molecule described above, such as, e.g., one encoding amino acid 1240P-1481G of SEQ ID NO:6.
Mammalian cells (such as BHK cells, VERO cells, CHO cells and the like) can also contain an expression vector (for example, one that harbors a nucleotide sequence encoding a VWF-A1 molecule described above, such as, e.g., one encoding amino acid 1240P-1481G of SEQ ID NO:6) for expression of a desired product. Expression vectors containing such a nucleic acid sequence linked to at least one regulatory sequence in a manner that allows expression of the nucleotide sequence in a host cell can be introduced via methods known in the art, as described above. To those skilled in the art, regulatory sequences are well known and can be selected to direct the expression of a protein of interest in an appropriate host cell as described in Goeddel (Gene Expression Technology (1990) Methods in Enzymology 185, Academic Press, San Diego, Calif.). Regulatory sequences can comprise the following: enhancers, promoters, polyadenylation signals, and other expression control elements. Practitioners in the art understand that designing an expression vector can depend on factors, such as the choice of host cell to be transfected and/or the type and/or amount of desired protein to be expressed.
Animal or mammalian host cells capable of harboring, expressing, and secreting large quantities of a VWF-A1 molecule (described above, such as, e.g., one encoding amino acid 1240P-1481G of SEQ ID NO:6) of interest into the culture medium for subsequent isolation and/or purification include, but are not limited to, Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., (1986) Som. Cell Molec. Genet, 12:555-556; Kolkekar et al., (1997) Biochemistry, 36:10901-10909; and WO 01/92337 A2), dihydrofolate reductase negative CHO cells (CHO/dhfr-, Urlaub et al., (1980)
Proc. Natl. Acad. Sci. U.S.A., 77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); human embryonic kidney cells (e.g., 293 cells, or 293 cells subcloned for growth in suspension culture, Graham et al., (1977) J. Gen. Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells (CV1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse sertoli cells (TM4; Mather (1980) Biol. Reprod., 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TR1 cells (Mather (1982) Annals NY Acad. Sci., 383:44-68); MCR 5 cells; FS4 cells. A cell line transformed to produce a VWF-A1 molecule described above, such as, e.g., one encoding amino acid 1240P-1481G of SEQ ID NO:6, can also be an immortalized mammalian cell line of lymphoid origin, which include but are not limited to, a myeloma, hybridoma, trioma or quadroma cell line. The cell line can also comprise a normal lymphoid cell, such as a B cell, which has been immortalized by transformation with a virus, such as the Epstein Barr virus (such as a myeloma cell line or a derivative thereof).
A host cell strain, which modulates the expression of the inserted sequences, or modifies and processes the nucleic acid in a specific fashion desired also may be chosen. Such modifications (for example, glycosylation and other post-translational modifications) and processing (for example, cleavage) of protein products may be important for the function of the protein. Different host cell strains have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. As such, appropriate host systems or cell lines can be chosen to ensure the correct modification and processing of the foreign protein expressed, which includes, for example, a VWF-A1 molecule described above, such as, e.g., one encoding amino acid 1240P-1481G of SEQ ID NO:6. Thus, eukaryotic host cells possessing the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Non-limiting examples of mammalian host cells include 3T3, W138, BT483, Hs578T, CHO, VERY, BHK, Hela, COS, BT2O, T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, MDCK, 293, HTB2, and HsS78Bst cells.
For protein recovery, isolation and/or purification, the cell culture medium or cell lysate is centrifuged to remove particulate cells and cell debris. The desired polypeptide molecule (for example, a VWF-A1 protein such as, e.g., one encoding amino acid 1240P-1481G of SEQ ID NO:6) is isolated or purified away from contaminating soluble proteins and polypeptides by suitable purification techniques. Non-limiting purification methods for proteins include: separation or fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on a resin, such as silica, or cation exchange resin, e.g., DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, e.g., Sephadex G-75, Sepharose; protein A sepharose chromatography for removal of immunoglobulin contaminants; and the like. Other additives, such as protease inhibitors (e.g., PMSF or proteinase K) can be used to inhibit proteolytic degradation during purification. Purification procedures that can select for carbohydrates can also be used, e.g., ion-exchange soft gel chromatography, or HPLC using cation- or anion-exchange resins, in which the more acidic fraction(s) is/are collected.
The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein. In the present invention, these terms mean a linked sequence of amino acids, which may be natural, synthetic, or a modification, or combination of natural and synthetic. The term includes antibodies, antibody mimetics, domain antibodies, lipocalins, targeted proteases, and polypeptide mimetics. The term also includes vaccines containing a peptide or peptide fragment intended to raise antibodies against the peptide or peptide fragment.
In the present invention, the term “antibody” means full immunoglobulin molecules, as well as to parts of such immunoglobulin molecules except Fab fragments, and encompasses naturally occurring antibodies as well as non-naturally occurring antibodies, including antibody-like molecules. The term “a Fab fragment” means a Fab fragment of an antibody. Full immunoglobulin molecules include IgMs, IgDs, IgEs, IgAs or IgGs, such as IgG1, IgG2a, IgG2b, IgG3 or IgG4. Antigen-binding fragments of full immunoglobulin include, for example, Fab′, F(ab′)2, Fv and rIgG. The term antibody further include single chain antibodies, chimeric, bifunctional and humanized antibodies. See also, e.g., Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.; Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York (1998). Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference. These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995); each of which is incorporated herein by reference). Antibody-like molecule include affibody, affilin molecule, adnectin, anticalin, designed ankyrin repeat protein (DARPin), domain antibody, evibody, a knottin, Kunitz-type domain, maxibody, nanobody, tetranectin, trans-body, or a V(NAR).
The term “antibody” includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). The term also refers to recombinant single chain Fv fragments (scFv). As set forth above, the term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described in, e.g., Kostelny et al. (1992) J Immunol 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579, Hollinger et al., 1993, supra, Gruber et al. (1994) J Immunol:5368, Zhu et al. (1997) Protein Sci 6:781, Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.
Typically, an antibody has a heavy and light chain. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”). Light and heavy chain variable regions contain four “framework” regions interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs”. The extent of the framework regions and CDRs have been defined. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional space.
The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
“VH” refer to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab. “VL” refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.
The phrase “single chain Fv” or “scFv” refers to an antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain. Typically, a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site.
A “chimeric antibody” is an immunoglobulin molecule in which (a) the constant region, or a portion thereof; is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
A “humanized antibody” is an immunoglobulin molecule that contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol 2:593-596 (1992)). Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-3′27 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
“Epitope” or “antigenic determinant” refers to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). A preferred method for epitope mapping is surface plasmon resonance, which has been used to identify preferred granulation inhibitors recognizing the same epitope region as the IIA1 antibody disclosed herein.
The phrase “specifically (or selectively) binds” or when referring to protein-protein interaction, refers to a binding reaction between two molecules that is at least two times the background and more typically more than 10 to 100 times background molecular associations under physiological conditions. When using one or more detectable binding agents that are proteins, specific binding is determinative of the presence of the protein, in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein sequence, thereby identifying its presence.
Peptides binding agents include receptor traps. A receptor trap is a decoy receptor that can comprise fusions between two distinct receptor components and the Fc region of an antibody molecule, which can result in the generation of a molecule with an increased affinity over single component reagents. This technology is available from Regeneron (Tarrytown, N.Y.) and is described in Wachsberger et al., (2007) Int J Radiat Oncol Biol Phys. 67(5):1526-37; Holash et al., (2002) Proc Natl Acad Sci USA. 2002 99(17):11393-8; Davis et al., (1996) Cell. 87(7):1161-9; U.S. Pat. No. 7,087,411; and in United States Publication Applications 2004/0014667, 2005/0175610, 2005/0260203, 2006/0030529, 2006/0058234, which are all hereby incorporated by reference in their entirety.
Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, antibodies raised against a particular protein, polymorphic variants, alleles, orthologs, and conservatively modified variants, or splice variants, or portions thereof; can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with, e.g., a VWF protein (or a portion thereof, such as the A1 domain) and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Methods for determining whether two molecules specifically interact are disclosed herein, and methods of determining binding affinity and specificity are well known in the art (see, for example, Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press, 1988); Friefelder, “Physical Biochemistry: Applications to biochemistry and molecular biology” (W.H. Freeman and Co. 1976)).
Furthermore, VWF binding agent (or a VWF-A1 domain binding agent) can interfere with the specific binding of a VWF and a platelet (or a protein in the platelet, such as, e.g., GPIb-alpha protein) by various mechanism. For purposes of the methods disclosed herein, an understanding of the mechanism by which the interference occurs is not required and no mechanism of action is proposed. An VWF binding agent (or a VWF-A1 domain binding agent), such as an anti-VWF antibody, an anti-VWF-A1 domain antibody, or Fab fragments thereof, is characterized by having specific binding activity (Ka) for a VWF protein, e.g., a polypeptide that includes amino acid 1240P-1481G of SEQ ID NO:6 or a functional equivalent thereof, or the VWF-A1 domain antibody, as appropriate, of at least about 105 mol−1, 106 mol−1, or greater, preferably 107 mol−1, or greater, more preferably 108 mol−1, or greater, and most preferably 109 mol−1, or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949).
Candidate agents include compounds that may be obtained from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., (1996) Tib Tech 14:60).
Methods for preparing libraries of molecules are well known in the art and many libraries are commercially available. Libraries of interest in the invention include peptide libraries, randomized oligonucleotide libraries, synthetic organic combinatorial libraries, and the like. Degenerate peptide libraries can be readily prepared in solution, in immobilized form as bacterial flagella peptide display libraries or as phage display libraries. Peptide ligands can be selected from combinatorial libraries of peptides containing at least one amino acid. Libraries can be synthesized of peptoids and non-peptide synthetic moieties. Such libraries can further be synthesized which contain non-peptide synthetic moieties, which are less subject to enzymatic degradation compared to their naturally-occurring counterparts. Libraries are also meant to include for example but are not limited to peptide-on-plasmid libraries, polysome libraries, aptamer libraries, synthetic peptide libraries, synthetic small molecule libraries and chemical libraries. The libraries can also comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Screening compound libraries listed above [also see Examples below and U.S. Patent Application Publication No. 2005/0009163, which is hereby incorporated by reference], in combination with dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination of these assays (for example, those assays described in EXAMPLES 1-5) can be used to identify modulators of VWF-A1 binding to GPIb-alpha, wherein the compound abbreviates or increases off-rate (koff) binding kinetics between VWF-A1 and GPIb-alpha by at least two-fold (Lew et al., (2000) Curr. Med. Chem. 7(6):663-72; Werner et al., (2006) Brief Funct. Genomic Proteomic 5(1):32-6).
Small molecule combinatorial libraries may also be generated. A combinatorial library of small organic compounds is a collection of closely related analogs that differ from each other in one or more points of diversity and are synthesized by organic techniques using multi-step processes. Combinatorial libraries include a vast number of small organic compounds. One type of combinatorial library is prepared by means of parallel synthesis methods to produce a compound array. A compound array can be a collection of compounds identifiable by their spatial addresses in Cartesian coordinates and arranged such that each compound has a common molecular core and one or more variable structural diversity elements. The compounds in such a compound array are produced in parallel in separate reaction vessels, with each compound identified and tracked by its spatial address. Examples of parallel synthesis mixtures and parallel synthesis methods are provided in U.S. Ser. No. 08/177,497, filed Jan. 5, 1994 and its corresponding PCT published patent application WO95/18972, published Jul. 13, 1995 and U.S. Pat. No. 5,712,171 granted Jan. 27, 1998 and its corresponding PCT published patent application WO96/22529, which are hereby incorporated by reference.
Examples of chemically synthesized libraries are described in Fodor et al., (1991) Science 251:767-773; Houghten et al., (1991) Nature 354:84-86; Lam et al., (1991) Nature 354:82-84; Medynski, (1994) BioTechnology 12:709-710; Gallop et al., (1994) J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., (1993) Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., (1994) Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., (1992) Biotechniques 13:412; Jayawickreme et al., (1994) Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., (1993) Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242, dated Oct. 14, 1993; and Brenner et al., (1992) Proc. Natl. Acad. Sci. USA 89:5381-5383.
Examples of phage display libraries are described in Scott et al., (1990) Science 249:386-390; Devlin et al., (1990) Science, 249:404-406; Christian, et al., (1992) J. Mol. Biol. 227:711-718; Lenstra, (1992) J. Immunol. Meth. 152:149-157; Kay et al., (1993) Gene 128:59-65; and PCT Publication No. WO 94/18318.
In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058; and Mattheakis et al., (1994) Proc. Natl. Acad. Sci. USA 91:9022-9026.
In one non-limiting example, non-peptide libraries, such as a benzodiazepine library (see e.g., Bunin et al., (1994) Proc. Natl. Acad. Sci. USA 91:4708-4712), can be screened. Peptoid libraries, such as that described by Simon et al., (1992) Proc. Natl. Acad. Sci. USA 89:9367-9371, can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994), Proc. Natl. Acad. Sci. USA 91:11138-11142.
Preferred candidate agents are small molecules. Small molecules can include any number of therapeutic agents presently known and used, or can be synthesized in a library of such molecules for the purpose of screening for biological function(s). Small molecules are distinguished from macromolecules by size. The small molecules of this invention usually have a molecular weight less than about 5,000 Daltons (Da), preferably less than about 2,500 Da, more preferably less than 1,000 Da, most preferably less than about 500 Da.
Preferred small molecules are relatively easier and less expensively manufactured, formulated or otherwise prepared. Preferred small molecules are stable under a variety of storage conditions. Preferred small molecules may be placed in tight association with macromolecules to form molecules that are biologically active and that have improved pharmaceutical properties. Improved pharmaceutical properties include changes in circulation time, distribution, metabolism, modification, excretion, secretion, elimination, and stability that are favorable to the desired biological activity. Improved pharmaceutical properties include changes in the toxicological and efficacy characteristics of the chemical entity.
Diversity libraries, such as random or combinatorial peptide or non-peptide libraries can be screened for small molecules and compounds that specifically bind to a VWF-A1 protein. Many libraries are known in the art that can be used such as, e.g., chemically synthesized libraries, recombinant (e.g., phage display) libraries, and in vitro translation-based libraries.
Any screening technique known in the art can be used to screen for agonist (i.e., compounds that promote platelet adhesion) or antagonist molecules (such as anti-thrombotics) directed at a target of interest (e.g. VWF-A1). The present invention contemplates screens for small molecule ligands or ligand analogs and mimics, as well as screens for natural ligands that bind to and modulate VWF-A1 binding to GPIb-alpha, such as via examining the degree of thrombus formation, platelet adhesion, coagulation, blood flow, vessel occlusion, or bleeding times. For example, natural products libraries can be screened using assays of the invention for molecules that modulate the activity of a molecule of interest, such as a VWF-A1 binding to GPIb-alpha.
Knowledge of the primary sequence of a molecule of interest, such as a VWF-A1, can provide an initial clue as to proteins that can modulate VWF-A1 binding to GPIb-alpha. Identification and screening of modulators is further facilitated by determining structural features of the protein, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination. These techniques provide for the rational design or identification of such modulators.
Screening the libraries can be accomplished by any variety of commonly known methods. See, for example, the following references, which disclose screening of peptide libraries: Parmley and Smith, (1989) Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, (1990) Science 249:386-390; Fowlkes et al., (1992) BioTechniques 13:422-427; Oldenburg et al., (1992) Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., (1994) Cell 76:933-945; Staudt et al., (1988) Science 241:577-580; Bock et al., (1992) Nature 355:564-566; Tuerk et al., (1992) Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., (1992) Nature 355:850-852; U.S. Pat. Nos. 5,096,815; 5,223,409; and 5,198,346, all to Ladner et al.; Rebar et al., (1993) Science 263:671-673; and PCT Pub. WO 94/18318.
The present invention provides methods for evaluating potential anti-thrombotic reagents in pre-clinical testing using a non-human transgenic animal. The animal may be any useful non-human laboratory or agricultural animal. For example, the animal may be selected from the group consisting of mouse, rat, hamster, guinea pig, rabbit, dog, goat, horse, and monkey.
There are at least three classes of antithrombotic drugs that can be screened using the transgenic mouse model of the invention: Anticoagulant drugs (such as Heparins; Vitamin K antagonists, which are currently the only anticoagulants that can be administered orally; and direct thrombin inhibitors), Antiplatelet drugs (such as cyclooxygenase inhibitors like aspirin; phosphodiesterase inhibitors like ticlopidine (Ticlid); adenosine diphosphate receptor inhibitors like clopidogrel (Plavix); tirofiban (Aggrastat); adenosine reuptake inhibitors, and inhibitors of integrins on platelets (for example, alpha IIb Beta3) like eptifibatide (Integrilin)), and Thrombolytic or fibrinolytic drugs (such as t-PA (alteplase Activase); reteplase (Retavase); urokinase (Abbokinase); streptokinase (Kabikinase, Streptase); tenectaplase; lanoteplase; and anistreplase (Eminase)).
The invention provides an in vivo model to test the efficacy of potential anti-thrombotic drugs against human platelets prior to FDA approval. To date, in vitro models of thrombosis do not accurately recapitulate the hemodynamic conditions, cell-cell interactions, or cell-protein interactions that occur at sites of vascular injury in a living animal. Thus, anti-thrombotics can be identified and their potential therapeutic effects can be assessed for treatment of abnormal thrombotic events associated with atherothrombotic arterial diseases and venous thrombotic diseases (such as abnormal bleeding and/or abnormal clotting).
Atherothrombotic arterial diseases can include, but is not limited to, coronary artery disease, (for example, acute myocardial infarction, acute coronary syndromes (such as unstable angina pectoris) and stable angina pectoris); mesenteric ischemia, “abdominal angina,” and mesenteric infarction; cerebral vascular disease, including acute stroke and transient ischemic attack; mesenteric arterial disease; as well as peripheral arterial disease, including acute peripheral arterial occlusion and intermittent claudication. Anti-thrombotic compounds identified by the pre-clinical testing method of the present invention can also be useful for the treatment of coronary artery disease (which includes, but is not limited to anti-thrombotic therapy during coronary angioplasty, anti-thrombotic therapy during cardiopulmonary bypass, and limiting of platelet activation during ischemia reperfusion) as well as venous thrombotic diseases (which include, but are not limited to deep venous thrombosis and pulmonary thromboembolism). Anti-thrombotic compounds identified by the pre-clinical testing method of the present invention can also be useful in anti-thrombotic therapy for pulmonary hypertension.
The candidate agents may be administered by a topical, oral, rectal, parenteral (such as subcutaneously, intramuscularly, intravenously, intraperitoneally, intrapleurally, intravesicularly or intrathecally), or nasal route. These compounds may also be applied topically or locally, in liposomes, solutions, gels, ointments, biodegradable microcapsules, or impregnated bandages. Compositions or dosage forms for topical application may include suspensions, dusting powder, solutions, lotions, suppositories, sprays, aerosols, biodegradable polymers, ointments, creams, gels, impregnated bandages and dressings, liposomes, and artificial skin.
The candidate agents may also be formulated in a pharmaceutical composition prior to administration. A “pharmaceutical composition” refers to a mixture of one or more of the compounds, or pharmaceutically acceptable salts, hydrates, polymorphs, or pro-drugs thereof, with other chemical components, such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. The pharmaceutical composition may contain other components so long as the other components do not reduce the effectiveness of the compound according to this invention so much that the therapy is negated. Some other components may have independent therapeutic effects. Pharmaceutically acceptable carriers are well known, and one skilled in the pharmaceutical art can easily select carriers suitable for particular routes of administration (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985).
Pharmaceutical carriers utilized by one skilled in the art which make up the foregoing compositions include petrolatum, polyethylene glycol, alginates, carboxymethylcellulose, methylcellulose, agarose, pectins, gelatins, collagen, vegetable oils, phospholipids, stearic acid, stearyl alcohol, polysorbate, mineral oils, polylactate, polyglycolate, polyanhydrides, polyvinylpyrrolidone, and the like.
A “pro-drug” refers to an agent which is converted into the parent drug in vivo. Pro-drugs are often useful because, in some situations, they are easier to administer than the parent drug. They are bioavailable, for instance, by oral administration whereas the parent drug is not. The pro-drug also has improved solubility in pharmaceutical compositions over the parent drug. For example, the compound carries protective groups which are split off by hydrolysis in body fluids, e.g., in the bloodstream, thus releasing active compound or is oxidized or reduced in body fluids to release the compound.
A compound of the present invention also can be formulated as a pharmaceutically acceptable salt, e.g., acid addition salt, and complexes thereof. The preparation of such salts can facilitate the pharmacological use by altering the physical characteristics of the agent without preventing its physiological effect. Examples of useful alterations in physical properties include, but are not limited to, lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate administering higher concentrations of the drug.
The term “pharmaceutically acceptable salt” means a salt, which is suitable for or compatible with the treatment of a patient or a subject such as a human patient or an animal.
The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compounds of the invention or any of their intermediates. Illustrative inorganic acids which form suitable acid addition salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable acid addition salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed and such salts exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds of the invention are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, are used, for example, in the isolation of compounds of the invention for laboratory use or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
The candidate agent that alters interactions between a VWF, such as the VWF which includes amino acids 1240P-1481G of SEQ ID NO:6, and a human platelet may be used to treat diseases disclosed herein. As used herein and as well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
Therapy dose and duration will depend on a variety of factors, such as the disease type, patient age, therapeutic index of the drugs, patient weight, and tolerance of toxicity. Initial dose levels will be selected based on their ability to achieve ambient concentrations shown to be effective in in vitro models (for example, a dose level used to determine therapeutic index), in vivo models, and in clinical trials. The skilled clinician using standard pharmacological approaches can determine the dose of a particular drug and duration of therapy for a particular patient in view of the above stated factors. The response to treatment can be monitored by analysis of body fluid or blood levels of the compound and the skilled clinician will adjust the dose and duration of therapy based on the response to treatment revealed by these measurements.
A “labeled” agent means an agent that has an attachment which renders the agent detectable. In the present invention, the labeled agent would aid in the detection of either the presence or absence of a thrombus. As set forth above, the labeled agent may comprise a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination thereof. A fluorophore, for example green fluorescent protein, (such as GFP, RFP, YFP and the like; see Johnson and Johnson, (2007) ACS Chem. Biol. 2(1):31-8) can be used as a biomarker. A quantum dot is a semiconductor nanocrystal, that can be as small as 2 to 10 nm or can 15-20 nm (for example, Q-dot nanocrystals; also see Kaji et al., (2007) Anal Sci. 23(1):21-4). Quantum dot fluorescence can be induced by exposure to ultraviolet light. Both a fluorescent protein and a quantum dot can be obtained commercially (for example, Molecular Probes—Invitrogen, Carlsbad, Calif. or Evident Technologies, Troy N.Y.). A fluorophore can also be generated in the laboratory according to molecular biology methods practiced in the art. A radiolabel is a radioactive isotope that can be used as a tracer. Non-limiting examples of radiolabels include: Technetium-99m, Iodine-123 and 131, Thallium-201, Gallium-67, Fluorine-18, -19, Indium-111, Xenon-I 33, and Krypton-81m. Radiolabels can be obtained commercially, for example, from SRI International (Menlo Park, Calif.). As set forth above, certain methods include the use of a nanoparticle, which may comprise a perfluorocarbon (PFC). Non-limiting examples of perfluorocarbons include perfluorobutane, perfluorohexane, perfluorooctane, perfluorodecal in, perfluoromethyldecalin, and perfluoroperhydrophenanthrene. These can be synthesized according to the method described in EXAMPLE 5 or according to Partlow et al. (FASEB J (2007) February 6 on-line publication). The perfluorocarbon molecules can also be obtained commercially (F2 Chemicals Ltd.; Lancashire, UK). The PFC nanoparticle can be coupled to a platelet receptor antibody (such as platelet receptor alpha-IIb beta3). In some embodiments, imaging can comprise a PET scan, a CT scan, an MRI, an IR scan, an ultrasound, nuclear imaging, or a combination thereof.
The invention provides methods for detecting an internal vascular injury site (occult bleeding) in a subject. This could be useful in emergency room (ER) settings or on the battlefield in order to quickly identify sites of internal bleeding. For instance, the method can entail: administering to a subject a targeted molecular imaging agent, wherein the molecule circulates for an effective period of time in order to bind to the injury site within the subject; tracking a deposition of the labeled thrombosis-indicating-molecule in the subject; and identifying the site of a thrombus formation in the subject by imaging the labeled targeted molecular imaging agent. Thus, the deposition of the targeted molecular imaging agent at the internal vascular injury site can be indicative of internal bleeding within a subject. For example, a targeted molecular imaging agent can recognize constituents of thrombi that comprise a lipid, a protein, a cellular molecule, or a combination thereof.
The invention also provides a method to test contrast agents for imaging of human platelets at sites of thrombosis. For instance, one could test the ability of nanoparticle contrast agents targeted to human platelets to identify areas of thrombosis or occult bleeding. In some embodiments, the prevention or reduction of thrombus formation at site of injury upon administration of a compound can be visually examined via tracking the localization of labeled platelets (such as with high resolution in vivo microscopy or MRI). In further embodiments, the platelets can be labeled with a nanoparticle, fluorophore, quantum dot, microcrystal, radiolabel, dye, or gold biolabel. The prevention or reduction of thrombus formation also can be readily determined by methods known to one skilled in the art, which include but are not limited to aggregometry, review of real-time video of blood flow in the animal, and determination of vessel occlusion, as well as by the examples provided below.
The invention also provides a method to correlate results obtained with an in vitro assay designed to measure the effects of antithrombotics or biomarkers of platelet activation in patients. For example, a biomarker is an indicator of a particular disease state or a particular state of an organism, such as when the subject experiences vascular vessel wall injury. Upon injury to the vessel wall and subsequent damage to the endothelial lining, exposure of the subendothelial matrix to blood flow results in deposition of platelets at the site of injury via binding to the collagen with the surface collagen-specific glycoprotein Ia/IIa receptor. This adhesion is strengthened further by the large multimeric circulating protein VWF, which forms links between the platelet glycoprotein Ib/IX/V and collagen fibrils. The platelets are then activated and release the contents of their granules into the plasma, in turn activating other platelets. For example, Glycoprotein VI (GP6) is a 58-kD platelet membrane glycoprotein that plays a crucial role in the collagen-induced activation and aggregation of platelets. The shedding of GP6 can act as a marker representing that a person is at risk of myocardial infarction. In one embodiment, platelets obtained from a subject determined to have an elevated biomarker level (for example, GP6) can be infused into the non-human transgenic animal described above according to previously described methods, wherein the occurrence of a thrombotic event can be evaluated. In another embodiment, platelets obtained from a subject undergoing an anti-thrombotic treatment can be infused into the non-human transgenic animal described above according to previously described methods, wherein the occurrence of a thrombotic event can then be evaluated.
The following examples are provided to further illustrate the methods of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.
The association and dissociation kinetics of the GPIb α-VWF-A1 bond and the impact of fluid shear and particle size on these parameters can be determined by measuring the frequency and duration of transient adhesive events, known as transient tethers, that represent the smallest unit of interaction observable in a parallel-platelet flow chamber.
The generation of recombinant VWF-A1 protein (residues 1238 to 1472 of the mature, recombinant VWF) and its subsequent coupling to microspheres is performed as previously described (Doggett, et al., 2002). Proper size, purity, and disulfide bonding of all proteins is assessed by coomasie-blue staining of SDS-PAGE gels run under reducing and non-reducing conditions. Mass spectrometry is also employed to evaluate size and disulfide bonding pattern.
The resulting recombinant proteins are bound to polystyrene microspheres (goat anti-mouse IgG (FC); Bangs Lab, Inc., Fishers, Ind.) that were initially coated with a saturating concentration of mouse anti-6-HIS antibody as previously described. This coating method was found to be superior to direct covalent coupling of the VWF-A1 to the beads because it prevents significant loss in protein function. Estimation of the amount of VWF-A1 bound to the beads is determined using monoclonal antibodies generated in the inventors' laboratory against the human and murine A1 domains, mAb AMD-1 and mAb AMD-2, respectively, and a calibrated microbead system (Quantum Simply Cellular; Flow Cytometry Standards Corp., San Juan, P.R.) following the manufacturer's instructions.
In flow assays involving protein-coated microspheres, human or murine platelets purified by gel filtration are incubated with 10 mM sodium azide (NaN3), 50 ng/ml prostaglandin E1, and 10 μm indomethacin (Sigma Immunochemicals, St. Louis, Mo.) to reduce the possibility of activation and potential alterations in expression and/or distribution of GPIb α on their surface. Platelets are subsequently allowed to settle in stasis on Fab fragments of monoclonal antibodies that recognize either human (i.e., mAb 7E3) or murine (i.e., mAb NAD-1) αIIb/β3 in order to form a reactive substrate. The use of platelets in lieu of recombinant proteins or transfected cells as the immobilized substrate enables evaluation of GPIb α in its native form (i.e. correct orientation and proper post-translational modification). Platelet coverage of <10% bound in this manner can remain relatively unactivated for up to 30 minutes as evident by morphology on light microscopic examination (
To reduce the possibility of multiple bond formation that would result in a prolongation in interaction times between the beads and immobilized platelets, the lowest site densities of VWF-A1 capable of supporting these brief interactions is used, a value that corresponds to about 30 molecules μm2. At this site density, the formation of transient tethers between this receptor-ligand pair has distribution of bond lifetimes that obey first order dissociation kinetics. The duration of these interactions are measured by recording images from a Nikon X60 DIC objective (oil immersion) viewed at a frame rate of 235 fps (Speed Vision Technologies, San Diego, Calif.) and subjected to wall shear stresses (WSS) ranging from 0.5 to 3.0 dyn cm−2. The cellular off-rates are determined by plotting the natural log of the number of VWF-A1 coated microspheres that interacted as a function of time after the initiation of tethering, the slope of the line=−koff (s−1) which is the inverse of the bond lifetime. The force acting on the i tether bond was calculated from force balance equations and koff plotted as a function of these forces. An example of the measurement of the duration of a transient tether and estimation of off-rates as a function of WSS is demonstrated for WT human VWF-A1 (
To determine the structure and function of murine VWF-A1, its adhesive interactions with murine and human GPIb α, and whether the kinetics of this interaction mimic those reported in studies of its human counterpart, the domain was initially cloned by PCR from purified mouse genomic DNA. For the purpose of generating a mouse with a genetically modified VWF-A1 domain, a 100-kb P1 clone was obtained from screening a 129/Svj DNA genomic library (Genomic Systems, St. Louis, Mo.) by polymerase chain reaction (PCR) using primers directed against a 200 by region of exon 28. Sequence analysis of flanking regions (10 kb in size) as well as the A1 domain itself was performed and compared to those obtained from a BLAST search to confirm the fidelity of the clone. The deduced single-letter amino acid sequence of mouse VWF-A1 domain (M VWF) is shown compared to its human counterpart (H VWF) and encompasses amino acids 1260 to 1480 (
Although the amino acid sequence homology between the human and mouse VWF-A1 domains is high (about 85% identity), preliminary studies suggest that functional differences do exist between human and murine VWF-A1 domains. In Ristocetin-induced platelet aggregation assays (RIPA), platelet GPIb a binding to wild type human VWF or mouse VWF was analyzed in the absence or presence of ristocetin as described previously (Inbal, et al., 1993). In this method, platelet rich plasma (PRP) is placed in a clear cuvette containing a stir bar and inserted into the aggregometer. Platelet aggregation is induced by the addition of ristocetin. In this RIPA, it was observed that concentrations of this modulator that are known to cause agglutination of human platelets (about 1.0 mg/ml) had no such effect using murine PRP (
To better evaluate the above interactions and to compare functional relationships between human and murine VWF with GPIb α, VWF from human and mouse plasma was purified and its ability to mediate platelet adhesion in flow was determined. Multimer gel analysis did not reveal any differences between the two species, especially with regard to high molecular weight components (
Recombinant protein was expressed using a bacterial expression vector under the control of an inducible promoter (pQE9, Qiagen). Insertion of the murine fragment containing the majority of the VWF A1-domain (encoding for amino acids 1233 to 1471) into pQE9 produces an amino-terminal fusion protein containing 10 amino acids (including 6Xhistidine) contributed by the vector. After induction, inclusion bodies were harvested, washed, and solubilized according to previously published methods (Cruz et al., 2000). The solubilized protein was diluted 40-fold in 50 mM Tris-HCl, 500 mM NaCl, 0.2% Tween 20, pH 7.8 and initially purified over a Ni2+-chelated Sepharose (Pharmacia) column. To increase the yield of functional protein, the material purified from the Ni2+ column was absorbed to and eluted from a Heparin-Sepharose column (Amersham Pharmacia Biotech).
The highly purified protein was dialyzed against 25 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.8. SDS-PAGE analysis revealed a prominent protein band of about 34,000 Da under non-reducing conditions (
The protein was subsequently used in a series of in vitro flow chamber assays to assess function. Washed human or murine platelets (5×107/ml) were infused through a parallel plate flow chamber containing glass cover slips coated with either human VWF-A1 or mouse VWF-A1 protein (100 μg/ml final concentration) at a shear rate of 800 s−1. After 5 minutes of continuous flow, adherent platelets were quantified.
As shown in
To demonstrate that the presence of the N-terminus His tag does not appear to affect the function of the recombinant protein, the ability of a tagged vs. a non-tagged M VWF-A1 to support mouse platelet adhesion in flow was compared. In the case of the latter, the murine A1 fragment was inserted into pET-11b (Stratagene) and purified as previously described (Miura et al., 2000). The purified bacterial non-His tag protein was analyzed by SDS-PAGE (12.5%) and found to migrate in an analogous manner to its tagged counterpart under non-reducing and reducing conditions (
The A1 domains of human and mouse serve an identical purpose: to mediate primary attachment and translocation of platelets in flow. The crystal structure of the mouse A1 domain was solved using recombinant proteins (Fukuda, et al., 2005). The main chain schematic of this domain, with β-strands (arrows) and helices (coils), is shown in
Support for this hypothesis is provided by mutagenesis studies. By analyzing the data obtained from the crystal structure of the murine VWF-A1 domain, the inventors have identified several residues that may participate in interactions with GPIb α (
Thus, all that remains is to demonstrate that the kinetics of the interaction between the murine GPIb α and murine VWF-A1 are similar to its human counterpart and that mutations in man that cause functional alterations in platelet adhesion with VWF have the identical impact on the biophysical properties of the murine receptor-ligand pair.
To determine whether the kinetics of the murine GPIb α interactions with the murine VWF-A1 domain is similar to that of the human receptor-ligand pair, the dissociation of transient tethering events was measured using VWF-A1 coated beads (7 μm diameter) interacting with surface-immobilized platelets. The use of beads with one uniform size and shape permits determination of the relationship between wall shear stress and the force directly acting on the GPIb α-VWF-A1 tether bond (Fb), a parameter difficult to estimate for discoid-shaped objects such as platelets. A coating concentration of VWF-A1 was chosen (5 μg/ml corresponding to 30 molecules/μm2) that supported tether bond formation at wall shear stresses ranging from 0.5 to 3 dyn cm−2. Estimation of the site density of murine VWF-A1 on beads was performed using a monoclonal antibody generated in the inventors' laboratory designated as AMD-2. This antibody was made by immunizing Fischer 344 rats (3-4 months old) with recombinant WT protein. Following several injections of murine VWF-A1, serum was collected and screened by ELISA for anti-VWF-A1 antibodies. Spleens from animals with the highest antibody titers were harvested and splenocytes fused with Sp2/0 mouse myeloma cells (Kohler, et al., 1975).
Supernatants of hybridomas were screened for reactivity to mouse VWF-A1 by ELISA (
Analysis of the distribution of interaction times between human or murine VWF-A1 coated beads and their respective platelet substrates, as measured by high temporal resolution video microscopy, indicates that >95% of all transient tether bonds events fit a straight line, the regressed slope of which corresponded to a single koff (
Based on these results, it appears that the dissociation kinetics of murine GPIb α interactions with murine VWF-A1 are nearly identical to its human counterpart.
It is possible that minor differences may exist between murine and human VWF that would preclude one from studying human platelet behavior in a mouse model of thrombosis. However, the findings above that the estimated off-rate values and structure of these domains are similar suggest that one can investigate the role of the biophysical properties of the GPIb α-VWF-A1 bond in regulating platelet-VWF interactions in vivo using a mouse model. Both murine and human A1 crystal structures can be exploited to 1) identify candidate residues involved in the binding site for murine GPIb α and to determine their impact on the kinetic properties of this receptor-ligand pair, 2) identify residues that confer species specificity, and 3) ascertain whether insertion of known point mutations that cause type 2M and 2B VWD in man alter the kinetic properties of the murine A1 domain in a similar manner. Critical residues can be classified in terms of their impact on the cellular association and dissociation rate constants. Information obtained from these studies can be used to generate mice with mutant A1 domains in order to establish the degree in alteration in the kinetics of the GPIb α-VWF-A1 bond that is necessary to perturb hemostasis.
Similar critical structural elements exist in murine A1 domain to those identified in its human counter-part that contribute to the biophysical properties of the bond formed with GPIb α. Thus, to identify structural elements within the murine VWF-A1 domain that impact on the kinetics of interaction with GPIb α, the hypothesis that only minor structural alterations in this domain are responsible for its reduced ability to support interactions with GPIb α receptor on human platelets will be tested.
Site-specific mutagenesis of murine VWF-A1 domain may be performed to define residues that contribute to GPIb α binding as well as those that regulate this interaction. Studies will initially focus on amino acids that differ between human and mouse A1 that lie within the vicinity of the proposed GPIb α binding pocket.
To better define residues within murine VWF-A1 that are critical for binding of GPIb α on mouse platelets, mutations into VWF-A1 cDNA using a PCR-based strategy will be introduced and the resulting DNA will be sequenced to confirm the presence of the desired mutation(s). Mutations will be based on the murine A1 crystal structure and amino acid substitutions known to affect human VWF function such as those associated with Type 2M or 2B vWD (Tables 1-3). Several surface exposed residues have been identified within the murine A1 domain likely to participate in GPIb α binding. These are non-conserved residues in comparison to the human domain. Thus, these residues will be converted at first singly (then doubly and triply), into the murine VWF-A1 to those found in human VWF-A1. Residues are chosen based on surface-exposure on the front and upper surfaces of the domain as understood by modeling and crystal structure analysis.
Residues that perturb but do not abrogate platelet binding in the human VWF-A1 protein are shown in Table 2 and
Residues chosen based on their ability to abrogate or enhance interactions between human VWF and GPIb α are shown in Table 3.
Type 2B mutations will also be combined with those that dramatically shorten the bond lifetime to determine (increase koff) if these function-enhancing mutations can restore adhesion to that observed for WT VWF-A1.
Laminar flow assays will be performed to assess the impact of various mutations on platelet adhesion as well as the degree in alteration in the kinetic properties of the GPIb α-VWF-A1 bond.
Murine platelets will be purified as described above and stored in Tyrode's buffer containing 0.25% BSA, pH 7.4. For studies requiring human platelets, blood will be collected by venopuncture from healthy donors and cells obtained from centrifugation of PRP. All platelets will be used within 2 hours of purification.
To evaluate the impact of mutations on platelet adhesion, both human and murine platelets are perfused over high concentrations of murine VWF-A1 proteins (100 μg per ml) absorbed to glass cover slips in a parallel plate flow chamber at wall shear rates ranging from 20 to 1600 s−1. An enzyme-linked immunosorbent assay is utilized to ensure that an equivalent amount of recombinant WT or mutant protein is immobilized. The number of platelets that attach per unit area per minute and their velocity of forward motion (μm/s), termed translocation, is recorded on Hi-8 videotape using an inverted Nikon microscope with a plan 10× or 40× objective, respectively. In addition, whether the incorporated mutations alter the requirement for a critical level of hydrodynamic flow, termed the shear threshold phenomenon, to support interactions of platelets with the reactive substrate can be determined. It has been previously demonstrated that human attachment to immobilized VWF-A1 requires a minimum of >85 s−1 of WSR to initiate and sustain this interaction. This phenomenon has also been well described for selectin-dependent adhesion and is believed to rely on a balance between the number of times a receptor encounters the ligand over a defined period of time and the rate at which a bond can form, parameters affected by shear rate, association rate constant, and receptor-ligand concentrations (Chen, et al., 2001, Greenberg, et al., 2002). Once attached, however, the bond lifetime influences the velocity at which the cell will move on the reactive substrate in response to shear-induced force (Chen, et al., 1999). Thus, it is likely that several mutations may perturb platelet accumulation on surface-bound VWF-A1 by altering the level of shear flow required to promote platelet attachment as well as the translocation velocities of these cells. For example, it has been shown that the type 2B mutation, Ile1309Val (1309I>V), promotes greater platelet attachment at low shear rates and reduces their translocation velocities as compared to the WT substrate. Similar results were observed upon incorporation of the identical mutation into murine VWF-A1. Demonstration that GPIb α on murine platelets is responsible for mediating interactions with recombinant A1 domains can be confirmed by antibody blocking experiments.
To better ascertain the alteration in kinetics associated with the proposed mutations, the tethering frequency, translocation velocities, detachment profile, and off-rates of VWF-A1 coated beads (7 μm diameter) interacting with surface-immobilized platelets can be measured. As stated before, the use of beads with one uniform size and shape will permit determination of the relationship between wall shear stress and the force directly acting on the GPIb α-VWF-A1 tether bond (Fb), a parameter difficult to estimate for discoid shaped objects such as platelets. Platelet coverage >90% of the glass surface area is used in determining the tethering frequency (on rate driven phenomenon), translocation velocities (correlates with off-rate) and resistance to detachment forces (measure of bond strength) of VWF-A1 coated beads in flow. It was demonstrated that comparison of cellular on-rates and apparent bond strengths between WT and mutant forms of VWF-A1 can be achieved by limiting the concentration of these molecules to prevent multiple bond formation, a process that can mimic an enhancement in either of these kinetic parameters. By using a similar strategy, whether the proposed mutations will alter the apparent on-rate of the GPIb α-VWF-A1 bond may be determined by evaluating the frequency of transient tethering events between microspheres coated with low site densities of VWF-A1 proteins and a platelet substrate. Results are expressed as the percentage of beads (per 10× field) that paused, but did not translocate, over a range of wall shear rates that support such interactions (20 to 400 s−1). Tethers per minute are divided by the flux of beads near the wall per minute to obtain the frequency of this adhesive interaction. Only one tethering event per bead is counted during the observation period.
For determining translocation velocities, beads (1×106/ml), coated with a saturating concentration of VWF-A1 protein are infused into the parallel-plate flow chamber at 1.0 dyn cm−2 and allowed to accumulate for 5 minutes. Subsequently, the wall shear stress is increased every 10 seconds to a maximum 36 dyn cm−2 and the velocities of the beads determined.
For detachment assays, beads (1×106/ml), coated with the minimum but equal amounts of VWF-A1 required to support translocation, are infused into the parallel-plate flow chamber at 1.0 dyn cm−2 and allowed to accumulate for 5 minutes. Subsequently, the wall shear stress is increased every 10 seconds to a maximum 36 dyn cm−2. The number of beads remaining bound at the end of each incremental increase in wall shear stress is determined and expressed as the percentage of the total number of beads originally bound. Using this strategy, it was found that type 2B mutations do not strengthen, and in fact may even weaken the interaction between GPIb α and VWF-A1 as suggested by the increase in reactive compliance as compared to the native complex (Table 1). In all studies, video images are recorded using a Hi8 VCR (Sony, Boston, Mass.) and analysis performed using a PC-based image analysis system (Image Pro Plus).
For determining the kinetics of dissociation, the duration of transient tethers between murine VWF-A1 coated microspheres and immobilized murine platelets is measured as described herein. MC simulations are run and estimates of koff fit to the Bell model by standard linear regression to obtain the intrinsic off-rate (k0off) and the reactive compliance σ. Results are compared for all mutations to determine their impact on these kinetic parameters (i.e.—increase or shorten the bond lifetime (k0off) and/or increase or decrease the susceptibility of the bond to hydrodynamic forces (σ).
These experiments complement recent work on identifying residues in human VWF-A1 domain critical for interacting with the GPIb α. Moreover, they allow for delineation of its binding site in murine VWF-A1. This work elucidates the role of the biophysical properties of this receptor-ligand pair in regulating platelet-VWF interactions in vivo. Furthermore, it will pave the way for the generation of mice with comparable types of human VWD (i.e. type 2B) and may even permit the study of human platelets in a mouse model of thrombosis.
Although there is no guarantee that the introduction of mutations will not significantly perturb protein structure and thus function, the ability of murine VWF-A1 specific mAbs to recognize mutant proteins should clarify this matter. Similarly, the gain/loss of function experiments involving swapping of residues between human and mouse VWF-A1 will also prove useful in avoiding this pitfall.
To know whether the regions flanking the mouse A1 domain are important in mediating interactions with GPIb α, full-length mouse VWF will be inserted into a mammalian expression vector and expressed in COS-7 cells (Cooney, et al., 1996). Mutations found to be critical for binding will be inserted into the full-length construct. A recombinant protein containing the A1-A2-A3 domains will be generated initially. This will be accomplished using a baculovirus expression system as demonstrated for GPIb α.
The recent results on the structure of GPIb α and its complex with VWF-A1 domain has not only confirmed the major binding site for this platelet receptor, but has shed new light into the mechanism by which type 2B mutations may enhance this critical interaction. As shown in
Notably, the cellular off-rates of these quantal units of adhesion for the WT human and mouse proteins (
Based on these results and the results in Example 1 above, it appears that the dissociation kinetics of murine GPIb α interactions with murine VWF-A1 are nearly identical to its human counterpart and that type 2B mutations also prolong the bond lifetime of this interaction as seen in man.
The type 2B mutation 1309I>V was incorporated into recombinant human VWF-A1 containing either the type 2M mutation Gly1324Ser (1324G>S) that completely abolishes adhesion or the function reducing mutation His1326Arg (1326H>R) and the ability of these doubly mutated proteins to support human platelet adhesion in flow was determined. In comparison to WT, an about 3-fold increase in wall shear rate is required to promote platelet attachment to human VWF-A1 containing Arg at 1326 (
To determine whether the 1309 mutation would also prolong the lifetime of the interaction with GPIb α, the distribution of interaction times was analyzed between VWF-A1 coated beads and surface-immobilized platelets at a wall shear stress of 1 dyn cm−2. Remarkably, a 2-fold reduction in koff was noted (from 15.6 to 8.5 s−1) as compared to the single, function-diminishing mutation (
VWF contributes to human health and disease by promoting adhesive interactions between cells (Whittaker, C. A., et al., 2002). The VWF-A1 domain is thought to play a critical role in hemostasis by initiating the rapid deposition of platelets at sites of vascular damage by binding to the platelet receptor glycoprotein Ib α (GPIbα) at high shear rates (Roth, G. J., et al., 1991, Cruz, M. A., et al., 1993, Sugimoto, M. et al., 1991, Pietu, G. et al., 1989). Although congenital absence of VWF in humans has established a role for this plasma glycoprotein in hemostasis (Sadler, J. E. et al. 2006), the contribution of its A1 domain in clot formation has been questioned in a mouse model of vascular injury (Denis, C. et al., 1998).
Murine plasma VWF or its A1 domain fails to support significant interactions with human platelets (and likewise human VWF with murine platelets) under flow conditions. Atomic models of GPIb α-VWF-A1 complexes suggest that the structural basis for this behavior arises primarily from an electrostatic “hot-spot” at the binding interface. Introduction of a single point mutation within this region of murine VWF-A1 is sufficient to switch its binding specificity from murine to human platelets. In addition, introduction of a single point mutation within the electrostatic “hot-spot” region of human VWF-A1 is sufficient to switch its binding specificity from human to murine platelets. Moreover, mice possessing the 1326R>H mutation in their VWF have a bleeding phenotype distinct from VWF-deficient animals, and can be corrected by the administration of human platelets. Mechanistically, mutant animals can generate but not maintain thrombi at sites of vascular injury, whereas those infused with human platelets can form stable thrombi, a process that relies on GPIb α-VWF-A1 interaction. Thus, interspecies differences at protein interfaces can provide insight into the biological importance of a receptor-ligand bond, and aid in the development of an animal model to study human platelet behavior and drug therapies.
Generation of VWF1326R>H mice. The VWF1326R>H targeting vector (
Analysis of VWF transcripts, antigen levels, multimers, and collagen binding. Detection of transcripts from the A1-A2-A3 domains of murine VWF was performed by RT-PCR. Briefly, mRNA was isolated from lung tissue harvested from either homozygous VWF-A11326R>H mice or aged-mated WT littermate controls (Oligotex™, Qiagen, Germantown, Md.). Generation of cDNA and PCR-amplification of desired transcripts was performed using SuperScript™. One-Step RT-PCR (Invitrogen Corp., Carlsbad, Calif.) and oligos specific for the A domains of VWF.
Functional factor VIII levels were determined by a mechanical clot detection method using the STA automated coagulation analyzer (Diagnostica Stago, Parsippany, N.J.). A log-log calibration curve was established by measuring the activated partial Thromboplastin time (aPTT) of varying dilutions of reference plasma. The aPTT of a 1:10 dilution of sample plasma mixed with factor VIII deficient plasma was determined, compared to the calibration curve, and the activity expressed as a percent of normal.
Evaluation of VWF antigen levels was performed as previously described (Denis, C. et al., 1998). For multimer analysis, plasma from sodium citrate treated whole blood was diluted 1:5 in electrophoresis sample buffer (final concentration 10 mM Tris-HCl pH 8.0, 2% SDS, 1 mM EDTA) and heated at 56° C. for 30 minutes. Electrophoresis was carried out overnight (64 volts, 15° C.) through a horizontal SDS-agarose gel in 1.2% agarose (Ruggeri, Z. M., et al., 1981). The gel was then electrophoretically transferred (150 mA, 90 minutes) to Immobilon (Millipore, Billerica, Mass.) followed by blocking (2 hours) with 5% powdered milk in TBST (Tris HCl pH 8.0, 0.15M NaCl, 0.05% Tween-20). The membrane was incubated with a 1:500 dilution of rabbit anti-human VWF antiserum (Dako, Fort Collins, Colo.) for 1 hour, washed in TBST, and then incubated with a 1:10,000 dilution of HRP-conjugated mouse anti-rabbit IgG (Calbiochem, Merck KGaA, Darmstadt, Germany). Bands were subsequently detected by a chemiluminescence system (GE Healthcare, Waukesha, Wis.). For comparison, a sample containing pooled human plasma from healthy individuals or patients with type 2B VWD was also loaded on the gel. Binding of VWF to surface-immobilized collagen was performed as previously described (Smith, C. et al., 2000). Briefly, 100 μg/ml of acid soluble type I collagen from human placenta (Sigma, St. Louis, Mo.) was added to a 96 well microtiter plate and allowed to incubate overnight (4° C.). After washing and blocking with TBS containing 3% BSA and 0.05% Tween 20, varying concentrations of platelet poor plasma harvested and pooled from WT, homozygous VWF1326R>H, and VWF deficient mice was added to the wells and incubated for 1 hour (37° C.). Wells were then washed and bound VWF detected by an ELISA as described above.
Ex vivo platelet adhesion studies. Experiments were performed in a parallel-plate flow chamber as previously described (Offermanns, S., et al., 2006). For studies involving plasma VWF, a polyclonal anti-VWF antibody (Dako) was absorbed overnight (4° C.) to a six well tissue culture plate. Subsequently, the plate was washed and non-specific interactions blocked by the addition of TBS containing 3% BSA, pH 7.4 (1 hour, 37° C.). Human or murine plasma obtained from heparinized whole blood was added and the plates placed at 37° C. for an additional 2 hours. Generation, purification, and surface-immobilization of recombinant VWF-A1 proteins was performed as previously described (Doggett, T. A. et al., 2002). Both human and murine VWF-A1 constructs consist of amino acid residues 1238 to 1471, with a single intra-disulfide bond formed between residues 1272 and 1458 and were generated in bacteria. Citrated whole blood (150 μl) collected via cardiac puncture from anesthetized homozygous VWF1326R>H or WT mice or from venopuncture from human volunteers was perfused over the immobilized substrates at a wall shear rate of 1600 s−1 for 4 minutes, followed by washing with Tyrode's buffer under the identical flow conditions. The number of platelets attached per unit area (0.07 mm2) and translocation velocities were determined by off-line analysis (Image-Pro Plus, Media Cybernetics). For GPIb α inhibition studies, the function-blocking mAb 6D1 (20 μg/ml) or mAb SZ2 (20 μg/ml; Beckman Coulter, Brea, Calif.) was added to anticoagulated human blood for 30 minutes prior to use. Experiments were performed in triplicate on two separate days. An ELISA was used to ensure equivalent coating concentration of plasma and recombinant proteins (Denis, C. et al., 1998).
In vivo thrombus formation. Administration of anesthesia, insertion of venous and arterial catheters, fluorescent labeling and administration of human platelets (5×108/ml), and surgical preparation of the cremaster muscle in mice have been previously described (Doggett, T. A. et al., 2002, Diacovo, T. G., et al., 1996). Injury to the vessel wall of arterioles (about 40-65 μm diameter) was performed using a pulsed nitrogen dye laser (440 nm, Photonic Instruments) applied through a 20× water-immersion Olympus objective (LUMPIanFI, 0.5 NA) of a Zeiss Axiotech vario microscope. Mouse platelet- and human platelet-vessel wall interactions were visualized using either bright field or fluorescence microscopy. The latter utilized a fluorescent microscope system equipped with a Yokogawa CSU-22 spinning disk confocal scanner and 488 nm laser line (Revolution XD, Andor™ Technology). The extent of thrombus formation was assessed for 2 minutes post injury and the area (μm2) of coverage determined (Image IQ, Andor™ Technology). For GPIb α or αIIb β3 inhibition studies, the function-blocking mAb 6D1 or 7E3 (20 μg/ml), respectively (from B. Coller, Rockefeller University), was added to purified human platelets for 30 min prior to administration.
Tail bleeding assay. Bleeding times were measured in 7-week old mice after amputating 1 cm of the tail tip as previously described (Denis, C. et al., 1998). In studies involving human platelets, platelet concentrates were obtained from Columbia Presbyterian Hospital Blood Bank, washed and resuspended in normal saline (1.5×109/300 μl) before administering through a catheter inserted into the right internal jugular vein. Tail cuts were performed 5 minutes after completion of the infusion of platelets. PLAVIX and ReoPro™ were obtained from the research pharmacy at Columbia University Medical Center. For studies involving PLAVIX, animals received a 50 mg/kg oral dose of the drug the day before and 2 hours prior to the administration of human platelets. ReoPro™ was given initially as an intravenous bolus (0.25 mg/kg) 5 minutes after the administration of human platelets, followed by a continuous infusion (0.125 μg/kg/min) as per the manufacturer's recommendations.
Structural Modeling. There are three crystal structures of the GPIb α-VWF-A1 complex: two are WT except for mutated N-glycosylation sites in GPIb a (Fukuda, K. et al., 2005), and one is a gain-of-function mutant (Huizing a, E. G. et al., 2002). The structures have only small differences that are not the result of the presence of mutations or botrocetin binding (Fukuda, K., et al., 2005). Both N-glycosylation sites in human GPIb α lie on the well-ordered upper ridge of the LRR, 18 Å and 27 Å (Cα-Cα) from the nearest VWF-A1 residue, so their absence is unlikely to affect the structure of the complex. Murine GPIb α has no predicted N-glycosylation sites.
Human GPIb α contains sulfated tyrosines implicated in binding VWF within an acidic loop just C-terminal to the sequence included in the crystal structures. Murine GPIb α has a predicted sulfation site in the same loop, so that the differential binding of human vs. murine GPIb α to VWF-A1 is also likely to be small. The interfacial regions are otherwise highly conserved between species, with the exception of three salt bridges (See
A consensus model of the human complex was used to build the murine model. Murine A1 onto human A1 was first overlaid by fitting the central β-sheets (RMSD 0.3 Å; within experimental error); the only notable difference is the location of helix α 4, which is shifted by 2-3 Å away from the GPIb α interface in the mouse owing to a larger residue on the buried face of this helix. For the GPIb α model, only the side-chains were altered, since the human and murine LRRs have identical lengths. Consensus rotamers with minimal steric clashes were chosen, followed by manual adjustment where necessary to create sensible van der waals interactions and H-bonding, using TURBOFRODO (Bio-Graphics, Marseille, France). Molecular overlays were optimized using LSQKAB (Collaborative Computational Project, 1994); molecular figures were created using MOLSCRIPT (Esnouf, R. M., 1997)) and OPENGL (Khronos Group, Beaverton, Oreg.)
Statistics. An unpaired, two-tailed Student t test was used for multiple comparisons.
Because the interaction between GPIb α and VWF-A1 is a prerequisite for effective thrombus formation in the arterial circulation, the ex vivo ability of surface-bound murine plasma VWF or its recombinant A1 domain (rVWF-A1) to support human platelet adhesion under physiologically relevant flow conditions was first tested using a parallel-plate flow chamber at a shear rate exceeding 1000 s−1 (Ruggeri, Z. M. et al., 2006). The adhesive properties of VWF are tightly regulated such that it preferentially binds to platelets only when immobilized to sites of vascular injury and under hydrodynamic conditions encountered on the arterial side of the circulation (Sakariassen et al., 1979, Ruggeri, Z. M. et al., 2006). Perfusion of human whole blood over murine plasma VWF or rVWF-A1 resulted in limited platelet deposition (10 to 25%, respectively) as compared with same-species controls (
In order to gain insight into the structural origins of this species incompatibility, models of murine-murine and human-murine GPIb α-VWF-A1 complexes were built based on the crystal structures of the human complex (Fukuda, K., et al., 2005, Dumas, J. J. et al., 2004, Huizing a, E. G. et al., 2002) and human and murine VWF-A1 (Fukuda, K., et al., 2005) (
The A1 domain comprises a Rossmann-like fold with a central, mostly parallel β-sheet flanked on both sides by α-helices (Fukuda, K., et al., 2005). Human and murine VWF-A1 share considerable sequence (86% identity) and structure homology; in fact, the β-sheets of both species are identical within experimental error (a root mean square difference of 0.33 Å for Cα-atoms). The only major difference is the location of helix a 4 (nomenclature as previously described in Dumas, J. J. et al., 2004), which is shifted 2-3 Å away from the GPIb α binding site in the mouse, owing to a difference in a buried hydrophobic residue (
In the complexes, the major contact region involves the “β-switch” region (residues 227 to 241 in the C-terminal flank of GPIb α), which forms a β-hairpin that augments the β-sheet of the VWF-A1 domain. On its other side, this region of GPIb α packs tightly against the concave face of the LRR, which highly constrains it movement. Residues in mouse and human are mostly invariant on both sides of this interface. Notable exceptions are at position 1326 in VWF-A1, which is histidine (H) in humans versus an arginine (R) in mouse, and at position 238 in GPIb α, which is alanine (A) in humans versus an aspartic acid (D) in mouse (
In the human GPIb α-murine VWF-A1 interspecies complex, it is believed that the two positively charged residues (GPIb α K231 and VWF-A1 R1326) create an electrostatic clash that impedes binding, owing to the lack of a negatively charged group at position 238 (
To explore the importance of the electrostatic mismatches in destabilizing the interspecies complexes, human residues were substituted into murine rVWF-A1 at positions 1326 (R>H), 1330 (E>G), and 1370 (S>G), and the ability of the mutant proteins to support human platelet accumulation under flow was analyzed. As expected, amino acid substitutions at positions 1330 (predicted to remove a salt-bridge) and 1370 (predicted to have no effect) failed to promote the interaction between murine rVWF-A1 and human GPIb α. However, the 1326R>H mutation, which eliminates the electrostatic clash with K231, rendered murine A1 capable of supporting interactions at a level comparable to its wild-type (WT) human counterpart (
In order to determine the ability of full-length murine VWF containing the 1326R>H mutation (VWF1326R>H) to support human platelet interactions and ultimately thrombus formation in vivo, mice were genetically modified to express VWF1326R>H (
Hemostasis relies on platelet adhesion and activation at sites of vascular injury, which ultimately results in the formation of a hemostatic plug. To demonstrate the importance of VWF-A1 in this process, bleeding times for mice possessing the 1326R>H mutation were measured by removing 1 cm of distal tail (
To gain insight into how the 1326R>H mutation alters hemostasis, murine platelet adhesion at sites of vascular damage was evaluated in vivo. A laser-induced vascular injury model was utilized to initiate platelet deposition in arterioles located in the microcirculation of the cremaster muscle of WT and homozygous mutant mice (Furie, B. et al., 2005). Although VWF1326R>H animals can initially form thrombi that fill the vessel lumen, they rapidly dissipated under the prevailing hydrodynamic conditions (
To demonstrate that the removal of the electrostatic clash between residues 1326 and 231 in murine VWF-A1 and human GPIb α, respectively, promotes substantial interactions between this chimeric receptor-ligand pair, human whole blood was perfused over surface-immobilized plasma VWF obtained from mice homozygous for VWF1326R>H. Remarkably, mutant murine VWF bound human platelets at levels comparable with its human counterpart (
By contrast, the antibody SZ2 that recognizes the anionic-sulfated tyrosine sequence of GPIb α (residues 276 to 282) had a minimal affect on platelet accumulation, results consistent with a previous report (Fredrickson et al., 1998). The A1 domain also possesses the ability to support the movement of attached platelets in the direction of the prevailing hydrodynamic force owing to rapid and reversible interactions with GPIb α (Savage et al., 1996, Doggett, T. A. et al., 2002). Translocation velocities of human platelets on either human or mutant murine VWF were therefore compared. Translocation velocities of human platelets on either homozygous mutant murine or native human plasma VWF were also similar (3.5±0.1 μm/sec vs. 3.2±0.1 μm/sec, respectively; mean±s.e.m, n=4), demonstrating that VWF1326R->H functions in a manner indistinguishable from its human counterpart.
The ability of murine VWF1326R>H to support human platelet adhesion in vivo was then tested. The ability of human platelets to preferentially support thrombus formation was monitored simultaneously by labeling purified human cells with BCECF ex-vivo, and mouse platelets with rhodamine 6G by intravenous administration. Fluorescently-labeled human platelets were infused continuously via a catheter inserted into the femoral artery, resulting in a high local concentration of these cells within the microcirculation of the cremaster muscle. Their behavior in response to laser-induced vascular injury was monitored in real-time using confocal intravital microscopy (Furie, B., et al., 2005). Upon induction of laser damage to the vessel wall of arterioles in mice homozygous for VWF1326R>H human platelets rapidly adhered to the site of injury, forming large thrombi composed mainly of human cells (91.7±1.2%; mean±s.e.m) (
Consistent with the critical role of platelet GPIb α in mediating interactions with VWF-A1, pre-treatment of human platelets with mAb 6D1 or with mAb AP-1 greatly reduced thrombus size in the vasculature of VWF1326R>H mice (265±125 μm2 and 198±103 μm2, respectively; mean±s.e.m.) (
Although GPIb α initiates platelet deposition at arterial shear rates, it is ultimately the platelet integrin αIIb β3 that supports thrombus growth by promoting platelet-platelet interactions. The contribution of human αIIb β3 in this process is demonstrated by the ability of the function blocking antibody 7E3 to also limit thrombus size (529±150 μm2) (
In summary, these studies demonstrate how one can effectively utilize atomic models of interspecies complexes to identify a binding hot spot where a disproportionate amount of the binding free energy is localized, such that a single amino acid substitution significantly affects the interaction (Bogan, A. A., et al., 1998), and in this case switches species specificity. Moreover, a subtle and localized change of this nature limits the possibility of inducing structural perturbations that impact on the function of other domains contained within VWF. These data show that human platelet adhesion to VWF1326R>H is dependent on GPIb α binding to VWF-A1, with other potential ligands for this receptor playing a subservient role in this process (Bergmeier, W. et al., 2006). The reliance of human thrombus formation on the integrin αIIb β3, as well as the ability of the FDA approved drugs PLAVIX and ReoPro™ to impair human platelet-mediated hemostasis indicate that downstream adhesive and activation events known to be critical for clot formation and stability are intact in the mutant VWF animals. Thus, the VWF1326R>H knock-in mice will prove useful in the preclinical evaluation of new antithrombotic therapeutics designed ultimately for human use. These results also have implications for advancing both knowledge of human platelet biology and in preclinical testing of antithrombotic therapies in vivo.
Kinetic evaluation of mutations associated with type 2B and platelet-type VWF suggests that the intrinsic properties of the GPIb α-VWF-A1 tether bond contribute to the regulation of platelet interactions with VWF. This is also supported by preliminary studies investigating the impact of botrocetin on the biophysical properties of this receptor-ligand pair. Thus, by using the information obtained in Example 2, mutations can be incorporated into the murine A1 domain of the VWF gene that increase or decrease the intrinsic on- and off-rates by varying degrees in order to truly understand the importance of these kinetic parameters in controlling platelet adhesion. Moreover, the role of the minor binding site, where the majority of type 2B mutations have been identified, can be further delineated by combining such mutations with those that significantly shorten the lifetime of interaction between GPIb α and VWF-A1. Results indicate that substitution of the murine residue Arg at position 1326 for His at the same location in the human A1 domain results in a diminished on-rate as manifested by the increased requirement for shear flow to promote attachment and a significant increase in koff (shortening of bond lifetime). Subsequent incorporation of the type 2B mutation 1309I>V into this mutant domain significantly reverses the functional defect in adhesion and returns the off-rate closer to that observed for the WT domain. Similar results have been obtained with murine VWF-A1 in which Arg was replaced by His at residue 1326. Thus, introduction of these two mutations separately and then together into the mouse VWF gene will be the initial focus.
Generation of mice that incorporate mutations into their A1 domain that significantly shorten the bond lifetime will be present with prolonged bleeding times and will be resistant to thrombus formation, while the additional incorporation of a type 2B mutation will correct these abnormalities by prolonging the tether bond lifetime to that observed for the WT domain. This should allow for sufficient time to form multiple bonds between platelets and VWF deposited at sites of vascular injury.
By performing a detailed kinetic analysis of mutant VWF-A1 domains prior to the generation of animals with the identical substitutions in amino acids, the likelihood of altering the interaction between platelets and VWF in a similar manner is greatly increased. The role of the intrinsic properties of the bonds formed between this receptor-ligand pair under complex hemodynamic conditions (i.e. in vivo) may be studied.
1309I>V single mutant or 1309I>V and 1326R>H double mutant mice were generated as follows. A 100-kb P1 clone containing the majority of the VWF gene (Genomic Systems, St. Louis, Mo.) was obtained. Digestion with Bam HI resulted in a about 5.3 kb fragment containing part of intron 28 (including the splice sites), all of exon 28 and part of intron 29 which was the inserted into the pSP72 vector (Promega Corp., Madison, Wis.). This was subsequently digested with Bam HI and Eco R5 to yield a 2.9 kb (including exon 28) and a 2.4 kb fragment, designated Arm 1 and 2, respectively, both of which were subcloned back into pSP72 vector. This facilitated site-directed mutagenesis of the A1 domain contained within Arm 1. In addition, the 3′ end of Arm 1 was extended 2 kb by PCR. Subsequently, Arms 1 and 2 were inserted into a lox P-targeting vector as shown below (
R1 embryonic stem cells derived from a 129/Sv X 129/Sv-CP F1 3.5-day blastocysts were electroporated with 25 μg of linearized targeting construct and selected in both G418 (26 μmol/L) and gancyclovir (0.2 μmol/L). Genomic DNA from resistant clones were digested with EcoRI or KpnI, and analyzed by Southern blot hybridization with probe “a” or “b”, respectively, to determine if the construct was appropriately targeted (
Other mutant mice may be generated using any of the vector targeting strategies disclosed herein.
For platelet counts, whole blood will be collected into heparinized tubes and 100 μl volumes will be analyzed on a Hemavet (CBC Tech, Oxford, Conn., USA) Coulter Counter. The multimeric structure of murine VWF will be assayed by using the Pharmacia Phast Gel System (Pharmacia LKB Biotechnology). Briefly, samples diluted in 10 mmol/L Tris/HCl, 1 mmol/L EDTA, 2% SDS, 8 mol/L urea, and 0.05% bromophenol blue, pH 8.0, will be applied to a 1.7% agarose gel (LE, Seakem, FMC Bioproducts) in 0.5 mol/L Tris/HCl, pH 8.8, and 0.1% SDS with a stacking gel consisting of 0.8% agarose (HGT, Seakem) in 0.125 mol/L Tris/HCl, pH 6.8, and 0.1% SDS. After electrophoresis the protein will be transferred to a polyvinylidene fluoride membrane (Immobilon P, Millipore) by diffusion blotting for 1 hour at 60° C. The membrane will be blocked with 5% nonfat dry milk protein solution for 1 hour at room temperature. After washing with PBS/T, pH 7.4, the blot will be incubated with a polyclonal antibody raised in rabbits against murine VWF at a dilution of 1:500, washed, and incubated with a goat anti-rabbit horseradish peroxidase (Sigma) diluted 1:2000 in PBS/T. After three washes with PBS/T, the membrane will be incubated with the substrate solution (25 mg 3,3′-diaminobenzidine tetrahydrochloride (Sigma) in 50 mL PBS with 10 μL 30% H2O2). The enzyme reaction will be stopped by washing the membrane with distilled water.
This assay provides an indirect measure of the ability of platelets and VWF to support hemostasis by interacting with the injured vessel wall. It also indirectly determines the function of multiple receptors and ligands on platelets that are required to form a hemostatic plug. That said, it provides direct evidence that the bleeding defect in the animals can be corrected by the administration of human platelets (
One goal of the work is to generate mice with mutant A1 domains that alter the kinetics of its interactions with GPIb α on mouse platelets. The first mutation introduced was the substitution of histidine for arginine at position 1326. This mutation was chosen based on the crystal structure analysis of the mouse and human A1 domains, which suggested that the location of this amino acid is central to GPIb α binding. Mice bearing this mutation are viable and demonstrate a bleeding phenotype, albeit not as severe as those lacking VWF (VWF KO) (
Blood will be collected by cardiac puncture from anesthetized mice and thrombin-mediated activation prevented by the addition of hirudin (160 U/ml, Sigma) (Andre, et al., 2002). Platelet adhesion to a glass cover slip coated with 100 μg/ml of equine tendon collagen (Helena Laboratories, Beaumont, Tex.) will be assessed in a parallel-plate flow chamber apparatus. Whole blood will be infused through the chamber at a wall shear rate of 1600 s−1 for 3 minutes. As platelet adhesion under these homodynamic conditions requires VWF deposition and subsequent interactions between its A1 domain and GPIb α, the extent of platelet coverage should provide a gross estimate of the degree of impairment between this receptor-ligand pair. In addition, plasma VWF will be purified from these animals to evaluate platelet attachment to this immobilized substrate in flow. The surface area covered by adherent platelets at the end of each experiment will be determined (Image Pro Plus software) and expressed as a percentage of platelet coverage using blood from WT littermates. To better isolate GPIb α-VWF A1 interactions, identical experiments can be performed using platelets isolated from αIIb β3 deficient animals and reconstituting them in platelet poor plasma from the mutant A1 knock-in mice.
In addition to the in vitro work, platelet-VWF interactions in vivo will also be studied using intravital microscopy (Falati et al., 2002). This is accomplished by using a murine model of thrombosis that involves laser-induced injury to micro-vessels contained within the mouse cremaster muscle. The surgical preparation of animals, insertion of lines for administration of cells and anesthesia, will be performed as previously described (Coxon et al., 1996). Human platelets will be collected and prepared, fluorescently labeled, perfused into a mouse model (such as the transgenic mouse of the current invention) via an intravenous injection (Pozgajova et al., 2006).
Insertion of lines for administration of cells and anesthesia. Briefly, the skin covering the scrotum will be incised and the intact cremaster muscle dissected free from the connections to the subcutis. The mouse will be placed on a custom-built plexiglass board, and the exposed muscle positioned on a heated circular glass coverslip (25 mm) for viewing. The muscle will be slit along the ventral surface (using a thermal cautery), the testis excised, and the muscle spread across the coverslip with attached sutures (6/0 silk) (
The segment of an arteriole will be visualized and recorded as “pre-injury”. Subsequently, endothelial damage will be induced via a pulsed nitrogen dye laser at 440 nm applied through the microscope objective using the Micropoint laser system (Photonics Instruments, St. Charles, Ill.). The duration of exposure of the endothelium to the laser light will be varied to produce either a mild injury that supports the formation of a platelet monolayer or significant injury resulting in thrombus formation. The region of interest will then be videotaped and analyzed as described below.
For example, vascular damage can subsequently be induced in arterioles contained within the cremaster muscle of mice by either 1) a pulsed nitrogen dye laser applied through the objective of an intravital microscope (
For studies analyzing the dynamic interactions between individual platelets and the injured vessel wall (attachment, translocation, and sticking), cells purified from genetically altered mice will be labeled ex-vivo with a derivative of carboxyfluorescein (BCECF, Molecular Probes) (Diacovo, et al., 1996). A human thrombus generated in the mutant mouse can also be visualized by this technique, thus allowing one to distinguish human platelets from endogenous circulating mouse platelets upon illumination with an appropriate laser light source (see
A role for GPIb α as well as the collagen (α2 β1) and the fibrinogen (αIIb β3) receptors can be evaluated by using function-blocking antibodies to these proteins. Moreover, FDA approved anti-thrombotics (such as clopidogrel and tirofiban) can be used to examine whether the drugs inhibit human platelets from forming a thrombus in vivo, validating the mouse model for use in pre-clinical screening. The effect that antibodies and drugs have on altering the interaction between GPIb α-VWF-A1 interaction is determined by evaluating whether thrombus formation in the proposed mice is reduced or augmented upon arteriolar injury (
For all experiments, the centerline erythrocyte velocity (Vrbc) is measured using an optical doppler velocimeter (Microcirculation Research Institute, Texas A&M College of Medicine, College Station, Tex.) prior to and after inducing the injury. Shear rate (SR) is then calculated based on Poiseulle's law for a Newtonian fluid: SR=8(Vmean/Dv), where Dv is the diameter of the vessel and Vmean is estimated from the measured Vrbc (Vmean=Vrbc/1.6).
Thrombus formation can be characterized as follows: (1) Early individual platelet interactions with the damaged vessel wall (number of fluorescently labeled human platelets that attach during the first minute post-injury); (2) time required for thrombus generation of >20 μm diameter; (3) the ability of thrombi to remain at the initial site of vascular injury and not break free (measure of stability); (4) time until vessel occlusion; and (5) site of vessel occlusion, that is, at the site of injury or downstream from it. Platelet-vessel wall interactions can be viewed through 40× or 60× water immersion objectives. To standardize in vivo conditions, the velocity of flowing blood (shear rate) pre-injury is determined by measuring the centerline erythrocyte velocity (Vrbc) using an optical doppler velocimeter. Shear rate (SR) can then be calculated based on Poiseulle's law for a Newtonian fluid: SR=8×(Vmean/Dv), where Dv is the diameter of the vessel and Vmean is estimated from the measured Vrbc (Vmean=Vrbc/1.6). Vessel and thrombus diameters are measured using imaging software (ImagePro Plus).
Function-blocking monoclonal antibodies 6D1 (anti-human GPIb α), 6F1 (anti-human α2 β1) and 7E3 (anti-human αIIb. β3) have been generously provided by Dr. Barry Cotler (Rockefeller University, NY). All antibodies are converted to F(ab′)2 fragments to limit Fc receptor interactions in vivo. An intravenous dose of 10 μg/g body weight is given approximately 10 minutes after the injection of human platelets but 30 minutes prior to inducing vascular injury. Non-function blocking antibodies to these receptors are used as negative controls and administered under identical conditions. To ensure optimal ligand availability for the collagen and fibrinogen receptors on human platelets, mice possessing the A1 domain mutation have been bred with animals genetically deficient in α2 β1 or αIIb β3. Thus, endogenous platelets in these animals not only have a reduced ability to interact with the VWF-A1 domain, but also are incapable of binding to collagen or fibrinogen, respectively. Although human platelets have been shown to circulate in mice for a maximum of 24 hours, we can ensure that an equivalent percentage of human platelets are present at the time of vascular injury under each experimental condition (Xu et al., 2006). Thus, 50 μl is obtained from an inserted venous catheter and flow cytometric analysis will be performed to determine the percentage of circulating fluorescently-labeled human platelets.
In comparison to aspirin, clopidogrel (Plavix) is the second most commonly used anti-thrombotic drug that targets one of the ADP receptors (P2Y12) on platelets, causing irreversible inhibition (Hankey, et al., 2003). ADP is a potent mediator of platelet activation and aggregate formation, and thus considerable effort and funds have been devoted to inhibiting this activation pathway in platelets. Clopidogrel was approved by the FDA in 1997 for clinical use and was found to be of benefit in the secondary prevention of major vascular events in patients with a history of cerebrovascular and coronary artery diseases and major cardiac events post coronary artery stent placement (Gachet et al., 2005). Disadvantages of this drug are: 1) It must be metabolized in the liver to generate an active metabolite, thus limiting its effectiveness in acute settings, and 2) irreversible inhibition that results in a marked prolongation of bleeding time.
Clopidogrel has been shown to reduce thrombus size and delay its formation in mice with a maximal effective dose of 50 mg/kg given the day before and 2 hours prior to experimentation (Lenain, et al., 2003). This drug will be obtained from the hospital pharmacy and tablets will be dissolved in sterile water for oral administration. Control animals will receive water in lieu of drug. The effectiveness of this treatment regime will be confirmed by first measuring the responsiveness of platelets isolated from drug-treated WT animals to ADP-induced aggregation using an optical aggregometer (Chrono-Log Corp.) as previously described (Leon et al., 1999). Because the mutant VWF-A1 domain mice also have a defect in platelet aggregation, these animals cannot be used for the purpose of testing to ADP-induced aggregation ex-vivo. However, this additional phenotype will be advantageous because it limits potential competition between human and mouse platelets for binding to ligands exposed at sites of vascular injury. Human platelets will be administered 30 minutes prior to vascular injury and 50 μl of blood drawn to determine the percentage of circulating cells as described above. Platelet rich plasma will also be purified from control and drug treated animals that receive human platelets to evaluate the effectiveness of clopidogrel on preventing ADP-induced aggregation of these cells ex-vivo.
Tirofiban (Aggrastat) is a non-peptide inhibitor of the fibrinogen receptor αIIb β3 that limits the ability of platelets to form aggregates, an event required for thrombus progression. It has a plasma half-life of approximately 2 hours but only remains bound to platelets for seconds, thus necessitating continuous intravenous administration. It is currently approved for short-term treatment of patients with acute coronary syndrome that require interventional catheterization. Thus, the animals will be dosed based on that given for interventional procedures such as angioplasty, which consists of a 25 μg/kg bolus over 3 minutes followed by a continuous maintenance infusion of 0.15 μg/kg/min until the completion of the experiment (Valgimigli et al., 2005). Human platelets will be administered 30 minutes prior to vascular injury and 50 μl of blood drawn to determine the percentage of circulating cells as described above.
Mice are used as platelet donors. A means to evaluate murine platelet interactions with wild type and mutant VWF-A1 proteins is via in vitro flow chamber assays. Blood from about 10 mice are required to purify adequate numbers of platelets per assay. Blood from donor animals is obtained from the retro-orbital plexus using a heparinized glass pipette. Mice will be anesthetized with Ketamine and Xylazine prior to the procedure and are euthanized by CO2 inhalation upon completion.
This assay is carried out as disclosed above.
For type 2B mutant VWF, its capacity to bind to platelet GPIb α in solution can be determined. Plasma is harvested from these mice and VWF purified. Various concentrations of the plasma glycoprotein will be indirectly labeled using a non-function blocking, 125I-labeled mAb to its A1 domain as previously described (Ribba et al., 1992). After a 30 minutes incubation, a quantity of this mixture will be incubated with platelets purified from β3 deficient mice so to prevent integrin-mediated binding to VWF. After an incubation period of 1 hour, an aliquot of this mixture will be added to a sucrose gradient and centrifuged to pellet the platelets. Radioactivity associated with the pellet vs. supernatant will be determined in a γ-scintillation counter, and the binding estimated as the percent of total radioactivity.
To better simulate human platelet mediated thrombosis in mice, an animal in which the majority of the A1 domain of von Willebrand factor (VWF) has been replaced with its human counterpart (amino acids 1240P through 1481G of the human VWF) was generated. The rationale for generating this animal is the ability to rapidly develop and determine the preclinical efficacy of therapies that specifically target the A1 domain of human VWF. There are 34 amino acid differences between the human and murine A1 domains of VWF (
Generating a mouse bearing the majority of the human VWF A1 domain required a targeting approach different from the VWF mutant mice disclosed in Examples 3 and 4, because the use of the original targeting vector did not result in transgenic animals. To generate a transgenic mouse containing a majority of the human VWF A1 domain, Arm 1 of the construct was extended to a total length of about 3.5 kb and >85% of the human A1 domain sequence was substituted for its murine counterpart. The targeting construct is shown in SEQ ID NO:11. Additional modifications from the process disclosed in Examples 3 and 4 required for the generation of the human VWF A1 domain bearing animal included: (i) splitting ES cells 1:6 (vol:vol) instead 1:3; (ii) harvesting ES cells at 50% confluence rather than over 70% confluence; (iii) replacing media for the ES cells 4 hours before harvesting for electroporation; (iv) devising a screening method (see below for details) to identify correctly targeted clones, which included long-range PCR to detect homologous recombination of the targeting arms. This permitted the use of primers external to the 5′ and 3′ regions of the targeting vector. Other modifications include (v) enriching for viable ES cell clones, which required plating cells on feeders and incubating at 37° C. for 20 minutes instead of the typical 45 minutes. Unattached cells were then aspirated away. The loosely attached ES cells were then washed off for use in microinjection. A further modification from the method used in Examples 3 and 4 included (vi) increasing the number of targeted ES cells for injection into blastocysts. 30 correctly targeted ES cells instead of the typical 15 ES cells were used. This permitted more ES cells to have contact with the inner cell mass in order to increase the percentage of ES cells that become incorporated into germ line cells. An additional modification from the method used in Examples 3 and 4 was (vii) the development of new Southern probes (SEQ ID NOs:19 and 20) to detect correctly targeted construct in transgenic mice, as well as (viii) a new PCR strategy to rapidly screen mice that possess the desired construct (for details, see below). The combination of these critical modifications was essential for the generation of a transgenic mouse bearing the majority of the human VWF A1 domain (amino acids 1240P to 1481G). The final vector used for the generation of the mice is shown in (
Identification of mice bearing the human VWF A1 domain was achieved by performing Southern blot analyses (
Evaluation of mice homozygous for the VWF-HA1 substitution revealed evidence of a significant bleeding diathesis as manifested by tail bleeding times of greater than 10 minutes versus a mean bleeding time of 180 seconds for WT controls (
To demonstrate that the defect in hemostasis was due to the inability of mouse platelets to interact with the humanized VWF, intravital studies that assessed the ability of thrombi to form at sites of laser induced injury in the microcirculation of the cremaster muscle in mice were performed. Endogenous circulating mouse platelets were unable to participate in thrombus formation in injured arterioles of VWF HA1 mice (
To further demonstrate that mouse VWF containing the majority of the human A1 domain cannot support interactions with mouse platelets, plasma VWF from these animals was surfaced immobilized onto a glass cover slip and incorporated into a parallel plate flow system. WT mouse blood was then infused over the immobilized substrate. GPIbα on mouse platelets could not support any significant interactions with plasma VWF containing the human A1 domain (
Table 7 below shows the reagents used for PCR. Other than the primers and the template DNA, the reagents (Roche extraLong PCR kit 11-732-650-001) were purchased from Roche (Nutley, N.J.).
The PCR program was set at 94° C. for 5 minutes to start; followed by 30 cycles at 94° C. for 1 minutes, 64° C. for 1 minute, and 68° C. for 4 minutes; and finally 68° C. for 8 minutes. The samples were stored at 4° C. The PCR products were then analyzed by gel eletrophoresis. The desired product is a 3.5 KB band for the downstream screen and an approximately 4 KB band for the upstream screen.
PCR Strategy to Rapidly Screen Mice that Possess the Desired Construct.
The reagents, other than the primers and the template DNA, were purchased from Fisher Scientific (Waltham, Mass.).
The PCR program was set at 95° C. for 5 minutes to start; followed by 25 cycles at 95° C. for 1 minute, 60° C. for 1 minute, and 72° C. for 2 minutes; and finally 72° C. for 8 minutes. The samples were stored at 4° C. The PCR products were then analyzed by gel eletrophoresis. The desired product from reactions using primers m9677F and m447R is a 550 by band, and the desired product from reactions using primers m3628R and H585F is a 1100 bp band.
The ability of a VWF-A1 mutant animal, such as the 1326R>H mutant mouse, or the mouse bearing the majority of the human VWF A1 domain (amino acids 1240 through 1481 of VWF), to generate thrombi composed of human platelets at sites of vascular injury in vivo, provides a means for developing imaging technologies designed to detect sites of occult bleeding or thrombus formation in humans. For example, such technologies may prove useful in expediting the discovery of sites of internal bleeding in humans as a result of injuries obtained form a motor vehicle accident. Similarly, it may be useful in detecting injuries obtained in a military battle. Suitable probes include antibodies, small molecules, peptides that recognize molecules expressed on human platelets or the various domains of VWF. However, coupling contrast agents directly to antibodies is cumbersome and insufficient for detection of such complexes in the body by various imaging modalities (i.e. MRI) due to low signal to noise output. Thus, an ideal candidate for detection would not only preserve the specificity associated with monoclonal antibodies, small molecules, or peptides but also have the following properties: 1) high signal-to-noise ratio, 2) long circulating half-life, 3) acceptable toxicity profile, 4) ease of use and production, and 5) compatibility with standard commercially available imaging modalities. Perfluorocarbon Nanoparticle (PNP) may provide the answer. This proposal will take advantage of a novel nanoparticle contrast agent that can be imaged by ultrasound, magnetic resonance, and nuclear imaging (Lanza et al., 2000, Lanza et al., 1997, Yu et al., 2000). This agent is a small (about 150-250 nanometer diameter), lipid encapsulated, perfluorocarbon emulsion that can be administered by vein. Importantly, monoclonal antibodies as well as small molecules and peptides that recognize platelets and/or VWF can be covalently coupled to PNPs. Moreover, PNPs can also be potentially used for targeted drug delivery (
PNPs have been shown to remain stable in the circulation with a half-life of >1 hour, which permits rapid binding and local contrast enhancement sufficient for diagnostic imaging within 30-60 minutes. PNPs are cleared by the liver and spleen, and are similar to “artificial blood” formulations used to enhance oxygen, which have acceptable safety profiles for clinical use at 10 times greater dose than would be required for targeted contrast enhancement. In addition, perfluorocarbon to be used in this study (perfluorooctylbromide) has an extensive track record for human safety in clinical trials (i.e. Oxygent, Alliance Pharmaceuticals). Thus, this nanoparticle platform provides an ideal opportunity to prove that contrast agents can be targeted specifically to sites of human thrombus formation.
The basic method for formulating perfluorocarbon nanoparticles comprised of perfluorooctyl bromide (40% w/v), a surfactant co-mixture (2.0%, w/v) and glycerin 9(1.7%, w/v) has been well described (Lanza et al., 2000, Lanza et al., 1997).
Briefly, the surfactant co-mixture is dissolved in chloroform/methanol, evaporated under reduced pressure, dried in a 50° C. vacuum oven, and finally dispersed into water by sonication. The suspension is combined with perfluorocarbon and then emulsified at 20,000 PSI. Fluorescent nanoparticles are manufactured by including in the lipid mixture 0.1 mole ° A) Fluorescence-FITC or PE prior to the emulsification step. Coupling of monoclonal antibodies involves the introduction of a sulflhydrl group onto the protein by modification of amines with N-succinimidyl S-acetylthioacetate (SATA), which then is reacted with nanoparticles containing activated maleimide. We coupled an antibody that recognizes the human, but not the mouse, platelet receptor allb β3 and determined the ability of FITC-labeled PNPs to detect a thrombus composed of human platelets at a site of laser-induced vascular injury in the cremaster muscle of a mouse homozygous for the 1326R>H mutation. These antibody-coupled PNPs rapidly and selectively accumulated at the site of the developing human thrombus (
Small molecules, often with molecular weights of 500 or below, have proven to be extremely important to researchers for exploring function at the molecular, cellular, and in vivo level. Such compounds have also been proven to be valuable as drugs to treat diseases, and most medicines marketed today are from this class (i.e. Aggrastat—see above). As the interaction between GPIb α and VWF-A1 is essential for the platelet deposition in damaged arterioles, it is a reasonable to assume that disruption of this adhesive event will inhibit or ameliorate thrombus formation. Moreover, it is believed that only partial inhibition is required to achieve this goal based on the phenotype of the mutant A1 domain mice, the inability to form stable thrombi in vivo.
Traditional approaches to small molecule discovery typically rely on a step-wise synthesis and screening program for large numbers of compounds to optimize activity profiles. Over the past decade, scientists have used computer models to aid in the development of new chemical agonists or antagonists as well as to better define activity profiles and binding affinities of such compounds. In particular, these tools are being successfully used, in conjunction with traditional research techniques, to examine the structural properties of existing compounds in order to predict their ability to alter the function of biologically relevant proteins. For this approach to be successful, one must have high quality crystal structures of the biological molecule(s) in order to generate an accurate 3-dimensional model so that it can then be used to identify binding regions for small molecules.
The structure of the binary complex formed when GPIb α binds to the A1 domain of VWF can be determined using such methods. For example, a mechanism by which the snake venom protein botrocetin enhances the interaction between GPIbα and the VWF-A1 in order to promote spontaneous platelet aggregation, resulting in death has been elucidated. Botrocetin was known to bind with high affinity to the A1 domain (crystallization data summary available from PDB access no. 1AUQ and Emsley et al., 1998, see also U.S. Patent Publication No. 2009/0202429, which is incorporated herein in its entirety, for the atomic coordinate data), but was not thought to interact directly with GPIb a. This snake venom has the capacity to form a small, but distinct interface with this platelet receptor so as to prevent its release from the A1 domain, thus facilitating platelet aggregation (
To demonstrate the feasibility of identifying potential small molecule inhibitors in silico, computational modeling software was utilized in conjunction with high-resolution crystal structure results to screen databases for existing compounds that would bind to the A1 domain where it interfaces with botrocetin (exogenous ligand binding site). Several small molecules predicted to bind with sub-micromolar IC50s (concentration of drug required to inhibit the activity by 50%) and that could also severely disrupt binding of this snake venom protein were identified. Thus, potential candidate small molecules can be identified that may interfere with the interaction between GPIb α and the A1 domain of VWF.
Although the use of computational modeling is a state-of-the-art method for identifying lead compounds, it is not without its limitations. Thus, an actual library of 20,000 small molecules manufactured by the Chembridge Corporation (San Diego, Calif.) will also be screened. The library consists of handcrafted drug-like organic molecules with molecular weights in a range of 25-550, which are soluble in DMSO at concentrations ranging from 10-20 mM. The structure and purity (>95%) of these compounds have been validated by NMR. The library is formatted in a 96 well plate for high throughput screening using instrumentation made available through the OCCC (under supervision of the Landry laboratory) and includes a robot plate reader (FLexStation II 384, Molecular Devices, Sunnyvale, Calif.), an 8-tip robotic pipettor (Multiprobe II Plus, Perkin Elmer, Shelton, Conn.), a 96-tip robotic pipettor (Mintrak, Perkin Elmer), and an automated 96 well plate washer (Perkin Elmer).
An ELISA based system will be used to screen for compounds that may inhibit the interaction between GPIb α and the VWF-A1 domain. Enzyme-Linked Immunosorbent Assay (ELISA) methods are immunoassay techniques used for detection or quantification of a substance. An example of this assay is demonstrated in
Assay system: Recombinant GPIb α and VWF-A1 proteins will be generated and purified as disclosed, with the latter containing a 6×His tag. Purified GPIb α will be absorbed overnight (4° C.) to PRO-BIND polystyrene 96-well assay plates (Falcon) at 10 μg/ml per well. Plates will be washed and non-specific binding sites blocked by the addition of TENTC buffer (50 mM Tris, 1 mM EDTA, 0.15 M NaCl, 0.2% casein, 0.05% Tween 20, pH 8.0) for 1 hour at room temperature. Subsequently, plates will be washed with and resuspended in TBS buffer (50 mM Tris, 150 mM NaCl, pH 8.0) and 1 test compound per well added at a final concentration of 10 μM (final DMSO concentration 0.5%). After 30 minutes, recombinant His tagged VWF-A1 protein will be added at a 1:1 Molar ratio to that of GPIb α and left to incubate for 1 hour before washing with TBS buffer. VWF-A1 bound to surface-immobilized GPIb α will be determined by the addition of HRP-conjugated anti-His tag antibody and the A1-antibody conjugate detected by the addition of LumiGlow reagent (KPL, Gaithersburg, Md.). The resulting fluorescence will be quantified by the number of luminescence emissions per second using a FLexStation II 384 plate reader. A sample will be considered positive when the luminescence (in counts per second) is more than 2 standard deviations above the mean value for negative-controls.
Negative controls: Addition of mAb 6D1 to certain wells to prevent VWF-A1 binding to GPIb α or no addition of VWF-A1 protein (
To demonstrate the feasibility of the VWF1326R>H mice to identify anti-thrombotic drugs capable of perturbing human platelet function in vivo, the ability of 2 FDA approved drugs, Plavix and ReoPro, to prevent human platelet-induced hemostasis was tested. As noted above, Clopidogrel has been shown to reduce thrombus size and delay its formation in mice with a maximal effective dose of 50 mg/kg given the day before and 2 hours prior to experimentation. Homozygous VWF1326R>H mice that received this dosing schema, were unable to produce a hemostatic clot when administered human platelets in contrast to homozygous VWF1326R>H mice that received saline in lieu of drug.
Because Plavix can also block the function of the ADP receptor on murine platelets (see
Intravital microscopic study was carried out to evaluate the ability of the VWF1326R>H mouse to determine the efficacy of anti-platelet therapies given to patients at risk or with active cardiovascular disease. The typical prophylactic dose of aspirin (ASA) of 81 mg did not prevent laser-injury induced human platelet thrombus formation in the genetically modified animal while increasing the daily dose to 162 mg was preventative (
Although the R1326H mutation in murine VWF can support human platelet mediated hemostasis and thrombosis, there still exists a major difference between the human and mouse A1 domains. Specifically, only the human form can be activated to bind to GPIb alpha on human platelets. Ristocetin is an antibiotic that was taken off the market due to its ability to cause von Willebrand factor to bind the platelet receptor GPIb alpha, so when ristocetin is added to normal blood, it causes platelet clumping. This is demonstrated in the following experiment in which human platelets were resuspended in platelet poor plasma from either VWFR1326H or VWFHA1 mice, and the ability of ristocetin to induce aggregation was determined by optical aggregometry. Whereas ristocetin could activate plasma from VWFHA1 mice so that it could cause human platelet aggregation comparable to its human counterpart, such was not the case with plasma obtained from VWFR1326H animals (
Jenkins et al. (1998) “Molecular Modeling of Ligand and Mutation Sites of the Type A Domains of Human von Willebrand Factor and Their Relevance to von Willebrand's Disease” Vol. 91, No. 6, Blood, pp. 2032-2044.
All documents cited in this application are hereby incorporated by reference as if recited in full herein.
Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention.
The present invention claims benefit to U.S. provisional application Ser. No. 61/662,896 filed Jun. 21, 2012, the entire contents of which are incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61662896 | Jun 2012 | US |
