Patent Application
20050086710
The present invention relates to modified or transgenic platelets, in particular transgenic mammalian platelets, and to the use of such platelets as a probe of platelet function, preferably in a blood vessel. Further, the invention relates to a method of determining platelet adhesion to a vessel wall and to a method of determining platelet aggregation. Moreover, the invention relates to a highly efficient method of preparing transgenic platelets, in particular transgenic mammalian platelets. The invention enables the identification of novel drug targets, notably for the treatment of vascular diseases, in particular atherosclerosis as well as venous and arterial thrombosis. Moreover, the present invention provides targets useful in the screeing for novel drug candidates. Moreover, the novel drug candidates may be validated in vivo or in vitro by using the transgenic platelets of the invention.
Coronary heart disease and stroke currently account for almost 50% of all deaths in the United States. The interaction of platelets with the vessel wall plays a central role in the development of these and similar diseases by inducing arterial or venous thrombosis with subsequent disturbance of macro- and microcirculation. During adhesion/aggregation platelets shed active mediators, which have a decisive impact on the function of the vessel wall, on other blood cells and on the surrounding tissues. These are pivotal steps during the initiation and further propagation of a vessel thrombus. A prolonged presence of these mediators in blood vessels triggers arteriosclerosis of their walls. Thereby, platelets play a key role not only in the development of coronary heart disease or vessel disease per se, but also for the development of arteriosclerotic complications.
Initially, circulating platelets are recruited to the vessel wall by their interaction with the endothelium. They trigger the formation of aggregates, which hinder or completely interrupt arterial blood flow and therefore disturb the nutritive perfusion of the dependent tissues. A thrombotic vessel closure leads to the clinical syndromes of acute myocardial infarction, ischemic stroke or infarction of other organs which are all characterized by the formation of ischemic tissue necrosis. These events can occur acutely, but also in a chronically repetitive way and contribute to tissue ischemia with progressive tissue damage (e.g. in chronically way and contribute to tissue ischemia with progressive tissue damage (e.g. in chronically ischemic heart disease with the resulting ischemic heart failure as a disease end point). Beside the acute or chronic impact on arterial blood flow, an increased interaction of platelets with the endothelium also leads to a chronic-inflammatory, proliferative issue response induced by the release of active mediators. Thereby, the interaction between platelets and the endothelium markedly contributes to the development of the morphological syndrome of arteriosclerosis. Platelets constitute a pivotal and early active component in the pathophysiology of vascular disease and therefore represent an attractive pharmacological target.
Platelets play a pivotal role in a variety of diseases, such as heart disease, stroke, renal infarction or peripheral artery disease and other vascular diseases and in various hematologic disorders. Existing antiplatelet compounds inhibit aggregation and some platelet signaling pathways, but they do not influence other platelet functions such as their shape change during activation or the secretion of vasoactive agents. Therefore, novel drug targets should be identified to improve the treatment of the above-mentioned diseases.
Currently existing antiplatelet compounds have markedly improved the prevention and therapy of acute thrombotic processes. Especially, glycoprotein IIb/IIIa antagonists are efficient but must be given intravenously and have to be strictly controlled. Moreover, vessel damage caused by the release of mediators from platelets cannot be influenced by existing anb-platelet strategies.
An important prerequisite for the development of attractive novel pharmacologic compounds is a more detailed understanding of platelet-endothelial interaction and of their subsequent activation and secretion. Especially, an understanding of the early phases of platelet activation would allow to find new approaches for specific and efficient intervention in platelet-dependent pathological processes, and thereby, a selective inhibition of the subpopulation of activated platelets (existing therapies non-specifically inhibit all platelets in the body).
Such innovative antiplatelet compounds would offer the potential of a thrombolysis with markedly reduced side effects, or of highly effective therapies against chronically arteriosclerotic disease and venous disease (such as venous thrombosis or pulmonary embolism), as much as for an effective prophylaxis in high risk vascular patients. The development of such novel antiplatelet therapies by using promising platelet targets intimately depends on a more detailed molecular understanding and characterization of biochemical and signal transduction pathways in platelets. The technology described on the following pages allows to characterize the above-mentioned mechanisms more exactly and to influence potential pharmacological targets selectively by using somatic gene transfer into megakaryocytes. Platelets themselves cannot be genetically modified, because—as nucleus-less cells—they do not possess a DNA-protein synthesis apparatus.
Current Methods to Identify Novel Target Proteins in Platelets
Out of the approximately 30 000 different proteins in every cell only a small part is functionally relevant at any given moment, depending on the respective pathophysiological condition. Therefore, the information offered by genomic investigation is rather static, whereas all cells—among them platelets—are rather dynamic units, in which proteins are regulated by disease conditions. Especially in the case of platelets, the development of novel therapeutics rather depends on a detailed understanding of protein function (“functional proteomics”) in pathophysiological conditions than on genomic investigations. Since the blueprint for all existing proteins, i.e. the genomic DNA sequence, has been identified by the human genome project, the challenge now is to define disease-relevant theraputic targets on the protein level.
One of the most straightforward approaches to study new drug target proteins is to overexpress them in the desired target tissues and cells, or to inhibit them by dominant negative mutant overexpression. Also target proteins in platelets could be investigated in a similar manner, and the effect of these interventions on platelet physiology and on disease endpoints could be studied. So far, however, platelets cannot be used for gene transfer directly, as they do not express foreign transgenes due to the absence of a nucleus in these cells. In particular, the present invention therefore relates to a method which allows for the study of the relevance of protein targets in human platelets ex vivo and in vivo by overexpressing these proteins. Thereby, the invention relates to methods of identifying compounds capable of treating the above-mentoned diseases (acute vessel diseases such as myocardial infarction, stroke, peripheral arterial disease, renal infarction and others; chronic atherosclerosis; venous disease and pulmonary embolism; or hematologic disorders).
Current research projects rely on the following techniques:
These methods generate a variety of hardly comparable single results (1) or are very time consuming (2, 3). Due to the vast amount of data generated by methods (2) or (3), their functional relevance often remains unclear. This problem still impedes the fast and efficient identification of novel compounds directed against platelet-induced disease.
Current Methods of Generating Recombinant Platelets
The use of recombinant platelets has only been investigated for the purpose of hematological research. The group of Choi and Hunt had shown that platelets can be generated from megakaryocytes in vitro, and that they have some characteristic morphological features of mature platelets (Choi E S, Nichol J L, Hokom M M, Homkohl A C, Hunt P. Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional. Blood 1995; 85:402-413). This group has not tested any expression of foreign transgenes. Reports on the ex vivo expansion of CD34+ PBPC for the preparation of a composition for the treatment of thrombocytopenia cannot be reworked (Kratz-Albers K. et al. Experimental Hematology, Vol. 28, (2000), 335-346). WO99/61588 discloses the production of megakaryocytes by coculturing human mesenchymal stem cells with CD34+ cells. The megakaryocytes may contain transgenes. WO99/61588 does not disclose the use of modified platelets in the context of cardiovascular disease.
For hematological purposes, an overexpression of transgenes in megakaryocytes was proposed by others either by retroviral gene transfer (Burstein S A, Dubart A, Norol F, Debili N, Friese P, Downs T, Yu X, Kincade P W, Villeval J L, Vainchenker W. Expression of a foreign protein in human megakaryocytes and platelets by retrovirally mediated gene transfer. Exp Hematology 1999; 27:110-116; Wilcox D A, Olsen J C, Ishizawa L, Bray P F, French D L, Steeber D A, Bell W R, Griffith M, White G C III. Megakaryocyte-targeted synthesis of the integrin β3-subunit results in the phenotypic correction of Glanzmann thrombasthenia. Blood 2000; 95:3645-3652) or adenoviral gene transfer (Faraday N, Rade J J, Johns D C, Khetawat G, Noga S J, DiPersio J F, Jin Y, Nichol J L, Haug J S, Bray P F. Ex vivo cultured megakaryocytes express functional glycoprotein IIb-IIIa receptors and are capable of adenovirus-mediated transgene expression. Blood 1999; 94:4084-4092). The use of tissue-specific promotors has been shown (Wilcox D A, Olsen J C, Ishizawa L, Griffith M, White GC III Integrin alpha IIb promotor-targeted expression of gene products in megakaryocytes derived from retrovirus-transduced human hematopoietic cells. Proc Natl Acad Sci USA 1999; 96: 9654-9659).
In all studies published to date, however, the use of gene transfer into the progenitor cells and subsequent use of recombinant platelets for studying vascular biology, and thereby investigating drug targets relevant for heart and circulatory diseases ex vivo and preferably in vivo has never been mentioned. Further, such applications of recombinant platelets have been out of reach so far due to insufficient platelet numbers achieved by cytokine-induced shedding of functional platelets.
Currently available technologies of finding drug targets in platelets rely on the study of peripheral human or animal platelets both in clinical and in experimental studies. This approach offers the advantage of measuring physiological parameters directly, for instance in response to a therapy with existing anti-platelet drugs. However, this approach very often does not allow to test specific molecular effects of novel signalling pathways nor to identify novel drug targets. This is partly due to the fact that existing compounds such as acetylsalicylic acid have a simultaneous impact on several biological pathways and target proteins in platelets. Therefore, a method to relate molecular mechanisms to the pathology of vascular and other diseases did not exist before.
It is therefore a problem of the invention to provide novel methods of identifying new drug targets preferably in platelets, especially for treating heart and vascular diseases.
It is another problem of the invention to provide new methods of determining platelet functions, notably in vivo.
It is another problem of the invention to provide new modified platelets.
It is another problem of the invention to provide new methods of generating modified platelets in larger amounts than possible with methods of the prior art.
It is another problem of the invention to provide a method of inducing highly efficient shedding of platelets from megakaryocytes.
This invention provides a composition comprising platelets having a combination of the following modifications:
The composition of the invention may be formulated as a dosage form for parenteral administration. Specifically, the composition of the invention may be in the form of a liquid for injection or infusion. Accordingly, the composition may further contain an aqueous solvent, preferably water, preservatives and/or osmotically active components such as NaCl. Alternatively, the composition of the invention may be a freeze-dried powder.
The platelets are preferably obtained or obtainable by genectic modification of mammalian platelet precursor cells, in particular precursor cells of a human, mouse, rat or rabbit.
The composition of the invention preferably does essentially not contain platelet precursor cells such as megakaryocytes. The compositions of the invention may be organised as a library comprising at least two compositions each containing platelets having a unique modification of the protein constituents which is obtained or obtainable by genetic modification of platelet precursor cells.
The present inventors have found that the composition of the invention may be used as a probe for platelet function. Notably, said composition may be used as a probe for platelet function in a model animal, preferably in a blood vessel of a model animal. Therefore, the composition may be used for studying the function of a gene or its gene product (protein) expressed in platelet precursor cells and present in platelets in the context of the vascular system and in the context of vascular diseases. This invention allows for the first time to investigate and to determine the function of proteins in platelets in vivo. The identification of functions of platelet proteins notably in vivo is of utmost importance for identifying new drug targets for treating vascular diseases like atherosclerosis, thrombosis, heart infarction, stroke, bleeding diseases etc.
Genetically modified or recombinant platelets are already known in the art However, these platelets were created for academic purposes and the methods used for creating these platelets were inefficient. Recombinant platelets have never before been used to screen for drug targets for treating diseases involving platelets. For the first time, this invention makes available sufficient amounts of ex vivo generated modified platelets for such studies. In particular, the present invention provides a composition suitable for administration by injection or infusion to an animal for the investigation of modified platelets in vivo.
Platelet functions to be probed by said composition of the invention may be any function which involves platelets or platelet components, whereby said functions may involve further factors or components of the vascular system like endothelial cells or factors of the blood coagulation cascade (e.g. fibrinogen). Said platelet functions are preferably functions platelets have in vivo. Examples of platelet functions to be probed are platelet activation, platelet aggregation, platelet interaction with or adhesion to endothelium, secretion of factors from platelet granula, activation of endothelial cells by platelets or by platelet-secreted factors etc. Most preferred functions to be probed involve platelet aggregation and platelet adhesion to endothelial cells.
The invention discloses methods of determining platelet functions in vivo using said composition comprising platelets. Specifically, the invention discloses a method of determining platelet adhesion to a vessel wall, whereby the method comprises the steps of
Further, the invention specifically discloses a method of determining platelet aggregation in a blood vessel, whereby the method comprises the steps of
Said methods are preferably carried out in vivo in a blood vessel of a model animal. Said blood vessel may be a healthy blood vessel of a healthy model animal. Said blood vessel may also be a blood vessel affected by a vascular injury or a vascular disease. Preferably, said blood vessel is an ischemic blood vessel or an atherosclerotic blood vessel. Said blood vessel may be an artery or a vein. An artery is preferred. More preferred is a carotid artery or an intestinal capillary bed. However, said blood vessel may be any blood vessel which is accessible experimentally for observing platelet aggregation or adhesion of platelets to a vessel wall, notably in vivo.
Said model animal should be related to humans with respect to their vascular system. Preferably, said model animal is a mammal. Further, said model animal should be suitable for in vivo experimentation. Preferred examples of such animals are mice, rats, rabbits etc. Most preferred are mice.
Said model animal may be genetically modified, i.e. it may be transgenic or it may have been subjected to gene therapy. Said genetic modification may be a deletion or a knock-out of a natural gene or it may be (over)expression of a native or mutated gene. Preferably, said genetically modified animal is a transgenic mouse, e.g. an ApoE-deficient mouse.
Further, said model animal may be a disease model in order to carry out said methods in a disease model blood vessel. Preferably, said model animal is a model for a vascular injury or a vascular disease like thrombosis, atherosclerosis, ischemia/reperfusion, myocardial infarction, acute coronary syndrome etc. Said disease model may be created by way of genetic modification or by way of subjecting an animal to certain conditions or physical treatment. Further, a disease model in a model animal may be created by breeding methods. Such disease models and methods of their creation are known in the art. An example of a physical treatment producing vascular injury is given in example 14. An example of a disease model created by genetic modification is said ApoE-deficient mouse mentioned above.
Said methods of determining platelet functions in a model animal preferably comprise anesthetising and sacrificing said model animal.
Said methods of determining platelet functions in a model animal may comprise administration of said composition of the invention into the vascular system of said model animal. Preferably, said composition is administered intravenously making use of a catheter. Platelet function, notably platelet aggregation and/or adhesion of platelets to endothelium, may be visualised by intravital videofluorescence microscopy in a blood vessel by way of said detectable, preferably fluorescent, label of the platelets of the invention. A function of the administered platelets of the invention may be compared to a function of platelets carrying said detectable label but no modification of the protein constituents of the platelets. Alternatively, a function of the platelets of the invention in a disease model blood vessel may be compared to a reference blood vessel not affected by said disease. Further, platelet functions may be compared in a model animal of any state or medical or environmental condition like age, sex, drug medication, nutrition, exposure to cigarette smoke etc. with a reference model animal.
Once a drug target protein has been identified according to the invention, drug candidates may be tested using analogous methods. The invention also discloses an assay for screening for drug candidates, wherein a drug target identified using a method for determining platelet function according to the invention is used. Further, an assay for testing new drug candidates by using the composition of the invention is disclosed.
Said composition of the invention may further be used for determining protein function in platelets in vitro. A preferred platelet function to be studied in vitro is platelet aggregation or secretion of active mediators from platelets (cf. examples 11 and 12). Further, flow cytometry may be used to determine the amounts of proteins present in said platelets, preferably on their surface, in response to a modification of the protein constituents (cf. example 9).
The platelets of the composition of the invention have a modification of their protein constituents which is obtained or obtainable by genetic modification of platelet precursor cells. Said precursor cells are preferably megakaryocytes. However, precursor cells of megakaryocytes, notably CD34+ cells, may also be genetically modified to obtain modified megakaryocytes and platelets therefrom.
Protein constituents of the platelets refers to the proteome of the said platelets, i.e. all types of proteins which may be present in platelets including their abundance. Said proteome may be that of normal healthy platelets or that of platelets affected by a genetic disorder.
Said modification of the protein constituents of the platelets is obtained or obtainable by genetic modification of platelet precursor cells. Said modification may affect a single or several proteins of platelets. Preferably, said modification affects one protein. Said modification of the protein constituents may relate to (i) the absence or inhibition of one or more proteins; (ii) the overexpression of one or more proteins; and/or (iii) to the presence of a foreign protein capable of modifying an existing protein.
For the pupose of the invention, “absence of a protein” also comprises reduction of the amount of said protein normally present in platelets. Absence of one or more proteins in platelet precursor cells may be achieved by genetic engineering according to known methods. For example, the gene coding for said protein may be deleted fully or partly or may be replaced by a non-functional (e.g. mutated or truncated) form of said protein. Said genetic engineering may make use of homologous recombination. Further, expression of said protein may be suppressed by way of an anti-sense or ribozyme mechanism. Inhibition of a protein may be achieved by introducing a mutation reducing a functionality of said protein or by dominant negative inhibition by expressing a non-functional form of said protein (see below).
Methods of overexpressing one or more proteins in animal cells and in particular in platelet precursor cells are known in the art (cf. examples 7 and 8). Viral vectors like adenovirus-based vectors or retrovirus-based vectors are frequently used for this purpose. The protein to be expressed may be any native mammalian protein or a foreign protein. Preferably, said protein to be (over)expressed is a mammalian protein that is expressed in platelets either under normal, healthy conditions or under the condition of a disease. Said protein is preferably native to humans or native to said model animal. Said foreign protein may be a protein that does not exist in humans e.g. a microbial (bacterial, fungal) gene. Said foreign protein may alter the amount or functionality of a native protein e.g. by cleaving said native protein. Preferably, said cleavage is specific for a particular native protein under investigation.
As a further modification, said platelets of said composition have incorporated a detectable label. Said detectable label may be present internally in said platelets or it may be attached to the outer membrane of said platelets. Said detectable label may be a fluorescent dye or a fluorescent protein. Numerous such dyes and proteins are commonly used in cell biology/microscopy. Among fluorescent dyes, fluorescein, phycoerythrine, and rhodamine may be mentioned. Labeling of biological objects like macromolecules (e.g. antibodies) or cells with such dyes is widely used in cell biology/fluorescence microscopy. Among fluorescent proteins, green fluorescent protein (GFP) and its blue, yellow, orange and red derivatives and relatives of various emission maxima may be mentioned (e.g. those discussed in Nature Biotechnology (2002) 20, 28-29 and references cited therein). Such a fluorescent protein may be expressed in platelet precursor cells according to known methods such that they are present in platelets. Said detectable label may be used for visualising said platelets when determining the function of a modification of the protein constituents of platelets.
The compositions of the invention contain at least two modifications which are absent in naturally occurring platelets of a healthy animal. At least one of the modifications has a genetic basis in hematopoietic progenitor cells.
The functions of the modification of the protein constituents of the platelets (a) and the modification by incorporation of a detectable label (b) are mutually independent. Specifically, the modification (a) provides a functional platelet wherein the modification of the protein constituents shows the same function in the presence or absence of the specific modification (b). In particular, the presence of modification (b) does not interfer with the function of modification (a) to such an extend that the function of modification (a) is eliminated, significantly changed or replaced by another function such that the function of modification (a) observed in the absence of modification (b) cannot be used for the purpose of the invention. The functions of the modifications (a) and (b) are mutually dependent, e.g. in case the detectable label impairs the function of a surface-expressed platelet protein. Specifically, the invention does not relate to the case when the label attaches to the protein modification (a) via an antibody, since the antibody then prevents interaction of said protein with other interaction partners. The invention does not relate to the case of platelets obtainable or obtained by genetic modification of platelet precursor cells wherein the gene of a protein constituent is replaced by a gene for a protein such as GFP used as a fluorescent label.
If the detectable label is a protein, it may be expressed in platelet precursor cells from a different expression cassette than the protein responsible for said modification (a). However, the same promoter may be used in these expression cassettes. Alternatively, the protein which is the detectable label and a protein which is responsible for said modification of the protein constituents may also be expressed as a fusion protein as long as the functions of said proteins are mutually independent. Whether the functions of the proteins making up a fusion protein are mutually independent may be tested by comparing said functions with the case wherein these proteins are expressed separately (not as a fusion protein). A negligible influence of one modification of said platelets on the other modification may, however, be tolerated.
This invention further discloses a method of preparing platelets for said composition of the invention, wherein the method comprises the following steps:
Hematopoietic progenitor cells may be prepared from peripheral blood e.g. as described in example 2 or from bone marrow as described in example 5. Said progenitor cells may be prepared from humans or from said model animal, e.g. from an animal species as used for the above methods of determining platelet adhesion or platelet aggregation, specifically from a mammal. Hematopoietic progenitor cells may be enriched according to methods known in the art (cf. examples 3 and 4). Megakaryocytes may then be generated based on the hematopoietic progenitor cells of step (a) (cf. example 6).
In the next step of said method of preparing platelets, a gene of interest is selected from a gene library. Said gene may be selected based on any criteria, e.g. based on information already known in the art.
Subsequently, the megakaryocytes are transformed or transfected with a vector containing the gene of interest selected in step (c) for achieving (over)expression of said gene of interest. Viral vectors like adenovirus-based vectors or retrovirus-based vectors are frequently used for this purpose (cf. examples 7 and 8).
After extensive investigations, the inventors found a unique cytokine cocktail for inducing shedding of platelets from megakaryocytes (step (e)). This cytokine cocktail comprises of thrombopoietin, interleukin-1β, stem cell factor and interleukin-6. These cytokines are preferably used in the following concentration ranges: thrombopoietin (TPO; 10±5 ng/μl), interleukin-1β (IL-1β; 10±5 ng/μl); stem cell factor (SCF; 50±20 ng/μL) and interleukin-6 (IL-6; 10±5 ng/μL). In contrast to previous publications, this unique cytokine cocktail allows for optimized generation of modified platelets, as tested by directly comparing this protocol to conventional ones proposed in the existing relevant literature (
Platelets containing expression products of the gene of interest are then isolated, preferably by centrifugation.
In the next step, said detectable label may be incorporated into the platelets obtained in the previous step. Types of detectable labels and methods of incorporation into platelets are discussed above and are also known in the art.
The modified platelets of the invention can be used for the measurement of different physiological parameters which proved the excellent comparability of the modified platelets of the invention with freshly isolated human platelets. To clarify the pathophysiologic role of specific intracellular proteins during platelet activation, modified platelets can be investigated by fluorescence-activated cell sorting (FACS) with specific antibodies which show alterations in relevant surface proteins by novel target proteins or their inhibition. The co-expression of e.g. green fluorescent protein (GFP) with a target protein allows to control the presence of the target protein in individual platelets. This approach allows to study the targets protein's effects e.g. on the expression and/or activation of fibrinogen receptors (glycoprotein IIb-IIIa), on the composition of storage vesicles and granules, the externalisation of internal proteins such as CD62P or CD40 ligand, on the degranulation of active compounds (VEGF, PDGF, β-thrombogloblin, platelet factor 4 and others). The aggregation of modified platelets could be investigated directly e.g. by fluorescence microscopy or by luminometric aggregometry, which also allowed to measure ATP release from dense granules. The effect of novel target proteins on these physiologic parameters can then be studied.
Effect of Modified Platelets on Human Endothelial Cells
The ex vivo generated modified human platelets may be used for different experiments to characterize the effects of platelet-released mediators and platelet adhesion proteins on the function of human endothelial cells e.g. by measuring their chemotactic properties (e.g., release of monocyte chemoattractant protein-1), or their adhesive (e.g. expression of ICAM-1 and vitronectin receptors) or fibrinolytic potential (e.g. matrix metalloproteinases, uPAR) after incubation with activated modified platelets. Central biochemical functions of endothelial cells and important signal cascades can then be characterized e.g. by protein chemistry or FACS. Platelet-induced alterations of endothelial ex vivo adhesivity for monocytes, which play an important role in atherogenesis, may be investigated in parallel in cell adhesion assays (incubation of platelet-stimulated HUVECs with monocytes).
Preparation of Mice for Measurement of Novel Target Platelets and Measurement In Vivo
A major advantage of the invention is that it allows to clarify the functional significance of specific target proteins or signal transduction cascades for both platelet-endothelial cell interactions and platelet function in vivo. Importantly, this allows to define the role of specific platelet target proteins in pathophysiological settings including atherosclerosis and atherosclerotic tissue remodeling in animal models of disease (e.g. ApoE-deficient mice, cholesterol-fed rabbits) as well as ischemia-reperfusion.
Modified platelets can be used after intravenous administration to directly visualize and quantitatively assess their acute effects on arterial circulation/blood flow in animals by means of intravital videofluorescence microscopy. This can yield valuable data e.g. in the carotid artery which is a pivotal vessel in the pathogenesis of stroke, or in the postischemic intestinal capillary bed, which allows to study the role of platelet target proteins in ischemia-reperfusion, a major consequence of the atherosclerotic process. The invention enables a highly sensitive quantitative analysis of alterations of platelet adhesion to the vessel wall. It allows to study platelet adhesion to previously injured endothelium or atherosclerotic endothelium of Apo E−/− mice. Moreover adhesion to ischemic tissue can be studied (arterially and postcapillary), which also allows to indirectly measure secretion from platelets.
Freshly drawn blood from human volunteers was transferred into a Falcon tube and spun down for 20 min. at 1080 rpm at 20° C. The supernatant yielded the PRP (platelet-rich-plasma) and was transferred to a new tube and diluted 1:4 with wash-buffer (1×PBS (w/o CA2+ Mg+) with 0.1% sodium azide, pH 7.2+/−0.2, filtered through a 0.2 μm filter prior to use and stored at 4° C.). The mixture was spun down for 10 min. at 2100 rpm at 20° C. The pellet was resuspended in staining buffer (1×PBS (w/o CA2+ Mg+) with 0.1% sodium azide and 2% fetal bovine serum) in a volume of 40 μl.
The direct CD34 Progenitor Cell Isolation Kit (from Miltenyi Biotech, Bergisch Gladbach, Germany) contains MicroBeads directly conjugated to CD34 antibodies for magnetic labeling of CD34 expressing hematopoietic progenitor cells from peripheral blood, cord blood, bone marrow or apheresis harvest Hematopoietic progenitor cells, present at a frequency of about 0.05-0.2% in peripheral blood, 0.1-0.5% in cord blood and 0.5-3% in bone marrow, can be rapidly and efficiently enriched to a purity of about 6-98%.
Preparation of Peripheral Blood Mononuclear Cells:
The cell pellet was then resuspended in a final volume of 2 ml staining buffer (500 ml 1×PBS w/o Mg+/Ca+, 0.6% (3 ml) CPD-A, 0.5% BSA (35%)) per buffy (PBMC of about 500 ml blood). These cells were then used for magnetic labeling.
To avoid capping of antibodies on the cell surface during labeling all following steps were done rapidly. Cells were constantly kept cold, only cold solutions were used. All buffers were degassed prior to use.
Bone marrow cells were harvested by flushing the femurs and tibias of mice with 1×PBS (w/o Ca+, Mg+) with 0.6% CPD-A and 0.5% BSA (MACS-buffer). The cells were washed twice (spun down for 10 min., at 1800 rpm), aspirated through 20- and 25-gauge needles and filtered through Nytex mesh to break clumps and to ensure that mature MK's were destroyed. The remaining cells were layered over Percoll (15 mL). Mononuclear cells were separated by centrifugation for 30 min. at 1800 rpm, RT, without break and speeding. Cells were washed in Macs medium (1×PBS (w/o Ca+, Mg+) with 0.6% CPD-A, 0.5% BSA). For some experiments, cells were stained with SCA1 microbeads antibodies and positive cells were separated in a magnetic field according to the protocols of 4.3 and 4.4. Cells were then plated out in Optimem (Glutamax I) for 2 hours in 6 well-plates. Non-adherent cells were collected and resuspended in specific growth medium. The preceding steps were repeated after over-night culturing to remove adherent subset of populations.
For all following steps, the medium IMDM (from LifeTechnologies-Gibco; Wiesbaden, Germany) was used, to which stable glutamine (Glutamax II; LifeTechnologies-Gibco), 1.5% bovine serum albumin (BSA; Sigma, Deisenhofen), 300 μg/mL iron-saturated transferrin, 1 mM sodium pyruvate, 1×MEM vitamins (from Life Technologies-Gibco; Wiesbaden, Germany), 0.02 mg/mL L-aspargine, 0.01 mM of the antioxidant monothioglycerol, 1× non essential amino acids (Life Technologies); penicillin/streptomycin 10 ng/mL were added to the cells.
After extensive testing (see below) the following human or mouse cytokines (all from R+D systems, Wiesbaden) were used for differentiation: thrombopoietin (TPO; 10 ng/μl), interleukin-1β (IL-1β; 10 ng/μl); stem cell factor (SCF; 50 ng/μL) and interleukin-6 (IL-6; 10 ng/μL).
Comparing identical concentrations of cytokines on the same preparations in parallel, the new combination of TPO+SCF+IL-6+IL-1β resulted in 8.5±2×104 modified platelets per 105 megakaryocyte-differentiated stem cells per single harvest (
Fresh medium was added to all cells every 6-8 days: ˜300-400 μL of old medium was discarded from the wells by pipetting from the surface and exchanged by 300-400 μL fresh medium (drop by drop). Platelets were harvested by centrifugation (in undiluted medium) at 1600 rpm for 1¼ min. 20° C. The supernatant was transferred into a new tube (leaving ˜1 mL) and diluted 1:2 with 1×PBS w/o Mg+, Ca+, 0.5% FBS (in platelet wash buffer), then spun down 5 min at 1600 rpm. Megakaryocytes were replated after the separation of platelets with fresh growth medium in the same well.
Modified (E1/E3-deficient) flag-tagged adenoviruses for novel transgenes were generated that for expressing a transgene and green fluorescence protein (GFP) under control of two independent CMV promoters in a bi-cistronic system (He T C, Zhou S, da Costa L T, Yu J, Kinzler K W, Vogelstein B. A simplified system for generating modified adenoviruses. Proc Natl Acad Sci USA. 1998; 95:2509-14, purchased from Q Biogene, Heidelberg, Germany). As a control, Ad-GFP without further transgene was used. Large virus stocks were prepared and purified by centrifugation via cesium chloride density gradients. Adenoviral titers were determined using plaque titration and GFP expression titration in non-E1-expressing cells.
They were carefully pipetted from the surface with 150 μL Optimem, and 600 μL growth-medium was added. Typically, transgene expression occurred 48 hours thereafter and persisted for 3 weeks. Measurements could be done repeatedly during this period. Platelets could be harvested continuously.
Modified retrovirus were generated by using the pantropic retroviral expression system sold by Clontech, Heidelberg, Germany (order no. K1063-1). The HEK 293-based packaging cell line GP2-293 was propagated in complete medium according to the manufacturer's instructions. The genes of interest were cloned into a pLEGFP-C1 (Clontech cat. no. 6058-1), so that they were fused to GFP. Alternatively, HA-tagged genes were cloned into pLNCX-2 (Clontech, cat. no 6102-1). To produce infectious virus, the cells were cotransfected with pLNCX (Clontech) and pVSV-G (Clontech) by a calcium phosphate CalPhos transfection kit (Clontech, order no. K2051-1). The virus was harvested up to four times starting 48 hours after transfection and concentrated in a Centricon plus 80 column (Millipore, cat no. UFC5 LTK08) and titrated in NIH3T3 cells (mean titers of 5×105 infectious units/ml). For infection, retroviruses were applied for 3 hours at 37° C. to differentiating megakaryocytes 7-11 days after isolation (moi of 1-4 virus/cell), using Polybrene reagent (4 μg/ml) from Sigma (Deisenhofen, Germany). Alternatively, a coninfection protocol according to Burstein et al., 1999, was used.
For the following studies, antibodies against the following antigens were used:
The antibodies were coupled to the following fluorescence systems, where appropriate (either directly or via secondary antibodies) and measured by fluorescence-activated cell sorting (FACS):
Platelets which co-expressed the transgene GFP were gated for this marker by studying FITC fluorescence, and the same measurements were then exclusively related to this gate. The table shows that no significant differences in the activation (shift) of the various markers occurred between freshly isolated human platelets and stem cell-derived modified platelets which overexpressed GFP without further transgenes, although the basal absolute values differed somewhat (especially for CD 61).
Table of Results:
Modified Platelets Expressing GFP:
The content of nucleic acids was compared in freshly isolated and ex vivo generated platelets by a fluorochrome staining assay. A stock solution of dye mix containing 100 μg/mg acridine orange was prepared in PBS. Cells (1×105) were suspended at in staining medium. Two microliters of dye mix were added to 50 μl of cell suspension. Cells were stained for 20 minutes, then washed two times and analyzed by FACS at 530 nm (RNA) and 640 nm (DNA).
Washed platelets were resuspended in 50 μL at 0.9% NaCl. RED or RGD were added as required, where indicated, 10 μl each, and incubated for 5 min. at RT. Fibrinogen was added to the CaCl2 solution at a ratio of 5 mL: 1.25 mL CaCl2+1.5 mL. Fibrinogen (10 μg/μL) was used 20 μL/well. ADP was added in 10 μL. The mix was incubated at 37° C. and photographed under normal and fluorescent light (480 nm). All steps were done in a reaction volume of 100 μL in 96 well-plate flate-bottom-plate.
The measurements (
ELISA Assays for β-thromboglobulin (Asserachrom β-TG, Roche Diagnostics, Mannheim, Germany) and platelet-derived growth factor (PDGF; PDGF-AB Immunoassay, Qiagen, Hilden, Germany) were performed according to the manufacturers' instructions. By using these assays, the thrombin dependent release of both mediators from platelets was assessed.
Release of β-thromboglobulin from stem cell-derived platelets was 52±19 U/ml at basal conditions, 219±11 U/ml after addition of 0.2 U thrombin and 250±92 after 2 U thrombin (p<0.005 basal vs. thrombin). After infection with Ad-GFP, we detected a similar four-fold increase in βTG release. Also, in freshly isolated platelets, an increase from 59±6 to 115±10 U/ml occurred. The release of PDGF from stem cell-derived platelets was 280±95 pg/ml at basal conditions, 590±22 pg/ml after addition of 0.2 U thrombin, and 610 pg/ml after 2 U thrombin (p<0.05 basal vs. thrombin). After infection with Ad-GFP, an increase by 123±7% was determined. In freshly isolated platelets, basal PDGF release (214±11 pg/ml) was similarly increased to 566±10 pg/ml by 0.2 U thrombin, and to 664±12 pg/ml by 1 U thrombin.
Modified platelets (50×106) are resuspended in 0.25 ml of 38 mM citric acid/75 mM trisodium citrate/100 mM dextrose. Carboxyfluorescein diacetat succinimidyl ester (10 ng/ml DCF, Molecular probes, USA) is added to label platelets in vitro. Alternatively, GFP-expressing platelets were used and studied for fluorescence microscopy without prior labelling, because they emitted at the wavelength used to detect FITC emission. After 5 min of incubation, the suspension is centrifuged repeatedly at 2000×g for 10 min. The platelet pellet is resuspended in 0.25 ml phosphate-buffered saline (PBS, Seromed®, Berlin, Germany).
To investigate the role of potential platelet target proteins in diseases such as myocardial infarction, acute coronary syndrome or intestinal ischemia, modified platelets are investigated in intestinal ischemia and reperfusion injury in mice. To this end, fluorescent platelets are infused after intestinal I/R and visualized in the postischemic microcirculabon by intravital fluorescence microscopy. Six-week-old inbred C57BL6/J mice are anaesthetized by inhalation of isoflurane-N2O (0.35 FiO2, 0.015 liter/liter isoflurane; Forene®; Abbott GmbH). The animals are placed on a heating pad (Effenberger), and polyethylene catheters (Portex) are implanted into the left carotid artery and left jugular vein for continuous recording of mean arterial blood pressure and infusion of fluorescent platelets, respectively. After laparotomy, a segment of the jejunum is exteriorized and constantly superfused with 37° C. Ringer's lactat. Labeled, modified platelets (50×106) are infused as bolus via the venous catheter into the acceptor mouse. Segmental jejunal ischemia is induced for 60 min by occluding the supplying vessels with microsurgical clips. Prior to induction of ischemia (baseline conditions) and after reperfusion, the intestinal segment is exposed on a mechanical stage and platelet-platelet (platelet aggregation) and platelet-endothelial cell interactions in the postischemic microvasculature are investigated by intravital microscopy. Prior to induction of ischemia and following the reperfusion, 10 non-overlapping regions of interest from the submucosal vessel of the ischemic/reperfused segment are randomly selected in each mouse and observed for 30 s with a modified microscope (Leitz). The microscopic images with a final magnification on the video screen of 450× are recorded by a CCD camera (FK6990, Cohu; Prospective Measurements) connected to a video recording system (Sony Corp.). Fluorescence is measured with adequate filters, e.g. with filters for FITC, GFP or rhodamine. For analysis of platelet-platelet and platelet-endothelial cell interactions, a computer-assisted image analysis program (CAP IMAGE; Dr. Zeintl, University of Heidelberg, Heidelberg, Germany) and frame-to-frame analysis of the videotapes are used. All experiments are performed in a blinded manner, and adherence of platelets to the surface of arterioles and venules (vessel diameter, 15-85 μm) and formation of platelet aggregates in capillaries (diameter, 15 μm), arterioles, and venules (vessel diameter, 15-85 μm) are quantified. The number of adherent platelets is assessed by counting the platelets that do not move or detach from the endothelial surface within 15 s. Platelet adhesion is presented per square millimeter of endothelial surface; The number of occluding and non-occluding aggregates is quantified within arterioles and venules and is presented per 100 vessels. To determine platelet aggregation in the capillary bed, the length (centimeter) of capillaries occluded by fluorescent platelets is measured and calculated per square centimeter of tissue cross-sectional area.
To define the role of specific platelet target proteins for platelet adhesion to the vascular wall under physiological and pathophysiological conditions, such as atherosclerosis, modified platelets were also investigated in the carotid arteries of healthy or atherosclerotic mice. This approach allows to study their effects in cerebral vessels which play a pivotal role in the pathophysiology of stroke. Wild type C57BL6/J or atherosclerosis-prone ApoE-deficient mice are anesthetized by intraperitoneal injection of a solution of xylazine (5 mg/kg body weight, AnaSed; Lloyd Laboratories) and ketamine (80 mg/kg body weight, Ketaset; Aveco Co, Inc). Polyethylene catheters (Portex, Hythe, England) are implanted into the right jugular vein and fluorescent modified platelets (50×106/250 μl, see example 13) are infused intravenously. The right common carotid artery is dissected free for in situ assessment of platelet vessel wall interactions. Fluorescent platelets are visualized by in vivo videomicroscopy of the right common carotid bifurcation. We monitor platelet-endothelial cell interactions using a Zeiss Axiotech microscope (20× water immersion objective, W 20×/0.5, Zeiss) with a 100 W HBO mercury lamp for epi-illumination. All video-taped images are evaluated using a computer-assisted image analysis program (Cap Image 7.1, Dr. Zeintl, Heidelberg, Germany). Transiently adherent platelets are defined as cells crossing an imaginary perpendicular through the vessel at a velocity significantly lower than the centerline velocity; their numbers are given as cells per mm2 endothelial surface. The number of adherent platelets is assessed by counting the cells that did not move or detach from the endothelial surface within 20 seconds. The number of platelet aggregates at the site of vascular injury was also quantified and is presented per mm2.
To characterize the significance of specific platelet target proteins for platelet adhesion and aggregation on the subendothelial matrix following disruption of the endothelium, e.g. following balloon angioplasty or following spontaneous rupture of the atherosclerotic plaque, the adhesion and aggregation dynamics of transgene platelets are analyzed in a model of vascular injury of the mouse common carotid artery. Wild type C57BL6/J mice are anesthetized by intraperitoneal injection of a solution of xylazine (5 mg/kg body weight, AnaSed; Lloyd Laboratories) and ketamine (80 mg/kg body weight, Ketaset; Aveco Co, Inc). Polyethylene catheters (Portex, Hythe, England) were implanted into the right jugular vein and transgene platelets (50×106/250 μl) are infused intravenously. The right common carotid artery is dissected free and ligated vigorously near the carotid bifurcation for 5 min to induce vascular injury. This leads to complete loss of endothelial cells at the site of injury. Prior to and following vascular injury the fluorescent platelets are visualized in situ by in vivo video microscopy of the right common carotid bifurcation. Again, we monitor platelet-endothelial cell interactions using a Zeiss Axiotech microscope (20× water immersion objective, W 20×/0.5, Zeiss) with a 100 W HBO mercury lamp for epi-illumination. The definitions for transient and firm platelet adhesion were outlined above.
| Number | Date | Country | Kind |
|---|---|---|---|
| 02003352.8 | Feb 2002 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP03/01450 | 2/13/2003 | WO |
