Research Project
6184692
The integrin alphaIIbbeta3 is of key importance in the process of platelet thrombus formation, but the sequence of events that modulate its distinct functions on resting and activated platelets is not yet fully understood. The long term objective of this proposal is to gain insight into the dynamic regulation of the adhesive forces mediated by alphaIIbbeta3 under defined functional conditions. The first experimental model will evaluate receptor interaction with surface-bound ligand in a rapid (spin-on) adhesion assay not involving shear forces. Another method will measure directly the adhesive strength between a cell expressing recombinant alphaIIbbeta3 and a surface bound ligand using the single cell micropipette system. Lastly, the effects of varying shear forces on the functional state of alphaIIbbeta3 will be assessed using the parallel plate laminar flow model system. Recombinant expression of alphaIIbbeta3 on Chinese hamster ovary cells (A5 cells) will allow the detailed study of structure-function relationships by evaluating the effects of specific targeted mutations in relevant domains of the receptor. Moreover, the GP Ib-IX complex has been successfully coexpressed in the same cell with alphaIIbbeta3, thus making it possible to evaluate the synergistic role of these two receptors in supporting adhesion under high shear stress. Two external agonists, an activating monoclonal antibody (AP5 or anti-LIBS-6) and divalent ions, will be used to modulate different functional states of the receptor. To gain insight into the progression of alphaIIbbeta3 through its pathway of activation, the following specific aims are proposed: 1) To define the functional properties of each state of recombinant alphaIIbbeta3; 2) to examine the ligand binding specificities of this receptor with respect to immobilized and soluble ligands and discrete ligand domains; 3) to characterize the functional properties of the receptor under conditions of flow; 4) to establish the role of distinct domains of the receptor, such as cation binding sites, ligand binding sites and cytoplasmic tail sequences, in maintaining the distinct functional states of recombinant alphaIIbbeta3. The results obtained from these studies will contribute to elucidating the mechanism of alphaIIbbeta3 activation; this, in turn, will clarify an important aspect of platelet function during thrombogenesis.
