(Applicant's list of Aims) Specific Aim 1: To elaborate the mechanism by which TF is transferred to the platelet surface. We have documented that during ex-vivo thrombus formation platelets become TF-positive as judged by light-and electron- microscopy. Because platelets with bound TF would be an extremely thrombogenic species, we plan to study the detailed mechanism by which this transfer takes place. Initial studies will focus on the interaction of platelets and leukocytes in order to define the molecules required for this transfer. The transfer of TF will be evaluated morphometrically following shearing of leukocyte derived microparticles with fresh human platelets. We also have utilized FACS analysis for quantitative assessment of the transfer. Both techniques indicate that the CD-15 p-selectin ligand-receptor pair is involved as is TF,itself. Because we and others have identified TF- positive neutrophils (PMNs) in thrombi and in tissues, we propose to investigate the ability of these cells to synthesize TF.
Specific Aim 2 : Purification and characterization of a Tissue Factor Binding Protein from platelets: Our hypothesis is that the interaction of leukocyte-derived TF with platelets is an essential step in ex-vivo thrombus formation and these experiments are designed to provide the tools to test this hypothesis. Because we have shown that a monoclonal antibody to TF blocks this transfer, we believe that there likely is a tissue factor binding protein on platelets. We have initiated the purification of this protein using a combination of classical and affinity steps. As judged from ligand blots of proteins electro-tranferred from SDS gels, we have identified a candidate protein with an apparent mol wt of approximately 200,000, unreduced, and approximately 80,000 when reduced. We propose to purify and characterize this protein. We will identify the protein from amino acid sequence data performed on eluates from SDS gels. If the protein is present in the Brookhaven or other national databases, we will search for any known functions. If the cDNA is available, we propose to express it in an appropriate cell line. If not, we propose to clone the cDNA coding for it and then express it. Further, the extra-cellular domain of TF (sTF, TF1-218) binds to platelets as judged by enzyme kinetics utilizing sTF and factor VIIa and estimating the velocity of factor X activation. These kinetic experiments indicate that sTF and platelets interact to form an active catalytic complex. We propose to evaluate this hypothesis by directly measuring the binding by using fluorescence techniques. Knowing the equilibrium binding constant for this interaction will enable us to interpret the kinetic results and to test our hypothesis relating to the activity of TF bound to platelets.
Specific aim 3 : To Describe ex-vivo thrombus growth in detail. The order in which platelets, fibrin and TF are deposited is largely unknown and our hypothesis regarding the incorporation of circulating TF in thrombi can be tested by the experiments described in this aim in which we address the microscopic details of thrombus growth. Specifically, we propose to develop a system in which we can visualize in real time the deposition of fibrin, platelets and TF as ex vivo thrombi develop. We also propose to examine the permeability of thrombi to proteins. And, lastly, we plan to develop simulations of the diffusion of proteins generated at the vessel wall, e.g., factor Xa, into an overlying platelet mass by real-time observation of the deposition of these species. Three monoclonal antibodies or their fragments, which do not inhibit thrombus growth, will be labeled with different fluorophores. Thrombi will be formed by perfusion of blood over a collagen-coated glass slide in a parallel plate chamber. Our existing equipment will enable us to visualize three fluorophores each five seconds. (To be done in collaboration with Dr. Eric Grabowski, Harvard Medical School and Dr. Scott Diamond, Institute for Engineering and Medicine, U. Penn).
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