The purpose of the proposal is to characterize further cell interactions with fibrinogen and fibrin. We will identify and characterize the fibrinogen binding sites on the glycoprotein IIb/IIIa complex. We have observed that limited proteolysis of platelet membranes results in the exposure of fibrinogen receptors and in the release of a GP IIIa-derived, 80kDa component and a 66kDa component which appear to represent the C-terminal portion of the molecule. This suggests that fibrinogen binding sites are located in the NH2-terminal portion of the GPIIIa molecule. The GPIIIa and 80kDa components bind to insolubilized anti-GPIIb/IIIa monoclonal antibody, which blocks fibrinogen binding to platelets, whereas the 66kDa component does not interact with this antibody. We propose to isolate the fragment of GPIIIa associated with the fibrinogen binding domain and to study its effect on platelet- fibrinogen interaction. We also propose to test the hypothesis that limited proteolysis of the NH2-terminal domain of GPIIIa, or its reduction, may change the conformation of this molecule and lead to the exposure of fibrogen receptors. Furthermore we intend to produce recombinant DNA trigramin; trigramin is a low molecular weight peptide (72 amino acids) purified to homogeneity in our laboratory from T. gramineusu snake venom. It inhibits specifically the binding of 125I-fibrinogen to the GPIIb/IIIa complex with a Ki of 10-8 M. Trigramin contains the RGD (arg-gly- ser) sequence but its biological activity depends on secondary and tertiary structure. lt is also 500 times more active than RGDS. We propose, by isolating active peptide fragments of trigramin and by site directed mutagenesis, to study the relationship between the structure and function of this molecule. We further propose: to use trigramin to investigate platelet-fibrinogen interactions; to study the effect of trigramin on the adhesion of platelets and cultured cells to sutstrata cultured with adhesive proteins; and to explore the potential application of trigramin in the prevention of the formation of platelet thromboemboli in vivo. Finally, we ill compare the interaction of fibrinogen and of polymerizing fibrin with platelets. We propose to test the hypothesis that polymerizing fibrin monomers bind to platelet surface receptors other than those associated with the GPIlb/IIIa complex.
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