The purpose of this investigation is to develop a highly selective proteolytic inhibitor of a-thrombin that prevents its interaction with platelet receptors but does not interfere wit a-thrombin cleaving other substrates. The hypothesis of this proposal is that bradykinin-related sequences are inhibitors of a -thrombin cleaving certain physiologic substrates. Developing a highly selective thrombin inhibitor directed to the cloned thrombin receptor on cells would have applicability to prevent arterial thrombosis due to platelet thrombus. A new generation of selective a-thrombin inhibitors needs to be developed since nonspecific proteolytic inhibitors of a-thrombin, e.g. hirudin and hirulog, are associated with increased cerebral hemorrhage. This project will create a new class of selective a-thrombin inhibitors. These agents will be the first of a second generation a-thrombin inhibitors that selectively block a-thrombin s ability to activate platelets without interfering with its ability to activate factor V and VIII and clot fibrinogen The specific aims of this proposal are as follows: 1. The mechanism of selective inhibition of a-thrombin by RPPGF will be characterized. Investigations will be performed to determine if RPPGF binds to platelets and insect cells which express the clone thrombin receptor. Further, investigations will be performed to determine if RPPGF binds to Protein C and Protein S. 2. The structural requirements for RPPGF to inhibit a-thrombin will be characterized with the aim towards the development of a novel class of selective, a-thrombin inhibitors. 3. Animal experiments will be performed in rabbits to determine the efficacy of these peptides to prevent arterial thrombosis in vivo models. These proposed investigations will determine the mechanism of inhibition and efficacy of using RPPGF and its congers as selective anti-thrombins. These investigations should characterize a new class of a-thrombin inhibitors which inhibit this enzyme's ability to cleave the cloned thrombin receptor. The importance of these studies is that they could introduce a novel pharmacologic approach to prevent arterial thrombosis.
