The long-term objective of this research proposal is to understand the mechanisms by which the principal two plasma inhibitors of thrombin, antithrombin and heparin cofactor II, are activated by heparin-like glycosaminoglycans. Such an understanding will help explain dysfunction which leads to familial thrombophilia and aid in the development of novel therapeutic antithrombotic agents. This objective will largely be fulfilled through the successful completion of the specific aims which follow from the general hypotheses that: i) antithrombin and heparin cofactor II circulate in a kinetically inactive state; ii) binding to and activation by glycosaminoglycans involves a dramatic conformational change to a kinetically active state; iii) the shared serpin mechanismn of protease inhibition plays a critical role in facilitating release of the final complex from glycosaminoglycan and fibrin to allow for clearance and in situ degradation.
The specific aims fall under three general questions: I) How does heparin accelerate antithrombin inhibition of thrombin? II) How is antithrombin activated towards factor Xa? and, III) How is heparin cofactor II activated towards thrombin? A combination of protein X-ray crystallographic and biochemical studies will be employed to answer these questions.
The specific aims are: 1) To determine the structures of antithrombin and thrombin bound to heparin. 2) To determine the structure of the initial antithrombin-thrombin-heparin complex. 3) To determine the structure of the final antithrombin-thrombin complex. 4) To determine the structure of native and pentasaccharide-activated antithrombins. 5) To investigate the role of surface electrostatics in the allosteric activation of antithrombin. 6) To determine the structure of native and glycosaminoglycan bound heparin cofactor II. 7) To determine the structure of the initial heparin cofactor Il-thrombin complex. 8) To investigate the conformational link between glycosaminoglycan binding and release of the N-terminal tail. 9) To determine the conformational basis for cleavage of the N-terminal tail from the final heparin cofactor II-thrombin complex.
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