Six specific aims are identified. These include: 1) investigating the mechanism by which heparin facilitates dissociation between thrombin the HCII mutant Leu444 Arg; 2) to determine the affects of synthetic reactive site peptides on the ability of heparin cofactor 2 to serve as an inhibitor or substrate; 3) to identify structures in dermatan sulfate required for high-affinity binding to heparin cofactor II; 4) to crystallize heparin cofactor II and its complexes with dermatan sulfate for x-ray analysis; 5) to determine the location of endogenous heparin cofactor II in human and murine tissues by immunohistochemistry; 6) to prepare by homologous recombination, homozygous heparin cofactor II-deficient mice. The first Specific Aim is based on an observation by the investigator that for HCII Leu444 Arg, the complex with thrombin can be reversed if heparin is present, while the complex is stable in the presence of dermatan sulfate or in the absence of the added cofactor. The data obtained thus far suggests a model which involves the initial reversible complex of inhibitor and enzyme to an initially reversible covalent bond and subsequently to an SDS irreversible covalent bond. The studies anticipated with this mutant will allow elucidation and confirmation of this attractive mechanism. In the second Specific Aim, peptides which mimic the reactive site loop present in all serpins will be evaluated with respect to their ability to influence HCII inhibitory functions. Various loop peptides incorporating radiolabels with different sequences will be used to evaluate the interaction between the reactive site loop and the beta sheet will be permissible. Using binding and activity measurement. The peptide will also permit evaluation of the influence of glycosaminoglycans on the conformational-linkage between the various states of HCII. An evaluation of thrombin inhibition by binary complexes of synthetic peptide and HCII in the presence and absence of glycosaminoglycans will be evaluated using activity for the formation of covalent complexes with 125[I] thrombin. The third Specific Aim, tetra- and penta-sulfated hexasacchrides from porcine skin dermatan sulfate will be evaluated with respect to their structure and their ability to influence HCII binding and function. The techniques of approach are fairly common in the identification of glycosaminoglycan structure involved classical carbohydrate chemistry. In the further Specific Aim HCII and its complexes will be subjected to hanging drop crystallization techniques in an attempt to produce crystals of both the protein and its complexes suitable for crystallographic evaluation. The fifth and sixth Specific Aims involve the generation of a murine HCII deficiency model. Studies involve histochemistry in both human and murine systems to identify the extravascular sites of HCII. The development of a murine deletion model by homologous recombination in embryonic stem cells using the now standard techniques.
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