Thrombotic and thromboembolic diseases are associated with excessive activation of the coagulation system. The assembly of a protease with a cofactor is a key regulatory mechanism to increase the catalytic activity of the bound protease. The mechanism of the catalytic enhancement is unclear, but an understanding of the rules of activation of the protease domain and the reduction to a structural model can lead to the development of highly specific and novel therapeutic strategies. This application proposes to study the domain structure of a prototypic serine protease, factor VIIa (VIIa), its macromolecular assembly with cofactor tissue factor (TF) and with substrates. Site directed mutagenesis will be used to introduce cysteine residues at predicted surface positions in the VIIa light and heavy chain domains. These single free cysteine residues will provide a tag for specific modification with thiol-reactive reagents and crosslinking from these defined position in VIIa will be used to search for nearest neighbor residues in order to (1) establish the interdomainal organization in VIIa and its change upon catalytic activation of VIIa following cofactor assembly (2) identify complementary interactive sites of VIIa and TF (3) analyze the relative contribution of VIIa light and heavy chain regions to macromolecular assembly of substrate. The conclusions from crosslinking experiments will be tested for consistency with monoclonal antibody analysis as independent probes for the surface of VIIa, as well as, with the mapping of interactive areas by scanning alanine mutagenesis which will be guided by preliminary structural models. These independent approaches will provide conclusive evidence in the assignment of functional regions in VIIa. This analysis will further our understanding of the structural mechanism for macromolecular assembly and acquisition of catalytic function of VIIa and of other homologous proteases. The structural understanding of VIIa function will ultimately improve the therapy of thrombotic complications associated with malignancy and occlusive vascular lesions, and of excessive activity of the extrinsic coagulation pathway which contributes to lethality in gram negative septic shock.
Showing the most recent 10 out of 20 publications
