Previous studies from our laboratory as well as work from other laboratories have focused attention upon coagulation factor XI (FXI) and its interactions with other plasma proteins (i.e., factor XIIa [FXIIa], high molecular weight kininogen [HMrK] and factor IX [FIX], with the platelet plasma membrane and with various inhibitory molecules (i.e., alpha1protease inhibitor [alpha1PI] and a secreted platelet FXIa inhibitor ([PXIaI) in the initiation and regulation of blood coagulation. The long- term goals of the present proposal are to elucidate the molecular mechanisms involved in the interaction of FXI/XIa with FXIIa, HMrK, FIX, alpha1PI and PXaI, and to define the biological significance of these reactions in the regulation of normal hemostasis. Ultimately we hope to exploit the resultant structural information to develop novel therapeutic approaches to the prophylaxis and treatment of thromboembolic disorders. Specifically we propose: 1. To utilize monoclonal antibodies (MoAbs) to identify functional domains within the FXI/XIa molecule important in its interactions with FXIIa, HMrK, FIX, alpha1PI and PXIaI. 2. To map epitopes within the FXI/XIa molecule recognized by these MoAbs using fragments of FXI cDNA inserted into the coliphage expression vector lambdagt11 to make short fusion peptides to be screened for binding to MoAbs. 3. To fragment the FXI molecule both chemically and enzymatically in order to identify and sequence domains that interact with FXIIa, HMrK, FIX, alpha1PI and PXIaI. 4. To employ protein footprinting techniques to identify and characterize domains within the FXI/XIa molecule that interact with inhibitory MoAbs and with FXIIa, HMrK, FIX, alpha1PI and PXIaI. 5. To prepare synthetic peptides representing FXI/XIa domains containing binding sites for FXIIa, HMrK, FIX, alpha1PI and PXIaI. 6. To characterize two secreted platelet FXIa inhibitors (PXIaI) both biochemically and functionally and to determine their biological significance as regulators of FXIa in comparison with alpha1PI. These proposed studies will enable us to utilize combine immunochemical, biochemical, and molecular biological techniques to provide definitive information about the structure of FXI/XIa domains essential for these important intermolecular interactions and to define the structure-function relationships involved in the initiation and regulation of intrinsic coagulation.
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