Previous studies supported by NIH grant HL46213 (""""""""Molecular Interactions of Factor XI""""""""), for which continued support is sought, have focused attention on FXI and its interactions with other plasma proteins, i.e., thrombin, FXIa, FXIIa, prothrombin, high molecular weight kininogen (HK), and FIX, with the platelet plasma membrane and with various inhibitory molecules, e.g., protease nexin II (PN2), in the initiation and regulation of blood coagulation. These studies, utilizing NMR and x-ray crystallography, provide three separate structures, one of the catalytic domain of FXIa in complex with the KPI domain of PN2, one of the Apple 4 (A4) domain dimer and one of the zymogen FXI dimer that provide the basis for the future proposed investigations. The long-term goals of the present proposal are to elucidate the molecular mechanisms involved in the interaction of FXI/FXIa with protein and cell surface ligands involved in its activation and with plasma protein and cell surface ligands involved in the expression and regulation of FXIa enzymatic activity. Specifically these studies propose to determine the mechanism and physiological importance of FXI homodimer formation by a mutational analysis of the A4 dimer interface to prepare a monomeric FXI molecule; to study the role of the A4 domain in FXI dimerization in equilibrium unfolding studies; and to determine whether the dimeric structure of FXI is required for FXI activation to FXIa by thrombin or by FXIIa in the presence or absence of activated platelets or glycocalicin. They will examine the hypothesis that the activation of FXI to FXIa is accompanied by a major conformational change that promotes the efficient activation of FIX by exposing a substrate-binding site in the heavy chain (A2 and/or A3 domains) of FXIa for efficient binding and activation of FIX. Finally they will determine whether the dimeric structure of FXIa is required for efficient FIX-activation and to differentiate between alternative mechanisms to account for the failure of monomeric FXIa to activate FIX on the activated platelet surface. Recent observations resulting in the recognition of the essential role of FXI in hemostasis and thrombosis concern the relationship of its domain structure to its biological function, its molecular and cellular interactions, the elucidation of pathways for activation of FXI, and the expression and regulation of its enzymatic activity. It is hoped that information gleaned from the proposed studies will result in novel approaches to targeting FXI/XIa in the development of new anticoagulants that prevent thrombosis (heart attacks, strokes and pulmonary emboli) without bleeding complications. ? ? ?
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