The activation of the coagulation pathways is essential for the pathophysiology of inflammation and thrombosis. Coagulation proteases activate soluble plasma proteins leading to clot formation, as well as, trigger cellular responses through protease activated receptors. Central to the understanding of the structural basis for function of the coagulation proteases is their dependence on cell surface localized co- factors that enhance catalytic activity and direct substrate specificity. The availability on high resolution structural data for the TF-VIIa complex in conjunction with an extensive mapping of VII's functional surface accomplished in the previous funding period provides an opportunity to progress an understanding of the molecular details of allosteric regulation of this co-factor-dependent serine protease. The preliminary data indicate specific conformational linkages that involve the catalytic cleft, the co-factor binding site and the macromolecular substrate exosite. VIIa retains zymogen-like features and requires co- factor to be """"""""switched"""""""" to an active enzyme conformation.
Aim 1 proposes to define the molecular basis for this labile zymogen to enzyme transition of VIIa that may reveal generalizable rules for the molecular details of co-factor-dependent, allosteric regulation of serine protease. The identification of a unique conformation-sensitive antibody that detects a """"""""crosstalk"""""""" between active site occupancy and the macromolecular substrate exosite is the basis for Aim 2 that analyzes the importance of conformational flexibility of this exosite for substrate cleavage. Experiments in Aim 3 define the structural basis for substrate specificity by dissecting pro-coagulant and signaling functions of VIIa. This knowledge enables the design of a """"""""signaling- defective"""""""" VIIa molecular that allows analysis of the biological function of VIIa's cell signaling in vivo. The detailed insight into allosteric regulation and determinants of substrate specificity in VIIa may provide novel strategies for therapeutic intervention in inflammatory and thrombotic diseases.
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