Regulation of blood coagulation by the ZPI/PZ anticoagulant system

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Regulation of blood coagulation by the ZPI/PZ anticoagulant system Protein Z-dependent protease inhibitor (ZPI) is a serpin superfamily protein that rapidly inhibits activated factor X (FXa) in the presence of protein Z (PZ), procoagulant phospholipids and calcium, or directly inhibits activated factor XI (FXIa) in the absence of these cofactors. ZPI and PZ circulate in plasma as a tight complex. PZ is a vitamin K-dependent protein, structurally homologous to factors II, VII, IX, X, but lacking protease activity due to the absence of serine and histidine residues of the catalytic triad. The importance of ZPI and PZ as regulators of hemostasis has been demonstrated from the findings that combined deficiencies of ZPI or PZ with factor V Leiden (FVL), result in a severe thrombosis phenotype in mice as well as in humans. Importantly, we have found that ZPI/PZ is a physiologically relevant inhibitor of FXa bound in prothrombinase (FXa/FVa complex), complementary to prothrombinase regulation by the activated protein C system which proteolytically degrades FVa, and by tissue factor pathway inhibitor α (TFPIα) inhibition of an intermediate prothrombinase-bound FXa complex at the initial stage of blood coagulation. The proposed studies will characterize the physiologic relevance and mechanism by which ZPI/PZ regulates prothrombinase activity. We hypothesize that ZPI/PZ dynamically inhibits prothrombinase-bound and unbound FXa on a procoagulant membrane through lipid-mediated bridging interactions of the ZPI-PZ complex with catalytic and Gla domains of FXa. FVa binding to FXa attenuates the rate of ZPI/PZ inhibition by interfering with ZPI exosite interactions with complementary exosites on FXa, whereas prothrombin further attenuates ZPI/PZ inhibition by acting as a competitive or noncompetitive substrate. Heparin is another important cofactor of ZPI in promoting ZPI inhibition of FXa and FXIa in the absence of PZ and lipid/Ca2+ cofactors. We have identified a new extended heparin-binding site on ZPI that is critical for heparin stimulation of ZPI anticoagulant function and that lies adjacent to the PZ binding site. The new heparin-binding site will be mapped on ZPI and we will elucidate the mechanism of heparin and PZ cofactors competing for ZPI to allow ZPI to act as a multi-purpose inhibitor of both membrane-associated FXa and FXa that escapes from a membrane. In vivo and in vitro studies by us and other research groups have shown that hemostasis is therapeutically restored in hemophilic plasma and in hemophilic mice when ZPI/PZ anticoagulant function is blocked. We have thus developed a novel ZPI- antagonist with high potency and specificity, a human ZPI mutant that binds PZ >20-fold tighter than wild-type ZPI but is devoid of ZPI activity, thereby blocking plasma ZPI/PZ anticoagulant activity at nM concentrations. We will further develop the concept of anti-ZPI/PZ therapy for hemophilia treatment by i) testing the efficacy of our novel ZPI/PZ antagonist in promoting hemostasis in hemophilia mice bleeding models and ii)developing and optimizing ZPI/PZ antagonists.