Regulation of blood coagulation by the ZPI/PZ anticoagulant system

ZPI/PZ 抗凝系统对凝血的调节

• 批准号: 10266229
• 负责人: Xin Huang
• 金额: $ 48.37万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-23 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10266229
• 关键词: Anticoagulants; Anticoagulation; Attenuated; Binding; Binding Proteins; Binding Sites; Biological Assay; Blood Coagulation Disorders; Blood coagulation; Calcium; Clinical Treatment; Complex; Crystallization; Disease; Docking; Down-Regulation; Factor X; Factor XI; Factor XIa; Factor Xa; Generations; Goals; Hemophilia A; Hemorrhage; Hemostatic function; Heparin; Heparin Binding; Histidine; Human; In Vitro; Lipids; Maps; Mediating; Membrane; Modeling; Mus; Mutagenesis; Mutate; N-terminal; Pathway interactions; Peptide Hydrolases; Phenotype; Phospholipids; Physiological; Plasma; Protease Inhibitor; Proteins; Prothrombin; Recombinants; Regulation; Research; Role; Serine; Serpin Superfamily; Site; Specificity; Structure; Surface; System; TFPI; Tail; Testing; Therapeutic; Thrombin; Thromboplastin; Thrombosis; Triad Acrylic Resin; Vitamin K; activated Protein C; base; cofactor; efficacy testing; factor V Leiden; improved; in vivo; inhibitor/antagonist; mouse model; mutant; novel; plasma protein Z; prothrombinase complex

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.

ZPI/PZ抗凝系统对血液凝固的调节作用 蛋白Z依赖性蛋白酶抑制剂（ZPI）是一种丝氨酸蛋白酶抑制剂超家族蛋白，可快速抑制 在蛋白Z（PZ）、促凝血磷脂和钙的存在下活化因子X（FXa），或直接 在不存在这些辅因子的情况下抑制活化因子XI（FXIa）。ZPI和PZ在血浆中循环， 复杂. PZ是一种维生素K依赖性蛋白，结构上与因子II、VII、IX、X同源，但缺乏 由于催化三联体中缺乏丝氨酸和组氨酸残基，因此具有蛋白酶活性。的重要性 ZPI和PZ作为止血调节剂已被证明是从研究结果， ZPI或PZ与因子V Leiden（FVL）的结合，在小鼠以及人中导致严重的血栓形成表型。 重要的是，我们发现ZPI/PZ是凝血酶原酶中FXa结合的生理相关抑制剂， (FXa/FVa复合物），与活化的蛋白C系统的凝血酶原酶调节互补， 蛋白水解降解FVa，并通过组织因子途径抑制剂α（TFPIα）抑制中间体 凝血酶原酶结合的FXa复合物在血液凝固的初始阶段。拟议的研究将 表征ZPI/PZ调节凝血酶原酶活性的生理相关性和机制。我们 假设ZPI/PZ动态抑制促凝血剂上凝血酶原酶结合和未结合FXa 通过脂质介导的ZPI-PZ复合物与催化和Gla结构域的桥接相互作用 关于FXa FVa与FXa的结合通过干扰ZPI外位点相互作用而减弱ZPI/PZ抑制的速率 与FXa上的互补外切位点，而凝血酶原通过充当一个外切位点进一步减弱ZPI/PZ抑制， 竞争性或非竞争性底物。肝素是ZPI促进ZPI的另一重要辅因子 在不存在PZ和脂质/Ca 2+辅因子的情况下抑制FXa和FXIa。我们发现了一种新的 ZPI上的肝素结合位点，对肝素刺激ZPI抗凝功能至关重要， 与PZ结合位点相邻。新的肝素结合位点将被定位在ZPI上，我们将阐明 肝素和PZ辅因子竞争ZPI以使ZPI作为多用途抑制剂的机制 膜相关的FXa和从膜逃逸的FXa。我们进行的体内和体外研究， 其他研究小组已经表明，在血友病血浆中止血是治疗性恢复的， 血友病小鼠ZPI/PZ抗凝功能被阻断。因此我们开发了一种新的ZPI- 具有高效力和特异性的拮抗剂，与PZ结合比野生型紧密>20倍的人ZPI突变体 ZPI但缺乏ZPI活性，从而在nM浓度下阻断血浆ZPI/PZ抗凝活性。 我们将进一步发展抗ZPI/PZ治疗血友病的概念，通过i）测试以下药物的疗效： 我们的新型ZPI/PZ拮抗剂在血友病小鼠出血模型中促进止血和ii）开发 优化ZPI/PZ拮抗剂。