Novel Anticoagulant Mechanisms

描述（由申请人提供）：长期目标是增加对调节凝血机制的了解，以便开发新的抗血栓治疗和预防措施。凝血调节的缺陷导致血管疾病风险增加，这是死亡的主要原因。研究将集中在一种新的凝血调节剂，蛋白Z依赖性蛋白酶抑制剂（ZPI），在蛋白Z，Ca 2+和磷脂（PL）的存在下，迅速灭活因子Xa（FXa）。我们的研究表明，ZPI/蛋白Z也抑制FIXa。低蛋白Z水平与卒中相关，提示生理相关性。我们假设：A）ZPI和蛋白Z是FXa和FIXa的显著下调剂; B）ZPI抑制的机制不同于大多数丝氨酸蛋白酶抑制剂的机制; D）ZPI通过不同的机制抑制FIXa和FXa;和C）定义ZPI机制和蛋白-蛋白相互作用的位点可能导致新的抗血栓形成疗法。具体目的是：1）评估ZPI和蛋白Z是否是FXa和FIXa的显著抑制剂。我们将确定在ZPI/蛋白Z、凝血酶原和FX的血浆水平下是否发生FXa和FIXa的抑制。我们将评估ZPI抑制FXa相对于FlXa的重要性，并确定联合抑制作用是否是累加的。我们将发现FXa-ZPI或FlXa-ZPI是否是当FIXa或FXa加入血浆时形成的主要复合物。2)定义ZPI/蛋白Z抑制FXa的机制。大多数丝氨酸蛋白酶抑制剂不需要蛋白质辅因子、PL或Ca 2+，并且丝氨酸蛋白酶抑制剂-蛋白酶复合物通常是共价的和不可逆的。ZPI对FXa的抑制似乎在这些方面有所不同。我们将阐明ZPI不寻常的机制。我们将发现是否发生FXa对ZPI的切割，以及是否可以形成ZPI-蛋白质Z-FXa的三元复合物。3)确定ZPI抑制FIXa和FXa的机制有何不同。ZPI对FXa的抑制严格需要蛋白Z，FVa部分保护FXa免受ZPI的影响。我们将扩展表明ZPI抑制FIXa不需要蛋白Z和FVIIIa增强ZPI抑制FIXa的发现。我们将找到差异的原因，并发现ZPI对FIXa的抑制是否与FXa在二元/三元复合物、复合物的共价性或可逆性方面不同。4)阐明导致FIXa或FXa抑制的ZPI和蛋白Z的结构-功能关系。我们将定义这些蛋白质之间的相互作用的一些关键区域进行功能研究与修改后的蛋白质，选定的突变体的rZPI和肽代表候选序列的相互作用。我们将发现我们确定的ZPI多态性是否影响功能。

DESCRIPTION (provided by applicant): The long-term goal is to increase knowledge of mechanisms that regulate blood coagulation so that new antithrombotic therapies and preventative measures can be developed. Defects in coagulation regulation lead to increased risk of vascular disease, a major cause of death. Studies will focus on a novel coagulation regulator, protein Z-dependent protease inhibitor (ZPI) that rapidly inactivates Factor Xa (FXa) in the presence of protein Z, Ca 2+ and phospholipids (PL). Our studies show that ZPI/protein Z also inhibits FIXa. Low protein Z levels are associated with stroke, suggesting physiologic relevance. We hypothesize that: A) ZPI and protein Z are significant down-regulators of both FXa and FIXa; B) Mechanisms of ZPI inhibition differ from those of most serpins; D) ZPI inhibits FIXa and FXa by different mechanisms; and C) Defining ZPI mechanisms and sites of protein-protein interaction may lead to new antithrombotic therapies. Specific aims are: 1) Assess whether ZPI and protein Z are significant inhibitors of FXa and FIXa. We will determine if inhibition of both FXa and FIXa occurs at plasma levels of ZPI/protein Z, prothrombin and FX. We will assess the importance of ZPI inhibition of FXa vs. FlXa and determine if combined inhibitory effects are additive. We will find if FXa-ZPI or FlXa-ZPI are major complexes formed when FIXa or FXa are added to plasma. 2) Define mechanisms for ZPI/protein Z inhibition of FXa. Most serpins do not require a protein cofactor, PL or Ca 2+, and serpin-protease complexes are usually covalent and irreversible. Inhibition of FXa by ZPI seems to differ in these respects. We will elucidate ZPI's unusual mechanisms. We will find if cleavage of ZPI by FXa occurs and if ternary complexes of ZPI-protein Z-FXa can form. 3) Determine how mechanisms of ZPI inhibition of FIXa and FXa differ. FXa inhibition by ZPI strictly requires protein Z and FXa is partly protected from ZPI by FVa. We will extend findings suggesting that ZPI inhibition of FIXa does not require protein Z and that FVIIla enhances ZPI inhibition of FIXa. We will find the reasons for the differences and discover if ZPI inhibition of FIXa differs from that of FXa with respect to binary/ternary complexes, covalency of complexes, or reversibility. 4) Elucidate structure-function relationships for ZPI and protein Z that lead to FIXa or FXa inhibition. We will define some of the regions crucial for interaction between these proteins by performing functional studies with modified proteins, selected mutants of rZPI and peptides representing candidate sequences for interactions. We will find whether a ZPI polymorphism we identified affects function.