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疗法和预防措施。凝血调节的缺陷会增加血管疾病的风险，而血管疾病是导致死亡的主要原因。研究将集中在一种新型的凝血调节剂，即蛋白Z依赖的蛋白酶抑制物(ZPI)，它能在蛋白Z、钙和磷脂(PL)存在的情况下快速灭活Xa因子(FXA)。我们的研究表明，ZPI/Protein Z也抑制FixA。低蛋白Z水平与中风有关，提示与生理相关。我们假设：A)ZPI和蛋白Z对FXA和FIXA都有显著的下调作用；B)ZPI的抑制机制不同于大多数蛇；D)ZPI通过不同的机制抑制FXA和FXA；以及C)确定ZPI的机制和蛋白质相互作用的位置可能导致新的抗血栓治疗。具体目标是：1)评估ZPI和蛋白Z是否是FXA和FixA的显著抑制剂。我们将在血浆ZPI/蛋白Z、凝血酶原和FX水平上确定是否同时抑制FXA和FIXA。我们将评估FXA和FlXa的ZPI抑制的重要性，并确定联合抑制效应是否是相加的。我们将发现FXA-ZPI或FlXa-ZPI是当FIXA或FXA加入到血浆中时形成的主要络合物。2)明确ZPI/Protein Z抑制FXA的机制。大多数蛇类不需要蛋白质辅因子PL或钙，而且蛇胶-蛋白酶复合体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的不寻常机制。我们将发现ZPI是否被FXA切割，以及是否能形成ZPI-蛋白质Z-FXA的三元复合体。3)确定FIXA和FXA抑制ZPI的机制有何不同。ZPI对FXA的抑制严格要求蛋白Z，FVA对FXA有部分保护作用。我们将扩大研究结果，表明抑制ZPI的FixA不需要蛋白质Z，而FVIIla增强了FixA的ZPI抑制。我们将找出差异的原因，并发现FixA和FXA的ZPI抑制是否在二元/三元络合物、络合物的共价性或可逆性方面不同。4)阐明ZPI和蛋白Z的结构-功能关系导致FIXA或FXA抑制。我们将通过对修饰的蛋白质、rZPI的选定突变体和代表候选相互作用序列的多肽进行功能研究，来定义这些蛋白质之间相互作用的一些关键区域。我们将发现我们发现的ZPI多态是否会影响功能。

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.