Physiology and Molecular Biology of Factor XI

描述（由申请人提供）：因子XI （fXI）是血浆蛋白酶（fXIa）的酶原，通过因子IX的蛋白水解激活有助于止血。这种反应是在血管损伤部位持续产生凝血酶以巩固凝血所必需的。FXIa与血栓性疾病的发病机制有关。在动脉损伤模型中，FXI缺乏可以保护小鼠免于血管闭塞，并保护蛋白C缺乏小鼠免于弥散性凝血导致的围产期死亡。FXI在结构上与其他凝血蛋白酶明显不同。该蛋白是一种同源二聚体，缺乏凝血蛋白酶特有的磷脂结合玻璃结构域。FXI与激肽前体高分子量激肽原（HK）复合物在血浆中循环。每个fXI多肽含有一个n端非催化“重链”区域和一个c端蛋白酶结构域。重链包含四个重复序列，称为苹果结构域（指定为Al到A4），其中包含对蛋白酶功能至关重要的结合位点。fXIa二聚体结构在蛋白酶活性中的作用尚未明确描述。对于fXIa重链上因子IX的结合位点也存在分歧。同样，关于fXI和HK的结合也存在矛盾的数据，这是fXI在血小板上激活所必需的相互作用。在本提案的目标1中，我们将使用标准SDS-PAGE、显色底物测定和稳态各向异性来研究fXIa对因子IX的激活，并将结果与fXIa单链变体（fXIa/PKA4）的结果进行比较。我们还将识别包含底物结合外源性位点的apple结构域，以及识别与fXIa结合的因子IX gla结构域区域。在Aim 2中，我们将描述fXI Al结构域与血小板上fXI激活所需的蛋白质（凝血酶原和凝血酶）之间的结合相互作用，并确定这些结合位点突变对fXI激活和血小板表面活性的影响。分析将包括对血浆蛋白a2糖蛋白1在调节血小板表面fXI活化中的可能作用的研究。最后，在Aim 3中，我们将使用正常和缺乏fXI的小鼠来确定fXI是否在内毒素输注诱导的弥散性血管内凝血的发病机制中起作用。这项工作将产生大量关于凝血蛋白酶的激活和活性的新信息，该酶与多种血栓性疾病有关，并可能确定fXIa作为消耗性凝血病治疗抑制的潜在靶点。

DESCRIPTION (provided by applicant): Factor XI (fXI) is the zymogen of a plasma protease (fXIa) that contributes to hemostasis through proteolytic activation of factor IX. This reaction is required for sustained thrombin generation at a site of vascular injury to consolidate coagulation. FXIa has been implicated in the pathogenesis of thrombotic disease. FXI deficiency protects mice from vascular occlusion in arterial injury models, and protects protein C deficient mice from perinatal death due to disseminated coagulation. FXI differs significantly in structure from other coagulation proteases. The protein is a homodimer that lacks the phospholipid binding Gla-domain characteristic of coagulation proteases. FXI circulates in plasma in complex with the kinin precursor high molecular weight kininogen (HK). Each fXI polypeptide contains an N-terminal non-catalytic "heavy chain" region and a C-terminal protease domain. The heavy chain contains four repeats called apple domains (designated Al to A4), that contain binding sites critical to protease function. The role of the dimeric structure of fXIa in protease activity has not been clearly delineated. There is also disagreement regarding the binding site for factor IX on the fXIa heavy chain. Similarly, there is conflicting data regarding binding of fXI and HK, an interaction that is necessary for fXI activation on platelets. In Aim 1 of this proposal we will use standard SDS-PAGE, chromogenic substrate assays and steady state anisotropy to investigate factor IX activation by fXIa, and compare the results to those from a single chain variant of fXIa (fXIa/PKA4). We will also identify the apple domain that contains the substrate binding exosite, and identify the area of the factor IX Gla-domain that binds to fXIa. In Aim 2 we will characterize binding interactions between the fXI Al domain and proteins thought to be required for proper fXI activation on platelets (prothrombin and thrombin), and determine the effects of mutations in these binding sites on fXI activation and activity on the platelet surface. The analysis will include an investigation into a possible role for the plasma protein a2 Glycoprotein 1 in regulating fXI activation on the platelet surface. Finally, in Aim 3, using normal and fXI-deficient mice, we will determine if fXI plays a role in the pathogenesis of disseminated intravascular coagulation induced by endotoxin infusion. The work will generate substantial new information regarding the activation and activity of a coagulation protease that is implicated in a variety of thrombotic disorders, and may identify fXIa as a potential target for therapeutic inhibition in consumptive coagulopathies.