Physiology and Molecular Biology of Factor XI

描述（由申请人提供）：因子Xi（fXi）是同源二聚体血浆蛋白酶因子XIa（fXIa）的酶原，其通过将因子IX（fIX）转化为蛋白酶fIXa而有助于止血。虽然fXI缺乏导致相对轻度的出血性疾病，但越来越多的证据表明，这种蛋白质实质上有助于病理过程，如血栓形成和弥散性血管内凝血。除了其二聚体结构之外，fXIa还具有与维生素K依赖性蛋白酶不同的结构特征。在这项提案中，我们解决有关fXI激活机制的问题，以及fXIa与fIX和血小板的相互作用，在止血。通常，fXI通过因子XIIa（fXIIa）转换为fXIa。然而，fXII缺乏不会导致异常出血。- 凝血酶在溶液中激活fXI，但这发生在血浆中的前提最近受到了挑战。因为它是同源二聚体，fXIa每个分子有两个活性位点。最近，我们表明，fXI到fXIa的转换收益通过一个物种与一个活性位点（1/2-fXIa），功能上类似于fXIa。在目的1中，我们解决的假设，fXI激活凝血酶在血浆中，和其他产品的凝血酶原激活，如meizothrobin和凝血酶，激活fXI。我们将检查每个fXI亚基的激活动力学，比较激活fXIIa，凝血酶，凝血酶，meizothrobin，凝血酶与阴离子结合exosites的突变，并将研究的重要性fXI二聚体结构的激活在纯化和血浆系统。FIX活化涉及顺序蛋白水解裂解，首先在Arg 145后产生中间体fIX，然后在Arg 180后产生fIXa。我们已经表明，fXIa重（非催化）链上的外位点是fIX转化为fIXa所需的。新的证据表明，第二个网站，可能在催化域，但不同的活性位点，也是必要的。我们假设，这第二个网站是必需的fIX的fIX的转换，而重链外位点作出贡献的两个裂解。在目标2中，我们将研究外切位点相互作用在fXIa激活fIX中的作用，描述fXI重链和催化结构域上的外切位点对两个fIX激活位点裂解的重要性，并确定Arg 145处的裂解是否是Arg 180处裂解的先决条件。FXI与血小板糖蛋白1b（GP 1b）结合。虽然有人提出这种相互作用促进fXI激活，但现在还不确定。初步工作表明，fXI还结合血小板上的载脂蛋白E（ApoER 2）的受体。ApoER 2可以与血小板上的GP 1b形成复合物，两者可能是fXI结合位点的组成部分。我们假设fXI-ApoER 2相互作用涉及fXI上的结合位点，该结合位点不同于与GP 1b结合所需的结合位点。在目标3中，我们将评估fXI和活性物质fXIa和1/2-fXIa与血小板的相互作用，研究血小板对基于流动的系统中fXI活化和活性的重要性，并鉴定fXI上参与与血小板ApoER 2相互作用的结合位点。公共卫生相关性：血浆因子XIa对血管损伤部位的正常血液凝固（止血）有适度的贡献，但似乎对病理过程（如血管血栓形成）中的凝固有不成比例的更大贡献。因此，这种蛋白酶是新型抗血栓治疗的合理靶点，与目前可用的抗凝剂相比，这种抗血栓治疗应具有较低的危及生命的出血风险。

DESCRIPTION (provided by applicant): Factor XI (fXI) is the zymogen of a homodimeric plasma protease, factor XIa (fXIa) that contributes to hemostasis by converting factor IX (fIX) to the protease fIXa. While fXI deficiency causes a relatively mild bleeding disorder, there is mounting evidence that this protein contributes substantively to pathologic processes such as thrombosis and disseminated intravascular coagulation. In addition to its dimeric structure, fXIa has structural features that are distinct from those of vitamin K-dependent proteases. In this proposal, we address questions regarding mechanisms of fXI activation, and the interactions of fXIa with fIX and platelets, during hemostasis. Classically, fXI is converted to fXIa by factor XIIa (fXIIa). However, fXII deficiency does not cause abnormal bleeding. -thrombin activates fXI in solution, but the premise that this occurs in plasma has been challenged recently. Because it is a homodimer, fXIa has two active sites per molecule. Recently we showed that conversion of fXI to fXIa proceeds through a species with one active site (1/2-fXIa) that is functionally similar to fXIa. In Aim 1, we address the hypotheses that fXI is activated by -thrombin in plasma, and that other products of prothrombin activation, such as meizothrombin and -thrombin, activate fXI. We will examine the kinetics of activation of each fXI subunit, comparing activation by fXIIa, -thrombin, -thrombin, meizothrombin, and -thrombin with mutations in anion binding exosites; and will study the importance of the fXI dimeric structure to activation in purified and plasma systems. FIX activation involves sequential proteolytic cleavage, first after Arg145 producing the intermediate fIX, and then after Arg180 generating fIXa. We have shown that an exosite on the fXIa heavy (non-catalytic) chain is required for fIX conversion to fIXa. New evidence indicates a second site, probably on the catalytic domain but distinct from the active site, is also required. We hypothesize that this second site is required for conversion of fIX to fIX, while the heavy chain exosite makes contributions to both cleavages. In Aim 2 we will study the role of exosite interactions in fIX activation by fXIa, delineate the importance of exosites on the fXI heavy chain and catalytic domains to cleavage of the two fIX activation sites, and determine if cleavage at Arg145 is a prerequisite for cleavage at Arg180. FXI binds to platelet glycoprotein 1b (GP1b). While it was proposed that this interaction facilitates fXI activation, this now is uncertain. Preliminary work indicates fXI also binds to a receptor for apolipoprotein E (ApoER2) on platelets. ApoER2 can form a complex with GP1b on platelets, and the two may be components of a fXI binding site. We hypothesize that the fXI-ApoER2 interaction involves a binding site on fXI distinct from the one required for binding to GP1b. In Aim 3 we will assess the interaction of fXI and the active species fXIa and 1/2-fXIa with platelets, study the importance of platelets to fXI activation and activity in a flow-based system, and identify the binding site on fXI involved in interactions with platelet ApoER2. PUBLIC HEALTH RELEVANCE: Plasma factor XIa makes modest contributions to normal blood coagulation (hemostasis) at a site of vessel injury, but appears to make a disproportionately greater contribution to coagulation in pathologic processes such as vascular thrombosis. This protease, therefore, is a legitimate target for novel anti- thrombotic therapies that should carry a lower risk of life-threatening bleeding, when compared to currently available anticoagulants.