Physiology and Molecular Biology of Factor XI

描述（由申请人提供）：因子XI （fXI）是一种同型二聚体血浆蛋白酶，因子XIa （fXIa）的酶原，通过将因子IX （fIX）转化为蛋白酶fIXa来促进止血。虽然fXI缺乏会导致相对轻微的出血性疾病，但越来越多的证据表明，这种蛋白质在血栓形成和弥散性血管内凝血等病理过程中起着重要作用。除了二聚体结构外，fXIa还具有不同于维生素k依赖性蛋白酶的结构特征。在本研究中，我们探讨了止血过程中fXI的激活机制，以及fXI与fIX和血小板的相互作用。经典地，fXI通过因子XIIa （fXIIa）转换为fXIa。然而，缺乏fXII不会引起异常出血。-凝血酶在溶液中激活fXI，但这种情况发生在血浆中的前提最近受到了挑战。因为它是同型二聚体，所以fXIa每个分子有两个活性位点。最近，我们发现fXI向fXIa的转化是通过一个具有一个功能类似于fXIa的活性位点（1/2-fXIa）的物种进行的。在Aim 1中，我们提出了fXI被血浆中的-凝血酶激活的假设，以及凝血酶原激活的其他产物，如减数凝血酶和-凝血酶，可以激活fXI。我们将研究每个fXI亚基的激活动力学，比较fXIIa、-凝血酶、-凝血酶、减数凝血酶和-凝血酶在阴离子结合外源性突变下的激活；并将研究fXI二聚体结构在纯化和等离子体系统中活化的重要性。FIX激活涉及序列蛋白水解裂解，首先在Arg145产生中间FIX之后，然后在Arg180产生fIXa之后。我们已经证明，fIX转化为fIXa需要fXIa重（非催化）链上的外源位点。新的证据表明，可能在催化结构域上但与活性位点不同的第二个位点也是必需的。我们假设这第二个位点是fIX向fIX转化所必需的，而重链外源位点对这两个裂解都有贡献。在Aim 2中，我们将研究外源位点相互作用在fXIa激活fIX中的作用，描述fXI重链和催化结构域上的外源位点对两个fIX激活位点裂解的重要性，并确定Arg145裂解是否是Arg180裂解的先决条件。FXI结合血小板糖蛋白1b （GP1b）。虽然有人提出这种相互作用促进了fXI的激活，但现在还不确定。初步研究表明，fXI还能与血小板上的载脂蛋白E受体（ApoER2）结合。ApoER2可以在血小板上与GP1b形成复合物，两者可能是fXI结合位点的组分。我们假设fXI- apoer2相互作用涉及fXI上的一个与GP1b结合所需的结合位点不同的结合位点。在Aim 3中，我们将评估fXI和活性物种fXIa以及1/2-fXIa与血小板的相互作用，研究血小板在血流系统中对fXI激活和活性的重要性，并确定fXI上与血小板ApoER2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凝血（止血）贡献不大，但在血管血栓形成等病理过程中对凝血的贡献似乎不成比例地大。因此，与目前可用的抗凝剂相比，这种蛋白酶是新型抗血栓治疗的合理靶点，它应该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.