Physiology and Molecular Biology of Factor XI

描述（由申请人提供）：因子Xi（fXi）是胰蛋白酶样蛋白酶（fXIa）的酶原，其通过催化因子IX（fIX）活化而促进凝血酶生成。尽管在止血方面发挥的作用有限，但现在各种数据令人信服地表明，fXI有助于动脉和静脉血栓形成，因此是治疗或预防血栓栓塞性疾病的靶向治疗的有吸引力的候选药物。FXI具有许多与维生素K依赖性凝血蛋白酶不同的结构特征。该蛋白是两个相同亚基的二聚体，每个亚基有两个结合血小板受体的位点， 两个阴离子结合位点。这些特征的功能重要性仍有待确定，在血浆凝血试验（如活化部分凝血活酶时间（aPTT）试验）中，fXI功能正常不需要这些特征。我们认为fXI的二聚体结构及其与血小板和聚阴离子的相互作用是调节流动血液中fXI活化和fXIa活性的关键。在aPTT试验中，fXI被因子XIIa（fXIIa）激活，fXIa反过来激活fIX。在fXII缺乏症中不存在异常出血表明其他蛋白酶必须能够在体内激活fXI。例如，凝血酶可以激活fXI。然而，有越来越多的证据表明，fXI激活fXIIa有助于血栓形成。FXII缺陷型和fXI缺陷型小鼠对损伤诱导的动脉和静脉闭塞具有抗性，并且在用抗fXI或fXII抗体处理的狒狒中已经进行了类似的观察。我们假设fXI和fXII通过凝血酶依赖性过程促进流动的人血液中闭塞性血栓的生长。此外，我们提出，需要fXI和血小板或血小板产物之间的相互作用来支持血栓在fXIa的生成下流动生长。FXI活化通过从血小板致密颗粒释放的无机磷酸盐聚合物（聚-P）增强。聚-P似乎与fXI上的两个阴离子结合位点结合，增强其通过凝血酶、fXII和自激活的激活，并且可以下调丝氨酸蛋白酶抑制剂对fXIa的抑制。在目的1中，我们将使用离体流动模型来研究fXI和fXII对全血中血栓形成的重要性。该模型还将用于研究fXI二聚体、fXI血小板结合位点和fXI阴离子结合位点对血栓形成的重要性，使用重组fXI分子的充分表征的面板。在目标2中，我们将研究fXI阴离子结合位点对小鼠动脉血栓形成模型和静脉血栓栓塞模型中fXI活性的重要性;并将评估阴离子结合位点通过与血管壁糖胺聚糖相互作用参与建立fXI非循环池的可能性。在目标3中，我们将确定fXI阴离子结合位点对血浆丝氨酸蛋白酶抑制剂对fXIa的肝素依赖性调节的重要性，并评估聚P下调丝氨酸蛋白酶抑制剂介导的抑制的能力。本提案中提出的研究结果将阐明fXI和fXII促进血栓形成的机制，并可能确定可用于治疗目的的新过程。

DESCRIPTION (provided by applicant): Factor XI (fXI) is the zymogen of a trypsin-like protease (fXIa) that contributes to thrombin generation by catalyzing factor IX (fIX) activation. Despite playing a limited role in hemostasis, a variety of data now make a compelling case that fXI contributes to arterial and venous thrombosis and is, therefore, an attractive candidate for targeted therapy to treat or prevent thromboembolic disease. FXI has a number of structural features that distinguish it from vitamin K-dependent coagulation proteases. The protein is a dimer of two identical subunits, each of which has two sites for binding to platelet receptors, and two anion-binding sites. The functional importance of these features remains to be established, and none are required for normal fXI function in plasma clotting assays such as the activated partial thromboplastin time (aPTT) assay. We propose that the dimeric fXI structure and interactions with platelets, and polyanions are key to regulation of fXI activation and fXIa activiy in flowing blood. In the aPTT assay, fXI is activated by factor XIIa (fXIIa), and fXIa in turn activates fIX. The absence of abnormal bleeding in fXII deficiency indicates other proteases must be capable of activating fXI in vivo. For example, ¿-thrombin can activate fXI. However, there is mounting evidence that fXI activation by fXIIa contributes to thrombosis. FXII deficient and fXI deficient mice are resistant to injury-induced arterial and venous occlusion, and similar observations have been made in baboons treated with anti-fXI or fXII antibodies. We hypothesize that both fXI and fXII contribute to growth of an occlusive thrombus in flowing human blood through thrombin-dependent processes. Furthermore, we propose that an interaction between fXI and the platelet or a platelet product is required to support thrombus growth under flow through generation of fXIa. FXI activation is enhanced by polymers of inorganic phosphate (poly-P) released from platelet dense granules. Poly-P appears to bind to both of the anion binding sites on fXI, potentiating its activation by thrombin, fXII and autoactivation, and may down-regulate fXIa inhibition by serpins. In Aim 1, we will use an ex vivo flow model to study the importance of fXI and fXII to thrombus formation in whole blood. This model will also be used to study the importance of the fXI dimer, fXI platelet binding sites, and fXI anion binding sites to thrombus formation, using a well characterized panel of recombinant fXI molecules. In Aim 2, we will study the importance of the fXI anion binding sites to fXI activity in two models of arterial thrombosis, and one model of venous thromboembolism, in mice; and will assess the possibility that the anion binding sites are involved in establishing non-circulating pool of fXI through interactions with vessel wall glycosaminoglycans. In Aim 3, we will determine the importance of the fXI anion binding sites to heparin-dependent regulation of fXIa by plasma serpins, and evaluate the capacity of poly-P to down-regulate serpin-mediated inhibition. Results from the studies presented in this proposal will elucidate mechanisms by which fXI and fXII contribute to thrombus formation, and may identify novel processes that can be exploited for therapeutic purposes.