Physiology and Molecular Biology of Factor XI

描述(由申请人提供)：凝血因子XI(FXI)是一种类胰蛋白酶(FXIa)的酶原，它通过催化凝血因子IX(FIX)的激活来促进凝血酶的生成。尽管FXI在止血方面的作用有限，但现在的各种数据表明，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具有许多区别于维生素K依赖的凝血酶的结构特征。这种蛋白质是由两个相同的亚基组成的二聚体，每个亚基都有两个与血小板受体结合的位置，以及 两个阴离子结合部位。这些功能的功能重要性仍有待确定，在血浆凝血试验(如活化的部分凝血活酶时间(APTT)测定)中，正常的FXI功能不是必需的。我们认为，FXI的二聚体结构及其与血小板的相互作用和多阴离子是调节流动血液中FXI活性和fXIa活性的关键。在aPTT实验中，FXI被因子XIIa(FXIIa)激活，而FXIa又激活FIX。FXII缺乏症患者未见异常出血，提示体内一定有其他蛋白水解酶能够激活FXI。例如，β-凝血酶可以激活FXI。然而，越来越多的证据表明，fXIIa激活FXI有助于血栓形成。FXII缺陷和FXI缺陷小鼠对损伤诱导的动脉和静脉闭塞具有抵抗力，用抗FXI或FXII抗体治疗的狒狒也有类似的观察结果。我们假设FXI和FXII都通过凝血酶依赖的过程在流动的人类血液中促进闭塞血栓的生长。此外，我们认为FXI与血小板或血小板产物之间的相互作用是通过fXIa的产生来支持血栓生长所必需的。血小板致密颗粒释放的无机磷聚合物(Poly-P)可增强FXI的活性。Poly-P似乎与FXI上的两个阴离子结合部位结合，通过凝血酶、FXII和自激活加强其激活，并可能下调蛇类对FXIa的抑制。在目标1中，我们将使用体外流动模型来研究FXI和FXII在全血血栓形成中的重要性。该模型还将用于研究FXI二聚体、FXI血小板结合部位和FXI阴离子结合部位对血栓形成的重要性，使用一组具有良好特性的重组FXI分子。在目标2中，我们将在两种动脉血栓形成模型和一种静脉血栓栓塞症模型中研究FXI阴离子结合部位对FXI活性的重要性；并将评估阴离子结合部位通过与管壁糖胺多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阴离子结合位点在血浆蛇毒对fXIa的肝素依赖调节中的重要性，并评估Poly-P下调丝氨酸介导的抑制的能力。这项建议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.