Molecular Basis of Blood Coagulation Regulation

丝氨酸蛋白酶抑制剂家族凝血蛋白酶抑制剂、抗凝血酶及其糖胺聚糖激活剂、肝素 和硫酸乙酰肝素，包括维持止血所必需的关键抗凝机制 在脊椎动物中。有趣的是，抗凝血酶最近已被证明具有抗血管生成和抗肿瘤 其生理作用仍有待确定。为了推进我们的长期目标， 抗凝血酶发挥其生理相关功能的分子机制，我们建议 追求三个具体目标：1）我们以前已经表明，介导的分子决定因素， 肝素活化抗凝血酶对凝血蛋白酶的特异性存在于暴露的反应性 丝氨酸蛋白酶抑制剂的中心环和环外的外部位点区域。我们建议绘制这些外来岩区的地图 对抗凝血酶和因子Xa和因子IXa的影响，并确定外切位点 介导肝素与蛋白酶的抗凝血酶反应的速率增强; 2）抗凝血酶在 一种不寻常的低活性构象，其中反应环部分埋在β-折叠A中，但 由肝素诱导成类似于其他丝氨酸蛋白酶抑制剂的高活性构象，其中反应性 线圈从板中排出。我们建议确定这个反应环的分子决定因素 构象转换，并描绘肝素结合位点中的分子事件序列， 触发构象转换; 3）当抗凝血酶表达出有效的抗血管生成活性时， 经历自发发生的或由反应中的裂解诱导的构象变化， 环，并导致反应性环埋入β-折叠A。我们将确定 抗凝血酶的切割的、潜伏的和新的预潜伏形式的抗血管生成活性的基础。的作用 这种活性中的特异性内皮细胞结合位点以及这些结合位点是否涉及蛋白质受体 和/或硫酸乙酰肝素蛋白聚糖的特定结构域。拟议的研究是 预计将提供更多的了解在分子水平上的功能，一个关键的自然 抗凝血和抗血管生成血蛋白。这种理解有望促进理性的 设计新一代抗凝血和抗肿瘤药物，用于治疗和预防 心血管疾病和癌症。

The serpin family coagulation protease inhibitor, antithrombin and its glycosaminoglycan activators, heparin and heparan sulfate, comprise a key anticoagulant mechanism which is essential for maintaining hemostasis in vertebrates. Intriguingly, antithrombin has been recently shown to possess antiangiogenic and antitumor functions whose physiologic role remains to be determined. To advance our long-term goal of elucidating the molecular mechanisms by which antithrombin carries out its physiologically relevant functions, we propose to pursue three specific aims: 1) We have previously shown that the molecular determinants which mediate the specificity of heparin-activated antithrombin for blood clotting proteases reside both in an exposed reactive center loop of the serpin and in exosite regions outside the loop. We propose to map these exosite regions on antithrombin and on factor Xa and factor IXa and determine the mechanism by which the exosites mediate heparin rate enhancement of antithrombin reactions with the proteases; 2) Antithrombin circulates in an unusual low-activity conformation in which the reactive loop is partially buried in beta-sheet A, but is induced by heparin into a high-activity conformation resembling that of other serpins in which the reactive loop is expelled from the sheet. We propose to identify the molecular determinants of this reactive loop conformational switch and delineate the sequence of molecular events in the heparin binding site which trigger the conformational switch; 3) Antithrombin expresses a potent antiangiogenic activity when it undergoes conformational changes which occur spontaneously or are induced by cleavage in the reactive loop and which result in the burial of the reactive loop into beta-sheet A. We will determine the structural basis for the antiangiogenic activity of cleaved, latent and a novel prelatent form of antithrombin. The role of specific endothelial cell binding sites in this activity and whether these binding sites involve a protein receptor and/or specific domains of a heparan sulfate proteoglycan will be determined. The proposed studies are expected to provide increased understanding at the molecular level of the function of a key natural anticoagulant and antiangiogenic blood protein. This understanding is expected to facilitate the rational design of a new generation of anticoagulant and antitumor drugs for the treatment and prevention of cardiovascular diseases and cancer.