INITIATION AND REGULATION OF BLOOD COAGULATION

组织因子（TF）依赖性凝血在血液凝固中起主要作用。 组织损伤后止血和发病机制。因子结合 VII转化为TF是TF依赖性血液凝固的初始步骤。一个关键 反应必须遵循：激活结合因子VII， 催化活性VIIa/TF复合物。人们对这一现象知之甚少 因子VII的活化和随后的VIIa/TF-催化活性， 在体内调节以及在何种程度上抗凝活性细胞表面 硫酸肝素蛋白聚糖在这些调节中起作用， TF诱导凝血的关键步骤。此外，哪个特定的氨基 VIIa和TF中的酸性侧链参与组织识别 因子途径抑制剂（TFPI）和VIIa/TF的细胞生物学仍然存在 未知本提案的主要重点是获得重要的 有关这些未解答问题的信息。具体目标是：（1） 在体外和体内测试一个假设，细胞表面肝素 硫酸化蛋白聚糖限制与表达TF结合的因子VII的活化 并由此调节TF诱导的凝血（2）测试a 假设底物因子X的识别结构存在于 VIIa/TF复合物有助于增强Xa的作用， VIIa/TF/TFPI/Xa的四元复合物的形成（3）检验假设， 细胞表面TF在与VIIa、Xa和 TFPI并定义TFPI在此过程中的作用（4）检验假设 TFPI/Xa与细胞表面VIIa/TF的结合影响 细胞表面VIIa/TF复合物的内化，并阐明 内化的VIIa和TF的细胞内运输。拟议 研究，各种成熟的技术，如放射性配体结合， 凝血测定，电子显微镜，和兔血管内 将使用凝血模型系统。从这些来源获得的信息 研究将有助于理解重要的临床问题，例如为什么 输注凝血因子VIIa可有效控制 血友病患者上述研究将进一步加深我们对 VIIa/TF/TFPI/Xa大分子组装结构机理等 理解可能导致治疗的设计和应用， 旨在治疗出血性疾病和血栓性疾病的措施 并发症

Tissue factor (TF)-dependent blood coagulation plays a primary role in hemostasis after tissue injury and also in pathogenesis. Binding of factor VII to TF is the initial step in TF-dependent blood coagulation. A key reaction must then follow: activation of the bound factor VII to give rise to catalytically active VIIa/TF complexes. Little is known about how the activation of factor VII and the subsequent VIIa/TF-catalytic activity are regulated in vivo and to what extent anti-coagulantly active cell surface heparin sulfate proteoglycans play a role in the regulation of these critical steps in TF-induced coagulation. Further, which specific amino acid side chains in VIIa and TF are involved in the recognition of tissue factor pathway inhibitor (TFPI) and the cell biology of VIIa/TF remain unknown. The primary focus of the present proposal is to obtain important information on these unanswered questions. The specific goals are: (1) Test a hypothesis, both in vitro and in vivo, that cell surface heparin sulfate proteoglycans limit activation of factor VII bound to TF expressed on cell surfaces and thereby regulated TF-induced coagulation (2) Test a hypothesis that recognition structures for substrate factor X presents in VIIa/TF complex contribute to the enhancing effect of Xa during the quaternary complex formation of VIIa/TF/TFPI/Xa (3) Test a hypothesis that cell surface TF redistributes upon complex formation with VIIa, Xa and TFPI and define the role of TFPI in this process (4) Test a hypothesis that the binding of TFPI/Xa to cell surface VIIa/TF affects internalization of cell surface VIIa/TF complexes and elucidate the intracellular trafficking of internalized VIIa and TF. In the proposed studies, various well established techniques, such as radioligand binding, coagulation assays, electron microscopy, and a rabbit intravascular coagulation model system will be used. Information obtained from these studies will aid in understanding important clinical issues, such as why infused factor VIIa is effective in controlling the bleeding in hemophiliacs. The above studies will further our understanding of the structural mechanism of macromolecular assembly of VIIa/TF/TFPI/Xa. Such an understanding may lead to the design and application of therapeutic measures aimed at the treatment of hemorrhagic disease and thrombotic complications.