INITIATION AND REGULATION OF BLOOD COAGULATION

组织因子(TF)依赖的血液凝固在脑出血中起主要作用 组织损伤后的止血也在发病机制中起作用。因子结合力 Vi到Tf是Tf依赖的血液凝固的初始步骤。一把钥匙 然后必须进行反应：激活结合因子vii以产生 具有催化活性的VIIa/Tf络合物。人们对此知之甚少 因子VII的激活和随后的VIIa/Tf-催化活性是 体内调节以及在多大程度上抗凝活性细胞表面 硫酸肝素蛋白多糖在这些调节中发挥作用 转铁蛋白诱导凝血的关键步骤。此外，哪种特定的氨基 VIIa和Tf中的酸性侧链参与组织识别 因子途径抑制物(TFPI)与VIIa/Tf的细胞生物学 未知。本提案的主要重点是获得重要的 关于这些悬而未决的问题的信息。具体目标是：(1) 在体外和体内验证一种假设，即细胞表面肝素 硫酸盐蛋白多糖限制因子VII与转铁蛋白结合的激活 从而调节TF诱导的凝血(2)试验a 假设底物因子X的识别结构存在于 VIIa/Tf复合体对Xa的增强作用 VIIa/Tf/TFPI/Xa的四元络合物形成(3)检验一个假设 细胞表面Tf与VIIa、Xa和 TFPI和定义TFPI在这一过程中的作用(4)检验假设 TFPI/Xa与细胞表面VIIa/Tf结合的影响 细胞表面VIIa/Tf复合体的内化及其机制 内化的VIIa和Tf的细胞内运输。在建议的 研究，各种成熟的技术，如放射性配基结合， 凝血试验、电子显微镜和兔血管内 将使用混凝模型系统。从这些网站获得的信息 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能有效控制出血 血友病患者。上述研究将进一步加深我们对 VIIa/Tf/TFPI/Xa大分子组装的结构机理是这样的 对治疗的理解可能会导致治疗的设计和应用 针对出血性疾病和血栓形成的治疗措施 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.