PROTHROMBIN ACTIVATION ON BLOOD MONONUCLEAR CELLS

单核/巨噬细胞在炎症和动脉粥样硬化中起关键作用 在几种情况下，在血管外组织进行的过程 以纤维增殖性反应和纤维蛋白为特征 证词。由于凝血酶是这些反应的有力效应者，因此 总体假设是单核/巨噬细胞产生凝血酶 表面提供了一个重要的生物调节效应分子在这些 网站。目标是定义约束参数和动力学参数 控制导致凝血酶表达的分子事件 在单核/巨噬细胞表面，并开始阐明细胞 调节促凝血剂表型表达的机制。 特别强调的是单核/巨噬细胞表面 膜通过调节凝血酶原酶复合体的组装和功能 辅因子Va与酶结合位点的表达 凝血因子Xa与激活膜结合蛋白的表达 原辅因子V到因子Va。平衡结合研究使用 放射性标记因子V、Va和Xa将被耦合到动力学分析 凝血酶原激活与结合和功能相关。蛋白水解酶 将使用电泳法检测因子V和Va的修饰， 放射自显影技术。凝血因子Xa与单核细胞结合 与因子Va无关，并生成因子IX(将研究到 确定这种相互作用如何调节Xa因子的蛋白分解活性 以及它参与凝血酶原和凝血因子IX的激活。这个 细胞活化和细胞与胞外黏附的机制 基质蛋白成分增强了这些不同的结合事件，并 蛋白质分解活性将被定义。此外，该地区的 介导其独立结合的因子Va和因子Xa分子 将使用分离的蛋白质片段来确定对单核细胞的影响， 模拟已知序列的合成肽及其抗肽 抑制性多肽抗体。最后，亲和层析 技术将被用来分离单核/巨噬细胞“受体” 因子Va。这个项目的总体目标是提供一个详细的 了解凝血酶的生成是如何在 单核细胞表面通过一系列与整体相关的事件。这个 凝血酶在单核/巨噬细胞表面的形成似乎是 这些细胞是生理和病理生理功能的关键 当它们定位于血管和血管外组织部位时提供。这个 单核/巨噬细胞参与和调节 导致凝血酶形成的分子事件是 它们在动脉粥样硬化中的生理和病理生理学作用， 慢性炎症和伤口修复。此外，由于单核细胞与 血管内皮细胞、血小板和间充质细胞 这些细胞对凝血酶的反应方式多种多样，似乎很可能 单核/巨噬细胞膜表面凝血酶的产生 提供一种重要的生物调节效应器分子 地点。这一假说是基于凝血酶可以 是细胞-细胞信号转导中的重要效应分子 重塑细胞外环境，重要的活动 在已知单核细胞的各种(病理)生理环境中 参与其中。 我们的成就包括证明1)就像单核细胞一样， 来源于人脑微血管的周细胞可以激活和 通过外在途径(即， 组织因子/因子VIIa复合体随后是凝血酶原酶复合体)， 然而，与单核细胞不同的是，所需酶复合体的活性 可对激动剂刺激作出差异调节；2) 单核/巨噬细胞通过以下途径维持其表面凝血酶的生成 活化蛋白保护辅因子Va不被失活 C；3)组织蛋白酶G对因子V的蛋白分解产生一个辅因子 对Xa因子的亲和力降低，从而限制凝血酶的生成。我们的 目前的计划是：1)完成结构/功能分析和 弹性蛋白酶和组织蛋白酶G切割Va因子的辅因子活性；2) 通过组织因子/因子确定因子IX的激活途径 VIIa复合体组装在单核细胞上，以及3)通过以下方式确定其机制 单核细胞通过活化蛋白C抑制Va因子失活。

Monocytes/macrophages play key roles in inflammatory and atherosclerotic processes, which in several instances proceed at extravascular tissue sites and are characterized by a fibroproliferative response and fibrin deposition. Since thrombin is a potent effector of these responses, the overall hypothesis is that thrombin production at the monocyte/macrophage surface provides an important bioregulatory effector molecule at these sites. The objectives are to define the binding and kinetic parameters governing the molecular events which result in the expression of thrombin at the monocyte/macrophage surface and to begin to elucidate the cellular mechanisms which regulate the expression of a procoagulant phenotype. Particular emphasis is placed on how the monocyte/macrophage surface membrane regulates prothrombinase complex assembly and function through the expression of binding sites for the cofactor factor Va and the enzyme factor Xa, and the expression of membrane-bound proteases which activate the procofactor factor V to factor Va. Equilibrium binding studies using radiolabeled factors V, Va and Xa will be coupled to kinetic analyses of prothrombin activation to correlate binding and function. Proteolytic modification of factors V and Va will be detected using electrophoretic, autoradiographic techniques. Factor Xa binding to monocytes, which occurs independently of factor Va and generates factor IX( will be studied to determine how this interaction regulates factor Xa proteolytic activity and its participation in prothrombin and factor IX activation. The mechanisms by which cell activation and cell adherence to extracellular matrix protein components enhances these various binding events and proteolytic activities will be defined. In addition, the regions of the factor Va and factor Xa molecules which mediate their independent binding to monocytes will be determined using isolated protein fragments, synthetic peptides mimicking their known sequences and anti-peptide antibodies of inhibitory peptides. Finally, affinity chromatographic techniques will be used to isolate the monocyte/macrophage "receptor" for factor Va. The overall goal of this project is to provide a detailed understanding of how thrombin generation is effected and regulated at the monocyte surface through a series of integrally related events. The formation of thrombin at the monocyte/macrophage surface appears to be pivotal to the physiological and pathophysiological functions these cells provide as they localize to vascular and extravascular tissue sites. The ability of monocytes/macrophages to participate in and regulate the molecular events leading to thrombin formation is an integral part of their physiological and pathophysiological roles in atherosclerosis, chronic inflammation and wound repair. Also, since monocytes interact with vascular endothelium, platelets and mesenchymal cells, and since all of these cells respond in a variety of ways to thrombin, it seems likely that the production of thrombin at the monocyte/macrophage membrane surface provides an important bioregulatory effector molecule at precise locations. This hypothesis is based on the observations that thrombin can be an important effector molecule in cell-cell signaling and in remodelling the extracellular environment, activities which are important in a variety of (patho)physiologic settings in which monocytes are known to participate. Our accomplishments included demonstrating that 1) like monocytes, pericytes derived from human brain microvessels, can activate and propagate the coagulant response through the extrinsic pathway (i.e. a tissue factor/factor VIIa complex followed by a prothrombinase complex), however, unlike monocytes the activities of the required enzyme complexes can be differentially regulated in response to agonist stimulation; 2) monocytes/macrophages sustain thrombin generation at their surface by protecting the cofactor factor Va from inactivation by activated protein C; and 3) proteolysis of factyor V by cathepsin G yields a cofactor with a reduced affinity for factor Xa, thus limiting thrombin generation. Our current plans are: 1) to complete the structure/function analyses and cofactor activities of factor Va cleaved by elastase and cathepsin G; 2) to define the activation pathway of factor IX by the tissue factor/factor VIIa complex assembled on monocytes, and 3) to define the mechanism by which monocytes inhibit factor Va inactivation by activated protein C.