VON WILLEBRAND FACTOR, FIBRINOLYSIS, & THE ENDOTHELIUM

内皮素在原发性高血压的调节中起着重要作用。 止血、纤维蛋白溶解和血管紧张素。 这些细胞 合成血管性血友病因子（vWF）、组织型纤溶酶原 纤溶酶原激活剂（tPA）、纤溶酶原激活剂抑制剂（PAI）和vWF 和tPA似乎经常在体内一起释放， 例如运动、静脉阻塞或加压素（AVP）等刺激。 这些调查的目的是为了更好地了解 内皮合成和释放vWF、tPA和PSI， 内皮细胞对AVP的反应。 实验将在 从脐静脉获得的培养的人内皮细胞， 成人动脉和静脉以及脂肪组织微血管。 细胞内vWF结构和释放。 储存细胞器 vWF（韦伯-帕拉德体）将通过胶体二氧化硅纯化 密度梯度离心法 vWF多聚体分布 将通过琼脂糖凝胶的免疫印迹进行评价， 血浆vWF的水平。 其他蛋白质包含在相同的 分泌颗粒将被表征;特别是，tPA是否 与vWF的分泌细胞器是否相同。 链激酶（SK）治疗与快速增加， 血浆vWF。 释放的确切刺激将由以下因素决定： 测试SK诱导的纤维蛋白溶解状态的组分（即SK， 纤溶酶和纤维蛋白原降解产物）的能力， 诱导培养细胞释放vWF。 右旋糖酐另一方面 降低vWF水平。 为探讨其作用机制， 对内皮蛋白合成、vWF合成、vWF释放和 将检测激动剂（即凝血酶）对细胞的激活作用。 纤维蛋白溶解的内皮调节。 PAI将从 人内皮细胞条件培养基和氨基酸 序列获得。 因为PAI水平非常高 怀孕，类固醇激素对合成率的影响将 下定决心。 活化的蛋白C对PAI的失活作用， 以确定PAI是否被蛋白水解降解。 内皮加压素受体。 AVP刺激vWF的释放， tPA、血小板活化因子与"内皮源性舒张" 因子"将检查3H-AVP与内皮细胞的结合， 确定是否存在特异性高亲和力结合位点， 在什么类型的血管中，它们是V1还是V2受体， 是否产生胞质钙或cAMP第二信使。

Endothelium plays a major role in the regulation of primary hemostasis, fibrinolysis, and vasomotor tone. These cells synthesize von Willerbrand factor (vWF), tissue-type plasminogen activator (tPA), and plasminogen-activator inhibitor (PAI), and vWF and tPA often seem to be released together in vivo in response to such stimuli as exercise, venous occlusion, or vasopressin (AVP). The goal of these investigations is to better understand the endothelial synthesis and release of vWF, tPA and PSI and the endothelial response to AVP. Experiments will be performed on cultured human endothelial cells obtained from umbilical veins, adult arteries and veins, and adipose tissue microvasculature. Intracellular vWF Structure and Release. The storage organelle for vWF (Weibel-Palade body) will be purified by colloidal silica density gradient centrifugation. The vWF multimer distribution will be evaluated by immunoblotting of agarose gels and compared to that of plasma vWF. Other proteins contained in the same secretory granule will be characterized; in particular, whether tPA is the same secretory organelle with vWF will be determined. Streptokinase (SK) therapy is associated with a rapid increase in plasma vWF. The exact stimulus for release will be determined by testing components of the SK-induced fibrinolytic state (ie SK, plasmin, and fibrinogen degradation products) for the ability to induce vWF release from cultured cells. Dextran on the other hand lowers vWF level. To evaluate the mechanism, the effect of dextran on endothelial protein synthesis, vWF synthesis, vWF release, and cell activation by agonist (ie thrombin) will be examined. Endothelial Regulation of Fibrinolysis. PAI will be purified from conditioned media of human endothelial cells and amino acid sequence obtained. Because PAI levels are very high during pregnancy, effect of steroid hormones on the rate of synthesis will be determined. The inactivation of PAI by activated protein C will be examined to determine whether PAI is proteolytically degraded. Endothelial vasopressin receptors. AVP stimulates release of vWF, tPA, platelet activating factor and "endothelium-derived relaxing factor." 3H-AVP binding to endothelial cells will be examined to determine whether specific, high-affinity binding sites exist and in what types of vessels, whether they are V1 or V2 receptors, and whether cytosolic calcium or cAMP second messengers are generated.