VON WILLEBRAND FACTOR, FIBRINOLYSIS, & THE ENDOTHELIUM

内皮细胞在原发性高血压的调节中起主要作用 止血、纤溶和血管舒缩。这些细胞 合成血管性血友病因子、组织型纤溶酶原 激活物(TPA)、纤溶酶原激活物抑制物(PAI)和vWF 和tPA似乎经常在体内一起释放，以响应 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 血管内皮细胞合成和释放vWF、tPA和PSI及 血管内皮细胞对AVP的反应。实验将在 培养的人脐静脉内皮细胞， 成人动脉和静脉，以及脂肪组织微血管。 细胞内vWF的结构和释放。一种储藏细胞器，用于储存 VWF(Webel-Palade Body)将用胶体二氧化硅提纯 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估并比较 与血浆vWF相比。同一分子中包含的其他蛋白质 分泌颗粒的特征；特别是tPA是否 是否与vWF具有相同的分泌细胞器将被测定。 链激酶(SK)治疗与血浆中 血浆vWF。释放的确切刺激将由以下因素决定 测试SK诱导的纤溶状态的成分(即SK， 纤溶酶和纤维蛋白原降解产物) 诱导培养细胞释放vWF。另一方面，右旋糖苷 降低vWF级别。为了评价右旋糖苷的作用机制， 血管内皮细胞蛋白合成、vWF合成、vWF释放和 将检查激动剂(即凝血酶)对细胞的激活作用。 纤溶的内皮调节。PAI将从 人内皮细胞和氨基酸的条件培养液 获得的序列。因为PAI水平在 妊娠，类固醇激素对合成Will速度的影响 要下定决心。活化蛋白C对PAI的灭活作用 进行检查以确定PAI是否被蛋白质降解。 内皮血管加压素受体。AVP刺激vWF的释放， 组织型纤溶酶原激活剂、血小板活化因子与“内皮源性舒张” 与内皮细胞的~3H-AVP结合将检测 确定是否存在特定的高亲和力结合位点，以及 在什么类型的血管中，无论它们是V1还是V2受体，以及 无论是胞内钙还是cAMP第二信使都会产生。

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.