WEIBEL PALADE BODY FORMATION AND ROLE IN HEMOSTASIS

在血管损伤后或炎症期间， 迅速释放出它们的储存颗粒，称为韦伯， Palade机构（WPB）。 这些颗粒的主要可溶性成分是 血管性血友病因子（vWf）。 vWf是一种大的多聚体糖蛋白 它在止血中具有双重作用：它促进血小板粘附， 它保护因子VIII（FVIII）免受蛋白水解。 合成vWf 具有指导多聚体形成的前序列， 未知机制WPB的形成与vWf的作用 后分泌是本提案的主要议题。 我们将 我们制备的vWf缺陷小鼠的存在极大地帮助了我们 在最后一个补助期间。 该提案有四个具体目标。（一） 韦伯-帕拉德身体形成。 我们将研究vWF是否 前序列具有二硫键异构酶酶活性， 解释其在vWf多聚化中的作用。 我们将继续我们的研究 关于颗粒形成的分子机制。 二、vWf的作用 在正常生理学中。 我们将评估vWF缺乏对 FVIII半衰期和分泌。 我们将检查是否存在 vWf或其前序列调节VIII的反馈机制 生物合成 我们将研究vWf在血小板血栓形成中的作用 使用新的活体显微镜模型进行体外和体内形成 我们已经开发了。 vWf的重要性将与 其它血小板粘附分子，即，纤维蛋白原和β 3整联蛋白。 III）vWf在各种疾病中的作用。 我们将测试vWf的作用， 血小板被认为起重要作用的疾病，例如， 感染性休克中风和动脉粥样硬化 （四）生成和 新突变小鼠的特征。 我们将生产 通过将vWf缺陷小鼠与其他突变体杂交来产生遗传缺陷 菌株 我们将评估vWf基因剂量对 通过生产两倍于正常水平的小鼠来维持体内平衡和疾病 的vWF。我们的研究将采用各种技术，包括 蛋白质化学，细胞培养，活体显微镜，动物 实验，最后，目标基因的基因工程 复制在老鼠。 了解WPB涉及的机制 形成和分泌是临床相关的，作为主要治疗 vWf低的患者依赖于释放储存的蛋白池。 了解vWf在血小板粘附中的确切作用 和血栓形成可能导致新的抗血栓形成疗法， 可以应用于血小板粘附和/或 聚集是疾病过程的一部分。

Following a vascular injury or during inflammation, endothelial cells rapidly release the contents of their storage granules, called Weibel- Palade bodies (WPBs). The major soluble component of these granules is von Willebrand factor (vWf). vWf is a large multimeric glycoprotein that has a dual role in hemostasis: it promotes platelet adhesion and it protects factor VIII (FVIII) against proteolysis. vWf is synthesized with a prosequence which directs multimer formation through an as yet unknown mechanism. The formation of the WPBs and the function of vWf after secretion are the main topics of this proposal. We will be greatly assisted by the existence of vWf-deficient mice that we prepared during the last grant period. The proposal has four specific aims. I) Weibel-Palade body formation. We will examine whether the vWf prosequence has a disulfide isomerase enzymatic activity, which would explain its role in vWf multimerization. We will continue our studies on the molecular mechanisms involved in granulogenesis. II) Role of vWf in normal physiology. We will evaluate the effect of vWf-deficiency of FVIII half life and secretion. We will examine whether there is a feedback mechanism by which vWf or its prosequence modulates FVIII biosynthesis. We will study the role of vWf in platelet thrombus formation in vitro and in vivo using a new intravital microscopy model we have developed. The importance of vWf will be compared with that of other platelet adhesion molecules, i.e., fibrinogen and beta3 integrins. III) Role of vWf in various diseases. We will test the role of vWf in diseases in which platelets are thought to play an important part, e.g., septic shock, stroke and atherosclerosis. IV) Generation and characterization of new mutant mice. We will produce combinations of genetic defects by crossing the vWf-deficient mice with other mutant strains. We will evaluate the effect of gene dosage of vWf in homeostasis and disease by producing mice with twice the normal level of vWf. Our research will employ a variety of techniques including protein chemistry, cell culture, intravital microscopy, animal experimentation, and finally, genetic engineering of a targeted gene duplication in mice. Learning about the mechanisms involved in WPB formation and secretion is clinically relevant, as the main treatment of patients with low vWf relies on releasing the stored protein pool. An understanding of the exact roles that vWf plays in platelet adhesion and thrombus formation may lead to new anti-thrombotic therapies which could become applicable in diseases where platelet adhesion and/or aggregation is part of the disease process.