ENDOTHELIAL CELL INJURY AND PROCESSING OF ANNEXIN II

动脉粥样硬化的形成可以概念化为对血管细胞损伤的反应 或者是压力。内皮细胞构成了一个抗血栓的表面，即 与流动的血液密切相关。然而，微扰的 内皮细胞通过几种刺激中的任何一种提供更多的血栓前状态， 易于细胞成分涌入的粘附性表型， 有丝分裂原的释放和脂质的积累。我们最近确定了 钙调节磷脂结合蛋白，膜联蛋白II，作为一种 两种纤溶蛋白的内皮细胞表面受体， 纤溶酶原和组织型纤溶酶原激活剂。因为膜联蛋白II似乎 对这两种配体有不同的结合域，我们有 假设它调解了它们的组装以及 多功能丝氨酸蛋白酶，纤溶酶。我们的进一步研究表明 纯化的天然膜联蛋白II提高了t-PA的催化效率- 依赖纤溶酶原激活的能力提高了60倍。尽管有几项研究 已经证明内皮细胞上存在膜联蛋白II，以及 其他细胞，它被转移到细胞表面的机制 是未知的，因为它缺乏典型的疏水信号序列。我们的 初步数据表明，膜联蛋白II的易位是一个缓慢的过程 在静息细胞中，但它对 特定形式的细胞“应激”，如热休克或病毒感染。 更多研究表明，全长膜联蛋白II必须是 在完全发挥纤溶功能之前对其进行蛋白水解性修饰 受体，尿激酶和t-PA代表候选膜联蛋白II 激活剂。这项建议的具体目的是：(1)确定 内皮细胞将膜联蛋白II转运到血管内皮细胞 静息条件下的细胞表面，(2)识别对此的修改 对细胞损伤或应激做出反应的过程，以及(3) 描述了膜联蛋白II被激活以成为完全 功能性催化受体。拟议研究的成功将 依赖于与计划其他成员的关键互动 项目。具体来说，罗伊·西尔弗斯坦博士将协助准备 稳定转染293个细胞系，苏曼·F·A·皮尔斯博士将进行 突变型膜联蛋白的结构分析。David Hajjar博士和Robert博士 Kaner将合作进行涉及膜联蛋白II加工的研究 病毒应激的设定。我们假设交通工具和 在某些条件下，膜联蛋白II的加工可能会受到严重干扰 这会导致细胞增殖和脂质堆积，这是 动脉粥样硬化。

Atherogenesis may be conceptualized as a response to vascular cell injury or stress. The endothelium constitutes a thromboresistant surface that is intimately associated with flowing blood. However, perturbation of the endothelial cell by any of several stimuli confers a more prothrombotic, adhesive phenotype that predisposes to influx of cellular elements, release of mitogens, and lipid accumulation. We recently identified the calcium-regulated phospholipid-binding protein, annexin II, as an endothelial cell surface receptor for two fibrinolytic proteins, plasminogen and tissue plasminogen activator. Since annexin II appears to possess distinct binding domains for these two ligands, we have hypothesized that it mediates their assembly as well as generation of the multi-functional serine protease, plasmin. Our further studies have shown that purified native annexin II enhances the catalytic efficiency of t-PA- dependent plasminogen activation by 60-fold. Although several studies have demonstrated the presence of annexin II on endothelial cells, and other cells, the mechanism by which it is translocated to the cell surface is unknown since it lacks a typical hydrophobic signal sequence. Our preliminary data suggest that annexin II translocation is a slow process in resting cells, but that it is significantly more rapid in response to specific forms of cellular "stress" such as heat shock or viral infection. Additional studies indicate that full-length annexin II must be proteolytically modified before it can function fully as fibrinolytic receptor, and that urokinase and t-PA represent candidate annexin II activators. The specific aims of this proposal are to (1) determine the mechanism by which the endothelial cell translocates annexin II to the cell surface under resting conditions, (2) identify modifications of this process that occur in response to cellular injury or stress, and (3) delineate the mechanism by which annexin II is activated to become a fully functional catalytic receptor. The success of the proposed studies will depend upon critical interactions with other members of the Program Project. Specifically, Dr. Roy Silverstein will assist in preparation of stably transfected 293 cell lines, and Dr. Suman F. A. Pearce will conduct structural analyses of mutant annexins. Drs. David Hajjar and Robert Kaner will collaborate on studies involving processing of annexin II in the setting of virus-induced stress. We hypothesize that transport and processing of annexin II may be significantly perturbed under conditions that lead to cellular proliferation and lipid accumulation, the hallmarks of atherosclerosis.