CONTROL MECHANISMS IN HEMOSTASIS

这项建议的目的包括三个主要目标： 1.确定狼疮的作用机制(S) 抗凝剂。(Lac‘s)及其止血意义 阴离子磷脂的循环抗体：我们将 研究LAC与活化的血小板的结合以及对 内皮细胞暴露于IL-1、内毒素或肿瘤坏死 因子，并与磷脂酰丝氨酸暴露的结合相关。 我们将确定结合诱导的‘凝血酶原酶’的改变 血小板和内皮细胞的功能及其对细胞因子诱导合成的影响 EC对PGI2的影响及EC-血栓调节蛋白-凝血酶- 诱导蛋白C的激活我们将研究LAC的结合 其他对IL-1或肿瘤坏死因子敏感的细胞，以及PS的暴露。 我们将研究低凝血酶原血症的机制，见 许多LAC患者，通过做125I-凝血酶原转换 学习。 2.研究内皮细胞GPIB在内皮细胞功能中的作用： 我们将研究vWF和asialo-vWF与静止的EC和 以IL-1、肿瘤坏死因子或内毒素刺激内皮细胞。我们将衡量 用单抗检测静息和刺激内皮细胞中GPIB的表达 GPIB抗体，并确定这些抗体是否，或 本实验室生产的多克隆抗体可抑制vWF或 AS-vWF绑定。我们将考察欧共体与欧盟之间的关系 静息细胞和暴露细胞中的细胞骨架和EC GPIB 细胞因子。我们将研究EC GPIB-αmRNA的调控。 使用5‘-1.1kb探针和3’-1.3kb探针的细胞因子 相关变化与膜GPIB的表达。我们将研究一下 电子显微镜观察食道手术前后GPIB的分布 细胞因子和其他因素的刺激。我们将进行调查 血小板与刺激内皮细胞的结合及其可能的作用 VWF、AS-vWF、纤维蛋白原，可能还有其他黏附蛋白 这样的互动。 3.调查GPIB可能存在的结构异常或 假性红斑狼疮患者血小板细胞骨架成分的研究 VWD：利用聚合酶链式反应，我们将扩增出mRNA 从患者的血小板中提取并搜索结构性的 使用血小板GPIB-α探针进行异常检测。我们将研究一下 静息和静息后患者血小板的细胞骨架成分 聚集剂的刺激以及相互作用 在膜GPIB和细胞骨架之间。我们将决定 暴露条件对细胞骨架成分的影响 激活血小板钙蛋白酶，如钙离子载体、凝血酶 +胶原蛋白，以及麻醉剂地布卡因和丁卡因。

The aims of this proposal cover three major objectives: 1. To determine the mechanism(s) of action of lupus anticoagulants. (LAC's) and the hemostatic significance of circulating antibodies to anionic phospholipids: We shall investigate binding of LAC's to activated platelets and to endothelial cells (EC) exposed to IL-1, endotoxin or tumor necrosis factor, and correlate binding with exposure of phosphatidylserine. We shall determine binding-induced alterations in 'prothrombinase' capacity of platelets and EC, effects on cytokine-induced synthesis of PGI2 by EC, and alterations in EC-thrombomodulin-thrombin- inducec activation of protein C. We shall investigate LAC binding to other IL-1- or TNF-sensitive cells, as well as exposure of PS. We shall investigate the mechanism of hypoprothrombinemia, seen in many patients with LAC's, by doing 125 I-prothrombin turnover studies. 2. To study the role of endothelial cell GpIb in EC function: We shall examine binding of vWF and asialo-vWF to resting EC and to EC stimulated with IL-1, TNF or endotoxin. We shall measure GpIb expression in resting and stimulated EC, using monoclonal antibodies to GpIb, and determine whether these antibodies, or polyclonal antibodies produced in our laboratory, inhibit vWF or AS-vWF binding. We shall examine the relationship between the EC cytoskeleton and EC GpIb in resting cells and cells exposed to cytokines. We shall study modulation of EC GpIb-alpha mRNA by cytokines using a 5'-1.1 Kb probe and a 3'-1.3 Kb probe and correlate changes with membrane GpIb expression. We shall examine GpIb distribution by electron microscopy before and after EC stimulation by cytokines and other agents. We shall investigate the binding of platelets to stimulated EC and the possible role of vWF, AS-vWF, fibrinogen and, possibly, other adhesive proteins in such interactions. 3. To investigate possible structural abnormalities of GpIb or of cytoskeletal components in platelets from patients with pseudo- vWd: Using the polymerase chain reaction, we shall amplify mRNA from patient platelets and shall search for structural abnormalities using platelet GpIb-alpha probes. We shall examine cytoskeletal components of patient platelets, resting and after stimulation with aggregating agents, as well as the interaction between membrane GpIb and the cytoskeleton. We shall determine the effect on cytoskeletal components of exposure to conditions activating platelet calpain, such as calcium ionophore, thrombin + collagen, and the anesthetics dibucaine and tetracaine.