INTERACTION OF GPIIB-IIIA WITH THE PLATELET CYTOSKELETON

描述（改编自申请人摘要）：本修订提案 旨在检验talin和黏着斑蛋白在 将GPIIb-IIIa通过与以下物质的相互作用附着于血小板细胞骨架： 这种整合素的胞质结构域。 GPIIb-IIIa是血小板 纤维蛋白原和vWF受体。 由于这些约束力， 分子，GPIIb-IIIa与血小板细胞骨架结合， 这可能有助于GPIIb-IIIa在凝块收缩中的作用，酪氨酸 磷酸化、受体聚集和再分布。 申请人 发现talin、vinculin和actin与GPIIb-IIIa通过RGD共纯化 亲和层析并与GPIIb-IIIa共分布 凝血酶刺激的血小板 本建议的具体目标是：（1） 证明纯化的talin或黏着斑蛋白与活化的 GPIIb-IIIa。 这将通过测量同时结合 纤维蛋白原和talin或黏着斑蛋白与GPIIb-IIIa的双标记实验。 GPIIb-IIIa将被抗LIBS 6的RGD肽预处理激活。 此外，还将进行免疫沉淀研究，以检测相互作用 talin和vinculin与GPIIb-IIIa的结合。 2)定位特定的决定因素 在GPIIb-IIIa的胞质结构域内介导与 细胞骨架蛋白 这将通过重组GPIIb-IIIa完成 在胞质结构域中截短或突变。 3)审查 GPIIb-IIIa与血小板结合的功能意义 细胞骨架，试图抑制这种相互作用，在透化 具有模拟细胞质序列的合成肽或具有 抗胞质结构域抗体，和4）为了研究 纤维蛋白原与GPIIb-IIIa的结合改变了 受体胞质结构域。 这将通过LIBS来实现 识别GPIIb-IIIa胞质结构域的抗体。

DESCRIPTION (Adapted from Applicant's Abstract): This revised proposal is designed to test the hypothesis that talin and vinculin play a role in attaching GPIIb-IIIa to the platelet cytoskeleton via an interaction with the cytoplasmic domains of this integrin. GPIIb-IIIa is the platelet fibrinogen and vWF receptor. As a consequence of binding to these molecules, GPIIb-IIIa becomes associated with the platelet cytoskeleton, which may contribute to the role of GPIIb-IIIa in clot retraction, tyrosine phosphorylation, receptor clustering and redistribution. The Applicant found that talin, vinculin and actin co-purify with GPIIb-IIIa by RGD affinity chromatography and co-distribute with GPIIb-IIIa in thrombin-stimulated platelets. The Specific Aims of this proposal are 1) To demonstrate direct binding of purified talin or vinculin to activated GPIIb-IIIa. This will be done by measuring simultaneous binding of fibrinogen and talin or vinculin to GPIIb-IIIa in double label experiments. GPIIb-IIIa will be activated by RGD peptide pretreatment of anti-LIBS6. Also, immunoprecipitation studies will be performed to detect interactions of talin and vinculin with GPIIb-IIIa. 2) To localize specific determinants within the cytoplasmic domains of GPIIb-IIIa mediating interactions with cytoskeletal proteins. This will be done with recombinant GPIIb-IIIa truncated or mutated in the cytoplasmic domain. 3) To examine the functional significance of GPIIb-IIIa association with the platelet cytoskeleton by attempting to inhibit this interaction in permeabilized cells with synthetic peptides mimicking the cytoplasmic sequences or with anti-cytoplasmic domain antibodies, and 4) To investigate the possibility that fibrinogen binding to GPIIb-IIIa changes the conformation of the receptor cytoplasmic domains. This will be accomplished with LIBS antibodies that recognize the cytoplasmic domains of GPIIb-IIIa.