INTERACTION OF GPIIB-IIIA WITH THE PLATELET CYTOSKELETON

描述(改编自申请者摘要)：本修订方案为 旨在测试Talin和vinculin在 通过与GPIIb-IIIa相互作用将GPIIb-IIIa连接到血小板细胞骨架上 这种整合素的细胞质结构域。GPIIb-IIIa是血小板 纤维蛋白原和vWF受体。由于绑定到这些 分子，GPIIb-IIIa与血小板细胞骨架相关， 这可能与GPIIb-IIIa在凝块回缩、酪氨酸 磷酸化、受体聚集和重新分布。申请人 RGD法发现Talin、vinculin和肌动蛋白与GPIIb-IIIa共纯化 亲和层析及其与GPIIb-IIIa共分布的研究 凝血酶刺激的血小板。这项建议的具体目的是1) 证明纯化的Talin或vinculin直接结合到激活的 GPIIB-IIIa。这将通过测量同时绑定 纤维蛋白原和talin或vinculin在双标记实验中与GPIIb-IIIa结合。 抗LIBS6的RGD多肽将激活GPIIb-IIIa。 此外，还将进行免疫沉淀研究，以检测相互作用 他林和纽蛋白与GPIIb-IIIa结合。2)定位特定的决定因素 在GPIIb-IIIa胞质结构域内介导与 细胞骨架蛋白。这将通过重组的GPIIb-IIIa来完成 在细胞质区域被截断或突变。3)检查 GPIIb-IIIa与血小板相关的功能意义 细胞骨架通过试图抑制这种相互作用在渗透性 具有模拟细胞质序列的合成肽的细胞或具有 抗细胞质区抗体，以及4)研究其可能性 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胞浆结构域的抗体。

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.