PROTEINS THAT REGULATE INTEGRIN FUNCTIONS IN PLATELETS

整合素αIIbeta3介导了血小板的两个主要功能： 活化形式结合黏附配体并促进血小板聚集， 而连接形式会触发生化反应， 细胞骨架重组和血小板扩散。生物化学 αIIbeta3激活和整合素介导的通路 对信号的描述很差。这一点的中心假设是 建议是特定的蛋白质直接与短链相互作用 αIIbeta3的细胞质尾巴调节结合和信号转导 整合素的功能。一个具体的目标是确定 Rho家族的GTP酶在AlphaIIbeta3激活。因此， 结构性活性或显性-负性抑制形式 和Rho将被结合到渗透的血小板和他们的 流式细胞术检测纤维蛋白原受体功能的影响 能够监测AlphaIIbeta3激活和 整合素聚类法。第二个具体目标是确定 整合素最早触发信号反应的分子基础 结扎术。蛋白酪氨酸激酶pp72Syk被激活 当纤维蛋白原与αIIbeta3结合时立即。因此， Cdc42的构成活性或显性-负性抑制形式， RAc和Rho将被加入到渗透性血小板中，他们的 流式细胞术检测纤维蛋白原受体功能的影响 能够监测AlphaIIbeta3激活和 整合素聚类法。第二个具体目标是确定 整合素最早触发信号反应的分子基础 结扎术。蛋白酪氨酸激酶pp72Syk被激活 当纤维蛋白原与αIIbeta3结合时立即。因此， 免疫沉淀、免疫印迹和体外检测将 进行以确定在Syk中是否涉及Src家族激酶 以及其他分子，如p52Shc或 P140-145SHIP是新生的整合素信号单位的成员。 在CHO细胞中的瞬时转基因实验将尝试 重述整合素介导的酪氨酸磷酸化模式 在血小板中。第三个具体目标是直接关注 AplhaIIβ和Beta3的细胞质尾巴。合成多肽衍生 从这些尾巴进入通透性的血小板 检测它们对激动剂诱导的整合素激活的影响。 细胞质尾肽也将被衍生化，化学交叉- 将使用通透性血小板的链接策略来 在原位识别与尾巴相互作用的蛋白质。此外，由于 最近的证据表明，Beta3细胞质尾巴可能会变成 激活的血小板中酪氨酸磷酸化的酵母双杂交 系统将被修改以筛选相互作用的人类蛋白质 选择性地与酪氨酸磷酸化的Beta3尾巴结合。加在一起， 这些研究应该识别和表征那些能够 调节AlphaIIbeta3的粘附性和信号功能。

Integrin alphaIIbeta3 mediates two major functions in platelets: the activated form binds adhesive ligand and promotes platelet aggregation, while the liganded form triggers biochemical reactions required for cytoskeletal reorganization and platelet spreading. The biochemical pathways responsible for alphaIIbeta3 activation and integrin-mediated signaling are poorly characterized. The central hypothesis of this proposal is that specific proteins interact directly with the short cytoplasmic tails of alphaIIbeta3 to regulate the binding and signaling fucntions of the integrin. One specific aim is to establish the role of the Rho family of GTPases in alphaIIbeta3 activation. Therefore, constitutively-active or dominant-negative inhibitory formsCdc42, Rac and Rho will be incorporated into permeailized platelets and their effects on fibrinogen receptor function measured using flow cytometric assays that are capable of monitoring alphaIIbeta3 activation and integrin clustering. The second specific aim is to determine the molecular basis for the earliest signaling reactions triggered by integrin ligation. The protein tyrosine kinase, pp72Syk, becomes activated immediately upon binding of fibrinogen to alphaIIbeta3. Therefore, constitutively-active or dominant-negative inhibitory forms of Cdc42, Rac and Rho will be incorporated into permeabilized platelets and their effects on fibrinogen receptor function measured using flow cytometric assays that are capable of monitoring alphaIIbeta3 activation and integrin clustering. The second specific aim is to determine the molecular basis for the earliest signaling reactions triggered by integrin ligation. The protein tyrosine kinase, pp72Syk, becomes activated immediately upon binding of fibriogen to alphaIIbeta3. Therefore, immunoprecipitation, immunoblotting and in vitro assays will be carried out to establish whether Src family kinases are involved in Syk activation in platelets and whether other molecules, such as p52Shc or p140-145SHIP, are members of the nascent integrin signaling unit. Transient transfection experiments in CHO cells will attempt to recapitulate the pattern of integrin-mediated tyrosine phosphorylation in platelets. A third specific aim is to focus directly on the roles of th cytoplasmic tails of aplhaIIbeta and beta3. Synthetic peptides derived from these tails will be introduced into permeabilized platelets to examine their effects on agonist-induced integrin activation. Cytoplasmic tail peptides will also be derivatized, and chemical cross- linking strategies will be employed using permeabilized platelets to identify proteins that interact with the tails in situ. In addition, since recent evidence indicates that the beta3 cytoplasmic tail may become tyrosine-phosphorylated in activated platelets, a yeast two-hybrid system will be modified to screen for human proteins that interact selectively with the tyrosine-phosphorylated beta3 tail. Taken together, these studies should identify and characterize those platelet proteins that regulate the adhesive and signaling functions of alphaIIbeta3.