PROTEINS THAT REGULATE INTEGRIN FUNCTIONS IN PLATELETS

整合素α II β 3在血小板中介导两种主要功能： 活化形式结合粘附配体并促进血小板聚集， 而配体形式触发生物化学反应， 细胞骨架重组和血小板扩散。 生化 负责α II β 3激活和整合素介导的 信号的特征很差。 这个问题的核心假设是 一种新的假说是，特定的蛋白质直接与短链相互作用， 调节α II β 3的结合和信号传导 整合素的功能 一个具体的目标是确立 α II β 3激活中GTP酶Rho家族。 因此，我们认为， 组成型活性或显性阴性抑制型Cdc 42、Rac 和Rho将被掺入透化的血小板中， 使用流式细胞术测量对纤维蛋白原受体功能的影响 能够监测α II β 3活化的测定， 整联蛋白聚类。 第二个具体目标是确定 整合素引发的最早信号反应的分子基础 结扎 蛋白酪氨酸激酶pp 72 Syk被激活， 在纤维蛋白原与α II β 3结合后立即。 因此，我们认为， Cdc 42的组成型活性或显性阴性抑制形式， Rac和Rho将被掺入透化血小板中， 使用流式细胞术测量对纤维蛋白原受体功能的影响 能够监测α II β 3活化的测定， 整联蛋白聚类。 第二个具体目标是确定 整合素引发的最早信号反应的分子基础 结扎 蛋白酪氨酸激酶pp 72 Syk被激活， 在纤维原与α II β 3结合后立即发生。 因此，我们认为， 免疫沉淀、免疫印迹和体外测定将被 进行研究以确定Src家族激酶是否参与Syk 血小板活化以及其他分子，如p52 Shc或 p140- 145 SHIP是新生整合素信号传导单位的成员。 CHO细胞中的瞬时转染实验将试图 概括整合素介导的酪氨酸磷酸化的模式 在血小板中。 第三个具体目标是直接关注 aplhaII β和β 3的胞质尾。 合成肽 将从这些尾部引入透化血小板中， 检查它们对激动剂诱导的整合素活化的作用。 细胞质尾肽也将被衍生化，并进行化学交叉修饰。 将采用使用透化血小板的连接策略， 在原位鉴定与尾部相互作用的蛋白质。 另外由于 最近的证据表明，β 3细胞质尾区可能成为 激活血小板酪氨酸磷酸化，酵母双杂交 系统将被修改，以筛选人类蛋白质， 选择性地与酪氨酸磷酸化的β 3尾结合。 综合起来看， 这些研究应该鉴定和表征那些血小板蛋白， 调节α II β 3的粘附和信号传导功能。

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.