STRUCTURAL STUDIES OF PLATELET GPIIB/IIIA ACTIVATION

在激动剂刺激后，可溶性纤维蛋白原和/或Willebrand因子结合到整合素alphaIIbbeta3上，使血小板发生同型聚集。由于血浆纤维蛋白原和血管性血液病因子（vWF）的浓度大大超过饱和alphaIIbbeta3所需的浓度，循环血小板维持alphaIIbbeta3处于非活性状态，以防止自发血小板聚集。该项目的目的是阐明alphaIIbbeta3活化的机制，并描述alphaIIbbeta3成为盲溶配体所经历的变化。该项目包括两个特定目标，它们是正在进行的工作的延伸，代表了一种综合的细胞生物学，分子和生物物理学方法来理解伴随alphaIIbbeta3激活的结构变化。研究的重点是位于alphaIIb的基序，基于数据表明，alphaIIb中的序列负责大鼠alphaIIbbeta3对含rgd肽的抑制的相对抗性。为了确定赋予RGD抗性的序列，将确定人类和大鼠蛋白质之间一系列序列交换的结果。然后，通过圆二色光谱、分析超离心和x射线晶体学，将结果与alphaIIb氨基端对应的多肽的二级和三级结构相关联。特异性Aim 2解决了位于alphaIIb和beta3细胞质和跨膜段的序列基序，这些基序参与了alphaIIbbeta3的激活。细胞质结构域中介导细胞骨架约束alphaIIbbeta3功能的序列基序将通过位点定向诱变进行鉴定。为了证明alphaIIbbeta3的激活是由alphaIIb和beta3细胞质和/或跨膜结构域之间的特定相互作用引起的，对alphaIIb和β a3的跨膜和近膜区域进行“二硫和天冬酰胺扫描”对alphaIIb和β a3功能的影响将被测试，并与生物物理研究结果进行比较，这些研究结果与膜样环境中alphaIIb和β a3的跨膜螺旋和细胞质结构域对应的肽的结构和寡聚平衡。

Following agonist stimulation, soluble fibrinogen and/or Willebrand factor binds to the integrin alphaIIbbeta3, enabling platelets to undergo homotypic aggregation. Because the plasma concentration of fibrinogen and von Willebrand factor (vWF) are substantially in excess of those required to saturate alphaIIbbeta3, circulating platelets maintain alphaIIbbeta3 in an inactive state to prevent spontaneous platelet aggregation. The objectives of this project are to clarify the mechanisms of alphaIIbbeta3 activation and to characterize the changes that alphaIIbbeta3 undergoes that enable to be blind soluble ligands. The project consists of two Specific Aims that are an extension of ongoing work and represent an integrated cell biological, molecular, and biophysical approach to understanding the structural changes that accompany alphaIIbbeta3 activation. The studies focus on motifs located in alphaIIbased on data indicating that sequences in alphaIIb are responsible for the relative resistance of rat alphaIIbbeta3 to inhibition by RGD-containing peptides. To identify the sequences conferring RGD resistance, the consequences of a series of sequences exchanges between the human and rat proteins will be determined. The results will then be correlated with the secondary and tertiary structure of polypeptides corresponding to the amino terminus of alphaIIb as determined by circular dichroism spectroscopy, analytical ultracentrifugation, and x-ray crystallography. Specific Aim 2 addresses the sequence motifs located in the cytoplasmic and transmembrane segments of alphaIIb and beta3 that are involved in alphaIIbbeta3 activation. Sequence motifs in the cytoplasmic domains that mediate cytoskeletal constrain of alphaIIbbeta3 function will be identified using site-directed mutagenesis. To address the hypothesis that alphaIIbbeta3 activation results from specific interactions between the alphaIIb and beta3 cytoplasmic and/or transmembrane domains, the consequences of "disulfide and asparagine scanning" of the transmembrane and the juxta-membrane regions of alphaIIb and beta3 on alphaIIbbeta3 function will be tested and compared to the results of biophysical studies of the structure and oligomeric equilibria of peptides corresponding to the transmembrane helix and cytoplasmic domains of alphaIIb and beta3 in a membrane-like environment.