STRUCTURAL STUDIES OF PLATELET GPIIB/IIIA ACTIVATION

激动剂刺激后，可溶性纤维蛋白原和/或Willebrand因子与整合素αIIbbeta3结合，使血小板发生同型聚集。由于血浆中纤维蛋白原和血管性假血友病因子(VWF)的浓度远远超过饱和AlphaIIbbeta3所需的浓度，循环中的血小板保持AlphaIIbbeta3处于非活动状态，以防止自发的血小板聚集。本项目的目标是阐明AlphaIIbbeta3激活的机制，并表征AlphaIIbbeta3经历的使其成为盲溶配体的变化。该项目包括两个具体目标，这两个目标是正在进行的工作的延伸，代表了一种综合的细胞生物学、分子和生物物理方法，以了解伴随AlphaIIbbeta3激活而发生的结构变化。这些研究集中在位于AlphaII的基序上，数据表明AlphaIIb中的序列与大鼠AlphaIIbbeta3对含RGD的多肽的相对抵抗力有关。为了确定与RGD抗性有关的序列，将确定人和大鼠蛋白质之间一系列序列交换的后果。然后将结果与通过圆二色谱、分析超速离心法和X射线结晶学确定的与AlphaIIb氨基末端相对应的多肽的二级和三级结构相关联。特异性目标2解决了位于αIIb和β3胞质和跨膜片段中的序列基序，这些序列与αIIbbeta3的激活有关。通过定点突变，将确定胞质区域中介导alphaIIbbeta3功能的细胞骨架约束的序列基序。为了解决这一假设，即AlphaIIb和Beta3细胞质和/或跨膜结构域之间的特定相互作用导致了AlphaIIb和Beta3的激活，将测试跨膜以及AlphaIIb和Beta3膜旁区域的“二硫键和天冬酰胺扫描”对AlphaIIbbeta3功能的影响，并与在膜状环境中与AlphaIIb和Beta3的跨膜螺旋和细胞质结构域相对应的肽的结构和寡聚平衡的生物物理研究结果相比较。

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.