Characterization of regulatory proteins for β3 integrins.

β3整合素含有α ib β3和αvβ3，在动脉粥样硬化和血栓形成中起重要作用。因此，阐明整合素功能的调控机制是控制这些血管事件的关键问题。在本研究项目中，我们尝试建立血小板以外的新的实验模型来研究β3整合素功能及其配体结合位点的调控。我们利用W3和W4环内的丙氨酸扫描诱变技术检测了αvβ-螺旋桨结构域内的配体结合位点。我们证明178Tyr和Asp128是配体结合的关键残基。此外，Ala215Tyr αv增加了整合素的亲和力。接下来，我们证明了在巨核细胞系中，CMK、CD42b（GPIb）阳性细胞在PMA刺激后可以激活αIIbβ3。在CMK成熟过程中，306个基因的表达水平增加，我们利用差异显示方法和/或cDNA芯片寻找负责整合素激活的分子。其中我们感兴趣的是β1-tublin、CKIP-1、cortacn、PKCδ、PKCβ1、WAVE-1、Rab27B等。利用苔藓虫抑制素-1，我们发现PKCα和β在整合素激活中起作用。利用脐带血原代巨核细胞，我们证实了在巨核细胞分化过程中，WAVE-1的表达增加，而WAVE-2或-3的表达没有增加。在粘附的血小板中，WAVE-1和WAVE-2都位于板足边缘，表明这些分子可能在血小板扩散过程中调节肌动蛋白重组。

β3 integrins, which contain αIIbβ3 and αvβ3, play critical roles in atherosclerosis as well as thrombosis.Therefore, the elucidation of the regulatory mechanisms for integrin function is a critical issue to control these vascular events. In this research project, we have tried to establish a new experimental model other than platelets to examine the regulation of β3 integrin function and their ligand-binding sites.We examined ligand-binding sites within the αvβ-propeller domain employing alanine-scanning mutagenesis within W3 and W4 loops. We demonstrated that 178Tyr and Asp128 are the critical residues for ligand-binding.. In addition Ala215Tyr αv increased the integrin affinity.We next demonstrated that in a megakaryocytic cell line, CMK,CD42b(GPIb)-positive cells could activate αIIbβ3 after PMA stimulation. We then searched molecules responsible for integrin activation employing differential display method and/or cDNA micrarray During CMK maturation expression levels of 306 genes increased. Among them we were interested in β1-tublin, CKIP-1, cortactin, PKCδ, PKCβ1, WAVE-1, Rab27B etc. Employing bryostatin-1 we suggested that PKCα and β play a role in integrin activation. Employing primary megakaryocytic cells derived from cord blood, we confirmed that WAVE-1 expression, but not WAVE-2 or -3, increased during megakaryocytic differentiation. In adhered platelets both WAVE-1 and WAVE-2 were localized at the edge of the lamellipodia, suggesting that these molecules may regulate actin reorganization during platelet spreading.