Regulatory mechanism of β3 integrin function via signaling molecules

β_3整合素含有α_<IIb>β_3和α_vβ_3，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，我们试图建立一个除血小板之外的新的实验模型来研究β_3整合素功能的调节。首先，我们对αvβ_3整联素进行了结构和功能分析。基于β-螺旋桨模型，我们利用丙氨酸扫描诱变技术检测了αv亚基的配体结合位点。我们证明了w32 -3环中的178Tyr是配体结合的关键残基之一。其次，我们检测了来自脐带血单核细胞和某些巨核细胞系的早期巨核细胞，并证明CD42b（GPIb）阳性细胞在TRAP和/或PMA刺激后几分钟内可以激活α_<IIb>β_3。我们利用cd42b阳性细胞和cd42b阴性细胞的差异展示方法，在某巨核细胞系中寻找负责整合素激活的分子。第三，我们研究了血小板中的Na^+/Ca^<2+>交换器，我们首次证明了它是整合素激活的重要信号分子。我们在血小板中发现了一种新的70 kD亚型，而不是110-kD亚型。70 kD NCX的功能意义正在研究中。

β_3 integrins, which contain α_<IIb>β_3 and α_vβ_3, 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 β_3 integrin function.Firstly, we have analyzed structural and functional analyses of αvβ_3 integrin. Based on the β-propeller model we have examined ligand-binding sites in the αv subunit employing alanine-scanning mutagenesis. We demonstrated that 178Tyr in the W3 2-3 loop is one of the critical residues for ligand-binding. Secondly, we have examined early megakaryocytes derived from umbilical cord blood mononuclear cells and certain megakaryocytic cell lines and demonstrated that CD42b(GPIb)-positive cells could activate α_<IIb>β_3 within several minutes after TRAP and/or PMA stimulation. We have been searching molecules responsible for integrin activation employing differential display method between CD42b-positive and CD42b-negative cells in a certain megakaryocytic cell line. Thirdly, we have examined Na^+/Ca^<2+> exchanger in platelets which we have firstly demonstrated as an important signaling molecule for integrin activation. We demonstrated a new 70 kD isoform in platelets instead of 110-kD form. The functional significance of the 70 kD NCX is under investigation.