Signaling through integrin alpha IIb beta3

在本研究项目中，我们研究了整合素α IIb β 3的由内而外和由外而外的信号传导。关于α IIb β 3通过由内而外信号传导的激活，我们进一步研究了我们的假设，即Na^+/Ca^2+交换器（NCX）可能作为一种常见的由内而外信号传导参与。我们克隆了血小板NCX cDNA，表明血小板NCX是BD型，而心脏NCX是BCDEF型。接下来，使用两种不相关的NCX抑制剂DCB和苄普地尔，我们详细研究了NCX在血小板活化中的作用。2这两种抑制剂都能抑制所有检测的激动剂诱导的血小板α IIb β 3活化。然而，这些抑制剂并不抑制蛋白磷酸化或细胞内钙离子浓度的增加，这些结果进一步支持了我们的假设，即NCX参与了细胞内的共同步骤，通过α IIb β 3的out信号传导。我们描述了一个日本的Glanzmann血小板无力症的变异体，其血小板表达正常量的α IIb β 3，并且新发现在α IIb亚基的Cys 146-Cys 167环中的两个氨基酸插入是这种表型的原因。此外，Asp 163是配体结合功能的关键残基。关于由外向内信号传导，我们发现α IIb β 3拮抗剂可以分为两组，第一组拮抗剂可以诱导LIBS（配体诱导的结合位点），而在组II中，拮抗剂可以诱导α IIb上的LIBS，但不能诱导β 3上的LIBS。有趣的是，只有I组拮抗剂可以启动α IIb β 3介导的细胞内Ca^<2+>信号传导。我们的研究结果有力地表明β 3 LIBS表达与α IIb β 3介导的细胞内Ca^<2+>信号传导之间存在关联。

In this research project, we have investigated both inside-out and outside-in signaling through the integrin alphaIIbbeta3.Concerning alphaIIbbeta3 activation via inside-out signaling, we have further investigated our hypothesis that Na^+/Ca^<2+> exchanger (NCX) may be involved as a common inside-out signaling.We cloned platelet NCX cDNA and showed that platelet NCX is BD type, while cardiac NCX is BCDEF.Next, employing two unrelated NCX inhibitors, DCB and bepridil, we investigated the role of NCX in platelet activation in detail.Both inhibitors abolished platelet alphaIIbbeta3 activation induced by all agonists examined.However, these inhibitors did not inhibit protein phosphorylation or increase in intracellular calcium concentration.These findings further support our hypothesis that NCX is involves in the common steps of inside-out signaling through alphaIIbbeta3.We characterized a Japanese variant of Glanzmann thrombasthenia whose platelets express normal amounts of alphaIIbbeta3 and newly showed that a two-amino acid insertion in the Cysl46-Cysl67 loop of the alphaIIb subunit is responsible for this phenotype.In addition, Asp163 is a critical residue for ligand binding function.Concerning outside-in signaling, we showed alphaIIbbeta3 antagonists can be divided into two groups.In group I, antagonists can induce LIBS (ligand-induced binding site) on both alphaIIb and beta3, while in group II, antagonists can induce LIBS on alphaIIb, but not on beta3.Interestingly, only group I antagonists can initiate alphaIIbbeta3-mediated intracellular Ca^<2+> signaling.Our findings strongly sugget the association between beta3 LIBS expression and alphaIIbbeta3-mediated intracellular Ca^<2+> signaling.