Signaling through integrin alpha IIb beta3

在本研究项目中，我们通过整合素alphaIIbbeta3研究了由内向外和由外向内的信号传导。关于alphaIIbbeta3通过内向外信号激活，我们进一步研究了Na^+/Ca^<2+>交换器（NCX）可能作为一种常见的内向外信号参与其中的假设。克隆血小板NCX cDNA，发现血小板NCX为BD型，心脏NCX为BCDEF型。接下来，我们使用两种不相关的NCX抑制剂DCB和bepridil，详细研究了NCX在血小板活化中的作用。两种抑制剂均可消除所有激动剂诱导的血小板α iibbeta3活化。然而，这些抑制剂不抑制蛋白磷酸化或增加细胞内钙浓度。这些发现进一步支持了我们的假设，即NCX参与了通过alphaIIbbeta3从内到外的信号传导的常见步骤。我们鉴定了一种日本变异的Glanzmann血栓性贫血，其血小板表达正常量的alphaIIbbeta3，并新发现在alphaIIb亚基的Cysl46-Cysl67环中插入两个氨基酸是导致这种表型的原因。此外，Asp163是配体结合功能的关键残基。关于外向内信号传导，我们发现alphaIIbbeta3拮抗剂可分为两组。在I组中，拮抗剂可以在alphaIIb和β a3上诱导LIBS（配体诱导结合位点），而在II组中，拮抗剂可以在alphaIIb上诱导LIBS，但不能在β a3上诱导LIBS。有趣的是，只有I组拮抗剂可以启动alphaiibbeta3介导的细胞内Ca^<2+>信号传导。我们的研究结果强烈提示β a3 LIBS表达与α iibbeta3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.