Identification and functional analysis of novel proteins regulating platelet activation which triggers arterial thrombosis.

包括急性冠脉综合征在内的动脉血栓形成的触发因素是血小板活化。然而，由于很难将分子生物学应用于这项研究，其机制在很大程度上是未知的。为了克服这一困难，我们开发了使用链溶素-0通透性的血小板分析颗粒分泌物聚集的测试系统(方法：Enzymo.2005年，受邀)。我们正在用这些系统研究这些机制。2003年，我们直接证明了PKCα对于聚合是必不可少的(JBC，2003)。2004年，我们证明了一种与整合素的酪氨酸磷酸化β3亚单位结合的适配蛋白SHCA对血小板聚集是必不可少的(BBRC，2004)。我们发现小的GTP酶Rab27调节致密颗粒的分泌(JBC，2004)。我们进一步用亲和的方法鉴定了Munc13-4，它是Munc13-1的非神经元同源物，它是一种神经递质释放的基本启动因子，在血小板胞浆中是GTP-Rab27结合蛋白，并证明Munc1-4调节血小板致密颗粒的分泌。这是第一个表明RabGTP酶直接调节Munc13成员的迹象。我们与日本一个研究噬血细胞综合征的研究小组合作，发现大约30%的家族性噬血细胞综合征是由Munc13-4基因突变(FHL3)引起的，并且FHL3患者比穿孔素无义突变(FHL2)患者表现出更温和的表型(布拉德，2005)。

The trigger of arterial thrombosis inclusing acute coronary syndrome is platelet activation. However, the mechanism is largely unknown since it has been difficult to apply molecular biology on this research. To overcome the difficulty, we have developed assay systems for analyzing aggregation of granule secretion using platelets permeabilized with streptolysin-0 (Methods Enzymol. 2005, invited). We are investigating these mechanisms with these systems. In 2003, we directly demonstrated that PKCα is essential for the aggregation (JBC, 2003). In 2004, we demonstrated that an adaptor protein ShcA which binds to tyrosine-phophorylated β3 subunit of integrin, is essential for platelet aggregation (BBRC, 2004). We found that small GTPase Rab27 regulates dense-granule secretion (JBC, 2004). We further identified Munc13-4, a non-neuronal homologue of Munc13-1, an essential priming factor in neurotransmitter release, in platelet cytosol as a GTP-Rab27 binding protein using an affinity method and demonstrated that Munc1-4 regulates dense-granule secretion in platelets. This is the first indication that a RabGTPase directly regulates a member of Munc13. In collaboration with a Japanese research group investigating hemophagocytic syndrome, we found that approximately 30% of familial hemophagocytic syndrome is caused by mutation of Munc13-4 gene (FHL3) and that FHL3 patients reveals milder phenotype than patients caused by non-sense mutation of perforin (FHL2) (Blood, 2005).