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

包括急性冠状动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，由于分子生物学研究的困难，其作用机制在很大程度上是未知的。为了克服这一困难，我们开发了一种检测系统，利用溶血素-0渗透的血小板来分析颗粒分泌的聚集（Methods enzymatic . 2005，应邀）。我们正在用这些系统研究这些机制。在2003年，我们直接证明PKCα对于聚集是必不可少的（JBC, 2003）。2004年，我们证明了与酪氨酸磷酸化的整合素β3亚基结合的接头蛋白ShcA对血小板聚集至关重要（BBRC, 2004）。我们发现小GTPase Rab27调节致密颗粒分泌（JBC, 2004）。我们进一步用亲和方法鉴定了血小板胞浆中与Munc13-1（神经递质释放的必要启动因子）非神经元同源物Munc13-4是GTP-Rab27结合蛋白，并证明Munc1-4调节血小板中致密颗粒的分泌。这是RabGTPase直接调控Munc13成员的第一个迹象。我们与日本研究组合作研究噬血细胞综合征，发现家族性噬血细胞综合征中约30%是由Munc13-4基因突变（FHL3）引起的，FHL3患者的表型较穿孔素无义突变（FHL2）引起的患者更为温和（Blood, 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).