Elucidation of the physiological significance of novel platelet proteins.

因为我们已经进行了大量的实验，我们想把重点放在以下两个项目上。在2005年，使用针对WAVE亚型的特异性抗体，我们证明了人血小板表达所有3种亚型。通过使用体外下拉技术，胰岛素受体底物p53（IRSp 53）的SRC同源3结构域比profilin I更有效地从血小板溶解产物中沉淀WAVE 2。WAVE 1的情况正好相反，两者都没有沉淀WAVE 3，这表明WAVE同种型对这些配体具有不同的亲和力，而abl的SH 3结构域结合所有3种同种型。我们还发现，所有3个WAVE异构体是钙蛋白酶在体内和体外的底物。虽然这3种亚型的部分通常分布在肌动蛋白和Arp 2/3丰富的边缘的板状伪足展开血小板，只有WAVE 2留在细胞边缘洗涤剂提取或固定的细胞。这些数据表明，3个WAVE亚型表现出共同的和不同的功能，并可能参与调节肌动蛋白细胞骨架在血小板。2006年，我们又发现血小板表达IRSp 53蛋白。我们发现，通过RNA干扰敲低IRSp 53减少了板状伪足的形成，而没有减少WAVE 2复合物的量。WAVE 2在细胞外周的定位在IRSp 53敲除细胞中得以保留。此外，激活Cdc 42而不是Rac减弱WAVE 2和IRSp 53之间的关联。当我们在体外测量Arp 2/3活化时，从细胞膜部分分离的WAVE 2复合物以IRSp 53依赖性方式完全活化，但从胞质溶胶分离的WAVE 2不是。纯化的WAVE 2和纯化的WAVE 2复合物被IRSp 53以Rac依赖性方式与含有PIP（3）的脂质体激活。因此，IRSp 53在活化的Rac和PIP存在下优化WAVE 2复合物的活性（3）。

Because we have performed numerous experiments, we would like to focus on two following projects..In 2005, using specific antibodies against isoforms of WAVE, we demonstrated that human platelets express all 3 isoforms. With the use of an in vitro pull-down technique, the src homology 3 domain of insulin receptor substrate p53 (IRSp53) precipitated WAVE2 from platelet lysates more efficiently than did profilin I. The opposite was true for WAVE1, and neither precipitated WAVE3, suggesting that WAVE isoforms have different affinities to these ligands, while the SH3 domain of abl binds to all 3 isoforms. We also found that all 3 WAVE isoforms are substrates for calpain in vivo and in vitro. Although portions of these 3 isoforms were commonly distributed in the actin-and Arp2/3-rich edge of the lamellipodia in spreading platelets, only WAVE2 remained in the cell fringe following detergent extraction or fixation of the cells. These data suggest that the 3 WAVE isoforms exhibit common and distinct features and may potentially be involved in the regulation of actin cytoskeleton in platelets. In 2006, we also found that platelets express IRSp53 protein. We found that the knockdown of IRSp53 by RNA interference decreased lamellipodium formation without a decrease in the amount of WAVE2 complex. Localization of WAVE2 at the cell periphery was retained in IRSp53 knockdown cells. Moreover, activated Cdc42 but not Rac weakened the association between WAVE2 and IRSp53. When we measured Arp2/3 activation in vitro, the WAVE2 complex isolated from the membrane fraction of cells was fully active in an IRSp53-dependent manner but WAVE2 isolated from the cytosol was not. Purified WAVE2 and purified WAVE2 complex were activated by IRSp53 in a Rac-dependent manner with PIP(3)-containing liposomes. Therefore, IRSp53 optimizes the activity of the WAVE2 complex in the presence of activated Rac and PIP(3).