Elucidation of the physiological significance of novel platelet proteins.

由于我们已经进行了大量的实验，我们希望将重点放在以下两个项目上：2005年，我们使用针对WAVE亚型的特异性抗体，证明了人类血小板表达所有3种亚型。使用体外下拉技术，胰岛素受体底物p53 （IRSp53）的src同源3结构域比profilin i更有效地从血小板裂解物中沉淀WAVE2，而WAVE1则相反，两者都没有沉淀WAVE3，这表明WAVE异构体与这些配体具有不同的亲和力，而abl的SH3结构域与所有3种异构体结合。我们还发现所有3种WAVE亚型在体内和体外都是钙蛋白酶的底物。虽然这3种同工异构体的一部分通常分布在扩张血小板板足的肌动蛋白和arp2 /3丰富的边缘，但在细胞洗涤或固定后，只有WAVE2留在细胞边缘。这些数据表明，3种WAVE亚型表现出共同而独特的特征，并可能参与血小板中肌动蛋白细胞骨架的调节。2006年，我们还发现血小板表达IRSp53蛋白。我们发现，通过RNA干扰敲低IRSp53可以减少片层基的形成，但不减少WAVE2复合物的数量。在IRSp53敲除的细胞中，WAVE2在细胞周围的定位得以保留。此外，活化的Cdc42而不是Rac削弱了WAVE2和IRSp53之间的关联。当我们在体外测量Arp2/3的激活时，从细胞膜部分分离的WAVE2复合物以irsp53依赖的方式完全活跃，而从细胞质中分离的WAVE2则不是。纯化的WAVE2和纯化的WAVE2复合物被IRSp53以rac依赖的方式与含有PIP(3)的脂质体激活。因此，在活化的Rac和PIP存在下，IRSp53优化了WAVE2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).