Kindlin-3 signaling in platelets

血小板中的整合素α IIb β 3活化是纤维蛋白原结合、血小板聚集 和初级止血。然而，血小板聚集的过度反应可导致危及生命的动脉粥样硬化。 血栓形成，终末期心血管疾病的标志。仔细控制激活调节 因此，α IIb β 3的状态对于血小板实现其止血功能的能力至关重要，同时 时间限制血栓形成风险。α IIb β 3的活化，像细胞粘附的整合素家族的其他成员一样， 和信号受体，是仔细调节的协会的胞质蛋白结合在一个细胞 整合素β 3亚基胞质结构域的激活依赖性方式。Kindlin-3是一个FERM结构域- 含有主要在造血细胞中表达的蛋白质。以前的研究已经表明 Kindlin家族的成员主要通过与整合素β胞质尾区结合来促进整合素活化 以及通过称为亲合力调节的过程介导整联蛋白聚集。Kindlin-3已被证明是 对于整联蛋白活化是必需的，如发现白细胞粘附缺陷III型患者， 一种以白细胞和血小板功能障碍和严重出血为特征的先天性遗传性疾病， kindlin-3基因中的致病性功能丧失突变。kindlin-3是如何机械地 然而，支持整合素α IIb β 3激活的蛋白质在很大程度上仍然未知。与整联蛋白聚集一致 假设，一个相关的kindlin，kindlin-2，已被证明形成一个FERM结构域交换的二聚体，我们有 获得的初步数据显示，kindlin-3也需要二聚化以促进亲合力调节 整合素α IIb β 3。然而，有趣的是，在小鼠中没有观察到高度纯化的kindlin-3的二聚体形式。 晶体或溶液中，这表明可能需要额外的组分来促进kindlin-3 二聚化和整联蛋白聚集。在这方面，我们最近发现，kindlin-3直接与 肌球蛋白必需轻链（ELC）是肌球蛋白的一种关键成分，通过连接到 肌球蛋白的近端颈部区域。重要的是，我们实验室获得的初步数据表明， ELC还有助于血小板中整合素α IIb β 3的亲合力调节。根据这些发现，目的 本申请的目的是检验kindlin-3二聚化和调节α IIb β 3的能力 活化涉及迄今未描述的与肌球蛋白ECL的相互作用。具体目标1： 通过采用多重免疫荧光技术确定Kindlin-3与肌球蛋白ECL相互作用的分子基础， 生物化学和结构方法。在具体目标2中，我们将确定kindlin-3/ECL的作用 在调节小鼠和人整合素α IIb β 3的亲和力和亲合力调节中的相互作用 血小板总之，我们相信这项研究的发现将建立详细的分子机制 kindlin-3可以通过这种方式微调血小板中整合素α IIb β 3的活化，这可能会导致新的机会， 开发更安全和更具体的抗血栓策略。

Integrin αIIbβ3 activation in platelets is an important step on the road to fibrinogen binding, platelet aggregation, and primary hemostasis. Overreaction of platelet accumulation, however, can lead to life-threatening arterial thrombosis, a hallmark of end-stage cardiovascular disease. Carefully controlled regulation of the activation state of αIIbβ3, therefore, is crucial for the ability of platelets to fulfill their hemostatic function while at the same time limiting thrombotic risk. The activation of αIIbβ3, like other members of the integrin family of cell adhesion and signaling receptors, is carefully regulated by the association of cytosolic proteins that bind in a cellular activation-dependent manner to the integrin β3 subunit cytoplasmic domain. Kindlin-3 is a FERM domain- containing protein that is primarily expressed in cells of hematopoietic origin. Previous studies have shown that members of the kindlin family promote integrin activation primarily by binding to the integrin β cytoplasmic tail and mediating integrin clustering via a process known as avidity modulation. Kindlin-3 has been shown to be essential for integrin activation, as exemplified by the finding that patients with leukocyte adhesion deficiency III, a congenital inherited disorder characterized by leukocyte and platelet dysfunction and severe bleeding, have causative loss-of-function mutations in the kindlin-3 gene. Precisely how kindlin-3 acts mechanistically to support integrin αIIbβ3 activation, however, remains largely unknown. Consistent with the integrin clustering hypothesis, a related kindlin, kindlin-2, has been shown to form a FERM domain-swapped dimer, and we have obtained preliminary data showing that dimerization is also required for kindlin-3 to promote avidity modulation of integrin αIIbβ3. Interestingly, however, dimeric forms of highly purified kindlin-3 have not been observed in either crystals or in solution, suggesting that extra components may be required to facilitate kindlin-3 dimerization and integrin clustering. In this regard, we have recently found that kindlin-3 directly interacts with the myosin essential light chain (ELC), a key component of myosin that exists as a dimer by attaching to the proximal neck regions of myosin. Importantly, preliminary data obtained in our lab has shown that the myosin ELC also contributes to avidity modulation of integrin αIIbβ3 in platelets. Based on these findings, the purpose of this application is to examine the hypothesis that the ability of kindlin-3 to dimerize and modulate αIIbβ3 activation involves a heretofore undescribed interaction with the myosin ECL. In Specific Aim 1, we will determine the molecular basis of kindlin-3 in interacting with the myosin ECL by employing multiple biochemical and structural approaches. In Specific Aim 2, we will determine the role of kindlin-3/ECL interactions in regulating the affinity and avidity modulations of integrin αIIbβ3 in both mouse and human platelets. Together, we believe that the finding from this study will establish the detailed molecular mechanism by which kindlin-3 fine-tunes integrin αIIbβ3 activation in platelets, which may lead to novel opportunities for developing safer and more specific anti-thrombotic strategies.