Kindlin-3 signaling in platelets - 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.
