On the Yellow Brick Road to Platelet GP IIb-IIIa (Integrin αIIbβ3) Activation.
Authors: Watanabe Naohide , Han Jaewon , Lim C. James , Soriani Alessandra , Puzon-McLaughlin Wilma , LaFuente Esther M. , Boussiotis Vassiliki A. , Ginsberg Mark H. , Shattil Sanford J. 
Published: October 2019

Abstract
In Oz, the yellow brick road led to dreams come true. In platelets, the road from agonist receptors to αIIbβ3 leads to thrombus formation, a response necessary for hemostasis and thrombosis. Upon platelet stimulation, agonist receptor pathways converge on the cytoplasmic and transmembrane domains of αIIbβ3, causing conformational changes that are propagated to the extracellular domain to effect high affinity ligand binding (activation). Numerous intracellular proteins have been implicated inαIIbβ3 activation, including protein kinase C (PKC), the Rap1 GTPase and talin. Indeed, talin recruitment to the β3 cytoplasmic domain is a final common step in αIIbβ3 activation. However, the relationships of these signaling proteins to each other, and the mechanism(s) of talin recruitment to β3 have not been established. Here we used forward, reverse, and synthetic genetics to engineer and order an αIIbβ3 activation pathway. In a CHO cell model system, increased expression of PKCαand talin to levels approximating those in platelets reconstituted αIIbβ3 activation in response to phorbol myristate acetate (PMA). This response was detectable by monitoring fibrinogen binding or the binding of a monovalent, ligand-mimetic antibody Fab fragment (PAC-1 Fab) that is specific for high-affinity αIIbβ3. PMA-induced activation of αIIbβ3 could be blocked by over-expression of Rap1GAP, which inhibits Rap1 GTPase activity. In contrast, over-expression of constitutively-active Rap1A(G12V) caused αIIbβ3 activation that bypassed the requirement for PKCα, indicating that Rap1 is downstream of PKCαin this integrin activation pathway. αIIbβ3 activation by Rap1 was promoted by a specific Rap1 effector, RIAM, an adapter molecule with a PH domain and proline-rich and EVH-1 binding regions. For example, over-expression of RIAM, but not another Rap1 effector (RapL), caused activation of αIIbβ3 and bypassed the requirement for PKCαand Rap1. Furthermore, siRNA-mediated knockdown of RIAM blocked αIIbβ3 activation by PMA or Rap1A(G12V), and this effect could be rescued by an siRNA-resistant form of RIAM. The integrin activating effects of PKCα, Rap1A(G12V) and RIAM were not observed if an integrin binding-defective talin mutant (W359A) was expressed instead of talin. In both CHO cells and platelets, activated Rap1 formed an integrin-associated complex with talin and RIAM, as assessed by deconvolution microscopy and co-immunoprecipitation. In addition, when RIAM was knocked down by siRNA in ES cell-derived murine megakaryocytes, αIIbβ3 activation in response to ADP or thrombin receptor agonists was impaired. These results identify and order a signaling pathway from agonist stimulation to αIIbβ3 activation. At the end of this road, Rap1 organizes an integrin activating complex composed of RIAM and talin. The ability to synthetically reconstruct an αIIbβ3 activation pathway and validate it in platelets and megakaryocytes now provides molecular targets to facilitate characterization of clinical platelet activation defects and development of better anti-platelet drugs. We are not in Kansas anymore.