Remodeling of the actin cytoskeleton is one of the critical events that allows platelets to undergo morphological and functional changes in response to receptor-mediated signaling cascades. Coronins are a family of evolutionarily conserved proteins implicated in the regulation of the actin cytoskeleton, represented by the abundant coronins 1, 2, and 3 and the less abundant coronin 7 in platelets, but their functions in these cells are poorly understood. A recent report revealed impaired agonist-induced actin polymerization and cofilin phosphoregulation and altered thrombus formation in vivo as salient phenotypes in the absence of an overt hemostasis defect in vivo in a knockout mouse model of coronin 1. Here we show that the absence of coronin 1 is associated with impaired translocation of integrin β2 to the platelet surface upon stimulation with thrombin while morphological and functional alterations, including defects in Arp2/3 complex localization and cAMP-dependent signaling, are absent. Our results suggest a large extent of functional overlap among coronins 1, 2, and 3 in platelets, while aspects like integrin β2 translocation are specifically or predominantly dependent on coronin 1.
Platelets undergo profound shape changes upon adhesion to damaged blood vessel walls that are mediated by reorganisation of the actin cytoskeleton in response to receptor-mediated signalling cascades. The highly conserved 56 kDa multidomain cyclase associated protein 1 (CAP1) works in concert with cofilin and profilin to modulate actin filament turnover by facilitating cofilin-mediated actin filament severing and depolymerisation and catalysing profilin-mediated regeneration of actin monomers for reutilisation in growing filaments. CAP1 is abundant in platelets but its roles remain unexplored. We report that in suspended platelets CAP1 localises predominantly at the cell cortex whereas in spread platelets it is uniformly distributed in the cytoplasm, with enrichment at the cell cortex and the periphery of actin nodules. Upon subcellular fractionation most CAP1 was found cytosolic but part associated to the membrane fraction in an actin-independent manner. Interestingly, upon stimulation with thrombin a significant proportion of the membrane-associated CAP1 translocates to the cytosol. This relocalisation was prevented by prior treatment with PGI2 or the nitric oxide donor GSNO, or by inhibition of GSK3. Our results place CAP1 at a crossroad of signalling pathways that control platelet activation by contributing to actin remodelling at the cell cortex and actin nodules during platelet spreading.
Rapid reorganization of the actin cytoskeleton in response to receptor-mediated signaling cascades allows platelets to transition from a discoid shape to a flat spread shape upon adhesion to damaged vessel walls. Coronins are conserved regulators of the actin cytoskeleton turnover but they also participate in signaling events. To gain a better picture of their functions in platelets we have undertaken a biochemical and immunocytochemical investigation with a focus on Coro1. We found that class I coronins Coro1, 2 and 3 are abundant in human and mouse platelets whereas little Coro7 can be detected. Coro1 is mainly cytosolic, but a significant amount associates with membranes in an actin-independent manner and does not translocate from or to the membrane fraction upon exposure to thrombin, collagen or prostacyclin. Coro1 rapidly translocates to the Triton insoluble cytoskeleton upon platelet stimulation with thrombin or collagen. Coro1, 2 and 3 show a diffuse cytoplasmic localization with discontinuous accumulation at the cell cortex and actin nodules of human platelets, where all three coronins colocalize. Our data are consistent with a role of coronins as integrators of extracellular signals with actin remodeling and suggests a high extent of functional overlap among class I coronins in platelets.
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