The β isoform of the catalytic subunit of protein phosphatase 2B restrains platelet function by suppressing outside-in αII b β3 integrin signaling

Background: Calcium-dependent signaling mechanisms play a critical role in platelet activation. Unlike calcium-activated protease and kinase, the contribution of calcium-activated protein serine/threonine Phosphatase in platelet activation is poorly understood.

Objective: To assess the role of catalytic subunit of protein Phosphatase 2B (PP2B) or Calcineurin in platelet function.

Results: Here, we showed that an increase in PP2B activity was associated with agonist-induced activation of human and murine platelets. Pharmacological inhibitors of the catalytic subunit of protein Phosphatase 2B (PP2B-A) such as cyclosporine A or tacrolimus (FK506) potentiated aggregation of human platelets. Murine platelets lacking the β isoform of PP2B-A (PP2B-Aβ(-/-) ) displayed increased aggregation with low doses of agonist concentrations. Loss of PP2B-Aβ did not affect agonist-induced Integrin αII b β3 inside-out signaling, but increased basal Src activation and outside-in αII b β3 signaling to p38 mitogen-activated protein kinase (MAPK), with a concomitant enhancement in platelet spreading on immobilized fibrinogen and greater fibrin clot retraction. Fibrinogen-induced increased p38 activation in PP2B-Aβ(-/-) platelets were blocked by Src Inhibitor. Both PP2B-Aβ(-/-) platelets and PP2B-Aβ-depleted human embryonal kidney 293 αII b β3 cells displayed increased adhesion to immobilized fibrinogen. Filamin A, an actin crosslinking phosphoprotein that is known to associate with β3 , was dephosphorylated on Ser(2152) in fibrinogen-adhered wild-type but not in PP2B-Aβ(-/-) platelets. In a FeCl3 injury thrombosis model, PP2B-Aβ(-/-) mice showed decreased time to occlusion in the carotid artery.

Conclusion: These observations indicate that PP2B-Aβ by suppressing outside-in αII b β3 Integrin signaling limits platelet response to vascular injury.

beta(3) integrin; fibrinogen; platelet aggregation; platelets; protein-serine-threonine phosphatase.