The β isoform of the catalytic subunit of protein phosphatase 2B restrains platelet function by suppressing outside‐in αIIbβ3 integrin signaling

Abstract

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 (CsA) 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 αIIbβ3 inside-out signaling, but increased basal Src activation and outside-in αIIbβ3 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 αIIbβ3 cells displayed increased adhesion to immobilized fibrinogen. Filamin A, an actin crosslinking phosphoprotein that is known to associate with β3 was dephosphorylated on Ser2152 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 suggest that PP2B-Aβ by suppressing outside-in αIIbβ3 integrin signaling limits platelet response to vascular injury.

Unlocking Aspirin's Chemopreventive Activity: Role of Irreversibly Inhibiting Platelet Cyclooxygenase-1

“…Blood was diluted 1:1 with PBS and PRP was isolated following centrifugation at 68×g for 10 minutes. Washed platelets were obtained following additional centrifugation of PRP as we have described before (26). Platelets were resuspended in RPMI-1640 with 0.01 U/ml of apyrase and counts adjusted to 2.5×10 8 platelets/ml.…”

“…PRP was further challenged with 2.5 microgram/ml of collagen or left untreated (basal). The degree of platelet activation was assessed by flow cytometry using anti-P-selectin FITC and isotype control antibodies, as published (26). …”

Functional cyclophilin D moderates platelet adhesion, but enhances the lytic resistance of fibrin

“…Therefore, we focused on the effects of CsA on the intracellular form of CypA resulting in inhibition of calcineurin. Despite earlier, variable results with calcineurin-inhibition in platelets 37 , 38 , recently, deletion of an isoform of the catalytic subunit of calcineurin as well as FK-506-treatment have been shown to increase ADP-triggered aggregation 25 . In line with this finding, FK-506 and CsA had similar effects in our aggregation assay.…”

“…In fact, its Cyclophilin A (CypA)-mediated inhibitory effect on phosphoprotein phosphatase 2B (calcineurin) accounts for the use of CsA as an immunosuppressant 23 , 24 . A study reporting that calcineurin restricts platelet activation in a murine model highlights the relevance of the latter pathway 25 . Given the thrombotic complications of patients treated with CsA 26 , it is crucial to determine the overlaps and discrepancies of molecular pathways through which CsA and CypD affect platelet function.…”

Fluorescent labeling of endogenous platelets for intravital microscopy: Effects on platelet function

“…Platelet isolation was performed as described previously. [13] Briefly, blood was diluted with an equal volume of DPBS and then subjected to 80 × g centrifugation at room temperature (RT). Platelet-rich plasma was collected and pelleted with 500 × g centrifugation.…”