Full activation of mouse platelets requires ADP secretion regulated by SERCA3 ATPase–dependent calcium stores

Abstract

AbstractThe role of the sarco-endoplasmic reticulum calcium (Ca2+) adenosine triphosphatase (ATPase) 3 (SERCA3) in platelet physiology remains poorly understood. Here, we show that SERCA3 knockout (SERCA3−/−) mice exhibit prolonged tail bleeding time and rebleeding. Thrombus formation was delayed both in arteries and venules in an in vivo ferric chloride–induced thrombosis model. Defective platelet adhesion and thrombus growth over collagen was confirmed in vitro. Adenosine 5′-diphosphate (ADP) removal by apyrase diminished adhesion and thrombus growth of control platelets to the level of SERCA3−/− platelets. Aggregation, dense granule secretion, and Ca2+ mobilization of SERCA3−/− platelets induced by low collagen or low thrombin concentration were weaker than controls. Accordingly, SERCA3−/− platelets exhibited a partial defect in total stored Ca2+ and in Ca2+ store reuptake following thrombin stimulation. Importantly ADP, but not serotonin, rescued aggregation, secretion, and Ca2+ mobilization in SERCA3−/− platelets, suggesting specificity. Dense granules appeared normal upon electron microscopy, mepacrine staining, and total serotonin content, ruling out a dense granule defect. ADP induced normal platelet aggregation, excluding a defect in ADP activation pathways. The SERCA3-specific inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone diminished both Ca2+ mobilization and secretion of control platelets, as opposed to the SERCA2b inhibitor thapsigargin. This confirmed the specific role of catalytically active SERCA3 in ADP secretion. Accordingly, SERCA3-dependent Ca2+ stores appeared depleted in SERCA3−/− platelets. Finally, αIIbβ3 integrin blockade did not affect SERCA3-dependent secretion, therefore proving independent of αIIbβ3 engagement. Altogether, these results show that SERCA3-dependent Ca2+ stores control a specific ADP secretion pathway required for full platelet secretion induced by agonists at low concentration and independent of αIIbβ3.