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

Abstract

The 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 59-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.