Full activation of mouse platelets requires ADP secretion regulated by SERCA3 ATPase-dependent calcium stores.
The role of the sarco-endoplasmic reticulum calcium (Ca(2+)) 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 Ca(2+) 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 Ca(2+) and in Ca(2+) store reuptake following thrombin stimulation. Importantly ADP, but not serotonin, rescued aggregation, secretion, and Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) stores control a specific ADP secretion pathway required for full platelet secretion induced by agonists at low concentration and independent of αIIbβ3.
INSERM Unité Mixte de Recherche-Santé 1176, Université Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France;
Ziane
Z
Elaïb
INSERM Unité Mixte de Recherche-Santé 1176, Université Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France;
Frédéric
F
Adam
INSERM Unité Mixte de Recherche-Santé 1176, Université Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France;
Eliane
E
Berrou
Unité d'Hémostase Clinique, Lyon, France; CeCILE-SFR/Centre Commun d'Imagerie de Lyon-Est-Structure Fédérative de Recherche, Université de Lyon, Lyon, France; and.
Jean-Claude
JC
Bordet
Kyoto Gakuen University, Kameoka, Japan.
Nicolas
N
Prévost
INSERM Unité Mixte de Recherche-Santé 1176, Université Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France;
Régis
R
Bobe
INSERM Unité Mixte de Recherche-Santé 1176, Université Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France;
Marijke
M
Bryckaert
INSERM Unité Mixte de Recherche-Santé 1176, Université Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France;
Jean-Philippe
JP
Rosa
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D001760:Bleeding Time; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D017353:Gene Deletion; D018056:Hemorheology / Q000187:drug effects; D006487:Hemostasis / Q000187:drug effects; D006736:Horses; D008810:Mice, Inbred C57BL; D015539:Platelet Activation / Q000187:drug effects; D010973:Platelet Adhesiveness / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000172:deficiency / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology; D013927:Thrombosis / Q000473:pathology
D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D012701:Serotonin; D000244:Adenosine Diphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium