STIM1 Deficiency Results In Impaired Platelet Procoagulant Activity and Protection From Arterial Thrombosis

Abstract 485

Platelet activation is a central event in thrombosis and hemostasis. We recently demonstrated that most aspects of platelet activation depend on synergistic signaling by two signaling modules: 1) Ca²⁺/CalDAG-GEFI/Rap1 and 2) PKC/P2Y12/Rap1. The intracellular Ca²⁺ concentration of platelets is regulated by Ca²⁺ release from the endoplasmic reticulum (ER) and store-operated calcium entry (SOCE) through the plasma membrane. Stromal interaction molecule 1 (STIM1) was recently identified as the ER Ca²⁺ sensor that couples Ca²⁺ store release to SOCE. In this study, we compared the activation response of platelets lacking STIM1−/− or CalDAG-GEFI−/−, both in vitro and in vivo. To specifically investigate Ca²⁺-dependent platelet activation, some of the experiments were performed in the presence of inhibitors to P2Y12. The murine Stim1 gene was deleted in the megakaryocyte/platelet lineage by breeding Stim flox/flox mice with PF4-Cre mice (STIM1^fl/fl). STIM1^fl/fl platelets showed markedly reduced SOCE in response to agonist stimulation. aIIbβ3 activation in STIM1^fl/fl platelets was significantly reduced in the presence but not in the absence of the P2Y12 inhibitor, 2-MesAMP. In contrast, aIIbb3 activation was completely inhibited in 2-MesAMP-treated CalDAG-GEFI−/− platelets. Deficiency in STIM1, and to a lesser extent in CalDAG-GEFI, reduced phosphatidyl serine (PS) exposure in platelets stimulated under static conditions. PS exposure was completely abolished in both STIM1^fl/fl and CalDAG-GEFI−/− platelets stimulated in the presence of 2-MesAMP. To test the ability of platelets to form thrombi under conditions of arterial shear stress, we performed flow chamber experiments with anticoagulated blood perfused over a collagen surface. Thrombus formation was abolished in CalDAG-GEFI−/− blood and WT blood treated with 2-MesAMP. In contrast, STIM1^fl/fl platelets were indistinguishable from WT platelets in their ability to form thrombi. STIM1^fl/fl platelets, however, were impaired in their ability to express PS when adhering to collagen under flow. Consistently, when subjected to a laser injury thrombosis model, STIM1^fl/fl mice showed delayed and reduced fibrin generation, resulting in the formation of unstable thrombi. In conclusion, our studies indicate a critical role of STIM1 in SOCE and platelet procoagulant activity, but not in CalDAG-GEFI mediated activation of aIIbb3 integrin.