OCaR1 regulates dense granules secretion to evoke platelet aggregation

Activation and subsequent aggregation of thrombocytes at the site of vascular injury is essential for primary haemostasis. A crucial step in platelet activation is the increase of intracellular Ca²⁺ concentration and simultaneous reduction of intracellular cAMP. Stimulation of PAR4 receptors on the plasma membrane of thrombocytes results in Ca²⁺ release from both IP₃-sensitives stores (DTS) as well as NAADP-dependent Ca²⁺ stores. Subsequently, cAMP concentrations are reduced upon stimulation of the P2Y12 receptor resulting in activation of phosphodiesterase. In pancreatic acinar cells, OCaR1 functions a regulator of Ca²⁺ release from TPC2 containing secretory and acid granules, and thereby confers exocytotic vesicles an autoregulatory competence to prevent Ca²⁺ release, exocytosis and pancreatic tissue damage. OCaR1 is found in platelet Proteome data, and thus we aimed to determine how OCaR1 is able to regulate Ca²⁺ homeostasis in primary haemostasis by controlling platelet aggregation.

Analysis of washed platelets stimulated with 18nM γ-thrombin, which activates the PAR4 receptor, revealed a reduction in aggregation of 92.9% and 93.8% in platelets of male and female of OCaR1-/- mice, respectively. Although global Ca²⁺ release and Ca²⁺ entry remained the same between thrombocytes of WT and OCaR1-/- mice, we observed a significant reduction in exocytosis after activation with γ-thrombin in OCaR1-/- cells. Specifically, secretion of delta granules, containing Ca²⁺, ATP, ADP and serotonin, was significantly impaired in platelets of OCaR1-/- mice. Released ADP serves as an autocrine and paracrine signalling by activating the P2Y12 receptor to reduce intracellular cAMP concentration. OCaR1-/- platelets were unable to reduce cAMP after stimulation with γ-thrombin, compatible with impaired aggregation and increased bleeding times in OCaR1-/- mice.

Taken together, these results indicate that OCaR1 critically contributes to primary haemostasis by regulating exocytosis of dense granules. No effect on global Ca²⁺ was observed but local Ca²⁺ release from acidic Ca²⁺ stores is currently being analysed using TPC2-GCaMP6 mice.