OCaR1 regulates dense granules secretion to evoke platelet aggregation

Roger Ottenheijm (Heidelberg)1, L. Yang (Heidelberg)1, J. E. Camacho Londoño (Heidelberg)1, M. Freichel (Heidelberg)1

1Universitätsklinikum Heidelberg Pharmakologisches Institut Heidelberg, Deutschland


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.

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