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, which can occur via Ca²⁺ release from Inositol trisphosphate (IP₃)- and nicotinic acid adenine dinucleotide (NAADP)-sensitive intracellular stores as well as via Ca²⁺ entry.  Ca²⁺ entry triggered by depletion of intracellular Ca²⁺ stores is known as store-operated Ca²⁺ entry (SOCE). In platelets, thromboxane A2 stimulation evokes IP₃ formation and subsequently Ca²⁺ release from the SERCA2b-dependent dense tubular system (DTS). NAADP-mediated Ca²⁺ release occurs from acidic Ca²⁺ stores depending on SERCA3 and is activated following stimulation of GPIb-IX-V and PAR-3/PAR-4 receptors with e.g. von Willebrand factor and γ-Thrombin, respectively. We aimed to determine the contribution of Orai1 channel proteins to both SOCE and receptor-operated Ca²⁺ entry (ROCE) and associated platelet aggregation.

Since mice with global inactivation of ORAI1 show perinatal lethality, we generated Orai1f^x/fx;Pf4^Cre-Pos (Orai1^plt-KO) mice by crossing Orai1^flox/flox mice with C57BL/6-Tg(Pf4-iCre)Q3Rsko/J (JAX) to delete the Orai1 gene selectively in megakaryocytes and platelets. Application of low concentration thapsigargin (200nM), which passively depletes the DTS, resulted in Ca²⁺ entry (SOCE), which was significantly reduced in Orai1^plt-KO. Depleting NAADP-sensitive Ca²⁺ stores by tBHQ (10µM) lead to Ca²⁺ entry (SOCE) which was also significantly reduced in Orai1^plt-KO. The amplitude of ROCE triggered by U46619 (1µM), a thromboxane A2 analogue leading to IP₃ generation, was significantly decreased in Orai1^plt-KO cells as well. In agreement with reduced Ca²⁺ entry, aggregation of washed platelets was reduced in Orai1^plt-KO upon stimulation with either thapsigargin (100nM-1µM), tBHQ (10µM) or U46619 (1µM).

Taken together, these results indicate that Orai1 channels critically contribute to Ca²⁺ entry by depletion of both IP₃-sensitive and SERCA2b-dependent as well as by depletion of NAADP-sensitive and SERCA3-dependent Ca²⁺ stores. Furthermore, Orai1 at least partially contributes to thromboxane A2-evoked ROCE.  These changes in Ca²⁺ entry critically contribute to platelet aggregation. Ongoing analysis of the role of ORAI1 channels in platelet activation following stimulation with PAR4 receptor agonists engaging acidic Ca²⁺ stores will be presented.