Reduction in SOCE and Associated Aggregation in Platelets from Mice with Platelet-Specific Deletion of Orai1

Abstract

Calcium signalling in platelets through store operated Ca²⁺ entry (SOCE) or receptor-operated Ca²⁺ entry (ROCE) mechanisms is crucial for platelet activation and function. Orai1 proteins have been implicated in platelet's SOCE. In this study we evaluated the contribution of Orai1 proteins to these processes using washed platelets from adult mice from both genders with platelet-specific deletion of the Orai1 gene (Orai1^flox/flox; Pf4-Cre termed as Orai1^Plt-KO) since mice with ubiquitous Orai1 deficiency show early lethality. Platelet aggregation as well as Ca²⁺ entry and release were measured in vitro following stimulation with collagen, collagen related peptide (CRP), thromboxane A2 analogue U46619, thrombin, ADP and the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor thapsigargin, respectively. SOCE and aggregation induced by Thapsigargin up to a concentration of 0.3 µM was abrogated in Orai1-deficient platelets. Receptor-operated Ca²⁺-entry and/or platelet aggregation induced by CRP, U46619 or thrombin were partially affected by Orai1 deletion depending on the gender. In contrast, ADP-, collagen- and CRP-induced aggregation was comparable in Orai1^Plt-KO platelets and control cells over the entire concentration range. Our results reinforce the indispensability of Orai1 proteins for SOCE in murine platelets, contribute to understand its role in agonist-dependent signalling and emphasize the importance to analyse platelets from both genders.

Keywords: Orai1 proteins; calcium signalling; platelet aggregation; platelet specific knockout mice; receptor-operated Ca2+ entry (ROCE); store operated Ca2+ entry (SOCE).