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 (Orai1flox/flox; Pf4-Cre termed as Orai1Plt-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 Orai1Plt-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.

A rare ORAI1 missense variant associates with risk of vascular diseases in White British adults

“…Additionally, we found associations of rs3741596 with elevated circulating platelet counts (β = 1.8, P = 0.016), reduced mean platelet (thrombocyte) volume (β = −0.038, P = 0.008), lower total triglyceride levels (β = −0.035, P = 0.017) but without association with obesity or diabetes, which are important risk factors for atherosclerotic vascular diseases. ORAI1 expression was previously reported in hematopoietic cells, including platelets 32 , and a role of ORAI1-mediated SOCE in platelet activation was identified, whereby defective SOCE in Orai1 -/- or Stim1 -/- mice resulted in impaired platelet activation and thrombus formation 37–39 . It was also previously reported that patients heterozygous for the loss of function R91W or G98S ORAI1 mutation were presented with low platelet counts 40 .…”

Platelet‐derived microvesicles induce intracellular calcium mobilization in human platelets

“…A recent research showed that defective SOCE reduced the phosphorylation of Src and PKC substrates after stimulating glycoprotein GPVI or PAR4 receptor, which was followed by decreased integrin activation (Reusswig et al, 2023). Studies have linked the SOCE to the control of agonist‐induced PS exposure, platelet aggregation as well as thrombus formation (Gilio et al, 2010; Varga‐Szabo et al, 2008; Yang et al, 2022). Collectively, cytosolic calcium level regulates how platelet activation decisions are made.…”

Physiological functions of calcium signaling via Orai1 in cancer

“…The researchers overcame this limitation by crossing these mice to outbred ICR (Crl:CD1) mice. The authors reported that Orai1 (−/−) mice were small in size, with eyelid irritation and sporadic hair loss resembling the cyclical alopecia observed in mice with keratinocyte-specific deletion of the Cnb1 gene, which encodes Calcineurin subunit Yang et al also reported that mice with ubiquitous Orai1 deficiency show early lethality [ 47 ]. They generated a platelet-specific Orai1-deficient mouse line by mating Orai1 flox/flox mice with Pf4-Cre mice [ 48 ].…”