Suppressed ORAI1-STIM1-dependent Ca2+ entry by protein kinase C isoforms regulating platelet procoagulant activity

Agonist-induced rises in cytosolic Ca²⁺ control most platelet responses in thrombosis and hemostasis. In human platelets, we earlier demonstrated that the ORAI1-STIM1 pathway is a major component of extracellular Ca²⁺ entry, in particular when induced via the ITAM-linked collagen receptor, glycoprotein VI (GPVI). In the present article, using functionally defective platelets from patients with a loss-of-function mutation in ORAI1 or STIM1, we show that Ca²⁺ entry induced by the endoplasmic reticulum ATPase inhibitor, thapsigargin, fully relies on this pathway. We demonstrate that both the GPVI-induced and thapsigargin-induced Ca²⁺ entry are strongly suppressed by protein kinase C (PKC) activation while leaving intracellular Ca²⁺ mobilization unchanged. Comparing the effects of a PKC inhibitory panel pointed to redundant roles of beta and theta PKC isoforms in Ca²⁺-entry suppression. In contrast, tyrosine kinases positively regulated GPVI-induced Ca²⁺ entry and mobilization. Label-free and stable isotope phosphoproteome analysis of GPVI-stimulated platelets suggested a regulatory role of bridging integrator-2 (BIN2), known as an important mediator of the ORAI1-STIM1 pathway in mouse platelets. Identified were 25 to 45 regulated phospho-sites in BIN2 and 16 to 18 in STIM1. Five of these were characterized as direct substrates of the expressed PKC isoforms alpha, beta delta, and theta. Functional platelet testing indicated that the downregulation of Ca²⁺ entry by PKC resulted in suppressed phosphatidylserine exposure and plasmatic thrombin generation. Conclusively, our results indicate that in platelets multiple PKC isoforms constrain the store-regulated Ca²⁺ entry via ORAI1-BIN2-STIM1, and hence downregulate platelet-dependent coagulation.