Platelet calcium signaling by G-protein coupled and ITAM-linked receptors regulating anoctamin-6 and procoagulant activity

Abstract

Most agonists stimulate platelet Ca²⁺ rises via G-protein coupled receptors (GPCRs) or ITAM-linked receptors (ILRs). Well studied are the GPCRs stimulated by the soluble agonists thrombin (PAR1, PAR4), ADP (P2Y₁, P2Y₁₂), and thromboxane A₂ (TP), signaling via phospholipase (PLC)β isoforms. The platelet ILRs glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2), and FcγRIIa are stimulated by adhesive ligands or antibody complexes and signal via tyrosine protein kinases and PLCγ isoforms. Marked differences exist between the GPCR- and ILR-induced Ca²⁺ signaling in: (i) dependency of tyrosine phosphorylation; (ii) oscillatory versus continued Ca²⁺ rises by mobilization from the endoplasmic reticulum; and (iii) smaller or larger role of extracellular Ca²⁺ entry via STIM1/ORAI1. Co-stimulation of both types of receptors, especially by thrombin (PAR1/4) and collagen (GPVI), leads to a highly enforced Ca²⁺ rise, involving mitochondrial Ca²⁺ release, which activates the ion and phospholipid channel, anoctamin-6. This highly Ca²⁺-dependent process causes swelling, ballooning, and phosphatidylserine expression, establishing a unique platelet population swinging between vital and necrotic (procoagulant 'zombie' platelets). Additionally, the high Ca²⁺ status of procoagulant platelets induces a set of additional events: (i) Ca²⁺ dependent cleavage of signaling proteins and receptors via calpain and ADAM isoforms; (ii) microvesiculation; (iii) enhanced coagulation factor binding; and (iv) fibrin-coat formation involving transglutaminases. Given the additive roles of GPCR and ILR in Ca²⁺ signal generation, high-throughput screening of biomolecules or small molecules based on Ca²⁺ flux measurements provides a promising way to find new inhibitors interfering with prolonged high Ca²⁺, phosphatidylserine expression, and hence platelet procoagulant activity.

Keywords: Calcium channels; GPCR; TMEM16F; glycoprotein VI; thrombin.