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

Most agonists stimulate platelet Ca 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 (P2Y, P2Y), and thromboxane A (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 Ca signaling in: dependency of tyrosine phosphorylation; oscillatory versus continued Ca rises by mobilization from the endoplasmic reticulum; and smaller or larger role of extracellular Ca entry via STIM1/ORAI1. Co-stimulation of both types of receptors, especially by thrombin (PAR1/4) and collagen (GPVI), leads to a highly enforced Ca rise, involving mitochondrial Ca release, which activates the ion and phospholipid channel, anoctamin-6. This highly Ca-dependent process causes swelling, ballooning, and phosphatidylserine expression, establishing a unique platelet population swinging between vital and necrotic (procoagulant 'zombie' platelets). Additionally, the high Ca status of procoagulant platelets induces a set of additional events: Ca dependent cleavage of signaling proteins and receptors via calpain and ADAM isoforms; microvesiculation; enhanced coagulation factor binding; and fibrin-coat formation involving transglutaminases. Given the additive roles of GPCR and ILR in Ca signal generation, high-throughput screening of biomolecules or small molecules based on Ca flux measurements provides a promising way to find new inhibitors interfering with prolonged high Ca, phosphatidylserine expression, and hence platelet procoagulant activity.