A role for TRPV1 in agonist-evoked activation of human platelets

Background: Platelets play a role in a number of inflammatory conditions including atherosclerosis; however, the mechanisms of platelet activation under these conditions are unclear. Objectives: To investigate the presence of the vanilloid receptor, TRPV1, which is stimulated by noxious stimuli and by inflammatory mediators, in human platelets. Methods: Platelets loaded with fura-2 or sodium-binding benzofuran isophalate acetoxymethyl ester (SBFI) were used to monitor cytosolic calcium or sodium concentrations. 5-HT secretion was determined by fluorescence assay after conjugation with o-phthaldialdehyde. ATP secretion was determined using luciferin-luciferase. Results: TRPV1 was identified by Western blotting using a specific anti-hTRPV1 antibody. The TRPV1 agonist, capsaicin, evoked both Ca2+ influx and Ca2+ release from intracellular stores, responses that were blocked in a dose-dependent manner by the TRPV1 antagonists, 5'-Iodo-resiniferatoxin (5'-Iodo-RTX) and AMG 9810. Capsaicin also increased platelet cytosolic [Na+]. Capsaicin-evoked Ca2+ release was abolished in the absence of extracellular Na+ or by the 5-HT2A receptor antagonist, ketanserin. Capsaicin evoked 5-HT release from platelets, a response abolished in the absence of extracellular Na+ or by 5'-Iodo-RTX. Thus capsaicin-evoked Ca2+ release appeared to be mediated by Na+-dependent 5-HT release. TRPV1-dependent 5-HT release also contributed to ADP- and thrombin-evoked Ca2+ entry and release. 5'-Iodo-RTX reduced ADP- and thrombin-evoked Ca2+ signals, effects not additive with those of ketanserin, and 5'-Iodo-RTX inhibited agonist-evoked 5-HT and ATP release. Conclusion: These results indicate that TRPV1 is present and functionally important in human platelets. The presence of this receptor may provide a link between inflammatory mediators and platelet activation in conditions such as atherosclerosis.