Differential involvement of thrombin receptors in Ca2+ release from two different intracellular stores in human platelets

摘要

pPhysiological agonists increase cytosolic free Casup2+/sup concentration to regulate a number of cellular processes. The platelet thrombin receptors, PAR (protease-activated receptor) 1 PAR-4 and GPIb-IX-V (glycoprotein Ib-IX-V) have been described as potential contributors of thrombin-induced platelet aggregation. Platelets present two separate Casup2+/sup stores, the DTS (dense tubular system) and acidic organelles, differentiated by the distinct sensitivity of their respective SERCAs (sarcoplasmic/endoplasmic-reticulum Casup2+/sup-ATPases) to TG (thapsigargin) and TBHQ [2,5-di-(tert-butyl)-1,4-hydroquinone]. However, the involvement of the thrombin receptors in Casup2+/sup release from each Casup2+/sup store remains unknown. Depletion of the DTS using ADP, which releases Casup2+/sup solely from the DTS, in combination with 10 nM TG, to selectively inhibit SERCA2 located on the DTS reduced Casup2+/sup release evoked by the PAR-1 agonist, SFLLRN, and the PAR-4 agonist, AYPGKF, by 80 and 50% respectively. Desensitization of PAR-1 and PAR-4 or pre-treatment with the PAR-1 and PAR-4 antagonists SCH 79797 and tcY-NHsub2/sub reduced Casup2+/sup mobilization induced by thrombin, and depletion of the DTS after desensitization or blockade of PAR-1 and PAR-4 had no significant effect on Casup2+/sup release stimulated by thrombin through the GPIb-IX-V receptor. Converse experiments showed that depletion of the acidic stores using TBHQ reduced Casup2+/sup release evoked by SFLLRN or AYPGKF, by 20 and 50% respectively, and abolished thrombin-stimulated Casup2+/sup release through the GPIb-IX-V receptor when PAR-1 and PAR-4 had been desensitized or blocked. Our results indicate that thrombin-induced activation of PAR-1 and PAR-4 evokes Casup2+/sup release from both Casup2+/sup stores, while activation of GPIb-IX-V by thrombin releases Casup2+/sup solely from the acidic compartments in human platelets./p