Actin polymerisation regulates thrombin-evoked Ca2+ signalling after activation of PAR-4 but not PAR-1 in human platelets

Abstract

The role of actin polymerisation in regulating thrombin-evoked Ca2+ signalling was investigated in human platelets. We have previously reported that cytochalasin D (Cyt D) inhibits thapsigargin-evoked store-operated Ca2+ entry (SOCE), which is believed to contribute a major component of thrombin-evoked Ca2+ entry in platelets. In contrast, Cyt D increased thrombin-evoked Ca2+ entry to 147.5 ± 9.2% and Sr2+ entry to 134.2 ± 6.4% of control. Similar results were obtained with latrunculin A. This potentiation was not affected if protein kinase C was inhibited using Ro-31-8220, suggesting that it did not involve PKC-dependent non-capacitative Ca2+ entry. Ca2+ entry evoked by the PAR-4 agonist, AYPGKF, was increased to 133.7 ± 12.8% of control by Cyt D, whereas Ca2+ signalling evoked by the PAR-1 agonist, SFLLRN, was unaffected. The PAR-4 antagonist, tcY-NH2, abolished the effect of Cyt D on thrombin-evoked Ca2+ entry. Biotinylation of cell-surface proteins showed that PAR-4 was internalised after stimulation by thrombin. Cyt D reduced this internalisation. These data suggest that Cyt D prevents the internalisation of PAR-4, which may lead to prolonged signalling from this receptor. This may mask a direct effect of Cyt D on the activation of SOCE after the activation of PAR-4.