Actin polymerisation regulates thrombin-evoked Ca(2+) signalling after activation of PAR-4 but not PAR-1 in human platelets.
The role of actin polymerisation in regulating thrombin-evoked Ca(2+) signalling was investigated in human platelets. We have previously reported that cytochalasin D (Cyt D) inhibits thapsigargin-evoked store-operated Ca(2+) entry (SOCE), which is believed to contribute a major component of thrombin-evoked Ca(2+) entry in platelets. In contrast, Cyt D increased thrombin-evoked Ca(2+) entry to 147.5 +/- 9.2% and Sr(2+) 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 Ca(2+) entry. Ca(2+) entry evoked by the PAR-4 agonist, AYPGKF, was increased to 133.7 +/- 12.8% of control by Cyt D, whereas Ca(2+) signalling evoked by the PAR-1 agonist, SFLLRN, was unaffected. The PAR-4 antagonist, tcY-NH(2), abolished the effect of Cyt D on thrombin-evoked Ca(2+) 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.
Harper
Matthew T
MT
Department of Physiology, University of Cambridge, Downing Street, Cambridge, UK. mth29@cam.ac.uk
Sage
Stewart O
SO
D000199:Actins / Q000819:agonists* / Q000378:metabolism; D019921:Biotinylation; D001792:Blood Platelets / Q000187:drug effects* / Q000502:physiology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000502:physiology; D016257:Fura-2; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D044463:Receptor, PAR-1 / Q000378:metabolism*; D044462:Receptors, Proteinase-Activated / Q000378:metabolism; D018179:Receptors, Thrombin / Q000378:metabolism*; D013324:Strontium / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D013917:Thrombin / Q000494:pharmacology
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D007211:Indoles; D009842:Oligopeptides; D010446:Peptide Fragments; D044463:Receptor, PAR-1; D044462:Receptors, Proteinase-Activated; D018179:Receptors, Thrombin; D013844:Thiazoles; D053778:Thiazolidines; C455901:alanyl-tyrosyl-prolyl-glycyl-lysyl-phenylalanine; C082835:thrombin receptor peptide (42-47); D015638:Cytochalasin D; D011493:Protein Kinase C; D013917:Thrombin; C037067:latrunculin A; D002118:Calcium; D016257:Fura-2; C064758:Ro 31-8220; D013324:Strontium