Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression.
In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.
Proteomic Unit at University of Bergen (PROBE), University of Bergen, Bergen, Norway; Department of Biomedicine, University of Bergen, Bergen, Norway.
Gyrid
G
Nygaard
Department of Biomedicine, University of Bergen, Bergen, Norway.
Lars
L
Herfindal
Department of Biomedicine, University of Bergen, Bergen, Norway.
Reidun
R
Kopperud
Department of Biomedicine, University of Bergen, Bergen, Norway; Molecular Biology Institute of Barcelona (IBMB, CSIC), Barcelona, Spain.
Anna M
AM
Aragay
Department of Biomedicine, University of Bergen, Bergen, Norway.
Holm
H
Holmsen
Department of Biomedicine, University of Bergen, Bergen, Norway.
Stein Ove
SO
Døskeland
Department of Biomedicine, University of Bergen, Bergen, Norway.
Rune
R
Kleppe
Proteomic Unit at University of Bergen (PROBE), University of Bergen, Bergen, Norway; Department of Biomedicine, University of Bergen, Bergen, Norway. Electronic address: Frode.Selheim@biomed.uib.no.
Frode
F
Selheim
D055252:Cell-Derived Microparticles / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004004:Dichlororibofuranosylbenzimidazole / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D019007:P-Selectin / Q000096:biosynthesis*; D010974:Platelet Aggregation / Q000187:drug effects*; D018179:Receptors, Thrombin / Q000502:physiology*; D013873:Thionucleotides / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology*; D013997:Time Factors
C104224:8-bromo-beta-phenylethenoguanosine 3',5'-cyclic monophosphate; D019007:P-Selectin; D018179:Receptors, Thrombin; D013873:Thionucleotides; C071644:5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-monophosphorothioate; D004004:Dichlororibofuranosylbenzimidazole; C055656:8-((4-chlorophenyl)thio)cyclic-3',5'-GMP; D000242:Cyclic AMP; D017869:Cyclic GMP-Dependent Protein Kinases; D013917:Thrombin; D006152:Cyclic GMP
PKG; Platelets; VASP; cAMP; cGMP; cGMP-analogues