Protein kinase A mediates inhibition of the thrombin-induced platelet shape change by nitric oxide.
The thrombin-induced platelet shape change was blocked by nitric oxide (NO), as revealed by scanning electron microscopy, light transmission, and resistive-particle volume determination. The inhibitory effect of NO was accompanied by an increase in levels of both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). However, the inhibition of the shape change was only mimicked by cAMP analogs (Sp-5,6-DClcBIMPS, 8-AHA-cAMP, and 8-CPT-cAMP) and not by cGMP analogs (8-Br-PET-cGMP, 8-Br-cGMP, and 8-pCPT-cGMP). The effect of NO on the thrombin-induced shape change was prevented by the protein kinase A (PKA) antagonists Rp-8-Br-cAMPS and Rp-cAMPS. The protein kinase G (PKG) antagonist Rp-8-CPT-cGMPS strongly inhibited PKG-mediated 46-kDa VASP Ser239 phosphorylation, but did not inhibit the thrombin-induced shape change or the PKA-mediated VASP Ser157 phosphorylation. Whereas an inhibitor of cyclic nucleotide phosphodiesterase (PDE) 3A (milrinone) mimicked the effect of NO, inhibitors of PDE2 (erythro-9-(2-hydroxy-3-nonyl)adenine) and PDE5 (dipyridamole) were poorly effective. We concluded that (1) NO was a potent and reversible inhibitor of the platelet shape change, (2) the shape change was reversible, (3) the inhibitory effect of NO was mediated through activation of PKA, (4) the onset of the NO effect coincided with VASP Ser157 phosphorylation, and (5) removal of NO and platelet shape change coincided with VASP Ser157 dephosphorylation. These findings are compatible with elevation of cGMP by NO in a compartment close to PDE3A, PKA, and VASP, leading to a local increase of cAMP able to block thrombin-induced shape change.
Jensen
Baard Olav
BO
Section of Biochemistry and Molecular Biology, Department of Biomedicine, University of Bergen, Jonas Liesvei 91, N-5009 Bergen, Norway.
Selheim
Frode
F
Døskeland
Stein Ove
SO
Gear
Adrian R L
AR
Holmsen
Holm
H
D001792:Blood Platelets / Q000166:cytology* / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000378:metabolism; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000032:analysis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D006152:Cyclic GMP / Q000032:analysis; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008840:Microfilament Proteins; D009569:Nitric Oxide / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D013917:Thrombin / Q000494:pharmacology*; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D009569:Nitric Oxide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D013917:Thrombin; D019204:GTP-Binding Proteins; D006152:Cyclic GMP