The thrombin-induced platelet shape change was blocked by nitric oxide (NO), as revealed by scanning electron microscopy, light transmission, and resistiveparticle 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-PETcGMP, 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-BrcAMPS and Rp-cAMPS. The protein kinase G (PKG) antagonist Rp-8-CPTcGMPS 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.
IntroductionWhen platelets are stimulated, they rapidly change shape from discoids to spheres possessing extensive pseudopodia. [1][2][3] This shape change precedes aggregation, secretion, and activation of the coagulation cascade. 1,2,4 In healthy individuals, 65% to 90% of circulating platelets are discoidal, 2 and increased numbers of spherical platelets have been associated with vascular diseases. 5 Shape change could therefore be an important step in hemostasis and thrombosis. Activation of the platelet shape change by thrombin has been extensively studied in vitro, but little is known about mechanisms inhibiting this process. Thrombin, a potent platelet agonist that induces shape change, aggregation, and secretion through many signaling systems 6 is formed in vivo, in significant amounts at the site of a vascular injury, stimulating both platelet activation and overall thrombus growth. [7][8][9][10][11][12] Regulation of platelet activation is an important physiologic function of the vascular endothelium including release of nitric oxide (NO), 13 a potent inhibitor of platelet aggregation, secretion, adhesion, and binding of fibrinogen to integrin glycoprotein IIbIIIa (GpIIbIIIa). [14][15][16][17] Conflicting results have, however, been reported about the effect of NO on shape change. [18][19][20][...