Cross-talk of cGMP- and cAMP-signaling pathways in human platelets

Endothelium-derived prostacyclin and nitric oxide (NO) inhibit platelets by increasing the cytosolic concentrations of cAMP and cGMP, respectively. The increase of these second messengers leads to the activation of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) and the subsequent phosphorylation of specific target proteins in platelets. One such protein is the vasodilator-stimulated phosphoprotein (VASP). VASP interacts with actin filaments and actin-binding proteins (zyxin, profilin) and is associated with focal adhesions in platelets. VASP is an in vitro substrate for both PKA and PKG, and within the 3 phosphorylation sites identified in VASP (Ser157, Ser239, Thr278), PKA has been reported to prefer the Ser157 site, and PKG the Ser239 site, suggesting that both enzymes might independently phosphorylate VASP in intact cells. Further support for a model of parallel and separate cAMP- and cGMP-signaling pathways in platelets came from a study of PKG-knockout mice demonstrating an absent cGMP-mediated VASP phosphorylation and platelet inhibition whereas the cAMP response was unchanged. Moreover, mice platelets lacking PKG showed an increased adhesion and aggregation during ischemia-reperfusion, indicating that an intact cGMP/PKG pathway was important to protect platelets in vivo (Massberg et al, J Exp Med. 1999;189:1255-1263).

The article by Li and colleagues (page 4423) shows surprisingly that the situation is different in human platelets. By using specific membrane-permeable activators and inhibitors of PKG and PKA, they show that VASP phosphorylation in human platelets is mediated not by PKG but by PKA. To rule out questions about the specificity of the drugs applied to intact cells, the authors elegantly used as a control system the phosphorylation of the extracellular signal-responsive kinase, which is stimulated only by PKG but not PKA in human platelets. The authors demonstrate: (1) that cGMP analogs stimulate the levels of intracellular cAMP (most likely through the well-known inhibition of the cAMP degrading phosphodiesterase III by cGMP in human platelets); (2) that cGMP analogs stimulate the phosphorylation of VASP at both sites (not only the Ser239 site but also the PKA preferred Ser157 site); and, most importantly, (3) that the cGMP-mediated VASP phosporylation is inhibited by specific inhibitors of PKA but not PKG. Moreover, they show that only PKA inhibitors, and not PKG inhibitors, reversed the cGMP– and NO-donor–mediated inhibition of platelet aggregation. It is therefore unlikely that substrates of PKG other than VASP might mediate platelet inhibition, and it seems that PKG is not involved in the NO-mediated inhibition of human platelets. Whether PKG plays a role in the stimulus-induced platelet activation as the authors suggest remains to be shown. The study demonstrates a cross-talk of cGMP- and cAMP-signaling pathways in human platelets and adds to the regulation of signaling networks in human platelets a layer of complexity that is absent in mice platelets.