cAMP- and cGMP-elevating agents inhibit GPIbα-mediated aggregation but not GPIbα-stimulated Syk activation in human platelets

Abstract

Background The glycoprotein (GP) Ib-IX-V complex is a unique platelet plasma membrane receptor, which is essential for platelet adhesion and thrombus formation. GPIbα, part of the GPIb-IX-V complex, has several physiological ligands such as von Willebrand factor (vWF), thrombospondin and distinct coagulation factors, which trigger platelet activation. Despite having an important role, intracellular GPIb-IX-V signaling and its regulation by other pathways are not well defined. Our aim was to establish the intracellular signaling response of selective GPIbα activation in human platelets, in particular the role of the tyrosine kinase Syk and its regulation by cAMP/PKA and cGMP/PKG pathways, respectively. We addressed this using echicetin beads (EB), which selectively bind to GPIbα and induce platelet aggregation. Methods Purified echicetin from snake Echis carinatus venom was validated by mass spectrometry. Washed human platelets were incubated with EB, in the presence or absence of echicetin monomers (EM), Src family kinase (SFK) inhibitors, Syk inhibitors and the cAMP- and cGMP-elevating agents iloprost and riociguat, respectively. Platelet aggregation was analyzed by light transmission aggregometry, protein phosphorylation by immunoblotting. Intracellular messengers inositolmonophosphate (InsP1) and Ca2+i were measured by ELISA and Fluo-3 AM/FACS, respectively. Results EB-induced platelet aggregation was dependent on integrin αIIbβ3 and secondary mediators ADP and TxA2, and was antagonized by EM. EB stimulated Syk tyrosine phosphorylation at Y352, which was SFK-dependent and Syk-independent, whereas Y525/526 phosphorylation was SFK-dependent and partially Syk-dependent. Furthermore, phosphorylation of both Syk Y352 and Y525/526 was completely integrin αIIbβ3-independent but, in the case of Y525/526, was partially ADP/TxA2-dependent. Syk activation, observed as Y352/ Y525/Y526 phosphorylation, led to the phosphorylation of direct substrates (LAT Y191, PLCγ2 Y759) and additional targets (Akt S473). PKA/PKG pathways inhibited EB-induced platelet aggregation and Akt phosphorylation but, surprisingly, enhanced Syk and LAT/PLCγ2 tyrosine phosphorylation. A similar PKA/PKG effect was confirmed with convulxin−/GPVI-stimulated platelets. EB-induced InsP1 accumulation/InsP3 production and Ca2+-release were Syk-dependent, but only partially inhibited by PKA/PKG pathways. Conclusion EB and EM are specific agonists and antagonists, respectively, of GPIbα-mediated Syk activation leading to platelet aggregation. The cAMP/PKA and cGMP/PKG pathways do not inhibit but enhance GPIbα−/GPVI-initiated, SFK-dependent Syk activation, but strongly inhibit further downstream responses including aggregation. These data establish an important intracellular regulatory network induced by GPIbα. Graphical abstract

Feedback Regulation of Syk by Protein Kinase C in Human Platelets

“…There is another line of evidence for the conclusion that PKC inhibition enhances Syk and Syk substrate tyrosine phosphorylation with the possible involvement of Syk S297 phosphorylation. We recently showed that both cAMP-and cGMP-elevating platelet inhibitors (iloprost, riociguat), enhance Syk tyrosine phosphorylation/Syk activity [28]. Now, we show that iloprost (PKA pathway) strongly inhibits cvx/EB-stimulated Syk S297 phosphorylation.…”

“…EB-/cvx-mediated Syk tyrosine phosphorylation is mediated by SFKs and Syk autophosphorylation [18,19,28]. Therefore, we evaluated the role of SFKs and Syk for Syk S297 phosphorylation.…”

“…Whole blood was collected in citrate tubes from healthy volunteers who did not take any medication at least 10 days before the experiments. Platelet washing and the isolation procedure were performed as previously described [28]. A modified washing procedure was used for the experiments with ADP stimulation.…”

“…Very recently, we established methods for the selective quantitative assessment of GPVI-and GPIbα-mediated activation and function of human platelet Syk [27,28]. We observed that cAMP-and cyclic guanosine monophosphate (cGMP)-elevating platelet inhibitors strongly inhibited GPIbα-/GPVI-mediated platelet activation but enhanced the initial Syk activation [28]. These phosphoproteomic and functional approaches suggest that there is a network of interacting protein kinases at the level of Syk in platelets [29,30].…”

“…Interestingly, ADP, which activates platelet Ca 2+ /calmodulin-dependent protein kinases such as PKC, but not iloprost, stimulated Syk S297 phosphorylation. Very recently, we established methods for the selective quantitative assessment of GPVI-and GPIbα-mediated activation and function of human platelet Syk [27,28]. We observed that cAMP-and cyclic guanosine monophosphate (cGMP)-elevating platelet inhibitors strongly inhibited GPIbα-/GPVI-mediated platelet activation but enhanced the initial Syk activation [28].…”