GPIb-V-IX- and GPVI-specific intracellular signaling and their regulation by PKA/PKG-dependent inhibitory pathways in washed human platelets
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Abstract
The von Willebrand factor and collagen are the main physiological ligands of two platelet receptors, GPIb-IX-V and GPVI, respectively, which play an essential role in platelet adhesion and thrombus formation. However, these agonists also bind to the integrins αIIbβ3 and α2β1, respectively, which prevents the analysis of specific GPIbα and GPVI signaling. C type lectin snake toxins are widely used as specific platelet receptor ligands. Echicetin is a selective GPIbα agonist when coated on polystyrene beads (EB), whereas convulxin (cvx) induces GPVI-specific signaling. Despite having this important role, intracellular GPIbα and GPVI signaling and their regulation by other pathways are not well defined. This limitation was addressed here using EB and cvx, in order to study the effects of GPIbα and GPVI activation on platelet aggregation, Syk tyrosine kinase, Syk substrates and the crosstalk with inhibitory pathways.
Echicetin purified from snake Echis carinatus sochureki venom was validated by mass spectrometry. Washed human platelets were stimulated with EB or cvx, in the presence or absence of echicetin monomers (EM), Src family kinase (SFK) inhibitors, Syk inhibitors and the cAMP-, cGMP-elevating agents, iloprost and riociguat respectively. Platelet aggregation was analyzed by light transmission aggregometry, protein phosphorylation by immunoblotting. Intracellular messengers inositol monophosphate (IP1) and Ca2+i were measured by ELISA and Fluo-3 AM/FACS, respectively.
GPIbα and GPVI-specific agonists, EB and convulxin, induced full platelet aggregation and strong phosphorylation of tyrosine and serine/threonine protein kinases and substrates such as Syk, PLCγ2 (a direct Syk substrate) and Akt (a Syk downstream effector). These activation mechanisms were SFK- and Syk-dependent, integrin αIIbβ3 independent and differentially required the secondary mediators ADP, TxA2. The activation of cAMP or cGMP pathways by iloprost or riociguat, respectively, strongly inhibited platelet aggregation and Akt phosphorylation but not EB/cvx-induced tyrosine phosphorylation of Syk and PLCγ2, which was often enhanced. For the first time in platelets, an important human model system for Syk regulation, a strong, stoichiometric but transient stimulation of Syk S297 phosphorylation by EB/GPIbα and cvx/GPVI was observed, which was abolished by PKC inhibition and by the PKA/PKG system. Both interventions caused Syk tyrosine hyperphosphorylation and increased activation. The Syk S297 phosphorylation site is located within the interdomain B, which is a hot spot for Syk regulation by multiple modifications.
The major inhibitory pathways PKA/PKG strongly inhibited EB/cvx-induced platelet aggregation and Akt phosphorylation but, in contrast, enhanced Syk and LAT/PLCγ2 tyrosine phosphorylation (hyperphosphorylation). These data suggest that Syk and Syk effector systems are differentially regulated by protein kinases PKA, PKG, and PKC. This is also supported by the data that EB/cvx-induced Ca2+-release was Syk-dependent, but only partially inhibited by PKA/PKG pathways.
Overall, the results of this project demonstrate EB and EM as specific agonists and antagonists, respectively, of GPIbα-mediated Syk activation modulating platelet aggregation. This is independent of the integrin alphaIIbbeta3 and GPVI activation. 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. GPIbα and GPVI affect distinct signaling pathways, which are similar but not identical. These data establish an important intracellular regulatory network induced by GPIbα and GPVI with Syk as central element. This essential kinase is controlled by multiple phosphorylation sites, modifications and binding proteins and has multiple substrates as effector system, which are not fully understood yet.