Summary: Platelet activation is an irreversible process resulting in platelet apoptosis and necrosis, and circulating platelets contain many components of the apoptotic machinery. Cyclic guanosine monophosphate (cGMP) generated by nitric oxide (NO) activated soluble guanylyl cyclase (sGC) plays a crucial role in preventing platelet activation. However, in addition to activation of sGC, cGMP-independent NO effects in platelets have been described. To differentiate between cGMP-dependent and -independent NO effects on platelet apoptosis and reactive oxygen species (ROS) production, we generated platelet-specific sGC-deficient mice (PS-GCKO). Platelet apoptosis was induced by a combination of thrombin/convulxin (Thr/Cvx) and assessed by phosphatidylserine (PS) surface exposure, and loss of the mitochondrial membrane potential. NO-induced inhibition of PS externalisation was mediated only by cGMP-dependent mechanisms. Inhibition of the mitochondrial membrane potential decrease at low NO concentration was also cGMP-dependent but became cGMP-independent at high NO concentrations. In contrast, inhibition of ROS formation at any NO concentration was mediated by cGMP-independent mechanisms, very likely due to direct radical scavenging. NO inhibits platelet apoptosis by cGMP-dependent mechanisms and ROS production by cGMP-independent mechanisms. The PS-GCKO mouse model is an important tool for the differentiation of cGMP-dependent and -independent NO effects on platelets.

The cell cycle is controlled by microtubule-associated serine/threonine kinase-like (MASTL), which phosphorylates the cAMP-regulated phosphoproteins 19 (ARPP19) at S62 and 19e/α-endosulfine (ENSA) at S67and converts them into protein phosphatase 2A (PP2A) inhibitors. Based on initial proteomic data, we hypothesized that the MASTL-ENSA/ARPP19-PP2A pathway, unknown until now in platelets, is regulated and functional in these anucleate cells. We detected ENSA, ARPP19 and various PP2A subunits (including seven different PP2A B-subunits) in proteomic studies of human platelets. ENSA-S109/ARPP19–S104 were efficiently phosphorylated in platelets treated with cAMP- (iloprost) and cGMP-elevating (NO donors/riociguat) agents. ENSA-S67/ARPP19-S62 phosphorylations increased following PP2A inhibition by okadaic acid (OA) in intact and lysed platelets indicating the presence of MASTL or a related protein kinase in human platelets. These data were validated with recombinant ENSA/ARPP19 and phospho-mutants using recombinant MASTL, protein kinase A and G. Both ARPP19 phosphorylation sites S62/S104 were dephosphorylated by platelet PP2A, but only S62-phosphorylated ARPP19 acted as PP2A inhibitor. Low-dose OA treatment of platelets caused PP2A inhibition, diminished thrombin-stimulated platelet aggregation and increased phosphorylation of distinct sites of VASP, Akt, p38 and ERK1/2 MAP kinases. In summary, our data establish the entire MASTL(like)–ENSA/ARPP19–PP2A pathway in human platelets and important interactions with the PKA, MAPK and PI3K/Akt systems.

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 snakeEchis carinatusvenom 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⁺_iwere measured by ELISA and Fluo-3 AM/FACS, respectively.Results: EB-induced platelet aggregation was dependent on integrin α_IIbβ₃and 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 α_IIbβ₃-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