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cAMP- and cGMP-elevating agents inhibit GPIbα-mediated aggregation but not GPIbα-stimulated Syk activation in human platelets.
Cell Commun Signal 2019; 17(1):122CC

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.

Authors+Show Affiliations

Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz of the Johannes Gutenberg University Mainz, Mainz, Germany.Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz of the Johannes Gutenberg University Mainz, Mainz, Germany.Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz of the Johannes Gutenberg University Mainz, Mainz, Germany. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia.Core Facility for Mass Spectrometry, Institute for Immunology, University Medical Center Mainz, Mainz, Germany.DKD HELIOS Klinik Wiesbaden GmbH, Wiesbaden, Germany.Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz of the Johannes Gutenberg University Mainz, Mainz, Germany.Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz of the Johannes Gutenberg University Mainz, Mainz, Germany. kerstin.jurk@unimedizin-mainz.de.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31519182

Citation

Makhoul, Stephanie, et al. "CAMP- and cGMP-elevating Agents Inhibit GPIbα-mediated Aggregation but Not GPIbα-stimulated Syk Activation in Human Platelets." Cell Communication and Signaling : CCS, vol. 17, no. 1, 2019, p. 122.
Makhoul S, Trabold K, Gambaryan S, et al. CAMP- and cGMP-elevating agents inhibit GPIbα-mediated aggregation but not GPIbα-stimulated Syk activation in human platelets. Cell Commun Signal. 2019;17(1):122.
Makhoul, S., Trabold, K., Gambaryan, S., Tenzer, S., Pillitteri, D., Walter, U., & Jurk, K. (2019). CAMP- and cGMP-elevating agents inhibit GPIbα-mediated aggregation but not GPIbα-stimulated Syk activation in human platelets. Cell Communication and Signaling : CCS, 17(1), p. 122. doi:10.1186/s12964-019-0428-1.
Makhoul S, et al. CAMP- and cGMP-elevating Agents Inhibit GPIbα-mediated Aggregation but Not GPIbα-stimulated Syk Activation in Human Platelets. Cell Commun Signal. 2019 Sep 13;17(1):122. PubMed PMID: 31519182.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - cAMP- and cGMP-elevating agents inhibit GPIbα-mediated aggregation but not GPIbα-stimulated Syk activation in human platelets. AU - Makhoul,Stephanie, AU - Trabold,Katharina, AU - Gambaryan,Stepan, AU - Tenzer,Stefan, AU - Pillitteri,Daniele, AU - Walter,Ulrich, AU - Jurk,Kerstin, Y1 - 2019/09/13/ PY - 2019/05/02/received PY - 2019/08/29/accepted PY - 2019/9/15/entrez PY - 2019/9/15/pubmed PY - 2019/9/15/medline KW - Glycoprotein receptor GPIb-IX KW - Platelet activation KW - Syk kinase KW - cAMP-dependent protein kinase KW - cGMP-dependent protein kinase SP - 122 EP - 122 JF - Cell communication and signaling : CCS JO - Cell Commun. Signal VL - 17 IS - 1 N2 - 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. SN - 1478-811X UR - https://www.unboundmedicine.com/medline/citation/31519182/cAMP-_and_cGMP-elevating_agents_inhibit_GPIbα-mediated_aggregation_but_not_GPIbα-stimulated_Syk_activation_in_human_platelets L2 - https://biosignaling.biomedcentral.com/articles/10.1186/s12964-019-0428-1 DB - PRIME DP - Unbound Medicine ER -