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CC BY-NC-ND 4.0 · Thromb Haemost
DOI: 10.1055/a-2311-0117
Cellular Haemostasis and Platelets

Multi-phased Kinetics and Interaction of Protein Kinase Signaling in Glycoprotein VI-Induced Platelet αIIbβ3 Integrin Activation and Degranulation

Pengyu Zhang
1   Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
2   Leibniz Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany
3   Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands
,
Saskia von Ungern-Sternberg
1   Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
,
Luisa Hastenplug
1   Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
,
Fiorella A. Solari
2   Leibniz Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany
,
Albert Sickmann
2   Leibniz Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany
4   Medizinische Fakultät, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany
5   Department of Chemistry, College of Physical Sciences, University of Aberdeen, Aberdeen, United Kingdom
,
Marijke J. E. Kuijpers 
3   Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands
,
Johan W. M. Heemskerk 
3   Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands
6   Synapse Research Institute Maastricht, Koningin Emmaplein, Maastricht, The Netherlands
,
Ulrich Walter
1   Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
,
Kerstin Jurk 
1   Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
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Abstract

Background Platelet glycoprotein VI (GPVI) stimulation activates the tyrosine kinases Syk and Btk, and the effector proteins phospholipase Cγ 2 (PLCγ2) and protein kinase C (PKC). Here, the activation sequence, crosstalk, and downstream effects of this Syk-Btk-PKC signalosome in human platelets were analyzed.

Methods and Results Using immunoblotting, we quantified 14 regulated phospho-sites in platelets stimulated by convulxin with and without inhibition of Syk, Btk, or PKC. Convulxin induced fast, reversible tyrosine phosphorylation (pY) of Syk, Btk, LAT, and PLCγ2, followed by reversible serine/threonine phosphorylation (pS/T) of Syk, Btk, and downstream kinases MEK1/2, Erk1/2, p38, and Akt. Syk inhibition by PRT-060318 abolished all phosphorylations, except Syk pY352. Btk inhibition by acalabrutinib strongly decreased Btk pY223/pS180, Syk pS297, PLCγ2 pY759/Y1217, MEK1/2 pS217/221, Erk1/2 pT202/Y204, p38 pT180/Y182, and Akt pT308/S473. PKC inhibition by GF109203X abolished most pS/T phosphorylations except p38 pT180/Y182 and Akt pT308, but enhanced most Y-phosphorylations. Acalabrutinib, but not GF109203X, suppressed convulxin-induced intracellular Ca2+ mobilization, whereas all three protein kinase inhibitors abolished degranulation and αIIbβ3 integrin activation assessed by flow cytometry. Inhibition of autocrine ADP effects by AR-C669931 partly diminished convulxin-triggered degranulation.

Conclusion Kinetic analysis of GPVI-initiated multisite protein phosphorylation in human platelets demonstrates multiple phases and interactions of tyrosine and serine/threonine kinases with activation-altering feedforward and feedback loops partly involving PKC. The protein kinase inhibitor effects on multisite protein phosphorylation and functional readouts reveal that the signaling network of Syk, Btk, and PKC controls platelet granule exocytosis and αIIbβ3 integrin activation.

Keywords

collagen receptor - kinase inhibitors - Bruton's tyrosine kinase - protein kinase C - secretion

Authors' Contribution

Conceptualization: K.J. and U.W.; methodology: P.Z., S.v.U.-S., L.H.; data curation: P.Z.; formal analysis: P.Z. and U.W.; writing—original draft preparation: P.Z., U.W., and K.J.; writing—review and editing: P.Z., U.W., K.J., F.A.S., A.S., J.W.M.H., and M.J.E.K.; funding and supervision: A.S. and K.J.


Supplementary Material



Publication History

Received: 31 October 2023

Accepted: 16 April 2024

Accepted Manuscript online:
23 April 2024

Article published online:
15 May 2024

© 2024. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/)

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