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

Pengyu Zhang; Saskia von Ungern-Sternberg; Luisa Hastenplug; Fiorella A. Solari; Albert Sickmann; Marijke J. E. Kuijpers; Johan W. M. Heemskerk; Ulrich Walter; Kerstin Jurk

doi:10.1055/a-2311-0117

Thrombosis and Haemostasis, Table of Contents

CC BY-NC-ND 4.0 · Thromb Haemost 2025; 125(05): 470-483
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

¹Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany

²Leibniz Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany

³Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands

,

Saskia von Ungern-Sternberg

¹Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany

,

Luisa Hastenplug

¹Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany

,

Fiorella A. Solari

²Leibniz Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany

,

Albert Sickmann

²Leibniz Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany

⁴Medizinische Fakultät, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany

⁵Department of Chemistry, College of Physical Sciences, University of Aberdeen, Aberdeen, United Kingdom

,

Marijke J. E. Kuijpers

³Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands

,

Johan W. M. Heemskerk

³Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands

⁶Synapse Research Institute Maastricht, Koningin Emmaplein, Maastricht, The Netherlands

,

Ulrich Walter

¹Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany

,

Kerstin Jurk

¹Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany

› Author Affiliations

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

Full Text

References

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Supplementary Material

Supplementary Material