PB0406 - Differential regulation of GPVI-stimulated Y/S phosphorylation of Syk and Btk by both PKC and PKA in human platelets

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Pengyu Zhang, PhD

PhD student
1Department of Protein Dynamics, Leibniz Institute for Analytical Sciences - ISAS-e.V., Dortmund, Germany; 2 Center for Thrombosis and Haemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Germany; 3Department of Biochemistry, CARIM, Maastricht University, Maastricht, the Netherlands
Dortmund, Nordrhein-Westfalen, Germany

Co-Author(s)

FS

Fiorella A. Solari

Research Associate
Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V. Dortmund, Germany
Dortmund, Nordrhein-Westfalen, Germany
UW

Ulrich Walter

Senior Professor
1 Center for Thrombosis and Haemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Germany;
Mainz, Rheinland-Pfalz, Germany
JW

Johan W. M. Heemskerk

Emeritus Professor
Synapse research insitute
MAASTRICHT, Limburg, Netherlands
AS

Albert Sickmann

Professor
Department of Protein Dynamics, Leibniz Institute for Analytical Sciences - ISAS-e.V., Dortmund, Germany; Medizinische Fakultät, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Germany; Department of Chemistry, College of Physical Sciences, University of Aberdeen, United Kingdom.
Dortmund, Nordrhein-Westfalen, Germany
Kerstin Jurk, PhD

Professor
Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University, Mainz, Germany
Mainz, Rheinland-Pfalz, Germany

Background: Platelet glycoproteins (GP), including GPIbα, GPVI, αIIbβ3 integrin and Clec-2, activate Src family kinases resulting in the membrane-recruitment of both spleen tyrosine kinase (Syk) and Bruton tyrosine kinase (Btk), followed by their Y- phosphorylation (Y352, Y525/526 and Y551, respectively) and activation. Activated Syk and Btk stimulate phospholipase Cγ2, elevate IP3, Ca2+, 1,2-diacylglycerol and increase protein kinase C (PKC) activity. Syk and Btk are crucial for platelet activation, but their regulation by other major signaling pathways is not well understood.

Aims: Here we investigated the regulation of Syk/Btk Y/S phosphorylation by PKC and protein kinase A (PKA) in GPVI-stimulated platelets.

Methods: Aggregation of washed human platelets was stimulated by convulxin and monitored by light transmission aggregometry. Platelet PKC was activated with phorbol dibutyrate (PDBu) or inhibited with GF109203X (GFX), while PKA was activated with iloprost. Phospho-antibodies against Syk/Btk phospho-sites were used to measure their time-dependent (10-300s) regulation in convulxin-stimulated platelets by western blotting.

Results: Convulxin induced strong platelet aggregation and a rapid, transient up-regulation of Syk at pY525/526, pY352, pS297 and of Btk at pY551, pY223, pS180. PDBu alone induced platelet aggregation, major increases of Syk pS297 and Btk pS180, but only minor changes in Btk pY551. GFX partially inhibited convulxin-induced aggregation, abolished convulxin-stimulated pS-sites, but increased convulxin-induced Syk/Btk pY-sites. Effects of PKA activation resembled those of PKC inhibition. Currently, additional proteins of the GPVI/Syk/Btk signalosome are analyzed.

Conclusion(s): In human platelets, GPVI-stimulated PKC isoforms mediate Syk S297 and Btk S180 phosphorylation and negatively affect their Y-phosphorylation. PKA stimulation suppresses the PKC-mediated phosphorylation events. This implies that the GPVI/Syk/Btk signalosome is regulated by serine/threonine protein kinases, thus strongly modulating the Syk/Btk activation signal in human platelets.
This study was approved by our local Ethics committee and followed the Declaration of Helsinki. P.Z. is supported by EU TICARDIO No. 813409.