Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets

Abstract

Bruton’s tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphorylated at S297 in glycoprotein VI (GPVI)-stimulated human platelets, which limits Syk activation. Here, we tested the hypothesis that protein kinases C (PKC) and A (PKA) and protein phosphatase 2A (PP2A) jointly regulate GPVI-induced Btk activation in platelets. The GPVI agonist convulxin caused rapid, transient Btk phosphorylation at S180 (pS180↑), Y223 and Y551, while direct PKC activation strongly increased Btk pS180 and pY551. This increase in Btk pY551 was also Src family kinase (SFK)-dependent, but surprisingly Syk-independent, pointing to an alternative mechanism of Btk phosphorylation and activation. PKC inhibition abolished convulxin-stimulated Btk pS180 and Syk pS297, but markedly increased the tyrosine phosphorylation of Syk, Btk and effector phospholipase Cγ2 (PLCγ2). PKA activation increased convulxin-induced Btk activation at Y551 but strongly suppressed Btk pS180 and Syk pS297. PP2A inhibition by okadaic acid only increased Syk pS297. Both platelet aggregation and PLCγ2 phosphorylation with convulxin stimulation were Btk-dependent, as shown by the selective Btk inhibitor acalabrutinib. Together, these results revealed in GPVI-stimulated platelets a transient Syk, Btk and PLCγ2 phosphorylation at multiple sites, which are differentially regulated by PKC, PKA or PP2A. Our work thereby demonstrated the GPVI–Syk–Btk signalosome as a tightly controlled protein kinase network, in agreement with its role in atherothrombosis.

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

“…Blood collection was performed as previously described. 28 This study was approved in accordance with the Declaration of Helsinki by the local Ethics Committee of the University Medical Center Mainz (study no. 837.302.12; 25.07.12; 2018–13290_1; 27.07.2018).…”

“…For immunoblotting and flow cytometry, human platelets were washed and isolated as previously described. 28 For the measurement of cytosolic Ca 2+ rises, the protocol was slightly modified based on our previous publication. 29 Briefly, platelet-rich plasma (PRP) was prepared via centrifugation at 260 × g for 10 minutes at room temperature (RT), supplemented with 1:10 vol/vol acid citrate dextrose (ACD) (80 mM trisodium citrate, 183 mM glucose, 52 mM citric acid).…”

“…Light transmission aggregometry (LTA) was performed as previously described. 28 Briefly, using an Apact4S Plus aggregometer (DiaSys, Flacht, Germany), washed human platelets (200 µL, 3 × 10 8 /mL) were preincubated with vehicle control (0.1% DMSO), 1 µM PRT-060318, 5 µM acalabrutinib or 5 µM GF109203X for 5 minutes at 37°C and then stimulated by 50 ng/mL convulxin under stirring. Samples for immunoblotting were collected at 0, 10, 30, 60, 120, and 300 seconds by adding 100 µL of 3× Lämmli buffer (200 mM Tris/HCl, 15% (v/v) glycerol, 6% (w/v) SDS, 0.06% (w/v) bromophenol blue, 1:10 β-mercaptoethanol), and boiled for 10 minutes at 95°C with gentle shaking.…”

“…SDS-polyacrylamide electrophoresis (SDS-PAGE), immunoblotting, and phosphoprotein analysis were performed as previously described. 28 Briefly, proteins in prepared samples were separated by 8% gels and transferred to polyvinylidene fluoride membranes. After blocking membranes with 2% BSA in 1× TBS-T for 1 hour at RT, the membranes were incubated overnight at 4°C with specific antibodies with 2% BSA in 1× TBS-T.…”

“…26 27 We also compared the differential regulation of Syk and Btk by PKC, PKA, and PP2A in human platelets and noted that PP2A does not directly affect Btk pS180. 28…”