Syk Activity Is Dispensable for Platelet GP1b-IX-V Signaling

Abstract

The binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein 1b-IX (GP1b-IX) leads to activation of platelets. GP1b was shown to signal via the FcRγ-ITAM (Fc Receptor γ-Immunoreceptor tyrosine-based activation motif) pathway, activating spleen tyrosine kinase (Syk) and other tyrosine kinases. However, there have been conflicting reports regarding the role of Syk in GP1b signaling. In this study, we sought to resolve these conflicting reports and clarify the role of Syk in VWF-induced platelet activation. The inhibition of Syk with the selective Syk inhibitors, OXSI-2 and PRT-060318, did not inhibit VWF-induced platelet adhesion, agglutination, aggregation, or secretion. In contrast, platelets stimulated with the Glycoprotein VI (GPVI) agonist, collagen-related peptide (CRP), failed to cause any aggregation or secretion in presence of the Syk inhibitors. Furthermore, GP1b-induced platelet signaling was unaffected in the presence of Syk inhibitors, but GPVI-induced signaling was abolished under similar conditions. Thus, we conclude that Syk kinase activity does not play any functional role downstream of GP1b-mediated platelet activation.

Keywords: GP1b receptor; platelets; spleen tyrosine kinase (Syk); von Willebrand factor (VWF).

Figure 1

Inhibition of Syk does not inhibit GP1b-mediated platelet aggregation, secretion, and signaling: Washed non-aspirin human platelets were pre-incubated for 5 min with either dimethyl sulfoxide (DMSO) as control, 2,3-dihydro-3-[(1-methyl-1H-indol-3-yl) methylene]-2-oxo-1H-indole-5-sulfonamide (OXSI-2) (1 µM), or 2-((1R,2S)-2-aminocyclohexylamino)-4-(m-tolylamino) pyrimidine-5-carboxamide (PRT-318) (1 µM) and stimulated with (A) ristocetin/von Willebrand factor (VWF) (250 and 6 µg/mL respectively); (B) Collagen related peptide (CRP) (10 µg/mL), for 5 min at 37 °C under stirred conditions in a lumi-aggregometer. The tracings are representative of data from three individual experiments. (The black tracings represent the platelet aggregation while the red lines represent the adenosine triphosphate (ATP) secretion). Washed non-aspirin human platelets were pre-incubated for 5 min with either DMSO (control), OXSI-2 (1 µM) or PRT-060318 (1 µM) and stimulated with (C) ristocetin/VWF (250 and 6 µg/mL respectively) for 4 min; (D) CRP (10 µg/mL), for 1 min at 37 °C under stirred conditions in a lumi-aggregometer. The reaction was stopped by using 6.6 N Perchloric acid and platelet lysates were prepared. Platelet proteins were separated by SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis), transferred on a membrane by Western-blotting, and probed for phospho Syk (Tyr352), PLCγ2 (Tyr759), Akt (Ser473), Erk1/2 (T202/Y204), and respective total proteins as lane loading controls. The Western blot shown is a representative of three independent experiments (Badolia et al.).

Figure 2

Effect of Syk inhibitors on platelet adhesion to VWF. (A) Whole human blood was pretreated with DMSO (control) or OXSI-2 (1 µM) or PRT-318 (1 µM) for 5 min and perfused over immobilized VWF in a flow chamber at an arterial shear rate of 1000 s⁻¹. After washing, stably adherent platelets were observed at 20× magnification and photographed using a confocal microscope; (B) Quantification of the percentage of area covered by using Image J software (Image Processing and Analysis in Java from National Institute of Health, https://imagej.nih.gov/ij/). Shown in the figure are representative pictures from three different experiments.