PRN1008 blocks GPVI-mediated aggregation and PLCɣ2 phosphorylation in XLA platelets at low agonist concentrations of agonist. (A-D) Healthy donor and XLA washed platelets (2 × 108/mL) were incubated with vehicle (0.02% DMSO) or indicated concentration (0.5 and 5 μM) of PRN1008 for 1 hour before platelet aggregation to snake venom toxin rhodocytin 300 nM (A), collagen 3 (B) and 10 μg/mL (C), and CRP (3 μg/mL) (D) was measured by lumi-aggregometry. Representative traces (i) and quantification (ii); n = 3 for healthy donor; n = 2 for XLA. (E-F) Healthy donor and XLA washed platelets (4 × 108/mL) were incubated with vehicle (0.02% DMSO) or indicated concentration (0.5 or 5 μM) of PRN1008 for 1 hour, then stimulated with snake venom toxin rhodocytin 300 nM (E) or CRP 10 μg/mL (F) for 180 seconds in the presence of eptifibatide (9 μM) and lysed with reducing sample buffer. Whole cell lysates were then separated by SDS-PAGE and western blotted for tyrosine phosphorylation of indicated proteins. Total LAT was used as a loading control. Representative western blots (n = 1). OD, optical density; Veh, vehicle.
