American Society of Hematology

Figure 1.

PRN1008 and PRN473 inhibit CLEC-2– and GPVI-mediated signaling. (A-B) Healthy donor–washed platelets (4 × 108/mL) were incubated with vehicle (0.02% DMSO) or indicated concentration (50, 200, 500, or 2000 nM) of Btk inhibitors PRN1008 and PRN473 for 1 hour, then stimulated with snake venom toxin rhodocytin 300 nM (A) or CRP 10 μg/mL (B) for 180 seconds in the presence of eptifibatide (9 μM) and lysed with reducing sample buffer. Whole cell lysates were then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotted for tyrosine phosphorylation of indicated proteins. Total LAT was used as a loading control. Representative western blots (i) and normalized densitometry quantification (ii). Mean ± standard error of the mean (SEM) of 4 identical experiments. Bas, basal; Veh, vehicle; pan-pY, panphosphotyrosine.

PRN1008 and PRN473 inhibit CLEC-2– and GPVI-mediated signaling. (A-B) Healthy donor–washed platelets (4 × 10⁸/mL) were incubated with vehicle (0.02% DMSO) or indicated concentration (50, 200, 500, or 2000 nM) of Btk inhibitors PRN1008 and PRN473 for 1 hour, then stimulated with snake venom toxin rhodocytin 300 nM (A) or CRP 10 μg/mL (B) for 180 seconds in the presence of eptifibatide (9 μM) and lysed with reducing sample buffer. Whole cell lysates were then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotted for tyrosine phosphorylation of indicated proteins. Total LAT was used as a loading control. Representative western blots (i) and normalized densitometry quantification (ii). Mean ± standard error of the mean (SEM) of 4 identical experiments. Bas, basal; Veh, vehicle; pan-pY, panphosphotyrosine.

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