PRN1008 inhibits CLEC-2– and GPVI–mediated platelet activation and CLEC-2–induced thrombus formation in whole blood. Citrated healthy donor whole blood was incubated with vehicle (0.02% DMSO) or indicated concentration (0.2, 0.5, 2, or 5 μM) of Btk inhibitors PRN1008 and PRN473 for 1 hour. Platelet activation, indicated by activated integrin αIIbβ3 (PAC-1) and P-selectin surface expression, was then assessed by flow cytometry in response to stimulation with snake venom toxin rhodocytin (100 and 300 nM) (A) or CRP (3 and 10 μg/mL) (B) or thrombin receptor activating peptide (TRAP; 30 μM and 100 uM) (C). Mean ± SEM (n = 4-8). (D) Citrated healthy donor whole blood was incubated with vehicle (0.02% DMSO) or indicated concentration (0.5 or 5 μM) of Btk inhibitors PRN1008 or PRN473 for 1 hour. Blood was then labeled with DiOC6 dye (10 minutes) and perfused over recombinant podoplanin-coated (10 μg/mL) channels at 150 per second for 8 minutes. Representative images (i); quantification of platelet surface area coverage (ii, iv); and mean aggregate size in images captured every 10 seconds (iii, v) are shown. Mean ± SEM (n = 3-4 per condition). Scale bar, 100 μm. Statistical analysis by 2-way analysis of variance (ANOVA) with Tukey correction for multiple comparisons. ∗P < .05; comparison with vehicle indicated by color.
