High throughput platelet calcium mobilization identifies novel indole derived PAR4 antagonists.

<p>A. To determine the Z-factor, and thus the usefulness of the assay as a screening tool, platelets were treated with either vehicle (calcium assay buffer) or 80 µM PAR4-AP in checkerboard fashion in a 384 well plate. Representative data from 1 donor is displayed as mean ± S.D. using 192 replicates of each condition in a single plate. Z-factor of the represented sample is 0.35 and a second volunteer (data not shown) was 0.31. B. Platelets were treated with 10 µM YD-3 for the indicated period of time followed by stimulation with 80 µM PAR4-AP. Data represented is an n of 2 volunteers, mean ± S.D. performed in triplicate. Means are significantly different where indicated. Unpaired one-tailed t-test: time points versus DMSO treated control ***p<0.0001. Unpaired one-tailed t-test: time points versus 12 minute time point ^#p<0.05. C. Representative tracings of platelets treated with DMSO or 10 µM YD-3 for 6 minutes followed by stimulation with 80 µM PAR4-AP. D. Compounds were screened by treating platelets with 10 µM compound or DMSO control followed by challenge with 80 µM PAR4-AP. Data represented as percent 80 µM PAR4 response n of 2 volunteers mean±S.D. Compounds that displayed greater than 50% inhibitory activity (below dashed line) were subject to further investigation.