PB1177 - Developing 384-well plate platelet aggregometry

Background: Many novel drugs are approved by the FDA each year but repurposing of approved drugs in new settings (“drug repositioning”) is a growing trend. Repositioned drugs reach the market sooner and cheaper as safety and toxicity have already been explored. With anti-platelet therapies currently centered around aspirin and P2Y12 antagonists, significant bleeding side-effects remain a concern. Therefore, there is opportunity to explore other efficacious drugs with reduced side-effects but high-throughput methods to assess platelet reactivity are lacking.

Aims: In this proof-of-concept study, we aimed to test the utility of 384-well plate aggregometry.

Methods: Blood was taken into sodium citrate (0.32%) and platelet rich plasma (PRP; 200xg, 10min) and platelet poor plasma (PPP; 1500xg, 15min) were obtained by centrifugation. PRP was incubated with aspirin (30µM) or vehicle for 30min. 36µl PRP was mixed with 4µl of ADP (10µM final) or TRAP-6 amide (1µM final) in 8 replicates. Plates were shaken (1200rpm, 5min; BioShake IQ) and absorbance was read at 595nm in a single or 5 different well locations (Spark®, Tecan). Aggregation was calculated with respect to PRP (0%) and PPP (100%). Statistical significance (P < 0.05) was determined using a student’s t-test.

Results: We were able to detect platelet aggregation in both ADP and TRAP-6 amide-stimulated PRP. Treatment with aspirin had no effect on ADP (30±16% vs 26±8%). However, we observed a significant (p=0.01) reduction in TRAP-6-amide induced aggregation (53±2% vs 45±7%). We also found an attenuation in observed aggregation after 5 readings within a well in both agonists (ADP:23±15%, TRAP:45±2%; P< 0.01).

Conclusion(s): We have confirmed that 384-well plate aggregometry is achievable. Further experiments must be conducted to determine whether the discrepancy in read location within the wells is due to the size of aggregates. We must also test a wider range of agonists and antiplatelet drugs before moving to higher throughput drug screening.