Patients undergoing neurointerventional procedures are routinely administered anti-platelet agents to manage haemostatic function and reduce incidence of periprocedural adverse outcomes. Currently, Multiplate® and ROTEM POC analysis are used to monitor both patient haemostatic capacity and anti-platelet agent efficacy. Establishment of a highly sensitive assay of both platelet activation and inhibition would complement existing methodology and provide greater mechanistic confirmation of individual patient response to anti-platelet therapy. Platelet activation, and subsequent inhibition by anti-platelet agents, was examined using flow cytometric analysis of antibodies targeting platelets (CD42b-APC), P-selectin (CD62P-PE) and GPIIbIIIa receptor (PAC-1-FITC) expression.
Assay development was undertaken prior to clinical sample testing where standardized platelet rich plasma was stimulated, with or without eptifibatide treatment (2µM), with ADP (5-40µM) or TRAP (10-40µM), prior to antibody incubation, fixation, washing and analysis with a BD SORP LSR II Fortessa flow cytometer (n=8). CD62P and PAC-1 expression were significantly increased upon ADP or TRAP stimulation, with maximal activation occurring at >5µM ADP or >10µM TRAP. Treatment with 10µM ADP or 20µM TRAP were indicated as optimal agonist concentrations to maximally activate platelets, with robust inhibition in the presence of eptifibatide.
Patient platelet P-selectin expression was significant reduced preoperatively and twenty-four hours post-operatively, compared to admission, following anti-platelet agent loading. Furthermore, both P selectin and PAC-1 expression significantly correlate with Multiple plate platelet function analysis with increasing marker expression associated with increasing Multiplate score. These data indicate that this assay, in conjunction with ROTEM and Multiplate® analysis, could further assist in determining individual patient response to anti-platelet agents.