Methods: Platelets in whole blood (WB) were stimulated with a range of different platelet agonists following which PAMFix or AGGFix was added and the platelets were analysed using flow cytometry at a later stage (within 9 days). Platelet activation was assessed as the level of P-selectin (CD62P) expression, platelet aggregation was measured by platelet counting (% fall in number of single platelets) and PLC was quantitated using flow cytometry (% CD62P +ve monocytes and neutrophils).  

Results: 1. Levels of P-selectin on platelets in WB were determined at different times after platelet stimulation and addition of PAMFix; excellent stability was demonstrated, first up to 9 days, and later up to 28 days. Similarly, measurements of PA were stable for at least 9 days after adding AGGFix to blood samples stimulated with a variety of agonists; PLC was stable for at least 3 days. 2. AGGFix was used in a study to investigate the effects on PA of an EP3 receptor antagonist, DG-041, administered to healthy volunteers with and without clopidogrel. DG-041 inhibited PA and clopidogrel added to this inhibition. Platelet P-selectin was measured in parallel and both approaches yielded very similar data. 3. Both platelet activation, aggregation and PLC formation can be studied in 96-well plate format. The wells of a flat bottom plate are coated with agonists and small aliquots WB treated with various inhibitors and antagonists are added to each well. For assessing platelet aggregation and PLC formation, the plate is then shaken at 1000 rpm for 5mins at 37oC after which AGGFix is added. PA and PLC were assessed in the same small volume sample and results were in line with the expected actions of agonists and inhibitors. For assessing platelet activation, the samples are stimulated without shaking and contain EDTA to prevent platelet aggregation. Multiple platelet inhibitors were used at a range of concentrations providing an IC50 measure for each compound.