Kinetics of Free Platelet Decrease After ADP: Effect of Fibrinogen Binding Inhibitors

Evaluation of platelet aggregation in vitro has generally been performed by the turbidometric method. This technique is largely insensitive to early events of aggregation which, are best evaluated by following the decrease in number of free-platelets after agonist addition. In this study the kinetics of free-platelet decrease after adenosine-5-diphosphate (ADP) addition was analyzed in platelet rich plasma and in whole blood samples obtained from 29 normal donors and 5 thrombocytopenic subjects. Analysis of experimental data using a single exponential decay equation allowed us to compute the maximal velocity and the rate constant of the reaction as well as the number of un-reactive platelets. The velocity of aggregation was positively correlated with platelet number and was markedly increased by the presence of red blood cells. The process was unaffected by platelet cyclooxygenase inhibition and was also independent of thrombospondin or von Willebrand factor binding to GpIIb-IIIa. Fibrinogen receptor blockade by either anti-GpIIb-IIIa monoclonal antibody or by the RGDS analogue, MK-852, reduced, in a dose dependent manner, the aggregation velocity. Simple decay estimations based on the difference between counts made before and 15 s after ADP addition, were used to evaluate the anti-aggregating effect of MK-852. Compared to turbidometry, the method provided a more accurate dose-response curve and gave, in addition, a significantly higher IC, value (0.21±0.05 vs 0.11±0.04 pmol/l p<0.01, means±SD). We conclude that the kinetics of free-platelet decay allows characterization of early events of the platelet response to ADP by quantitative parameters. In addition, this technique may represent a significant improvement over turbidometry in the laboratory monitoring of the anti-aggregating effect of fibrinogen binding inhibitors.