Multiple agonist induction of aggregation: an approach to examine anti-aggregating effects in vitro

We tested the hypothesis that utilisation of multiple agonists in physiological concentrations is more appropriate for platelet aggregation in vitro than a single agonist in high concentration. Utilising impedance aggregometry in whole human blood (normal subjects and patients with coronary artery disease) we observed potentiation of pro-aggregatory effects of ADP by the combination of adrenaline, serotonin and thrombin in subthreshold concentrations (multiple agonist approach). In blood samples from the patients, verapamil (Ver, an L-type Ca(2+) channel blocker), nitroglycerine (NTG, a stimulator of cGMP formation) and prostaglandin E(1) (PGE(1) a stimulator of CAMP formation) inhibited platelet aggregation in vitro. With NTG and PGE(1) there was increased sensitivity to multiple agonists in comparison with ADP alone. For example, inhibition of aggregation with 10(-4)M NTG increased from 37 ± 5% with ADP alone to 86 ± 13% (P < 0.01) with multiple agonists. Threshold effects of NTG were seen at 10(-6) M with ADP alone and 10(-7) M with multiple agonists; whilst threshold for PGE(1) was reduced from 10(-10) to 10(-11) M. However, responses to Ver were unchanged by multiple agonists, demonstrating that the potentiation of anti-aggregating effects utilising the multiple agonist approach is not a non-specific phenomenon. The ability of multiple agonists to enhance the anti-aggregating effects of NTG and PGE(1) provides an in vitro experimental method mimicking the in vivo situation.