Interaction of antiplatelet drugs in vitro: aspirin, iloprost, and the nitric oxide donors SIN-1 and sodium nitroprusside.
The interaction of three antiplatelet drugs was studied in vitro: aspirin, an inhibitor of the cyclooxygenase pathway of platelet activation; iloprost, a stable analog of prostacyclin that increases platelet cAMP; and the nitrix oxide donors SIN-1 and sodium nitroprusside (SNP), which both raise platelet cGMP. Platelet adhesion and aggregation evoked by collagen/ADP were measured in anticoagulated blood under physiological flow conditions using the new Thrombostat. Aggregation was also measured in platelet-rich plasma (PRP) upon stimulation by a low (2.5 micrograms/ml) and high (20 micrograms/ml) dose of collagen, ADP, or thrombin-receptor activating peptide (TRAP). We found a synergism between iloprost and aspirin in inhibiting platelet adhesion/aggregation in flowing blood and aggregation of PRP stimulated by collagen. The mean inhibitory concentrations (IC50) of iloprost in the presence of aspirin were much lower (0.7 nM and 0.5 nM in flowing blood and low-dose collagen-stimulated PRP, respectively) than in the absence of aspirin (3 and 3.6 nM, respectively). Synergism between SIN-1 and aspirin was observed in inhibiting platelet activation in flowing blood but was much less pronounced in inhibiting collagen-induced aggregation of PRP. SIN-1/SNP and iloprost synergistically inhibited the aggregation of PRP induced by collagen as well as platelet adhesion/aggregation in blood. We found that two protein substrates of cAMP- and cGMP-dependent protein kinases, rap1B and a 50 kD protein, were associated with the functional synergism between SIN-1 and iloprost and were synergistically phosphorylated by platelet treatment with both iloprost and SIN-1. Platelet inhibition by SIN-1, iloprost, and aspirin was synergistic when measured in blood. In contrast, only additive effects of SIN-1 and iloprost were observed when platelet aggregation was measured in aspirin-treated PRP stimulated by ADP, TRAP, or collagen. Our study defines the basis for a more effective antiplatelet therapy using a combination of cGMP- and cAMP-elevating and cyclooxygenase-inhibiting drugs. The results also emphasize the importance of using various methods for the evaluation of antiplatelet drugs.
Institut für Prophylaxe und Epidemiologie, Kreislaufkrankheiten b. d. Universität München, Munich, Germany.
E V
EV
Negrescu
B
B
Grünberg
M A
MA
Kratzer
R
R
Lorenz
W
W
Siess
D000255:Adenosine Triphosphate / Q000097:blood; D001241:Aspirin / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D000242:Cyclic AMP / Q000097:blood; D006152:Cyclic GMP / Q000097:blood; D004347:Drug Interactions; D006801:Humans; D016285:Iloprost / Q000494:pharmacology*; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology*; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects; D010973:Platelet Adhesiveness / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*
D007457:Iodine Radioisotopes; D010975:Platelet Aggregation Inhibitors; D009599:Nitroprusside; D009569:Nitric Oxide; C002385:linsidomine; D000255:Adenosine Triphosphate; D008981:Molsidomine; D000242:Cyclic AMP; D006152:Cyclic GMP; D016285:Iloprost; D001241:Aspirin