Abstract

Direct thrombin inhibitors (DTIs) have been shown to be very potent inhibitors of platelet function when platelets are activated with thrombin. This action does not occur by direct binding of the DTI to the platelet PAR-1/-4 receptor, but indirectly, by reducing thrombin concentrations and thereby reducing the interactions of thrombin with its receptor on the platelet. It was hypothesized that both thrombin and factor Xa inhibitors could inhibit platelet aggregation, if the stimulus to initiate aggregation was higher in the cascade than factor Xa, such as tissue factor. Thus, dabigatran, a DTI, and the direct factor Xa inhibitors, rivaroxaban and apixaban were tested. Free flowing whole blood (60 ml) was obtained from an antecubital vein using an 18 gauge needle from healthy human volunteers. Blood was collected in tubes containing 3.13% sodium citrate (1 in 10 dilution with whole blood). Blood was centrifuged at 200x g to obtain platelet rich plasma (PRP). Samples (300 μL PRP) were placed in a 6-channel aggregometer, equilibrated for 5 min at 37°C and calibrated with PPP from same individual (0–1 Volts). Photometric tracings were continuously digitally recorded over 5 min following the addition of tissue factor and curves were evaluated as AUC over this time interval. Each PRP sample was incubated with 2 mg/ml Pefabloc®FG (Gly-Pro-Arg-Pro) to prevent fibrin polymerisation, 5 mM CaCl2 and increasing concentrations of dabigatran or factor Xa inhibitor. Tissue factor stimulus (range, 5–27 μl of 10 ml Innovin solution) was tailored for each individual, so that the minimum concentration that resulted in maximum aggregation was used. As positive controls, aggregation was also performed after stimulating with ADP (10 μM), collagen (2 μg/ml), TRAP (20 μM) or ecarin (0.1 U/ml). All substances inhibited tissue factor-induced platelet aggregation in a concentration-dependent manner. Dabigatran was the most potent inhibitor of platelet aggregation among the substances tested, with an IC50 of 35 nM, rivaroxaban and then apixaban followed, with IC50s of 312 and 817 nM, respectively. All substances had no effect on platelet aggregation induced by ADP, collagen and TRAP. Dabigatran was a potent inhibitor of ecarin-induced platelet aggregation, while the factor Xa inhibitors had no effect, as expected from their mechanism of action. Thus, these studies demonstrate that both direct thrombin inhibitors (by inhibiting thrombin) and direct factor Xa inhibitors (by preventing thrombin generation) indirectly inhibit platelet aggregation, though dabigatran was more potent than rivaroxaban and apixaban under these experimental conditions. Thus, these substances may not only be effective in venous/stasis thrombotic episodes where fibrin formation plays an important role, but may also be effective in more platelet dominant, arterial thrombosis settings.

Author notes

Disclosure:Employment: All authors are employees of Boehringer Ingelheim.