Thromboxane (Tx) A2 receptor blockade and TxA2 synthase inhibition alone and in combination: comparison of anti‐aggregatory efficacy in human platelets
Authors: Watts I.S. , Wharton K.A. , White B.P. , Lumley P. 
Published: July 2012

The present study has compared the relative anti‐aggregatory effect of various compounds which interfere with thromboxane (Tx) A2‐dependent aggregation of human platelets in whole blood in vitro. These included the cyclo‐oxygenase inhibitor aspirin, the TxA2 synthase inhibitor dazoxiben, the TxA2 (TP‐) receptor blocking drug GR32191 and two compounds, R.68070 ((E)‐5‐[[[(3‐pyridinyl) [3‐(trifluoromethyl)phenyl]‐methylen] amino]oxy] pentanoic acid) and CV‐4151 ((E)‐7‐phenyl‐7‐(3‐pyridyl)‐6‐heptenoic acid), which possess both TP‐receptor blocking and TxA2 synthase inhibitory activities in the same molecule.

GR32191, R.68070 and CV‐4151 all antagonized aggregation to the TxA2 mimetic U‐46619, with pA2 values of approximately 8.2, 5.4 and 4.8 respectively. This effect was specific, platelet aggregation induced by adenosine 5′‐diphosphate (ADP) being unaffected by concentrations up to 10, 1000 and 300 μm respectively. In contrast, neither aspirin nor dazoxiben exhibited any measurable TP‐receptor blocking activity.

The rank order of potency (pIC50)for inhibition of TxA2 formation in serum was R.68070 (7.4) > CV‐4151 (6.9) > dazoxiben (5.7) > aspirin (5.3). In addition, all four drugs abolished collagen‐induced platelet TxA2 formation. In contrast, GR32191 produced no consistent inhibition of TxA2 formation in either system up to concentrations of 10–30 μm.

The specificity of R.68070, CV‐4151 and dazoxiben for TxA2 synthase was indicated by their ability to increase serum levels of prostaglandin E2 (PGE2) and PGD2 in parallel with decreases in TxA2 formation. This profile was not observed with aspirin or GR32191. However, high concentrations of R.68070 (100 μm) and CV‐4151 (1000 μm) necessary for maximum TP‐receptor blocking activity, produced substantially smaller increases in PGE2 and PGD2, consistent with an aspirin‐like effect of these compounds upon cyclo‐oxygenase. With dazoxiben (1000 μm), PGE2 and PGD2 levels remained elevated.

Aspirin inhibited collagen‐induced platelet aggregation, the effect correlating with inhibition of TxA2 formation. Dazoxiben, whilst also achieving maximal inhibition of TxA2 formation, produced significantly less inhibition of aggregation than aspirin. In contrast, GR32191 (0.1–10 μm), at concentrations specific for TP‐receptor blockade, produced a significantly greater antagonism of collagen‐induced platelet aggregation than aspirin. This additional effect of GR32191 was absent in platelets pretreated with aspirin, indicating the probable involvement of an endogenous anti‐aggregatory cyclo‐oxygenase product in response to collagen stimulation.

R.68070 and CV‐4151 also inhibited collagen‐induced aggregation, with very high concentrations of R.68070 (100 μm) producing an effect equivalent to that of GR32191.

In contrast, the combination of GR32191 with either dazoxiben, R.68070 or CV‐4151, at concentrations specific for TxA2 synthase, produced a synergistic inhibitory effect upon collagen‐induced platelet aggregation which was greater than that achieved with either aspirin or any of the compounds used alone. Pretreatment of platelets with aspirin reversed this synergistic effect, consistent with it being dependent upon the formation and action of anti‐aggregatory prostaglandins.

In conclusion, the present study has confirmed the superior platelet inhibitory profile of a combination of a TP‐receptor blocking drug and a TxA2 synthase inhibitor to that of either activity alone. However, the maximum inhibitory effect of the currently available compounds, R.68070 and CV‐4151, which possess both activities in the same molecule, appears to be no greater in vitro than that obtained with the potent TP‐receptor blocking drug, GR32191. This most probably reflects the inhibition by R.68070 and CV‐4151 of platelet cyclo‐oxygenase at the concentrations required for effective TP‐receptor blockade which results in a reduction in the formation of anti‐aggregatory prostanoids.