De Clerck F

Production of thromboxane and prostaglandins in human blood in the presence of thromboxane synthase inhibitors: a comparison of RIA and GC/MS determinations.

The radioimmunological determination (RIA) of primary prostaglandins (PGs) in serum and plasma was evaluated with gas chromatography / mass spectrometry (GC/MS). Human blood was stimulated in vitro in the presence or absence of the specific thromboxane synthase inhibitor ridogrel. TxB2, 6-keto-PGFlα, PGE2, PGD2 and PGF2α were determined with RIA and GC/MS on the same samples. For GC/MS analysis, prostanoids were extracted and derivatized to their methoximepentafluorobenzyl-esrers-trimethylsilyl-ethers. An excellent correlation was observed between levels of all eicosanoids determined by RIA or GC/MS (r = 0.996–0.999) when plasma was spiked with known amounts of PGs and TxB2 (2 - 500 ng/ml). In stimulated blood, with or without inhibition of thromboxane synthase, the correlation between RIA and GC/MS values remained good, except for 6-keto-PGFlα. RIA largely overestimated the levels of 6-keto-PGFlα and no significant correlation was found with levels detected by GC/MS. The results demonstrate the importance of corroboraring the reliability of RIA with GC/MS.

Arachidonic acid metabolites, ADP and thrombin modulate occlusive thrombus formation over extensive arterial injury in the rat.

Platelet-dependent occlusive thrombosis at sites of deep vessel wall injury elicited by electrical stimulation of rat carotid arteries was significantly reduced by thromboxane A2 (TXA2) synthetase inhibition and/or TXA2/prostaglandin endoperoxide receptor antagonism (ridogrel 1.25 mg/kg i.v.; dazoxiben 5 mg/kg i.v.; sulotroban 20 mg/kg i.v.), by inhibition of ADP-dependent platelet responses (ticlopidine 3 x 200 mg/kg orally) and by anticoagulation (heparin 250 U/kg i.v.; warfarin 1.25 mg/kg i.p.). This points to an involvement of arachidonic acid metabolites, ADP and thrombin as modulators of the thrombotic process. The antithrombotic effect of ridogrel (IC50 = 0.22 mg/kg i.v.) was abolished by cyclooxygenase inhibition (suprofen 5 mg/kg i.v.) but enhanced by cAMP phosphodiesterase inhibition (HL 725 6 micrograms/kg/min i.v.), demonstrating the importance of platelet inhibitory prostanoids such as PGD2, and prostacyclin formed after TXA2 synthetase inhibition. High doses of ridogrel (1.25 mg/kg i.v.) producing additional TXA2/prostaglandin endoperoxide receptor antagonism were more effective than lower doses (0.16 mg/kg i.v.) providing TXA2 synthetase inhibition alone. The antithrombotic effect of ridogrel, when combined with ticlopidine or heparin, exceeded that of the single compounds, pointing to interactions between arachidonic acid metabolites, ADP and thrombin in the formation of occlusive thrombosis at sites of arterial injury.