Receptor Mechanism of Thrombin-Induced Endothelium-Dependent and Endothelium-Independent Coronary Vascular Effects in Dogs

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Abstract
Summary We previously reported that thrombin produces endothelium-dependent relaxation and endothelium-independent constrictions in canine coronary arteries. To determine whether these opposing vascular effects of thrombin are mediated by the same receptor mechanism, but at different cell types, we investigated the effects of thrombin receptor agonist peptide (TRAP) on isolated canine coronary arteries with and without intact endothelium. In coronary arteries with intact endothelium, addition of 0.01–3.0 μM TRAP, a 14-amino acid residue peptide (SFLLRNPNDKYEPF) homologous to the newly exposed N-terminus after cleavage of the cloned human thrombin receptor, produced rapid, dose-dependent relaxation (Emax = −89.6 ± 2.3%, n = 26). Threshold concentration was 0.03 μM, and IC50 value was 0.3 μM. Mechanical disruption of the endothelium completely abolished the TRAP-induced relaxation; instead a dose-dependent contraction was observed. Expressed as a percentage of the maximum 70 mM KCl-induced contraction, the maximum contraction observed with 3 μM TRAP was 62.0 ± 4.1% (n = 32). Pretreatment of endothelium-intact coronary arteries with either 3 μM hemoglobin or 0.25 mM NG-monomethyl-L-arginine (L-NMMA), specific inhibitors of endothelium-derived relaxing factor or nitric oxide (EDRF/NO), also inhibited the relaxation and unmasked the constrictor effect. The pharmacokinetic characteristics of the opposing coronary vascular effects of TRAP are similar to those observed with thrombin, but specific thrombin inhibitors, such as hirudin and D-phenylalanyl-prolyl-L-arginine chloromethyl ketone (PPACK), which inhibit both thrombin relaxant and constrictor effects, had no effect on TRAP-induced responses. Furthermore, in thrombin-tolerant coronary arteries, after repeated and prolonged thrombin exposures, TRAP was still capable of eliciting similar relaxation and constrictor responses. Thus, our results demonstrate that the two opposing coronary vascular effects of thrombin are mediated by the same receptor mechanism. Interaction with endothelium leads to EDRF/ NO-dependent relaxation and the interaction with vascular smooth muscle cells leads to contraction in canine coronary arteries.

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