Inhibition of cellular action of thrombin by N3-cyclopropyl-7-{[4-(1-methylethyl)phenyl]methyl}-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine (SCH 79797), a nonpeptide thrombin receptor antagonist

doi:10.1016/S0006-2952(00)00460-3

Biochemical Pharmacology

Volume 60, Issue 10, 15 November 2000, Pages 1425-1434

https://doi.org/10.1016/S0006-2952(00)00460-3 Get rights and content

Abstract

A growing body of evidence suggests an important contribution of the cellular actions of thrombin to thrombosis and restenosis following angioplasty. Recently we reported on SCH 79797 (N3-cyclopropyl-7-{[4-(1-methylethyl)phenyl]methyl}-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine) and its analogs as new potent, nonpeptide thrombin receptor antagonists. This study further characterizes the biochemical and pharmacological actions of pyrroloquinazoline inhibitors of protease activated receptor-1 (PAR-1) in human platelets and coronary artery smooth muscle cells (hCASMC). SCH 79797 and its N-methyl analog (SCH 203099) inhibited binding of a high-affinity thrombin receptor-activating peptide ([³H]haTRAP, Ala-Phe(p-F)-Arg-ChA-HArg-[³H]Tyr-NH₂) to PAR-1 with ic₅₀ values of 70 and 45 nM, respectively. SCH 79797 inhibited [³H]haTRAP binding in a competitive manner. SCH 79797 and SCH 203099 inhibited α-thrombin- and haTRAP-induced aggregation of human platelets, but did not inhibit human platelet aggregation induced by the tethered ligand agonist for protease-activated receptor-4 (PAR-4), γ-thrombin, ADP, or collagen. SCH 203099 inhibited surface expression of P-selectin induced by haTRAP and thrombin, and it did not increase P-selectin expression or prevent thrombin cleavage of the receptor. Thrombin and TFLLRNPNDK-NH₂ (TK), a PAR-1-selective agonist, produced transient increases in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in hCASMC. This increase in [Ca²⁺]_i was inhibited effectively by SCH 79797. However, the Ca²⁺ transients induced by SLIGKV-NH_2, a PAR-2-selective agonist, were not inhibited by SCH 79797. Thrombin- and TK-stimulated [³H]thymidine incorporation also was inhibited completely by SCH 79797. The results of this study demonstrate that SCH 79797 and SCH 203099 are potent, selective antagonists of PAR-1 in human platelets and hCASMC. These data also suggest that the thrombin stimulation of Ca²⁺ transients and mitogenesis in hCASMC is mediated primarily through activation of PAR-1.

Section snippets

Reagents

HaTRAP (Ala-Phe(p-F)-Arg-ChA-HArg-Tyr-NH₂), SLIGKV-NH_2, Gly-Tyr-Pro-Gly-Gln-Val-NH₂ (a PAR-4 agonist), and TFLLRNPNDK-NH₂ were custom-synthesized by AnaSpec. Human α-thrombin and γ-thrombin were purchased from Enzyme Research Laboratories. Unless otherwise indicated, other chemicals were obtained from the Sigma Chemical Co.

Thrombin receptor [³H]haTRAP binding assay

The filtration binding assay was performed as previously described [35]. Briefly, human platelet membranes (40 μg/0.2 mL reaction mixture) were incubated with 10 nM [³

Inhibition of [³H]haTRAP binding by SCH 79797 and SCH 203099

SCH 79797 and its N-methyl analog, SCH 203099, competed with [³H]haTRAP for binding to thrombin receptor (PAR-1) on human platelet membranes with ic₅₀ values of 70 nM (K_i = 35 nM) and 45 nM (K_i = 22 nM), respectively (Fig. 1). Analysis of saturation binding of [³H]haTRAP in the presence and absence of SCH 79797 indicated that this compound is a competitive inhibitor of PAR-1 (Fig. 2).

Inhibition of platelet aggregation and P-selectin expression

SCH 79797 and SCH 203099 blocked platelet aggregation induced by the PAR-1-selective agonist haTRAP in a

Discussion

The thrombin receptor PAR-1 is distributed widely in various cell types [45], and activation of this receptor following vascular injury contributes to thrombosis, restenosis, and atherosclerosis 1, 2, 3, 4. Thus, the development of potent, small-molecule PAR-1 antagonists would allow us to test the potential therapeutic effect of blocking cellular actions of thrombin. SCH 79797 and SCH 203099 represent the first potent, nonpeptide class of competitive PAR-1 receptor antagonists. SCH 79797 and

Acknowledgements

We thank Dr. C. Strader for her support during the course of this study.

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Inhibition of cellular action of thrombin by N3-cyclopropyl-7-{[4-(1-methylethyl)phenyl]methyl}-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine (SCH 79797), a nonpeptide thrombin receptor antagonist

Abstract

Section snippets

Reagents

Thrombin receptor [3H]haTRAP binding assay

Inhibition of [3H]haTRAP binding by SCH 79797 and SCH 203099

Inhibition of platelet aggregation and P-selectin expression

Discussion

Acknowledgements

Am J Cardiol

Cell

J Biol Chem

J Biol Chem

Life Sci

Am J Cardiol

Biochem Pharmacol

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Eur J Pharmacol

Bioorg Med Chem Lett

Bioorg Med Chem Lett

Bioorg Med Chem Lett

Bioorg Med Chem Lett

Eur J Pharmacol

Thromb Res

J Biol Chem

Blood

J Biol Chem

Biochem Biophys Res Commun

Biochem Biophys Res Commun

Thromb Res

Hematol Oncol Clin North Am

Regulation of thrombin generation and functions

Semin Thromb Hemost

Thrombin is an important mediator of platelet aggregation in stenosed canine coronary arteries with endothelial injury

J Clin Invest

Characterization of a functional thrombin receptor. Issues and opportunities

J Clin Invest

Thrombin receptor [³H]haTRAP binding assay

Inhibition of [³H]haTRAP binding by SCH 79797 and SCH 203099