Bronchoconstrictor effect of thrombin and thrombin receptor activating peptide in guinea-pigs in vivo

Abstract

1. Several thrombin cellular effects are dependent upon stimulation of proteinase activated receptor-1 (PAR-1) localized over the cellular surface. Following activation by thrombin, a new N-terminus peptide is unmasked on PAR-1 receptor, which functions as a tethered ligand for the receptor itself. Synthetic peptides called thrombin receptor activating peptides (TRAPs), corresponding to the N-terminus residue unmasked, reproduce several thrombin cellular effects, but are devoid of catalytic activity. We have evaluated the bronchial response to intravenous administration of human alpha-thrombin or a thrombin receptor activating peptide (TRAP-9) in anaesthetized, artificially ventilated guinea-pigs. 2. Intravenous injection of thrombin (100 microkg(-1)) caused bronchoconstriction that was recapitulated by injection of TRAP-9 (1 mg kg(-1)). Animal pretreatment with the thrombin inhibitor Hirulog (10 mg kg(-1) i.v.) prevented thrombin-induced bronchoconstriction, but did not affect bronchoconstriction induced by TRAP-9. Both agents did not induce bronchoconstriction when injected intravenously to rats. 3. The bronchoconstrictor effect of thrombin and TRAP-9 was subjected to tolerance; however, in animals desensitized to thrombin effect, TRAP-9 was still capable of inducing bronchoconstriction, but not vice versa. 4. Depleting animals of circulating platelets prevented bronchoconstriction induced by both thrombin and TRAP-9. 5. Bronchoconstriction was paralleled by a biphasic change in arterial blood pressure, characterized by a hypotensive phase followed by a hypertensive phase. Thrombin-induced hypotension was not subject to tolerance and was inhibited by Hirulog; conversely, hypertension was subject to tolerance and was not inhibited by Hirulog. Hypotension and hypertension induced by TRAP-9 were neither subject to tolerance nor inhibited by Hirulog. 6. Our results indicate that thrombin causes bronchoconstriction in guinea-pigs through a mechanism that requires proteolytic activation of its receptor and the exposure of the tethered ligand peptide. Platelet activation might be triggered by the thrombin effect.

Figure 1

Bronchoconstriction induced by thrombin (a) and TRAP-9 (b). Thrombin (100 u kg⁻¹  i.v.) or TRAP-9 (1 mg kg⁻¹  i.v.) were injected for three consecutive times each 20 min. I, first administration; II, second administration; III, third administration. Each bar represents the mean of the response obtained from seven different animals. ^**P<0.01 versus first administration (Bonferroni's test).

Figure 2

Typical traces representing thrombin- and TRAP-9-induced bronchoconstriction (a and c) and changes in arterial blood pressure (b and d). Thrombin (100 u kg⁻¹) and TRAP-9 (1 mg kg⁻¹) were administered for three consecutive times each 20 min, bronchoconstriction and arterial blood pressure were evaluated. Histamine (10 μg kg⁻¹  i.v.) was previously administered to check animal responsiveness to a bronchoconstrictory agent.

Figure 3

Histological analysis of lungs obtained from control animals (a; ×250), and thrombin (b; ×125) or TRAP-9 (c; ×250) injected animals. In b and c it is evident a strong bronchoconstriction (arrows) and vasoconstriction (arrows).

Figure 3

Histological analysis of lungs obtained from control animals (a; ×250), and thrombin (b; ×125) or TRAP-9 (c; ×250) injected animals. In b and c it is evident a strong bronchoconstriction (arrows) and vasoconstriction (arrows).

Figure 3

Histological analysis of lungs obtained from control animals (a; ×250), and thrombin (b; ×125) or TRAP-9 (c; ×250) injected animals. In b and c it is evident a strong bronchoconstriction (arrows) and vasoconstriction (arrows).