Unproductive cleavage and the inactivation of protease-activated receptor-1 by trypsin in vascular endothelial cells

Abstract

1 Using fura-2 fluorometry of [Ca(2+)](i) in response to thrombin, trypsin and protease-activated receptor activating peptides (PAR-APs), we determined whether trypsin cleaves protease-activated receptor 1 (PAR1) and activates it in the endothelial cells of the porcine aortic valves and human umbilical vein. 2 Once stimulated with thrombin, the subsequent application of trypsin induced a [Ca(2+)](i) elevation similar to that obtained without the preceding stimulation with thrombin in the valvular endothelial cells. However, the preceding stimulation with trypsin abolished the subsequent response to thrombin, but not to bradykinin or substance P. 3 The response to PAR1-AP (SFLLRNP) was significantly (P<0.05) reduced by the preceding stimulation with thrombin and PAR1-AP in the valvular endothelial cells, while, importantly, it remained unaffected by the preceding stimulation with either trypsin or PAR2-AP (SLIGRL). The response to PAR2-AP was reduced by the preceding stimulation with trypsin and PAP2-AP. PAR1-AP attenuated the subsequent responses not only to thrombin and PAR1-AP but also to trypsin and PAR2-AP, while PAR2-AP specifically attenuated the subsequent responses to trypsin and PAR2-AP. 4 In human umbilical vein endothelial cells, a higher affinity PAR1-AP (haPAR1-AP) (Ala-pF-Arg-Cha-HArg-Tyr-NH(2)) specifically attenuated the responses to thrombin but not trypsin. On the other hand, the response to haPAR1-AP was significantly (P<0.05) attenuated by the preceding stimulation with thrombin but not trypsin. 5 In conclusion, trypsin cleaved PAR1 but did not activate it in the endothelial cells. Moreover, the trypsin-cleaved PAR1 was no longer responsive to thrombin.

Figure 1

[Ca²⁺]_i elevations induced by thrombin, trypsin, PAR1-AP and PAR2-AP in the in situ endothelial cells of the porcine aortic valve. (a) Representative recordings of the changes in [Ca²⁺]_i induced by 6 units ml⁻¹ thrombin, 100 nM trypsin, 30 μM PAR1-AP and 30 μM PAR2-AP in the strips of the porcine aortic valve. The reference response to 10 μM ATP was recorded at the beginning of each measurement. The levels of [Ca²⁺]_i at rest and at the peak response to 10 μM ATP were assigned to be 0% and 100%, respectively. (b) The concentration–response curves for the peak elevation of [Ca²⁺]_i induced by thrombin, trypsin, PAR1-AP and PAP2-AP. The data are the mean±s.e.mean (n=5). The concentration of thrombin was estimated based on the proteolytic activity, while assigning 1 unit ml⁻¹ to be 10 nM.

Figure 2

Cross-desensitization of the responses to thrombin and trypsin in the in situ endothelial cells of the porcine aortic valve. (a,b) Representative recordings showing the changes in [Ca²⁺]_i induced by 6 units ml⁻¹ thrombin (a) and 100 nM trypsin (b) after the preceding stimulation with 6 units ml⁻¹ thrombin and 100 nM trypsin, either in the presence or absence of 10 μM p-APMSF. The reference response to 10 μM ATP was recorded at the beginning of each measurement. The strips were sequentially stimulated at 15 min intervals in various combinations of thrombin and trypsin. The levels of [Ca²⁺]_i at rest and at peak response to 10 μM ATP were assigned to be 0% and 100% respectively. (c) Summary of five independent measurements. The data are the mean±s.e.mean. ^*P<0.05; n.s., not significant (P>0.05). (d) The concentration-dependent inhibition of the thrombin response by trypsin. The concentration of trypsin of the preceding stimulation was varied, and the response to the subsequent stimulation with thrombin was evaluated. The data are the mean±s.e.mean. ^*P<0.05; n.s., not significant (P>0.05).

Figure 3

Responsiveness to bradykinin and substance P after the preceding stimulation with trypsin in the in situ endothelial cells of the porcine aortic valve. (a,b) Representative traces showing the response to 10 nM bradykinin (a) and 10 nM substance P (b) with and without the preceding stimulations with 100 nM trypsin. The levels of [Ca²⁺]_i at rest and at peak response to 10 μM ATP were assigned to be 0% and 100%, respectively. The traces are representative of three independent experiments.

Figure 4

Response to the activating peptides, PAR1-AP and PAR2-AP, after the receptor cleavage by thrombin and trypsin in the in situ endothelial cells of the porcine aortic valve. (a,b) Representative traces showing the responses to 30 μM PAR1-AP (a) and 30 μM PAR2-AP (b) with and without the preceding stimulations with 6 units ml⁻¹ thrombin or 100 nM trypsin. The levels of [Ca²⁺]_i at rest and at peak response to 10 μM ATP were assigned to be 0% and 100%, respectively. (c) Summary of five independent measurements. The data are the mean±s.e.mean. ^*P<0.05, n.s., not significant (P>0.05).

Figure 5

Responses to proteolytic activation and non-proteolytic activation after the preceding stimulation with activating peptides in the in situ endothelial cells of the porcine aortic valve. (a–d) Representative traces showing the responses to 6 units ml⁻¹ thrombin (a), 100 nM trypsin (b), 30 μM PAR1-AP (c) and 30 μM PAR2-AP (d) after the preceding stimulations with PAR1-AP or PAR2-AP. The levels of [Ca²⁺]_i at rest and at peak response to 10 μM ATP were assigned to be 0% and 100%, respectively.(e) Summary of five independent measurements. The data are the mean±s.e.mean. ^*P<0.05, n.s., not significant (P>0.05).

Figure 6

Effects of high affinity PAR1-AP on the response to thrombin and trypsin in HUVEC. (a,b) Representative traces showing the responses to 6 units ml⁻¹ thrombin and 100 nM trypsin and after preceding stimulations with 10 μM haPAR1-AP (a), and 10 μM haPAR1-AP 15 min after preceding stimulations with 6 units ml⁻¹ thrombin or 100 nM trypsin (b). The levels of [Ca²⁺]_i at rest and at peak response to 10 μM ATP were assigned values of 0% and 100%, respectively. (c) Summary of five independent measurements. The data are the mean±s.e.mean. ^*P<0.05, n.s., not significant (P>0.05).

Figure 7

Effect of the preceding stimulation with trypsin on the thrombin-induced relaxation in the strips of the porcine coronary artery. Representative traces of change in tension induced by 6 units ml⁻¹ thrombin without (a) and with (b) the preceding stimulation with 100 nM trypsin during the sustained contraction induced by 100 nM U46619. The tension obtained at rest and during the sustained contraction induced by 118 mM K⁺ was assigned to be 0% and 100%, respectively. Shown are the representative traces of three independent experiments.