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

Abstract

1 Using fura-2¯uorometry 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 signi®cantly (P50.05) reduced by the preceding stimulation with thrombin and PAR1-AP in the valvular endothelial cells, while, importantly, it remained unaected 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 speci®cally attenuated the subsequent responses to trypsin and PAR2-AP. 4 In human umbilical vein endothelial cells, a higher anity PAR1-AP (haPAR1-AP) (Ala-pFArg-Cha-HArg-Tyr-NH 2 ) speci®cally attenuated the responses to thrombin but not trypsin. On the other hand, the response to haPAR1-AP was signi®cantly (P50.05) 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.

N-Linked Glycosylation Regulates Human Proteinase-activated Receptor-1 Cell Surface Expression and Disarming via Neutrophil Proteinases and Thermolysin

“…In contrast, when PAR 1 is fully glycosylated at Asn 75 , these cleavage sites are subsequently shielded by the attached oligosaccharide, and thus trypsin disarms WT hPAR 1 less efficiently. Interestingly, the previous study by Nakayama et al (9) reported that 100 nM trypsin cleaved PAR 1 but did not activate it in HUVEC cells, thus suggesting receptor disarming. We therefore assume that the glycosylation status of PAR 1 in HUVEC cells must be different from our PAR 1 -KNRK system.…”

“…1). Furthermore, these residues are suggested to be cleaved by trypsin much faster than the residues Arg 41 -Ser 42 (9). Given that these two potential trypsin cleavage sites are located close to Asn 75 , it is not surprising that in the N62Q/N75Q and N75Q mutants, trypsin can gain easy access to these cleavage sites and therefore disarm these mutant receptors.…”

Impaired Feedback Regulation of the Receptor Activity and the Myofilament Ca2+ Sensitivity Contributes to Increased Vascular Reactiveness after Subarachnoid Hemorrhage

“…The removal of the tethered ligand region of PAR 1 by proteinases converts PAR 1 to the inactive conformation, thereby terminating the irreversibly persisted signals (Holinstat et al, 2009). Trypsin is one such proteinase and it removes the tethered ligand region of PAR 1 (Nakayama et al, 2004;Nakayama et al, 2003). The inhibition of the thrombin-induced sustained contraction by trypsin thus suggests the requirement of the active conformation of PAR 1 for the thrombin-induced sustained contraction.…”

Trypsin IV or Mesotrypsin and p23 Cleave Protease-activated Receptors 1 and 2 to Induce Inflammation and Hyperalgesia

“…Trypsin can cleave fragments of PAR 1 at the thrombin cleavage site to activate this receptor (27), and high concentrations of bovine pancreatic trypsin can activate PAR 1 in cell lines (25,26). However, pancreatic trypsin also cleaves PAR 1 at sites distal to the tethered ligand, which would disable this receptor (27), and trypsin can inactivate PAR 1 in endothelial cells (54). Thus, trypsin IV may cleave PAR 1 at sites that would both activate and disable this receptor.…”