Trypsin IV or mesotrypsin and p23 cleave protease-activated receptors 1 and 2 to induce inflammation and hyperalgesia

Although principally produced by the pancreas to degrade dietary proteins in the intestine, trypsins are also expressed in the nervous system and in epithelial tissues, where they have diverse actions that could be mediated by protease-activated receptors (PARs). We examined the biological actions of human trypsin IV (or mesotrypsin) and rat p23, inhibitor-resistant forms of trypsin. The zymogens trypsinogen IV and pro-p23 were expressed in Escherichia coli and purified to apparent homogeneity. Enteropeptidase cleaved both zymogens, liberating active trypsin IV and p23, which were resistant to soybean trypsin inhibitor and aprotinin. Trypsin IV cleaved N-terminal fragments of PAR ₁, PAR₂, and PAR₄ at sites that would expose the tethered ligand (PAR₁ = PAR₄ > PAR₂). Trypsin IV increased [Ca²⁺]_i in transfected cells expressing human PAR₁ and PAR₂ with similar potencies (PAR₁, 0.5 μM; PAR₂, 0.6 μM). p23 also cleaved fragments of PAR₁ and PAR₂ and signaled to cells expressing these receptors. Trypsin IV and p23 increased [Ca²⁺] _i in rat dorsal root ganglion neurons that responded to capsaicin and which thus mediate neurogenic inflammation and nociception. Intraplantar injection of trypsin IV and p23 in mice induced edema and granulocyte infiltration, which were not observed in PAR₁^-/-(trypsin IV) and PAR₂^-/- (trypsin IV and p23) mice. Trypsin IV and p23 caused thermal hyperalgesia and mechanical allodynia and hyperalgesia in mice, and these effects were absent in PAR₂^-/- mice but maintained in PAR₁^-/- mice. Thus, trypsin IV and p23 are inhibitor-resistant trypsins that can cleave and activate PARs, causing PAR ₁- and PAR₂-dependent inflammation and PAR ₂-dependent hyperalgesia.