Proteinase-activated receptors, targets for kallikrein signaling

Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR₁ and PAR₄, the endogenous enzymes responsible for activating PAR₂ in settings other than the gastrointestinal system, where trypsin can activate PAR ₂, are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR₁, PAR₂, and PAR ₄; 2) PAR-dependent calcium signaling responses in cells expressing PAR₁, PAR₂, and PAR₄ and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR₄-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR₁, PAR₂, and PAR₄ and to disarm/inhibit PAR₁. Although hK5 and -6 were also able to activate PAR₂, they failed to cause PAR₄-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR₂-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR₁, PAR₂, and PAR₄.