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 PAR(1) and PAR(4), the endogenous enzymes responsible for activating PAR(2) 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 PAR(1), PAR(2), and PAR(4); 2) PAR-dependent calcium signaling responses in cells expressing PAR(1), PAR(2), and PAR(4) and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR(4)-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 PAR(1), PAR(2), and PAR(4) and to disarm/inhibit PAR(1). Although hK5 and -6 were also able to activate PAR(2), they failed to cause PAR(4)-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR(2)-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 PAR(1), PAR(2), and PAR(4).
Oikonomopoulou
Katerina
K
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada.
Hansen
Kristina K
KK
Saifeddine
Mahmoud
M
Tea
Illa
I
Blaber
Michael
M
Blaber
Sachiko I
SI
Scarisbrick
Isobel
I
Andrade-Gordon
Patricia
P
Cottrell
Graeme S
GS
Bunnett
Nigel W
NW
Diamandis
Eleftherios P
EP
Hollenberg
Morley D
MD
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001013:Aorta, Thoracic / Q000187:drug effects; D016367:Baculoviridae / Q000235:genetics; D001792:Blood Platelets / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects; D006801:Humans; D007610:Kallikreins / Q000138:chemical synthesis / Q000737:chemistry / Q000145:classification / Q000494:pharmacology*; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009126:Muscle Relaxation / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D044462:Receptors, Proteinase-Activated / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D013552:Swine; D013917:Thrombin / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D044462:Receptors, Proteinase-Activated; D011994:Recombinant Proteins; D007610:Kallikreins; D014357:Trypsin; D013917:Thrombin