Thrombin rapidly and efficiently activates gelatinase A in human microvascular endothelial cells via a mechanism independent of active MT1 matrix metalloproteinase.
Thrombin has been shown previously to activate gelatinase A in human umbilical vein endothelial cells. The activation is thought to be mediated by membrane-type 1 matrix metalloproteinase (MT1-MMP) on the cell surface, which generates the 62-kd intermediate and the 59-kd fully active forms. We used microvascular endothelial cells derived from human neonatal foreskin to investigate the mechanism of gelatinase A activation by thrombin. Gelatinase A was measured using zymography. Whereas activation by PMA generated both the 62-kd intermediate and the 59-kd fully active forms of gelatinase A after 24 hours, activation by thrombin produced only the 59-kd species rapidly (within 2 hours). Four findings indicate that MT1-MMP was not involved in thrombin-induced activation: (1) there was no up-regulation of MT1-MMP after 2 hours stimulation by thrombin, even though there was activation of gelatinase A; (2) the 62-kd intermediate species was never detected in response to thrombin; (3) tissue inhibitor of matrix metalloproteinase-2 completely prevented gelatinase A activation induced by PMA but not by thrombin; and (4) the metalloproteinase inhibitor 1,10-phenanthroline did not inhibit thrombin-induced activation. Together, these data demonstrate that activation of gelatinase A by thrombin is different from PMA and operating via a pathway independent of MT1-MMP. The ability of thrombin to rapidly and efficiently activate gelatinase A is likely to be a major contributing factor to its potent angiogenic activity.
Nguyen
M
M
Sutton Arthritis Research Laboratory, Royal North Shore Hospital, St. Leonards, Australia.
Arkell
J
J
Jackson
C J
CJ
D002462:Cell Membrane / Q000201:enzymology; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation; D018093:Gelatinases / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D020778:Matrix Metalloproteinase 2; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000187:drug effects / Q000378:metabolism*; D008833:Microcirculation; D012867:Skin / Q000098:blood supply; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism
D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; D013917:Thrombin; D018093:Gelatinases; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D013755:Tetradecanoylphorbol Acetate