title: Activation of pro-(matrix metalloproteinase-2) (pro-MMP-2) by thrombin is membrane-type-MMP-dependent in human umbilical vein endothelial cells and generates a distinct 63 kDa active species.
abstract: Thrombin, a critical enzyme in the coagulation cascade, has also been associated with angiogenesis and activation of the zymogen form of matrix metalloproteinase-2 (MMP-2 or gelatinase-A). We show that thrombin activated pro-MMP-2 in a dose- and time-dependent manner in cultured human umbilical-vein endothelial cells (HUVECs) to generate a catalytically active 63 kDa protein that accumulated as the predominant form in the conditioned medium. This 63 kDa thrombin-activated MMP-2 is distinct from the 62 kDa species found following concanavalin A or PMA stimulated pro-MMP-2 activation. Hirudin and leupeptin blocked thrombin-induced pro-MMP-2 activation, demonstrating that the proteolytic activity of thrombin is essential. However, activation was also dependent upon membrane-type-MMP (MT-MMP) action, since it was blocked by EDTA, o-phenanthroline, hydroxamate metalloproteinase inhibitors, tissue inhibitor of metalloproteinase-2 (TIMP-2) and TIMP-4, but not TIMP-1. Thrombin inefficiently cleaved recombinant 72 kDa pro-MMP-2, but efficiently cleaved the 64 kDa MT-MMP-processed intermediate form in the presence of cells. Thrombin also rapidly (within 1 h) increased cellular MT-MMP activity, and at longer time points (>6 h) it increased expression of MT1-MMP mRNA and protein. Thus signalling via proteinase-activated receptors (PARs) may play a role in thrombin-induced MMP-2 activation, though this does not appear to involve PAR1, PAR2, or PAR4 in HUVECs. These results indicate that in HUVECs the activation of pro-MMP-2 by thrombin involves increased MT-MMP activity and preferential cleavage of the MT-MMP-processed 64 kDa MMP-2 form in the presence of cells. The integration of these proteinase systems in the vascular endothelium may be important during thrombogenesis and tissue remodelling associated with neovascularization.
authors: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
authors: M A
authors: MA
authors: Lafleur
authors: M D
authors: MD
authors: Hollenberg
authors: S J
authors: SJ
authors: Atkinson
authors: V
authors: V
authors: Knäuper
authors: G
authors: G
authors: Murphy
authors: D R
authors: DR
authors: Edwards
mesh_terms: D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D018093:Gelatinases / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007700:Kinetics; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism*; D008970:Molecular Weight; D009842:Oligopeptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D018179:Receptors, Thrombin / Q000037:antagonists & inhibitors / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic; D014471:Umbilical Veins
chemical_list: D004792:Enzyme Precursors; D009842:Oligopeptides; D011480:Protease Inhibitors; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D013917:Thrombin; D018093:Gelatinases; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; C069860:progelatinase; D013755:Tetradecanoylphorbol Acetate