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 63kDa protein that accumulated as the predominant form in the conditioned medium. This 63kDa thrombin-activated MMP-2 is distinct from the 62kDa 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 72kDa pro-MMP-2, but efficiently cleaved the 64kDa MT-MMP-processed intermediate form in the presence of cells. Thrombin also rapidly (within 1h) increased cellular MT-MMP activity, and at longer time points (>6h) 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 64kDa 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.
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July 2001
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Research Article|
June 25 2001
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
Marc A. LAFLEUR;
Marc A. LAFLEUR
∗School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.
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Morley D. HOLLENBERG;
Morley D. HOLLENBERG
†Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta, Canada T2N 4N1
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Susan J. ATKINSON;
Susan J. ATKINSON
∗School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.
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Vera KNÄUPER;
Vera KNÄUPER
∗School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.
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Gillian MURPHY;
Gillian MURPHY
∗School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.
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Dylan R. EDWARDS
Dylan R. EDWARDS
1
∗School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.
1To whom correspondence should be addressed (e-mail dylan.edwards@uea.ac.uk).
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Publisher: Portland Press Ltd
Received:
July 07 2000
Revision Received:
November 13 2000
Accepted:
April 20 2001
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2001
2001
Biochem J (2001) 357 (1): 107–115.
Article history
Received:
July 07 2000
Revision Received:
November 13 2000
Accepted:
April 20 2001
Citation
Marc A. LAFLEUR, Morley D. HOLLENBERG, Susan J. ATKINSON, Vera KNÄUPER, Gillian MURPHY, Dylan R. EDWARDS; 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. Biochem J 1 July 2001; 357 (1): 107–115. doi: https://doi.org/10.1042/bj3570107
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