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

Thrombin, a critical enzyme in the coagulation cascade, has also been assoc iated with angiogenesis and activation of the zymogen form of matrix metall oproteinase-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 prote in 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 an d leupeptin blocked thrombin-induced pro-MMP-2 activation, demonstrating th at the proteolytic activity of thrombin is essential. However, activation w as also dependent upon membrane-type-MMP (MT-MMP) action, since it was bloc ked by EDTA, o-phenanthroline, hydroxamate metalloproteinase inhibitors, ti ssue 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 c ells. 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 appe ar to involve PAR1, PAR2, or PAP4 in HUVECs. These results indicate that in HUVECs the activation of pro-MMP-2 by thrombin involves increased MT-MMP a ctivity 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 t he vascular endothelium may be important during thrombogenesis and tissue r emodelling associated with neovascularization.