PROTEOLYTIC CLEAVAGE OF UROKINASE-TYPE PLASMINOGEN-ACTIVATOR BY STROMELYSIN-1 (MMP-3)

Matrix metalloproteinase-3 (MMP-3, or stromelysin-1) specifically hydr olyzes the Glu(143)-Leu(144) peptide bond in 45-kDa single-chain uroki nase-type plasminogen activator (scu-PA) and in its two-chain (tcu-PA) derivative, yielding a 17-kDa NH2-terminal domain comprising the u-PA receptor (u-PAR) binding site and a 32-kDa COOH-terminal moiety conta ining the serine proteinase domain of u-PA. The conversion is complete ly abolished in the presence of the MMP inhibitors EDTA or 1,10-phenan throline. Biospecific interaction analysis indicates that binding of M MP-3 occurs through the 32-kDa fragment. The 32-kDa fragment derived f rom scu-PA (scu-PA-32k) has a specific activity of less than or equal to 500 IU/mg, but it can be activated with plasmin to a two-chain deri vative (tcu-PA-32k) with a specific activity of 79 000 IU/mg, tcu-PA a nd tcu-PA-32k moieties derived from scu-PA-32k by plasmin or from tcu- PA by MMP-3 have comparable amidolytic activities toward the chromogen ic substrate S-2444 (k(cat)/K-m of 110 and 160 mM(-1) s(-1), respectiv ely) and similar plasminogen activating activities in a coupled chromo genic substrate assay. Specific binding of the 17-kDa NH2-terminal dom ain to THP-1 monocytoid cells is completely abolished by competition w ith scu-PA but is not affected by scu-PA-32k (residual binding of 88 /- 9% (mean +/- SEM; n = 3) with 25-fold molar excess). Thus, MMP-3 re moves a functional NH2-terminal u-PAR-binding domain from u-PA without affecting its enzymatic properties.