FIGURE 7.

Thrombin processing of pro-MMP-2 is increased significantly under cell-associated conditions. A, zymograms of 1 μg/ml pro-MMP-2 incubated with 50 nm thrombin under cell-free or cell-associated conditions (HBMEC) for the indicated times. The arrows indicate pro-MMP-2 and processed MMP-2. The bar graph shows the ratio of processed MMP-2 to pro-MMP-2. Data are represented as the mean and S.D. of n = 3 experiments. *, p < 0.05 cell-associated versus cell-free conditions. B, effect of 10 μm GM6001 on thrombin-mediated processing of pro-MMP-2 in HBMEC. Zymographic analysis was performed to analyze the incubation mixture. Data are represented as the mean and S.D. of n = 3 experiments. C, zymograms of 1 μg/ml pro-MMP-2 incubated with 50 nm thrombin under cell-free or cell-associated conditions (COS-1). The data shown represent the mean and S.D. of n = 3 experiments. *, p < 0.05 cell-associated versus cell-free conditions. D, Western blot analysis (IB) using an anti-Myc antibody of 20 μg/ml pro-MMP-2-Myc/His incubated with 50 nm thrombin under cell-free or cell-associated conditions (HBMEC) for the indicated times. Cleavage products (T-D1 and T-D2) are seen in thrombin-treated MMP-2. T-D2 was also generated by cleavage of MMP-2 at Arg⁴³³. The bar graph shows the ratio of the cleaved MMP-2 fragments (T-D1 + T-D2) to total MMP-2 (pro-MMP-2 + MMP-2). Data represent the mean and S.D. of n = 3 experiments. *, p < 0.05 cell-associated versus cell-free conditions. E, Western blot analysis using an anti-Myc antibody of 20 μg/ml pro-MMP-2-Myc/His E404A incubated with 50 nm thrombin under cell-free conditions for the indicated times. Note that cleavage products (T-D1 and T-D2) are also seen in thrombin-treated pro-MMP-2-Myc/His E404A (n = 2). F, Western blotting using an anti-Myc antibody of 20 μg/ml each of pro-MMP-2-Myc/His and pro-MMP-2-Myc/His R433A incubated with 50 nm thrombin under cell-associated conditions (HBMEC) for the indicated times (n = 3).