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Fig. 3. Quantification of plasma kallikrein- or prekallikrein-mediated MMP-1 activation and capillary tube regression in 3D collagen matrices. ECs were suspended in collagen matrices and placed in a quantifiable 384 micro-well regression assay (15 µl gels, n=8 gels/condition) as shown in Fig. 1B. At time zero, media containing varying doses of active plasma kallikrein (A) or 2 µg/ml plasma prekallikrein and varying doses of Factor XII (B) were added to cultures. Collagen gels were monitored for tube regression and gel contraction over time and percentage of gel contraction was calculated (see Materials and Methods for details). Conditioned media were collected at 50 hours and analyzed for MMP-1 expression and activation. (C) ECs were suspended in collagen matrices. Culture media contained prekallikrein at 2 µg/ml, along with varying concentrations of HMW kininogen as indicated and either no cations or 100 µM ZnCl₂ or MnCl₂. Triplicate cultures were examined after 24 hours of culture for evidence of capillary tube regression and gel contraction (indicated by +) and analyzed for MMP-1. For all western blot analyses, arrows indicate the position of MMP-1 zymogen and arrowheads indicate activated MMP-1.

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