Heparin binds Matrix Metalloproteinase-9 (MMP-9): effect on the proteolytic activity

Objectives: Matrix metalloproteinase-9 (MMP-9) is involved in the normal and pathologic inflammatory remodelling processes of the extracellular matrix. Previous works showed that heparin interfered with the activity assay of MMP-9 (1) and caused an inhibition of the collagenolytic activity of an MMP-9/MMP-2 mixture (2). Therefore, the aim of this study was to verify the inhibitory effect of heparin on MMP-9 activity. Moreover, we aimed to identify the site of interaction of heparin with MMP-9. This might provide further insights on the anti-inflammatory effects of heparin. Material and Methods: The activity of MMP-9 in the presence and absence of different concentrations of heparin (0.1, 0.01 and 0.001 mg/ml; 180 USP/mg) was determined by a modification of a commercially available activity assay system and by using a fluorescent peptide. The site of interaction of MMP-9 with heparin was determined by the mean of heparin-agarose affinity chromatography. Results and Discussion: We observed a decrease in the activity of MMP-9 increasing the concentration of heparin, with the maximum effect at 0.1 mg/ml. On the contrary, we did not observe any influence on the activity of MMP-9 when assayed with the fluorescent peptide, indicating that the active site of the enzyme was not shielded by the heparin. We observed, by the mean of heparin-agarose affinity chromatography, that the MMP-9 isoform lacking the C-terminal domain was not retained by the column. Therefore the hemopexin-like domain was necessary to the high affinity interaction of the enzyme with heparin. Conclusions: Our results suggest that heparin can regulate the MMP-9 activity and further that this effect may have a relevant role in the treatment with heparin of patients with inflammatory conditions. 1. Castellazzi M. et al., Clin Biochem 2007; 40: 1272-1276 2. Sasaki M. et al., Mediators Inflamm 2000; 9: 85-91