Background and Objectives: Matrix Metalloproteases (MMPs) are a family of proteolytic enzymes involved in both physiological and pathological processes. In particular, MMP-9 and MMP-2, belonging to the gelatinases class, play important roles in inflammatory diseases, including cancer and its progression. Heparin has been demonstrated to have anti-inflammatory properties ranging from the modulation of gene transcription to the alteration of enzyme structure/activity, including the inhibition of the collagenolytic activity of an MMP-9/MMP-2 mixture (1). Therefore, we aimed to evaluate the effect of heparin on the activity of gelatinases. Considering the potential therapeutic relevance of MMP-9, we also evaluated the interaction between MMP-9 and heparin. Materials and Methods: The activity of gelatinases in the presence or absence of unfractionated heparin (HMW-heparin) and low molecular weight heparin (LMW-heparin) was determined by using a fluorogenic gelatin (DQ-gelatin). The importance of sulfate groups of heparin for its inhibitory activity was determined by regioselective desulfation. The interaction site of heparin with MMP-9 was determined by incubating different enzymatic isoforms (82 kDa active MMP-9 and 67 kDa active MMP-9 lacking the C-terminal domain) with a small fluorogenic peptide and by the means of affinity chromatography with immobilized LMW-heparin. The mechanism of inhibition and Ki were determined through Michaelis-Menden kinetics. Results: The activity of both MMPs decreased by increasing the concentration of LMW-heparin. On the contrary, HMW-heparin was effective only on MMP-2 at relatively high concentrations. Our results demonstrated that the 6-O sulfate on LMW-heparin was essential to its inhibitory activity. Considering that the N-sulfate is required for the anticoagulant action, this result could have a large impact for the design of novel MMP-9-inhibiting drugs. The site of interaction on MMP-9 was not located in the C-terminal domain but rather within the catalytic or fibronectin II-like domain. Finally, the mechanism of inhibition followed an uncompetitive model, with a Ki two times higher for the 67 kDa isoform (105.8 U/ml) than for the 82 kDa MMP-9 (47.32 U/ml), despite the useless of the C-terminal for the interaction. Conclusions: Our results suggest that LMW-heparin is able to modulate the MMP-9 activity and this effect can be independent from the anticoagulant action of the molecule, opening new avenues for the development of anti-inflammatory drugs based on LMW-heparin. Therefore, the new documented effect of LMW-heparin may have a relevant role for the treatment of patients with inflammatory conditions.

Low Molecular Weight Heparin as inhibitor of gelatinases: possile anti-inflammatory role

TRENTINI, Alessandro;MANFRINATO, Maria Cristina;HANAU, Stefania;DALLOCCHIO, Franco Pasquale Filippo;BELLINI, Tiziana
2016

Abstract

Background and Objectives: Matrix Metalloproteases (MMPs) are a family of proteolytic enzymes involved in both physiological and pathological processes. In particular, MMP-9 and MMP-2, belonging to the gelatinases class, play important roles in inflammatory diseases, including cancer and its progression. Heparin has been demonstrated to have anti-inflammatory properties ranging from the modulation of gene transcription to the alteration of enzyme structure/activity, including the inhibition of the collagenolytic activity of an MMP-9/MMP-2 mixture (1). Therefore, we aimed to evaluate the effect of heparin on the activity of gelatinases. Considering the potential therapeutic relevance of MMP-9, we also evaluated the interaction between MMP-9 and heparin. Materials and Methods: The activity of gelatinases in the presence or absence of unfractionated heparin (HMW-heparin) and low molecular weight heparin (LMW-heparin) was determined by using a fluorogenic gelatin (DQ-gelatin). The importance of sulfate groups of heparin for its inhibitory activity was determined by regioselective desulfation. The interaction site of heparin with MMP-9 was determined by incubating different enzymatic isoforms (82 kDa active MMP-9 and 67 kDa active MMP-9 lacking the C-terminal domain) with a small fluorogenic peptide and by the means of affinity chromatography with immobilized LMW-heparin. The mechanism of inhibition and Ki were determined through Michaelis-Menden kinetics. Results: The activity of both MMPs decreased by increasing the concentration of LMW-heparin. On the contrary, HMW-heparin was effective only on MMP-2 at relatively high concentrations. Our results demonstrated that the 6-O sulfate on LMW-heparin was essential to its inhibitory activity. Considering that the N-sulfate is required for the anticoagulant action, this result could have a large impact for the design of novel MMP-9-inhibiting drugs. The site of interaction on MMP-9 was not located in the C-terminal domain but rather within the catalytic or fibronectin II-like domain. Finally, the mechanism of inhibition followed an uncompetitive model, with a Ki two times higher for the 67 kDa isoform (105.8 U/ml) than for the 82 kDa MMP-9 (47.32 U/ml), despite the useless of the C-terminal for the interaction. Conclusions: Our results suggest that LMW-heparin is able to modulate the MMP-9 activity and this effect can be independent from the anticoagulant action of the molecule, opening new avenues for the development of anti-inflammatory drugs based on LMW-heparin. Therefore, the new documented effect of LMW-heparin may have a relevant role for the treatment of patients with inflammatory conditions.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11392/2342924
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