The matrix-degrading
enzyme aggrecanase has been identified in cartilage and is largely responsible for cartilage breakdown. The present study determined the efficacy of different
heparin molecular weight fractions (HMWFs) and low molecular weight heparins (LMWHs) on
aggrecanase activity.
Aggrecanase activity was determined using biotinylated
peptide substrate, which was immobilized onto
streptavidin-coated 96-well plates;
aggrecanase enzyme was then added. Proteolysis of the substrate at the specific
amide bond was detected using specific antibody for the neoepitope generated. HMWFs ranging from 1,700 to 12,000 Da demonstrated a concentration-dependent inhibitory efficacy of
aggrecanase activity, with a Ki ranging from 5,000 nM down to 1 nM as a function of the molecular weight. The higher the molecular weight distribution, the greater the inhibitory efficacy of the
heparin fragments toward
aggrecanase activity. The absence or presence of
antithrombin did not alter the affinity of
heparin in inhibiting
aggrecanase. Additionally,
tissue factor pathway inhibitor at various levels did not alter the activity of
aggrecanase. LMWHs demonstrated different levels of potency in inhibiting
aggrecanase activity as a function of their average molecular weight distribution.
Tinzaparin (average molecular weight = 6,500 Da) and
enoxaparin (average molecular weight = 4,500 Da) demonstrated a Ki of 20 and 80 nM, respectively. The
aggrecanase inhibitory effect of
LMWH might contribute to blocking
inflammation and
tumor invasion by inhibiting
aggrecanase activity and maintaining an intact extracellular matrix barrier.