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Development of a novel heparanase activity assay and identification of potent heparanase inhibitors
Abstract number: PP-WE-506
Alban1 S., Schiemann1 S.
11Pharmaceutical Institute, Christian-Albrechts-University Kiel, Kiel, Germany
How-to-cite Alban S, Schiemann S. Development of a novel heparanase activity assay and identification of potent heparanase inhibitors. Journal of Thrombosis and Haemostasis 2009; Volume 7, Supplement 2: Abstract PP-WE-506
Human heparanase overexpression promotes tumour metastasis, angiogenesis and is associated with poor prognosis. Therefore, heparanase inhibitors (HI) are considered as promising candidates for tumour therapy. So far, no simple, rapid assay is available for testing potential HI. The aim of this study was to develop an assay based on the finding that fondaparinux represents a substrate for heparanase and on the idea to quantify the degradation of fondaparinux by means of its anti-FXa (aXa)-activity. Structurally defined semi-synthetic glucan sulfates (GS) were examined for their heparanase inhibitory activity. The assay was established as a two step micro-plate-assay. First, heparanase is incubated with the substrate fondaparinux and different concentrations of potential HI. After stopping the reaction by freezing and pH-elevation, the remaining substrate is quantified by its aXa-activity in a chromogenic substrate assay (bovine FXa, AT, S2222). Initially, the following assay parameters were evaluated and optimized: incubation time, concentrations of substrate and heparanase, heparanase stability and inactivation method, as well as aXa-assay conditions. The GS structure-dependently inhibited heparanase with IC50 values ranging between 0.006 and 1.7 μg/mL. Their activity increased with increasing degree of sulfation (DS) and decreased with increasing molecular mass (Mr). Beta-1.3-GS were ∼20 times more active than alpha-1.4/1.6-GS with similar DS and Mr, so that the glucan basic structure is the most important structural parameter. PS3, the most potent beta-1.3-GS, exhibited a more than 10 times higher activity than the known heparanase inhibitor suramin. In conclusion, a novel chromogenic heparanase activity micro-plate-assay suitable for high-throughput screening has been developed. By studies on structure-activity relationships using this assay, beta-1.3-GS were identified as potent HI, which may contribute to their proven antimetastatic activity.
Disclosure of interest: none declared.
To cite this abstract use the following format:
Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number
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