Novel heparanase activity assay based on a fluorescence sensor technology

Since tumors and other diseases are characterized by increased heparanase (HEP) levels, HEP is considered a diagnostic marker and a target for antitumor therapy. Therefore, methods are needed to measure HEP and to examine inhibitors. Based on previous findings that HEP degrades not only heparan-sulfate, but also the also the sulfated pentasaccharide fondaparinux (FPX) [1] and that this can be quantified by its effect on the fluorescence intensity (FI) of the sensor molecule Polymer-H [2], we aimed to develop a fluorimetric HEP activity assay.

Since the FPX degradation products proved to have no effect on the FI, the remaining FPX can be measured without separation. Optimization of various assay parameters led to the following two-step procedure: (1) Incubation of HEP containing solution with FPX (10µg/mL). (2) Sample dilution and FPX detection by adding Polymer-H (7.5µg/mL) and measuring the FI (λ_em330nm, λ_ex510nm). The FXP degradation showed to increase with the concentration of HEP. After 30min at 37°C, 1.5mIU/mL HEP led to complete FXP-degradation. By varying incubation time and FPX concentration, the LOD of 0.2mIU/mL HEP can be considerably decreased. Various HEP inhibitors demonstrated the suitability of the assay for inhibitor screening.

In conclusion, a rapid, simple and robust microplate HEP activity assay was developed. A major advantage is the use of FPX as substrate. In contrast to heparan-sulfate, it is chemically defined, well available and has not to be labeled with radioactive or other markers for detection. Moreover, its fluorimetric detection is much more convenient than by its anti-FXa-activity or HPLC-MS.

Keywords: pharmacology, heparanase, assay development, fluoescence sensor technology, heparanase inhibitors

References: 1. Alban S et al (2009)J Thromb Haemost 7, Suppl.2: PP-WE-506.

2. Lühn S, Schrader T, Sun W, Alban S (2010)J Pharm Biomed Anal 52: 1–8.