High-throughput turbidimetric screening for heparin-neutralizing agents and low-molecular-weight heparin mimetics

Safer heparin-neutralizing agents are currently required to replace protamine, the use of which causes adverse effects such as anaphylaxia. Low-molecular-weight (LMW) heparin mimetics that potentiate antithrombin III (AT) action are also valuable as anti-thrombotics. This paper describes a high-throughput assay for both heparin-neutralizing agents and LMW heparin mimetics without the use of blood preparations. The assay is based on turbidimetric measurement of a solution of collagen, heparin, and a test compound. Native collagen molecules spontaneously form insoluble fibrils when transferred to a physiological buffer, and this process is inhibited by heparin. In the presence of a heparin-neutralizing agent or an LMW heparin mimetic, the inhibitory effect of heparin is canceled and turbidity increase is retrieved. We demonstrated that this assay is effective in detecting potential agents with high reliability (Z′ factor=0.9). The screening of a chemical library (34400 compounds) was further performed in a 384-well format, and led to the identification of a novel heparin-neutralizing agent. Since this assay protocol is feasible for an automated highthroughput screening (HTS) system, it could enhance the lead seeking process for drugs related to heparin/ heparan sulfate (HS) functions.