RADIOIMMUNOASSAY FOR THE DETECTION OF ACTIVE-SITE SPECIFIC THROMBIN INHIBITORS IN BIOLOGICAL-FLUIDS .2. HEPARIN AFFECTS THE BINDING OF HIRUDIN TO ALPHA-THROMBIN

A radioimmunoassay for recombinant (r.) hirudin was used to study the effect of heparin and other glycosaminoglycans (GAG's) on interactions between hirudin and thrombin. Binding of I-125-r.hirudin to thrombin in the presence or absence of heparin (and other GAG's) was monitored by immunoprecipitation of thrombin-I-125-r.hirudin complexes with a monoclonal antibody to human a-thrombin. Heparin and dextran sulfate substantially reduced hirudin binding to thrombin in human plasma. Inhibition by heparin was restored by addition of increasing amounts of antithrombin (AT) to samples containing constant amounts of heparin, thrombin and I-125-r.hirudin and was not neutralized by protamine sulfate. At very low heparin concentrations competitive displacement of I-125-r.hirudin was observed, while in the presence of heparin-AT complexes some I-125-r.hirudin remained bound to thrombin, suggesting that two or more binding sites for hirudin on the thrombin molecule may be occupied simultaneously.