Interaction of heparin with fibrinogen using surface plasmon resonance technology: investigation of heparin binding site on fibrinogen.

Heparin is widely used as an antithrombotic drug, having excellent anticoagulant properties. However in certain clinical situations heparin's efficacy seems to be somewhat limited. Despite the administration of heparin there is a high incidence of reocclusion of coronary arteries following thrombolytic therapy, and it has been observed that a significant number of patients receiving heparin treatment still exhibit thrombus extension. Although it is well established that the in vitro and in vivo anticoagulant activities of heparin is mediated via the potentiation of the major coagulation inhibitor, antithrombin III (ATIII), some in vivo antithrombotic mechanisms are not fully understood. There is poor correlation between the anticoagulant activity of heparin as measured by in vitro assays and their in vivo antithrombotic efficacy. This may be due to heparin being targeted to many blood constituents whose resultant activities on the coagulation system have not been measured as yet. The antithrombotic activity of heparin as well as the pathogenesis of bleeding complications during heparin treatment cannot be completely explained by the inhibition of blood coagulation factors. Platelet dysfunction and acceleration of fibrinolytic process have been implicated as additional factors involved. Recently a number of reports have suggested that the inhibition of the antithrombotic activity of heparin in these clinical situations may be due to the interaction of heparin with other plasma proteins specifically with fibrin(ogen) present in the thrombus. Despite the possible pathophysiological significance of heparin-fibrin(ogen) interaction, little is known about the physicochemical aspects of this reaction. In this study an attempt was made to locate where heparin binds to fibrin(ogen), using various isolated structural domains from the plasmin-mediated digests of fibrinogen and the individual chains of fibrinogen. The BIALITE system (Pharmacia Biosensor AB, Uppsala, Sweden) was employed for such a study. This utilises the Surface Plasmon Resonance (SPR) phenomenon and allows a direct quantitative analysis of the label-free molecular interaction, in real-time, from which association and dissociation rate constants can readily be obtained.