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The rate of the reaction between thrombin and antithrombin III is greatly increased in the presence of heparin. Several different mechanisms has been suggested to explain this effect. One possible mechanism is that the binding of heparin to antithrombin III transforms it to a much more rapidly acting inhibitor. Another possible mechanism is that the heparin is bound to thrombin and that this complex then reacts with antithrombin III. This study was performed to clarify how heparin actually works. A commercial heparin preparation was fractionated by affinity chromatography on matrixbound antithrombin III into a high and a low affinity fraction. The high affinity fraction had strong anti-coagulant activity both when assayed by activated partial thromboplastin time and in purified thrombin-antithrombin III system. The low affinity heparin fraction had very low activity in both test systems. The strength of the binding of the two heparin fractions to antithrombin III and thrombin, respectively, was determined by a crossed Immunoelectrophoresis technique. The results obtained showed that the high affinity heparin was strongly bound to antithrombin III as expected. The low affinity heparin was also bound but with a much lower strength. In contrast thrombin had essentially the same affinity for both heparin fractions indicating that the mechanism suggested first is the correct one. Competion experiments using both heparin fractions and the thrombin-antithrombin III system also yielded results in good agreement with that. Thus heparin acts as anti-coagulant by forming complex with antithrombin III making it a rapidly acting inhibitor.

Studies on the Mechanism of the Rate Enhancing Effect of Heparin on the Thrombin – Anti-Thrombin III Reaction