Multiple complexes of thrombin and heparin.

Fluorescence polarization has been used to study the interaction of thrombin and heparin, and the catalysis by heparin of the combination of thrombin and antithrombin. At low ionic strength (20 mM Tris, pH 7.4), the addition of heparins of known molecular weights to thrombin led to the formation of large complexes (defined as 'complex 1'). Further addition of heparin led to a rearrangement of these large complexes to form smaller complexes (defined as 'complex 2'). The molar ratio of thrombin to heparin in complex 1 increased with increasing heparin molecular weight, and corresponded to one thrombin molecule for every heparin segment of Mr 3000. The stoichiometry of complex 2 was 1 heparin to 1 thrombin, irrespective of the heparin molecular weight. At higher ionic strength (150 mM NaCl) some complex 1 was still formed. However, by reversing the titration and adding thrombin to fluorescein-heparin the dissociation constant for complex 2 was estimated to be 1-3 microM and independent of the heparin molecular weight. The complex formed between thrombin and heparin, to which antithrombin was attached, has a dissociation constant of 1-2 microM, again irrespective of the heparin molecular weight. In the heparin-catalysed thrombin-antithrombin reaction, an increase in the size of heparin leads to a lowering of the observed Km for thrombin. A possible explanation is that thrombin, after initial binding to the heparin, moves rapidly to the site where it combines with antithrombin.