MOLECULAR MAPPING OF THE HEPARIN-BINDING EXOSITE OF THROMBIN

Thrombin contains electropositive patches at opposite poles of the mol ecule which represent potential exosites for the binding of macromolec ular ligands. The function of anion-binding exosite I, the fibrin(ogen ) recognition site, has been web described. Anion-binding exosite II, located near the carboxyl terminus of the molecule, has been proposed to bind heparin on the basis of chemical modification studies. To defi ne the functional heparin-binding site on thrombin, purified recombina nt alpha-thrombins were prepared with glutamic acid substitution for s elected basic amino acid residues in exosite II or exosite I. Heparin affinity was assessed by NaCl gradient elution from heparin-agarose, a nd second-order rate constants for inhibition by antithrombin III were determined in the absence and presence of heparin. Affinity for hepar in-agarose was reduced markedly by selected mutations in exosite II (R 89E, R245E, K248E, and K252E, numbered from the amino terminus of the B chain) but not by other mutations in exosite II(K174E, K247E) or by mutations in exosite I(R68E, K154E). All recombinant thrombins had sim ilar rate constants for inhibition by antithrombin III without heparin . However, affinity for heparin-agarose correlated directly with the r ate of inhibition by antithrombin III with heparin. These results demo nstrate that selected mutations in anion-binding exosite II define a f unctional heparin-binding site and support the template mechanism of h eparin action.