HEPARIA-DEPENDENT MODIFICATION OF THE REACTIVE CENTER ARGININE OF ANTITHROMBIN AND CONSEQUENT INCREASE IN HEPARIN-BINDING AFFINITY

Antithrombin, the principal plasma inhibitor of coagulation proteinase s, circulates in a form with low inhibitory activity due to partial in sertion of its reactive site loop into the A-beta-sheet of the molecul e. Recent crystallographic structures reveal the structural changes th at occur when antithrombin is activated by the heparin pentasaccharide , with the exception of the final changes, which take place at the rea ctive center itself. Here we show that the side chain of the P-1 Arg o f alpha-antithrombin is only accessible to modification by the enzyme peptidylarginine deiminase on addition of the heparin pentasaccharide, thereby inactivating the inhibitor, whereas the natural P-1 His varia nt, antithrombin Glasgow, is unaffected, indicating that only the P-1 Arg becomes accessible. Furthermore, the deimination of P-1 Arg conver ts antithrombin to a form with 4-fold higher affinity for the heparin pentasaccharide, similar to the affinity found for the P-1 His variant , due to a lowered dissociation rate constant for the antithrombin-pen tasaccharide complex. The results support the proposal that antithromb in circulates in a constrained conformation, which when released, in t his study by perturbation of the bonding of P-1 Arg to the body of the molecule, allows the reactive site loop to take up the active inhibit ory conformation with exposure of the P-1 Arg.