Heparin-dependent Modification of the Reactive Center Arginine of Antithrombin and Consequent Increase in Heparin Binding Affinity

Abstract

Antithrombin, the principal plasma inhibitor of coagulation proteinases, circulates in a form with low inhibitory activity due to partial insertion of its reactive site loop into the A-␤-sheet of the molecule. Recent crystallographic structures reveal the structural changes that occur when antithrombin is activated by the heparin pentasaccharide, with the exception of the final changes, which take place at the reactive center itself. Here we show that the side chain of the P 1 Arg of ␣-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 variant, antithrombin Glasgow, is unaffected, indicating that only the P 1 Arg becomes accessible. Furthermore, the deimination of P 1 Arg converts 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 antithrombinpentasaccharide complex. The results support the proposal that antithrombin circulates in a constrained conformation, which when released, in this 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 inhibitory conformation with exposure of the P 1 Arg.The plasma serpin (1), antithrombin, is the major inhibitor of the serine proteinases of the coagulation network, especially thrombin and factor Xa (2). This inhibitory activity is stimulated by the complexing of antithrombin with the sulfated polysaccharide, heparin. In particular, the activity against factor Xa is mediated by a core pentasaccharide component of heparin. Antithrombin has an initial low affinity for heparin, but this immediately changes to a high affinity on initial binding, the change occurring concomitantly with the activation of inhibitory function (3).The interaction of the heparin pentasaccharide with antithrombin and the associated mechanism of conformational activation of inhibition has recently been revealed in the crystal structure of a dimer of antithrombin complexed with the pentasaccharide (4). Linkage between the two antithrombin molecules in the dimer directly involves the reactive site loop of the inhibitory component, with a consequent constraint in the movement at its reactive site. Hence, while showing the commencement of the movement of the reactive site loop that results in activation, the crystal structure does not show the change that occurs at the reactive center itself.There is, however, a good model of the likely unconstrained active conformation of the reactive site loop of antithrombin. This is provided by the structure of the closely related serpin, ␣ 1 -antitrypsin (5), in which the loop is fixed in the optimal canonical inhibitory conformation present in all other families of serine proteinase inhibitors. The transition of antithrombin to this conformation would require a shift of the side chain of the P 1 arginine from an i...

Partial Activation of Antithrombin without Heparin through Deletion of a Unique Sequence on the Reactive Site Loop of the Serpin

“…These results suggested that the mutants have been properly folded. Furthermore, consistent with previous observation by others (7,17), a high affinity interaction with heparin suggested that mutants might have adopted activated conformations.…”

“…These results suggested that the mutants have been properly folded. Furthermore, consistent with previous observation by others (7,17), a high affinity interaction with heparin suggested that mutants might have adopted activated conformations.Inactivation of FXa and Thrombin-k 2 values for the association of the wild type and mutant serpins with fXa and thrombin in both the absence and presence of H 5 or H 70 are presented in Tables I and II. As expected from the purity on SDS-PAGE, rAT and pAT exhibited identical reactivities with both proteinases in either the absence or presence of cofactors.…”

Molecular Determinants of the Mechanism Underlying Acceleration of the Interaction between Antithrombin and Factor Xa by Heparin Pentasaccharide

“…The RCL of ATIII clearly changes conformation upon binding H5 from a partially inserted form in the absence of heparin to a fully expelled position, but the consequences of this to the rest of the RCL are not evident because of the constraints imposed on the RCL of the native molecule by its interaction in the dimer form. It has been postulated that the RCL of ATIII changes conformation upon binding H5 to a "canonical" conformation similar to that seen in the crystal structure of the related serpin, ␣ 1 -antitrypsin (8,7,40). Recent evidence suggests that although P1 Arg of ATIII may indeed reorientate upon binding H5, this alone does not explain the increased rate of interaction between the serpin and fXa (41,42).…”

Asp- and Glu-specific Novel Dipeptidyl Peptidase 11 of Porphyromonas gingivalis Ensures Utilization of Proteinaceous Energy Sources

“…First, the predominant proteolytic activities of Kgp and Rgps (22)(23)(24)(25) are believed to digest nutritional proteins into oligopeptides. Subsequently, oligopeptides are processed by dipeptidyl peptidase IV (EC 3.4.14.5, DPPIV) (26), DPP7 (27) and prolyl tripeptidyl peptidase-A (PTP-A) (28,29).…”