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Characterization of a heparin-binding site on the catalytic domain of factor XIa: mechanism of heparin acceleration of factor XIa inhibition by the serpins antithrombin and C1-inhibitor.

ABSTRACT: Heparin accelerates inhibition of factor XIa (fXIa) by the serpins antithrombin (AT) and C1-inhibitor (C1-INH) by more than 2 orders of magnitude. The mechanism of the heparin-mediated acceleration of fXIa inhibition by these serpins is incompletely understood, as heparin appears to interact with both the catalytic and noncatalytic domains of the protease. We replaced the basic residues of the fXIa 170 loop (Lys-170, Arg-171, Arg-173, Lys-175, and Lys-179; chymotrypsin numbering) with Ala, using an expression system that allows separation of the fXIa catalytic domain (CD) from noncatalytic domains. Heparin-mediated inhibition of 170 loop CD variants with AT was impaired 3-10-fold relative to that of the wild-type (CD-WT). In reactions with C1-INH, Arg-171 was the most critical residue contributing approximately 2-3-fold to heparin-mediated inhibition of CD-WT. A template mechanism did not fully account for the effect of heparin with either serpin, as the second-order inhibition rate constants did not exhibit a characteristic bell-shaped dependence on heparin concentration. Further studies revealed that the C1-INH inhibition of full-length fXIa containing Ala substitutions for basic residues of the 148 loop is not enhanced by heparin. Inhibition by AT of a full-length fXIa variant containing an Ala substitution for Arg-37 in the fXIa CD was approximately 5-fold greater than for wild-type fXIa in the absence of heparin. These results suggest that basic residues of the fXIa 170 loop form a heparin-binding site and that the accelerating effect of heparin on inhibition of fXIa by AT or C1-INH may be mediated by charge neutralization and/or allosteric mechanisms that overcome the repulsive inhibitory interactions of serpins with basic residues on the fXIa 148 and 37 loops.

SUBMITTER: Yang L

PROVIDER: S-EPMC2654577 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Characterization of a heparin-binding site on the catalytic domain of factor XIa: mechanism of heparin acceleration of factor XIa inhibition by the serpins antithrombin and C1-inhibitor.

Yang Likui L Sun Mao-fu MF Gailani David D Rezaie Alireza R AR

Biochemistry 20090201 7

Heparin accelerates inhibition of factor XIa (fXIa) by the serpins antithrombin (AT) and C1-inhibitor (C1-INH) by more than 2 orders of magnitude. The mechanism of the heparin-mediated acceleration of fXIa inhibition by these serpins is incompletely understood, as heparin appears to interact with both the catalytic and noncatalytic domains of the protease. We replaced the basic residues of the fXIa 170 loop (Lys-170, Arg-171, Arg-173, Lys-175, and Lys-179; chymotrypsin numbering) with Ala, using ...[more]

PMID: 19178150

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Project description:Essentials Heparin-binding site (HBS) variants of antithrombin (AT) are associated with thrombosis risk. HSB variants have, in general, normal progressive inhibitory activity but reduced heparin affinity. Thrombosis in HSB carriers has been primarily attributed to the loss of heparin cofactor activity. Results here demonstrate that HSB variants of AT also lack anti-inflammatory signaling functions. SUMMARY:Background Several heparin-binding site (HBS) variants of antithrombin (AT) have been identified that predispose carriers to a higher incidence of thrombosis. Thrombosis in carriers of HBS variants has been primarily attributed to a loss in their heparin-dependent anticoagulant function. Objective The objective of this study was to determine whether HSB mutations affect the anti-inflammatory functions of variants. Methods Two HBS variants of AT (AT-I7N and AT-L99F), which are known to be associated with a higher incidence of thrombosis, were expressed in mammalian cells and purified to homogeneity. These variants were characterized by kinetic assays followed by analysis of their activities in established cellular and/or in vivo inflammatory models. The possible effects of mutations on AT structure were also evaluated by molecular modeling. Results The results indicated that, whereas progressive inhibitory activities of variants were minimally affected, their heparin affinity and inhibitory activity in the presence of heparin were markedly decreased. Unlike wild-type AT, neither AT variant was capable of inhibiting activation of nuclear factor-?B or downregulation of expression of cell adhesion molecules in response to lipopolysaccharide (LPS). Similarly, neither variant elicited barrier protective activity in response to LPS. Structural analysis suggested that the L99F substitution locally destabilizes AT structure. Conclusions It is concluded that the L99F mutation of AT is associated with destabilization of the serpin structure, and that the loss of anti-inflammatory signaling function of the HBS variants may also contribute to enhanced thrombosis in carriers of HBS mutations.

Project description:Inactivation of plasmin by a 3-4-fold molar excess of antithrombin III follows pseudo-first-order kinetics and the apparent rate constants are proportional to the concentration of the inhibitor. Heparin accelerates the inactivation reaction without changing its pseudo-first-order character, and the apparent rate constants are also proportional to the concentration of the polysaccharide. Heparin results in a minimum 20-fold rate enhancement of the reaction between plasmin and antithrombin III when the concentrations of heparin and plasmin are approx. 0.5mum and 1mum respectively. Heparin at a molar concentration well below that of plasmin still accelerates the reaction: one molecule of the polysaccharide is able to facilitate the inactivation of about 100 molecules of plasmin. Heparin must bind to plasmin to accelerate the plasmin-antithrombin III reaction, since the modification of four to five lysine residues of the enzyme inhibits the rate-enhancement effect of heparin and the dissociation of heparin-plasmin complex decreases the inactivation rate of plasmin. Increasing the concentration of antithrombin III, at a constant amount of heparin, results in increase of the inactivation rate. By contrast, the effect of increasing the amount of plasmin in the presence of constant amount of heparin and antithrombin III is such that higher plasmin-to-heparin ratios are associated with lower rates of inactivation. It seems, therefore, that to obtain ;optimal' conditions for fast enzyme inactivation, the amount of heparin should be matched to plasmin rather than to antithrombin III. Arrhenius plots of the plasmin-antithrombin III reaction are linear both in the absence and presence of heparin, at concentrations of 1 or 2mug/ml, over a range of 26K. Under these experimental conditions, heparin increases activation entropy. The findings show that heparin seems to fulfil some criteria that are characteristic for biological catalysis: binding, reaction-rate enhancement (increasing activation entropy), recycling of heparin (effectiveness of non-stoichiometric amounts of the polysaccharide) and specificity.

Dataset Information

Characterization of a heparin-binding site on the catalytic domain of factor XIa: mechanism of heparin acceleration of factor XIa inhibition by the serpins antithrombin and C1-inhibitor.

Publications

Characterization of a heparin-binding site on the catalytic domain of factor XIa: mechanism of heparin acceleration of factor XIa inhibition by the serpins antithrombin and C1-inhibitor.

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