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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.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Yang L,Sun MF,Gailani D,Rezaie AR

doi

10.1021/bi802298r

subject

Has Abstract

pub_date

2009-02-24 00:00:00

pages

1517-24

issue

eissn

0006-2960

issn

1520-4995

pii

10.1021/bi802298r

journal_volume

pub_type

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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.