Characterization of Heparin Binding Site Residues on the Catalytic Domain of Factor XIa

Abstract

The basic residues of the 170-helix (chymotrypsinogen numbering) in the catalytic domain of the procoagulant serine proteases are known to interact with heparin and facilitate their rapid inhibition by specific plasma serpins. The homologous loop of factor XIa possesses three basic residues (Lys-170, Arg-171 and Arg-173). To investigate the role of these residues in the heparin-mediated inhibition of fXIa by C1-inhibitor (C1-INH) and antithrombin (AT), we expressed the monomeric light chain fragment of fXIa containing only the catalytic domain of the protease in wild-type and mutant forms in which the three basic residues of the 170-helix have been substituted individually, or in combinations, with Ala. The catalytic properties of fXIa derivatives were compared to wild-type fXIa (fXIa-WT) with respect to their ability to hydrolyze the chromogenic substrate S2366 and to undergo inhibition by C1-INH and AT in the absence and presence of heparin. All mutants exhibited normal amidolytic activity, hydrolyzing S2366 with catalytic efficiencies similar to fXIa-WT. All mutants were also inhibited by both C1-INH and AT with normal rate constants in the absence of heparin. However, the heparincatalyzed inhibition of mutants by both serpins differed to varying degrees relative to fXIa-WT, with the Arg-171 to Ala substitution mutant exhibiting ~3-fold lower rate of inhibition by C1-INH and requiring ~3-fold higher concentration of heparin for observing optimal inhibitory effect. In reactions with AT, all three basic residues appeared to make similar contributions to the heparin-catalyzed inhibition of fXIa by the serpin as evidenced by all three mutants exhibiting dramatically lower inhibition rate constants in the presence of heparin. A bell-shaped heparin concentration dependence for the fXIa inhibition by both serpins suggested that the template effect of heparin primarily accounts for the acceleration mechanism. However, a comparison of the heparin bell-shaped dependence inhibition of the dimeric plasma-derived wild-type fXIa with that of the isolated single chain catalytic domain suggested that the optimal concentration of heparin for the acceleration of the protease has been increased from ~50 nM for the dimeric protease to ~250 nM for the isolated catalytic domain, possibly suggesting further contribution to affinity of the fXIaheparin interaction from the Apple-3 domain of the non-catalytic domain on which a binding-site for heparin has been reported.