Role of P2 glycine in determining the specificity of antithrombin reaction with coagulation proteases

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Abstract

Structural data suggests that bulky hydrophobic residues at the S2–S4 sub-sites of factor Xa (fXa) restrict the preference of this pocket for small and non-polar residues like Gly at the P2 position of substrates and inhibitors. However, kinetic studies monitoring the cleavage specificity of 10-residue peptides by fXa have identified Phe as the most preferred P2 residue and Gln-Phe-Arg-Ser-Leu-Ser as the most preferred P3–P3′ residues for recognition by fXa. To determine whether this mechanism of specificity is also true for fXa reaction with antithrombin (AT), we prepared two AT mutants having either a Phe at the P2 or Gln-Phe-Arg-Ser-Leu-Ser at the P3–P3′ positions of the reactive center loop. Inhibition kinetic studies indicated that the reactivity of P2-Phe with fXa was significantly (∼5-fold) impaired, however, the P3–P3′ mutant exhibited 1.5-fold improved reactivity with the protease, suggesting cooperative effects between P3–P3′ residues influence the P2 specificity of AT. Substitution of Tyr-99 of fXa with a Gly dramatically impaired the reactivity of fXa with wild-type AT, but improved its reactivity with the serpin mutants in the absence, but not in the presence of pentasaccharide. AT with a P2-Phe inhibited thrombin with >150-fold impaired reactivity, however, the defect was restored by either pentasaccharide or by replacing Leu-99 of thrombin with a Gly. The P3–P3′ mutant rapidly inhibited factors VIIa and XIa independent of pentasaccharide. These results indicate that P2-Gly plays a key role in determining the S2 sub-site specificity and target protease selectivity of AT in circulation.

Introduction

Antithrombin (AT) is a serine protease inhibitor (serpin) in plasma that regulates the activities of the serine proteases of both intrinsic and extrinsic pathways of the blood coagulation cascade [1], [2], [3], [4]. AT inhibits its target coagulation proteases by a branched pathway, suicide substrate inhibition mechanism in which a Michaelis-type enzyme–inhibitor complex, formed in the first reaction step, is converted to a covalent acyl-enzyme intermediate complex in the second step of the reaction [5]. Similar to interaction of serine proteases with true substrates, a typical salt-bridge between Asp-189 at the S1 sub-site of coagulation proteases and an Arg at the P1 position (nomenclature of Schechter and Berger [6]) of the AT reactive center loop (RCL) accounts for the specificity of the initial interaction [5], [7]. However, unlike reaction with true substrates, attack of P1-Arg by the catalytic Ser-195 in the second step induces a conformational change in the serpin which traps the protease in the form of inactive acylated complex [8], [9]. Since most of the substrates and inhibitors of coagulation proteases contain an Arg at the P1 position, thus other residues surrounding the scissile bond must contribute to determinants of specificity of these proteases [10]. Structural and mutagenesis data have indicated that differences in the P3–P3′ residues of the scissile bonds are partly responsible for determining the specificity of coagulation reactions [11], [12], [13], [14]. The molecular basis for such a specificity is known to be due to existence of variant residues in the extended binding pocket of coagulation proteases which can interact with the P3–P3′ residues of the scissile bonds [10], [15]. In support of this hypothesis, two residues at positions 99 and 192 of coagulation proteases have been demonstrated to be critical for determining the P2 and P3 recognition specificity of procoagulant and anticoagulant proteases [16], [17]. Thus, mutagenesis of both of these residues of coagulation proteases or the P3–P3′ residues of substrates and inhibitors is known to alter the specificity of catalytic reactions [17], [18].

Structural and mutagenesis data have indicated that a Gly at the P2 position of the AT RCL is required for an effective interaction of the serpin with fXa in both the absence and presence of heparin [19], [20]. The preference for a small residue at the P2 position of AT appears to be due to presence of bulky residues including Tyr-99 and Phe-174 at the P2 binding pocket of fXa which spatially limits the recognition specificity of this pocket for only small and non-polar residues, like Gly [17], [21]. Consistent with this hypothesis, fXa cleavage sites on the physiological substrates including prothrombin and protease activated receptor-2 also possess Gly at the P2 positions. However, a recent kinetic study monitoring the specificity of the cleavage of 10-residue fluorogenic synthetic peptides with an Arg at the P1 position by fXa identified Phe as the most preferred P2 residue and Gln-Phe-Arg-Ser-Leu-Ser (AT-QFRSLS) as the most preferred P3–P3′ residues for fXa in these substrates [22]. To determine whether the results with the small peptide substrates hold true for interaction of fXa with natural macromolecules, we prepared two RCL mutants of AT in one of which P2-Gly was replaced with Phe and in the other P3–P3′ residues were replaced with optimal residues, identified by the amidolytic activity assay [22]. Characterization of these mutants in inhibition studies with fXa suggest that cooperative effects between P3–P3′ residues influence the P2 recognition specificity of AT and that a Gly at the P2 position is the most preferred residue for interaction with fXa in the presence of pentasaccharide. Further studies showed that AT-QFRSLS rapidly inhibits factors VIIa and XIa independent of pentasaccharide, but reacts slower with thrombin, suggesting a critical role for P3–P3′ residues in modulating the specificity of AT in reaction with different coagulation proteases in circulation.

Section snippets

Materials and methods

Expression and purification of recombinant proteins. Recombinant human AT was expressed in HEK-293 cells using the RSV-PL4 expression/purification vector system as described [13]. The RCL mutants of AT with a Phe substituting for the native Gly at the P2 position (AT-P2-Phe) and Gln-Phe-Arg-Ser-Leu-Ser (AT-QFRSLS) replacing the native P3–P3′ residues (AGRSLN) of AT were constructed by standard PCR mutagenesis methods and expressed using the same vector system. Wild-type and mutant serpins were

Results and discussion

Wild-type and mutant AT derivatives were expressed in HEK-293 cells and purified to homogeneity by a combination of HPC4 immunoaffinity and HiTrap-Heparin column chromatography as described [13]. SDS–PAGE analysis under non-reducing conditions suggested that the recombinant serpins have been purified to homogeneity and that both migrate with a relative molecular mass identical to that of wild-type AT (data not shown). Both mutants formed stable complexes with fXa in both the absence and

Acknowledgments

This work was supported by grants awarded by the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL 62565 and HL 68571 to ARR).

The authors have no conflict of interests to declare.

References (34)

Cited by (1)

  • Heparin affinity of factor VIIa: Implications on the physiological inhibition by antithrombin and clearance of recombinant factor VIIa

    2011, Thrombosis Research
    Citation Excerpt :

    We confirmed that activation of antithrombin by pentasacharide or heparin accelerates the antithrombin inhibition of rFVIIa by a conformational activation mechanism [32]. However, antithrombin shows some preference for other proteases such as FXa, thrombin or even FIXa, since it has already been stated that the residues of the reactive center loop within antithrombin are a determinant for the specificity towards different coagulation proteases [42]. Our results support the role that long chain heparins may have for the effective physiological inhibition of rFVIIa, the formation of rFVIIa-antithrombin complexes and the clearance of rFVIIa.

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