Abstract
The two plasma inhibitors, protein Z-dependent protease inhibitor (ZPI) and tissue factor pathway inhibitor (TFPI), effectively inhibit the activity of activated factor X (FXa); however, neither inhibitor exhibits any reactivity with the homologous protease activated factor IX (FIXa). In this study, we investigated the molecular basis for the lack of reactivity of FIXa with these plasma inhibitors and discovered that unique structural features within residues of the 39-loop are responsible for restricting the inhibitor specificity of FIXa. This loop in FXa is highly acidic and contains three Glu residues at positions 36, 37, and 39. On the other hand, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. We discovered that replacing residues of the 39-loop (residues 31-41) of FIXa with corresponding residues of FXa renders the FIXa chimera susceptible to inactivation by both ZPI and TFPI. Thus, the inactivation rate of the FIXa chimera by ZPI in the presence of protein Z (PZ), negatively charged membrane vesicles, and calcium ions approached the same diffusion-limited rate (>10(7) m(-1) s(-1)) that has been observed for the PZ-dependent inhibition of FXa by ZPI. Interestingly, sequence alignments indicated that, similar to FXa, residue 36 is a Glu in both mouse and bovine FIXa and that both proteases are also susceptible to inhibition by the PZ-ZPI complex. These results suggest that structural features within residues of the 39-loop contribute to the resistance of FIXa to inhibition by plasma inhibitors ZPI and TFPI.
Highlights
FXa and FIXa have homologous structures, but only FIXa is resistant to inhibition by plasma inhibitors
We evaluated the reactivity of the FIXa-FX39-loop chimera with the two specific inhibitors of FXa: Z-dependent protease inhibitor (ZPI) and tissue factor pathway inhibitor (TFPI), which are known to participate in the physiological regulation of FXa during both the initiation and amplification phases of the clotting cascade
In this study we have demonstrated that unique structural features within residues of the 39-loop make a key contribution to determining the specificity of human FIXa interaction with the FXa-specific inhibitors in plasma
Summary
FXa and FIXa have homologous structures, but only FIXa is resistant to inhibition by plasma inhibitors. Sequence alignments indicated that, similar to FXa, residue 36 is a Glu in both mouse and bovine FIXa and that both proteases are susceptible to inhibition by the PZZPI complex These results suggest that structural features within residues of the 39-loop contribute to the resistance of FIXa to inhibition by plasma inhibitors ZPI and TFPI. It binds to the active site of its target protease through an exposed reactive center loop followed by undergoing a conformational change that leads to entrapment of the protease in the form of an inactive covalent serpin-protease complex [5–7] Another serpin that contributes to regulation of the clotting cascade is protein Z (PZ)-dependent protease inhibitor (ZPI) [8]. These results suggest that the residues of the 39-loop contribute to the lack of reactivity of human FIXa with the ZPI-PZ complex
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