Von Willebrand factor-binding protein is a hysteretic conformational activator of prothrombin

Abstract

Von Willebrand factor-binding protein (VWbp), secreted by Staphylococcus aureus, displays secondary structural homology to the 3-helix bundle, D1 and D2 domains of staphylocoagulase (SC), a potent conformational activator of the blood coagulation zymogen, prothrombin (ProT). In contrast to the classical proteolytic activation mechanism of trypsinogen-like serine proteinase zymogens, insertion of the first 2 residues of SC into the NH(2)-terminal binding cleft on ProT (molecular sexuality) induces rapid conformational activation of the catalytic site. Based on plasma-clotting assays, the target zymogen for VWbp may be ProT, but this has not been verified, and the mechanism of ProT activation is unknown. We demonstrate that VWbp activates ProT conformationally in a mechanism requiring its Val(1)-Val(2) residues. By contrast to SC, full time-course kinetic studies of ProT activation by VWbp demonstrate that it activates ProT by a substrate-dependent, hysteretic kinetic mechanism. VWbp binds weakly to ProT (K(D) 2.5 microM) to form an inactive complex, which is activated through a slow conformational change by tripeptide chromogenic substrates and its specific physiological substrate, identified here as fibrinogen (Fbg). This mechanism increases the specificity of ProT activation by delaying it in a slow reversible process, with full activation requiring binding of Fbg through an exosite expressed on the activated ProT*.VWbp complex. The results suggest that this unique mechanism regulates pathological fibrin (Fbn) deposition to VWF-rich areas during S. aureus endocarditis.

Fig. 1.

Active site-specific labeling of ProT·VWbp(1-263) and ProT·VWbp(1-474) complexes assessed by SDS-gel electrophoresis. Fluorescence (A and C) and protein-stained (B and D) SDS gels for reactions containing 15 μM ProT and 50 μM VWbp(1-263) (A and B) or VWbp(1-474) (C and D). Reduced samples of ProT (lane 1) and VWbp (lane 2), ProT-VWbp inactivated with ATA-FPR-CH₂Cl (lane 3), and inhibited complex incubated with NH₂OH and 5-IAF (lane 4). Control samples were ProT incubated without VWbp (lane 5), omission of the NH₂OH step (lane 6), or the complex blocked with excess FPR-CH₂Cl before sequential incubation with ATA-FPR-CH₂Cl and 5-IAF (lane 7).

Fig. 2.

Pre 1 activation by NH₂-terminal truncation mutants of VWbp. Initial velocities (v_obs) of hydrolysis of 100 μM d-Phe-Pip-Arg-pNA are shown for mixtures of 1 nM Pre 1 as a function of VWbp(1-474) (filled circles), VWbp(2-474) (open circles), or VWbp(3-474) (filled triangles) concentration. The lines represent the least-squares fits of the quadratic binding equation.

Fig. 3.

Full time-course analysis of the kinetics of ProT activation by VWbp(1-263). (A) Progress curves for hydrolysis of d-Phe-Pip-Arg-pNA by mixtures of VWbp(1-263) and ProT, at 200 μM substrate (black points), contained 1 nM ProT and 0.025, 0.075, 0.15, 0.3, 0.6, 3, or 10 μM VWbp. Substrate depletion assays (red points) contained 1 nM ProT, 1 μM VWbp, and concentrations of 7, 20, 40, 78, or 116 μM substrate. (B) Progress curves for hydrolysis of 200 μM Tosyl-Gly-Pro-Arg-pNA (black points) contained 1 nM ProT and 0.1, 0.3, 0.5, 0.75, 1, 3, or 10 μM VWbp. Substrate depletion assays (red points) contained 1 nM ProT, 5 μM VWbp, and 25, 50, 75, 100, 150, or 500 μM substrate. Representative examples of the whole dataset are shown for each substrate, which contained 17–24 progress curves over 0.025–10 μM VWbp and 5–500 μM substrate. Data are shown as every tenth point, and the fit with the mechanism in Scheme 1 and parameters in Table 1 is represented by solid lines.

Scheme 1.

Fig. 4.

Competitive binding of native ProT to [5-F]Hir(54-65) and VWbp(1-263). Fractional change in fluorescence (ΔF/F_o) of 48 nM [5-F]Hir(54-65) as a function of total native ProT concentration ([ProT]_o) at 0 (filled circles), 10 (open circles), and 20 μM (filled triangles) VWbp(1-263). The lines represent the simultaneous fit by the cubic equation with the parameters given in the text.

Fig. 5.

Clotting of Fbg by mixtures of ProT and VWbp(1-263). Increase in turbidity for mixtures of 0.5 mg/mL Fbg and 10 nM thrombin (blue), 10 nM ProT (black), 10 nM ProT·SC(1-325) (red), or 10 nM ProT and VWbp concentrations of 0.1 (a), 0.3 (b), 0.5 (c), or 1 μM (d) (green).