Histone H4 promotes prothrombin autoactivation

Abstract

Recent studies have documented the ability of prothrombin to spontaneously convert to the mature protease thrombin when Arg-320 becomes exposed to solvent for proteolytic attack upon mutation of residues in the activation domain. Whether prothrombin autoactivation occurs in the wild-type under conditions relevant to physiology remains unknown. Here, we report that binding of histone H4 to prothrombin under physiological conditions generates thrombin by autoactivation. The effect is abrogated by mutation of the catalytic Ser-525 and requires the presence of the Gla domain. Fluorescence titrations document direct binding of histone H4 to prothrombin with an affinity in the low nm range. Stopped flow data and luminescence resonance energy transfer measurements indicate that the binding mechanism obeys conformational selection. Among the two conformations of prothrombin, collapsed and fully extended, histone H4 binds selectively to the collapsed form and induces a transition toward a new conformation where the distance between Ser-101 in kringle-1 and Ser-210 in kringle-2 increases by 13 Å. These findings confirm the molecular plasticity of prothrombin emerged from recent structural studies and suggest that different conformations of the inter-kringle linker domain determine the functional behavior of prothrombin. The results also broaden our mechanistic understanding of the prothrombotic phenotype observed during cellular damage due to the release of histones in the blood stream. Prothrombin autoactivation induced by histone H4 emerges as a mechanism of pathophysiological relevance through which thrombin is generated independently of activation of the coagulation cascade.

Keywords: Blood Coagulation Factors; Enzyme Mechanisms; Protein Conformation; Prothrombin; Thrombin.

FIGURE 1.

Histone H4 promotes autoactivation of prothrombin under physiological conditions. A, prothrombin (0.1 mg/ml) was incubated with histone H4 (2 μm) in 150 mm NaCl, 5 mm CaCl₂, 20 mm Tris, pH 7.4, at 37 °C, and the reaction was followed by SDS-PAGE under reducing conditions as a function of time as indicated (h). The generation of thrombin was confirmed by cleavage of prothrombin (ProT) at Arg-155 and Arg-284 to generate the intermediates prethrombin-1 (Pre-1, 50 kDa, N-terminal sequence detected SEGSS) and prethrombin-2 (Pre-2, 38 kDa, N-terminal sequence detected TFGSG), respectively, as revealed by N-terminal sequencing. A third lower band was also identified by N-terminal sequencing and assigned to fragment 1.2 (F1.2, 34 kDa, N-terminal sequence detected ANTFL). Autoactivation requires histone H4, the catalytic Ser-525 (S195) and the Gla domain, because it is not observed in Gla domainless prothrombin (GD-ProT), prethrombin-1 or prethrombin-2. Cleavage of histone H4 by thrombin explains why autoactivation does not reach completion even after 84 h. B, schematic representation of prothrombin and its auto-proteolytic products upon incubation with histone H4. Cleavage at Arg-155 generates prethrombin-1 (Pre-1) and fragment 1 (F1). Cleavage at Arg-284 produces prethrombin-2 (Pre-2) and fragment 1.2 (F1.2), and additional cleavage of prethrombin-2 at Arg-320 generates the A and B chains of the mature enzyme thrombin. Direct cleavage of Arg-320 generates the B chain and fragment 1.2.A (F1.2.A) of meizothrombin, where fragment 1.2 remains attached to the A chain. C, formation of active enzyme confirmed by spectrophotometric analysis. Time point aliquots of the prothrombin autoactivation experiments were tested for activity toward the thrombin specific chromogenic substrate FPR (filled circles). Initial velocities were transformed in concentrations by using a thrombin standard curve. No activity was detected in the absence of histone H4 (open circles).

FIGURE 2.

Histone H4 binding to prothrombin. A, prothrombin mutant S101C/S210C (20 nm) labeled with Alexa Fluor 647 at both Cys residues was excited at 650 nm in the absence (solid line) or presence (dotted line) of 0.5 μm histone H4. Upon complex formation, the fluorescence intensity decreases 30%. Addition of 3.5 μm unfractioned heparin (gray solid line) neutralizes the effect of histone H4 and restores the original spectrum of prothrombin. B, histone H4 binds to prothrombin to a single site (N = 1.2 ± 0.2) with an apparent equilibrium dissociation constant K_d,app = 9 ± 1 nm. Data refer to three different prothrombin concentrations (5 nm, open circles; 20 nm, gray circles; 200 nm, black circles) and are plotted as intrinsic fluorescence F normalized by F₀ to facilitate comparison. Data were analyzed simultaneously according to Equations 1 and 2 in the text with eight independent parameters and best fit values: K_d,app = 9 ± 1 nm, N = 1.2 ± 0.2 and (open circles), F₀ = 2.1 ± 0.1 MV (1 MV = 10⁶ volts); ΔF_max = −0.86 ± 0.02 MV (gray circles); F₀ = 6.6 ± 0.1 MV, ΔF_max = −2.6 ± 0.1 MV; (black circles) F₀ = 12 ± 1 MV, ΔF_max = −4.8 ± 0.1. Experimental conditions were as follows: 150 mm NaCl, 5 mm CaCl₂, 0.1% PEG8000, 20 mm Tris, pH 7.4, at 25 °C.

FIGURE 3.

Kinetic mechanism of histone H4 binding to prothrombin. A, kinetic traces in the 0–10-s time scale of histone H4 binding to the prothrombin mutant S101C/S210C labeled with Alexa Fluor 647 at both Cys residues. Shown are the traces obtained by mixing 20 nm prothrombin with 100 nm (red), 200 nm (cyan), and 300 nm (magenta) histone H4. Solid lines were drawn according to a double exponential fit. Experimental conditions were as follows: 150 mm NaCl, 0.1% PEG 8000, 50 mm Tris, pH 7.4, at 25 °C. The inset gives the distribution of residuals for double exponential fits to the kinetic trace for the mixing of prothrombin with 100 nm histone H4. Double exponential fits rectify deviations from the experimental data present in the single exponential fits. B, plots of the two independent relaxations, fast (red) and slow (cyan), derived from the double exponential fits as a function of histone H4 concentration. Solid lines were drawn according to Scheme 1 in the text depicting a conformational selection mechanism, using the expressions in Equation 6 of Ref. with best fit parameter values: k₁₂ = 0.6 ± 0.2 s⁻¹, k₂₁ = 0.5 ± 0.1 s⁻¹, k_on = 5.5 ± 0.5 μm⁻¹s⁻¹, and k_off = 0.04 ± 0.01 s⁻¹. The resulting value of the apparent equilibrium dissociation constant, K_d,app, for this mechanism was derived from Equation 3 in the text as 13 ± 1 nm, in agreement with the value derived from fluorescence titrations under identical solution conditions (see Fig. 2).

FIGURE 4.

LRET measurements of histone H4 binding to prothrombin. Semilog plot of lifetime data for the LRET donor-acceptor pair conjugated to residues 101 in kringle-1 and 210 in kringle-2 of the full-length prothrombin mutant S101C/S210C (150 nm) in the absence (A) or presence (B) of 0.9 μm histone H4. The donor quenching (red dots) and acceptor sensitization (gray dots) curves of prothrombin both fit to a triple-exponential decay (green line) and two populations, collapsed and fully extended (5). Decomposition of the triple exponential fit into its individual components (dotted lines) reveals a short relaxation with a lifetime τ₁ = 28 ± 3 μs associated with the collapsed conformation and a longer relaxation with τ₂ = 272 ± 8 μs associated with the fully extended conformation. The third, slowest relaxation with τ₃ = 603 ± 9 μs corresponds to the donor only control. Binding of histone H4 to prothrombin does not alter the short relaxation but selectively perturbs the longer relaxation that is replaced by a shorter decay with a lifetime of 170 ± 5 μs. The amplitudes A₁ for τ₁ and A₂ for τ₂, which define the distribution between the two conformations, also change in favor of the newly generated species bound to histone H4. In the absence of histone H4, the ratio between collapsed and fully extended conformations is 0.85, consistent with the 1.2 ratio measured by stopped-flow for the ProT₂:ProT₁ distribution (see Fig. 3). In the presence of saturating concentrations of histone H4, the fully extended conformation disappears and the collapsed conformation partially converts to the new conformation to a final 0.35 ratio (collapsed:new). Best fit parameters for the triple-exponential decay curves are as follows: A, A₁ = 13,400 ± 200, τ₁ = 28 ± 3 μs; A₂ = 950 ± 20, τ₂ = 272 ± 8 μs; A₃ = 367, τ₃ = 603 ± 9 μs; B, A₁ = 22,900 ± 200, τ₁ = 28 ± 3 μs; A₂ = 6950 ± 70, τ₂ = 170 ± 5 μs; A₃ = 650 ± 8, τ₃ = 603 ± 9 μs. Experimental conditions were as follows: 150 mm NaCl, 5 mm CaCl₂, 0.1% PEG 8000, 20 mm Tris, pH 7.4, at 37 °C. C, proposed mechanism of histone H4 binding to prothrombin based on rapid kinetics and LRET measurements. When free in solution, prothrombin exists in at least two conformations where the distance between Ser-101 in kringle-1 and Ser-210 in kringle-2 is either partially collapsed (≤34 Å, corresponding to ProT₂ in Scheme 1) or fully extended (54 Å, corresponding to ProT₁ in Scheme 1), as reported recently (5). Histone H4 selectively binds to the collapsed conformation of prothrombin and then induces a conformational change that increases the distance between the two reporters (yellow and cyan stars) to 47 Å. The donor-acceptor distances were derived from the Föster equation (Equation 6 in the text). The induced fit step in the kinetic mechanism detected by LRET measurements is not detected in the stopped flow measurements (see Fig. 3) because the associated relaxation is spectroscopically silent.