Histone H4 Promotes Prothrombin Autoactivation*

Background: The molecular origin of the prothrombotic properties of histones is unknown. Results: Histone H4 interacts with prothrombin and promotes autoactivation. Conclusion: The prothrombotic properties of histone H4 are due to direct interaction with prothrombin. Significance: Prothrombin autoactivation has pathophysiological relevance. 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.

Prothrombin, or coagulation factor II, is a 579-residue-long vitamin K-dependent zymogen that circulates in the blood at a concentration of 0.1 mg/ml (1). The protein has a multidomain structure comprising fragment 1 containing the Gla domain and kringle-1, fragment 2 containing kringle-2, and the protease domain containing the A chain and the catalytic B chain. In the penultimate step of the coagulation cascade, prothrombin is proteolytically converted to the active protease thrombin by the prothrombinase complex composed of the protease factor Xa and the cofactor Va assembled on the surface of platelets in the presence of Ca 2ϩ (2). The conversion involves cleavage at two residues: Arg-271 located in a linker region connecting kringle-2 to the A chain and Arg-320 in the activation domain of the A chain. Cleavage at Arg-271 sheds fragment 1 and fragment 2 and generates the inactive precursor prethrombin-2. The alternative cleavage at Arg-320 separates the A and B chains that remain connected through a disulfide bond and yields the active intermediate meizothrombin. Under physiological conditions on the surface of platelets, activation of prothrombin proceeds via the prethrombin-2 intermediate (3,4). The recent crystal structure of prothrombin supports this preferred pathway of activation based on the different solvent accessibility of the two sites of cleavage and the distinct electrostatic properties of the epitope recognizing factor Xa in prothrombin and prethrombin-2 (5). Of particular importance is the observation that Arg-320 in the activation domain is not accessible to proteolysis in solution. Burial of Arg-320 acts as a safety mechanism to prevent prothrombin autoactivation (6) and directs prothrombinase to cleave at Arg-271 first to generate thrombin via the inactive prethrombin-2 intermediate.
The recently discovered property of prothrombin and other inactive thrombin precursors prethrombin-1 and prethrombin-2 to autoactivate has mechanistic significance. Mutations in the activation domain of prethrombin-2, prethrombin-1, and prothrombin that expose Arg-320 to solvent elicit a spontaneous conversion of the zymogen to the mature protease. The conversion typically unfolds over several hours, but in some cases, it is extremely rapid and completes during purification (6). The conversion is initiated by the zymogen itself by virtue of the pre-existing equilibrium between inactive (E*) and active (E) forms (7) and is then propagated and amplified by the mature enzyme. Whether autoactivation takes place in the wild-type protein under the influence of specific cofactors, as observed in other zymogens (8,9), remains an unresolved issue of potential physiological significance because it would afford a mechanism of thrombin generation that bypasses activation of the coagulation cascade. Cofactor-induced autoactivation represents an alternative strategy for thrombin generation compared with that used by Staphylococcus aureus, an important human pathogen implicated in sepsis and endocarditis, whose cofactor staphylocoagulase activates prothrombin by inserting its N-terminal peptide into the activation pocket to allosteri-cally induce a functional catalytic triad without cleaving at Arg-320 (10). We therefore screened a number of molecules relevant to blood pathophysiology for the ability to specifically promote prothrombin autoactivation.
During cell necrosis, a broad range of modulators such as polyamines, RNA, and histones are released into the extracellular media where they interact with many players of inflammation, sepsis, and thrombosis. Direct administration of histones to mice elicits lethal prothrombotic responses, thereby suggesting a direct link with clotting factors (11). Histones promote activation of factor VII activating protease in vivo (8) and have been suggested to interact with fibrinogen (12,13), prothrombin (12), thrombomodulin, and protein C (14). Histones have been postulated to enhance thrombin generation indirectly by reducing the thrombomodulin-dependent activation of protein C anticoagulant pathway (14) or directly through platelet dependent mechanisms involving the Toll-like receptors TLR2 and TLR4 (15). Here, we show that histone H4 promotes prothrombin autoactivation by interacting directly with the zymogen, and we elucidate the underlying mechanism. The results broaden our understanding of the prothrombotic role of histone H4 and offer the first example of a physiological cofactor of prothrombin autoactivation.

MATERIALS AND METHODS
Numbering-All prothrombin residues in this study are numbered sequentially based on the sequence of the zymogen. The corresponding number according to chymotrypsinogen for residues in the protease domain (A and B chains) is noted by parentheses, e.g. the catalytic Ser is identified as Ser-525 .
Materials-Recombinant full-length prothrombin (residues 1-579) wild-type and mutants S525A (S195A) and S101C/ S210C, Gla domainless prothrombin (residues 45-579) and prethrombin-1 were cloned into the pNUT vector using restriction sites HindIII and NdeI, transfected into baby hamster kidney cells by lipofection and selected by methotrexate. Prethrombin-2 wild-type and mutant S195A were cloned into the pET-21-b vector using the same restriction enzymes and expressed in Escherichia coli as described elsewhere (16). The nucleotide sequence of the constructs was confirmed by DNA sequencing. Addition of vitamin K to the full-length prothrombin media ensured correct ␥-carboxylation of the Gla domain. Purification of the recombinant proteins was carried out by affinity chromatography, ion exchange chromatography, and size exclusion chromatography, as described previously (16 -20). Homogeneity and chemical identity of final preparations were verified by SDS-PAGE and by RP-HPLC mass spectrometry analysis, giving a purity of Ͼ98%. Recombinant histone H3 and histone H4 were purchased from New England Biolabs. Histones were thawed and buffer exchanged immediately before use. Long-chain synthetic polyphosphate was differentially solubilized as described previously (21). Polymer lengths ranged from ϳ50 to 1500 phosphates, with a modal length of ϳ650 phosphates. Polyphosphate concentration was expressed in terms of the concentration of phosphate monomers (monomer formula, NaPO 3 ). Spermidine was purchased from Sigma-Aldrich. Protein concentrations were determined by reading at 280 nm with molar extinction coefficients ⑀ ϭ 111,280 M Ϫ1 cm Ϫ1 for recombinant prothrombin (molecular mass, 72,000 kDa) and ⑀ ϭ 5,120 M Ϫ1 cm Ϫ1 for histone H4 (molecular mass, 11237 kDa).
Autoactivation of Prothrombin-Prothrombin at physiological concentration (0.1 mg/ml) was incubated at 37°C with spermidine (40 M), RNA (10 g/ml), DNA (10 g/ml), polyphosphate (10 M), and histones H3 and H4 (2 M) in buffer containing 150 mM NaCl, 5 mM CaCl 2 , 20 mM Tris, pH 7.4 (TBS-Ca 2ϩ ). Prothrombin autoactivation was followed for up to 150 h. The reaction leading to generation of mature enzyme and depletion of zymogen was quenched at different times with 15 l of NuPAGE LDS buffer containing ␤-mercaptoethanol as reducing agent. Samples were processed by SDS-PAGE. Gels were stained with Coomassie Brilliant Blue R-250. Identity of the bands was confirmed by N-terminal sequencing. The same samples were tested for activity using the thrombin specific chromogenic substrate H-D-Phe-Pro-Arg-p-nitroanilide (FPR). The release of p-nitroanline was followed at 405 nm, and the amount of active enzyme was quantified with a standard reference curve.
Fluorescence Titration Studies-The prothrombin mutant S101C/S210C was labeled with Alexa Fluor 647 after selective reduction of the engineered cysteines as described elsewhere (5). The labeled protein was separated from the unreacted dye and aggregates by size exclusion chromatography. Incorporation of the probe was confirmed by reading at 650 nm where Alexa Fluor 647 absorbs with an extinction coefficient ⑀ ϭ 239,000 M Ϫ1 cm Ϫ1 . The derivatization yield at the two sites was estimated by the ratio of readings at 280/650 nm and found to be 91%. The labeled mutant was tested in the prothrombinase complex assay and returned kinetic parameters comparable with the recombinant wild-type and plasma prothrombin (data not shown). Titration measurements were performed in a 300-l cuvette at a protein concentration of 5, 20, or 200 nM in the presence of 150 mM NaCl, 5 mM CaCl 2 , 0.1% PEG 8000, 20 mM Tris, pH 7.4, at 25°C. The protein was excited at 650 nm (slits 5/10, exposure time of 0.02 s), and the emission spectrum was recorded between 660 and 750 nm. No photobleaching was observed under these conditions. Measurements were perfomed on a Fluoromax-4 spectrofluorimeter (Horiba Scientific, Edison, NJ) and analyzed with Origin (version 8.1, OriginLab, Northampton, MA) using Equation 1, where F is the fluorescence reading, F 0 is the value of F in the absence of histone H4, ⌬F max is the total change in fluorescence upon saturation, and shows the fractional saturation defined in terms of the apparent equilibrium dissociation constant, K d,app , the total concentrations of prothrombin, p t , and histone H4, h t , and the histone H4:prothrombin stoichiometry N. Histone H4 binding curves obtained at different values of p t were analyzed simultaneously to derive the independent parameters K d,app and N.
Stopped Flow Studies-Stopped flow fluorescence measurements were carried out with an Applied Photophysics SX20 spectrometer, using an excitation of 650 nm and a cutoff filter at 665 nm. Samples of prothrombin mutant S101C/S210C labeled with Alexa Fluor 647 at a final concentration of 20 nM were mixed 1:1 with 60-l solutions of the same buffer (150 mM NaCl, 0.1% PEG 8000, 50 mM Tris, pH 7.4, at 25°C) containing variable concentrations of histone H4. Each trace was determined in quadruplicate. A base line, established by mixing 20 nM prothrombin with buffer in a 1:1 ratio, was subtracted from each kinetic trace to control negligible amounts of photodegradation. Fits to single and double exponential equations were carried out using software provided by Applied Photophysics. Final k obs values were taken as the average from a minimum of three independent titrations. Two independent relaxations were detected as a function of histone H4 concentration, and the resulting k obs values were fit simultaneously using Mathematica (version 9.0.1.0) to the conformational selection mechanism (22,23), where k on (M Ϫ1 s Ϫ1 ) is the second-order rate constant for histone H4 binding and k off (s Ϫ1 ) is the first-order rate of dissociation of the complex into the parent species. The strength of the interaction is quantified by the equilibrium dissociation constant K d (M) defined as the ratio k off /k on . The species ProT 1 and ProT 2 denote two pre-existing conformations of prothrombin with histone H4 binding exclusively to ProT 2 . The rate constants k 12 and k 21 refer to the transitions from ProT 1 to ProT 2 and backward, with the ratio r ϭ k 21 /k 12 quantifying the population of ProT 1 relative to ProT 2 . The apparent equilibrium dissociation constant, K d,app associated with Scheme 1, is given by the linkage relationship (22) shown in Equation 3, which indicates that the value measured experimentally always overestimates the intrinsic equilibrium dissociation constant, K d , for the interaction.

Luminescence Resonance Energy Transfer (LRET) Studies-
The prothrombin mutant S101C/S210C and its labeled forms with the donor (Eu 3ϩ )AMCA-DTPA and acceptor Alexa Fluor 647 were prepared as described in detail elsewhere (5). LRET 3 measurements were performed with a custom-built two-channel spectrofluorometer with a pulsed nitrogen laser (NL100, Stanford Research Systems, Sunnyvale, CA) as an excitation source (24). Measurements were performed in a 300-l cuvette in 20 mM Tris, 150 mM NaCl, 5 mM CaCl 2 , 0.1% PEG 8000, pH 7.4 at 37°C. The concentration of labeled proteins was 10 -75 nM. Donor emission was recorded at 617 nm using a 620 nm, 10-nm bandwidth interference filter (Oriel, Stratford, CT), and acceptor emission was recorded at 670 nm using a 670 nm, 10 nm bandwidth interference filter (Oriel). Decays for donoronly samples were mono-exponential and were analyzed according to Equation 4, where A is the amplitude of the decay and is the luminescence lifetime. Decays of donors in the presence of acceptor and decays of sensitized acceptor emission were triple-exponential in the absence or presence of histone H4. Donor and sensitized acceptor decay curves were fitted simultaneously using global nonlinear regression with Origin (version 8.1). The energy transfer (E) was calculated from measurements of luminescence lifetime of the donor in the absence ( d ) and presence of acceptor ( da ) as shown in Equation 5.
The distance R between donor and acceptor was calculated according to the Förster equation below, where R 0 is the distance where the energy transfer E ϭ 0.5 (50%) and is equal to 55 Å for a completely randomized orientation of the donor-acceptor pair used (24,25).

RESULTS
During response to traumatic injury, sepsis, inflammation, and cell necrosis, the levels of histones, RNA, and polyamines become elevated in the blood (8,9,11,14,15,26) and correlate with thrombosis, inflammation, and multiple organ dysfunction (27,28). We explored the possibility that some of these components express their prothrombotic function via direct interaction with prothrombin. Autoactivation of prothrombin at physiological concentration (0.1 mg/ml) was monitored following incubation with histone H3, histone H4, spermidine, polyphosphate, RNA, or DNA, mindful of the autoactivation effects elicited by some of these molecules on other zymogens (29). Histone H4 was unique in promoting conversion of prothrombin to the mature protease thrombin and produced three new bands in SDS electrophoresis (Fig. 1), similar to the results recently obtained upon mutations of the activation domain leading to exposure of Arg-320 and autoactivation (6). N-terminal sequencing identified these new bands as prethrombin-1, prethrombin-2, and fragment 1.2. Prothrombin samples incubated with histone H4 at different time intervals showed increasing activity levels against the thrombin specific chromogenic substrate FPR (Fig. 1). Control experiments with prothrombin without histone H4 or the prothrombin mutant S525A (S195A) in the presence of histone H4 failed to show autoactivation ( Fig. 1), ruling out contamination of the sample with trace amounts of thrombin or other proteases. The control with prothrombin S525A (S195A), with the catalytic Ser replaced by Ala, indicates that autoactivation is initiated by prothrombin itself through the intrinsic activity of the zymogen. This was confirmed by a strong dependence of autoactivation on the prothrombin concentration, as expected for a reaction initiated by the zymogen itself as a catalyst (data not shown). Histone H3 also promoted autoactivation but over a time scale three times longer than that observed with histone H4 (data not shown), in agreement with the observation that the procoagulant effect of histones is due mainly to histone H4 (14).
Previous studies have shown a requirement for the Gla domain in the binding of histones to vitamin K-dependent proteins (12,14). Accordingly, autoactivation of prothrombin by histone H4 required the presence of the Gla domain and was not observed with Gla domainless prothrombin, or the thrombin precursors prethrombin-1 and prethrombin-2 that lack fragment 1 or both fragments 1 and 2 (Fig. 1), respectively.
The disappearance of the band corresponding to histone H4 in the gel during prothrombin autoactivation was due to proteolytic cleavage by the mature enzyme thrombin because no degradation was observed with prothrombin S525A (S195A). Activated protein C is currently the only enzyme known to target histones in the blood and able to counter their prothrombotic effects and cytotoxicity (11). The uniqueness of this effect should be reconsidered in view of the data reported in Fig. 1. The ability of thrombin to degrade histone H4 explains why prothrombin autoactivation does not proceed to completion (Fig. 1).
The evidence that histone H4 interacts with prothrombin is compelling but indirect. Histones are positively charged and may interact non-specifically with the surface of prothrombin that is mostly negatively charged, as recently revealed by the x-ray crystal structure (5). Evidence of a direct interaction of histone H4 with prothrombin comes from fluorescence titrations using the prothrombin mutant S101C/S210C engineered recently for quantitative LRET measurements (5). The mutant labeled with the dye Alexa Fluor 647 at both Cys residues was used first for direct fluorescence titrations (Fig. 2). Upon interaction with histone H4, the fluorescence of prothrombin decreases 30% and produces binding curves with an apparent equilibrium dissociation constant K d,app ϭ 9 Ϯ 1 nM and a stoichiometry N ϭ 1.2 Ϯ 0.1 indicating binding to a single site. Binding of histone H4 is reversed completely by addition of heparin that is known to interact strongly with histones (30).
Information on the mechanism of interaction of histone H4 with prothrombin was obtained from rapid kinetics measurements. Binding of histone H4 to the prothrombin mutant S101C/S210C labeled with Alexa Fluor 647 induces a significant fluorescence change consistent with a double exponential relaxation to equilibrium (Fig. 3). Two relaxations accessible to experimental measurements show distinct dependence on the histone H4 concentration (Fig. 3): one is fast and increases linearly at high concentrations, and the other is slow and increases hyperbolically. The concentration dependence of the two relaxations is consistent with a binding mechanism obeying conformational selection (22, 23) as described in Scheme 1: prothrombin undergoes a pre-existing equilibrium between two conformations, ProT 1 and ProT 2 , of which only ProT 2 specifically interacts with histone H4. The scenario agrees well with the recent crystal structure of prothrombin documenting conformational flexibility of the zymogen due to extreme mobility of the linker domain connecting kringle-1 and kringle-2 (5). The data in Fig. 3 enable derivation of important parameters for the binding interaction and the linked conformational transitions (22,23). The slope of the straight line in the fast relaxation gives the second order rate constant of association k on ϭ 5.5 Ϯ 0.5 M Ϫ1 s Ϫ1 and the asymptotic value of the slow relaxation gives the value of k 12 ϭ 0.6 Ϯ 0.2 s Ϫ1 for the conversion of ProT 1 to ProT 2 . A global fit of the two relaxations to Equation 6 of Ref. 22 yields the remaining parameters in Scheme 1: the rate constant for the reverse ProT 2 to ProT 1 transition is k 21 ϭ 0.5 Ϯ 0.1 s Ϫ1 , and the rate of dissociation of the complex into the parent species ProT 2 and histone H4 is k off ϭ 0.04 Ϯ 0.01 s Ϫ1 . These values give a K d,app ϭ 13 Ϯ 1 nM (see Equation 3), which is very close to the value of K d,app ϭ 9 Ϯ 1 nM derived from fluorescence titrations under identical solution conditions (Fig. 2). The two conformations of prothrombin, ProT 1 and ProT 2 , detected by stopped flow are populated as k 21 /(k 12 ϩ k 21 ) ϭ 45% and k 12 /(k 12 ϩ k 21 ) ϭ 55%, respectively. Notably, this distribution is similar to that involving the collapsed and fully extended conformations of prothrombin identified recently by LRET measurements (5). However, stopped flow data are unable to address whether ProT 1 and ProT 2 in Scheme 1 have any connection with the fully extended and collapsed conformations of prothrombin.
To further explore the mechanistic implications of the results from rapid kinetics data, the interaction of histone H4 with prothrombin was investigated by LRET measurements of the mutant S101C/S210C labeled with the donor (Eu 3ϩ )AMCA-DTPA and acceptor Alexa Fluor 647 as dyes. The recent crystal structure of prothrombin along with LRET measurements have revealed that kringle-1 moves similar to a dumbbell relative to the rest of the zymogen due to the extreme flexibility of the linker connecting the two kringles (5). As a result, prothrombin assumes two alternative conformations in solution: a fully extended conformation with a population of 55% where Ser-101 in kringle-1 and Ser-210 in   DECEMBER 13, 2013 • VOLUME 288 • NUMBER 50 kringle-2 are separated by a distance of 54 Ϯ 2 Å, and a collapsed conformation with a population of 45% where Ser-101 in kringle-1 and Ser-210 in kringle-2 are separated by a distance of Յ34 Å or are at least 20 Å closer than in the fully extended conformation (5). LRET measurements carried out at physiological temperature in the absence or presence of a saturating concentration (0.9 M) of histone H4 show a drastic effect on the distribution of prothrombin conformers. Three independent relaxations are detected experimentally in each case (Fig. 4). In the absence of histone H4, the slowest lifetime of 603 s refers to the decay of the donor-only control, the short lifetime of 28 Ϯ 3 s with 45% population has a distance between Ser-101 in kringle-1 and Ser-210 in kringle-2 of Յ34 Å, and the long lifetime of 272 Ϯ 8 s with 55% population has a distance of 54 Ϯ 2 Å. These results are practically identical to those reported recently (5). Under saturating concentrations of histone H4 the lifetime of the collapsed conformation remains unchanged, but a new lifetime of 170 Ϯ 5 s corresponding to a distance between the probes of 47 Ϯ 2 Å replaces the one per-taining to the fully extended conformation. Binding of histone H4 occurs exclusively to the collapsed conformation, followed by an induced fit transition into a new more extended form where the distance between the probes in kringle-1 and kringle-2 increases by 13 Å. LRET measurements enable assignment of ProT 1 in Scheme 1 as the fully extended conformation of prothrombin and identify ProT 2 with the collapsed conformation. The relative distributions of these two conformations are close to 1:1 based on stopped flow and LRET measurements. Upon binding to ProT 2 , histone H4 causes prothrombin to assume a new conformation (Fig. 4). The overall mechanism of histone H4 recognition calls for a mixed kinetic scheme where conformational selection is followed by an induced fit step (Fig. 4) that is spectroscopically silent. These findings provide evidence that histone H4 interacts specifically with prothrombin by selecting the collapsed conformation and promoting autoactivation via an induced fit mechanism that optimizes the conformation of the initial prothrombin-histone H4 complex. . 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 M 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 1 ϭ 28 Ϯ 3 s associated with the collapsed conformation and a longer relaxation with 2 ϭ 272 Ϯ 8 s associated with the fully extended conformation. The third, slowest relaxation with 3 ϭ 603 Ϯ 9 s 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 Ϯ 5 s. The amplitudes A 1 for 1 and A 2 for 2 , 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 ProT 2 :ProT 1 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, A 1 ϭ 13,400 Ϯ 200, 1 ϭ 28 Ϯ 3 s; A 2 ϭ 950 Ϯ 20, 2 ϭ 272 Ϯ 8 s; A 3 ϭ 367, 3 ϭ 603 Ϯ 9 s; B, A 1 ϭ 22,900 Ϯ 200, 1 ϭ 28 Ϯ 3 s; A 2 ϭ 6950 Ϯ 70, 2 ϭ 170 Ϯ 5 s; A 3 ϭ 650 Ϯ 8, 3 ϭ 603 Ϯ 9 s. Experimental conditions were as follows: 150 mM NaCl, 5 mM CaCl 2 , 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 (Յ34 Å, corresponding to ProT 2 in Scheme 1) or fully extended (54 Å, corresponding to ProT 1 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 Fö ster 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.

DISCUSSION
Recent studies have documented the ability of thrombin precursors prothrombin, prethrombin-1 and prethrombin-2 to autoactivate (6,16). Autoactivation is started by the zymogen itself because it is specifically abrogated by replacement of the catalytic Ser-525 (Ser-195) with Ala. The reaction is then propagated by thrombin and rapidly amplified (6). Autoactivation is found in other zymogens such as proprotein convertases furin and kexin type 9 (31)(32)(33), plasma hyaluronan-binding protein (26), recombinant factor VII (34), and the membrane-bound matriptases (35,36). The pre-existing equilibrium of the trypsin fold between the E* (inactive) and E (active) forms (7,37) explains autoactivation in terms of the small intrinsic activity of the zymogen (6). However, in the case of prothrombin, the question remains as to the physiological relevance of autoactivation given that Arg-320 (Arg-15) is not accessible to proteolytic attack in the wild-type (5). The results reported in this study demonstrate that autoactivation of prothrombin can be triggered in the wild-type by histone H4 over a time scale of ϳ8 h. The effect is slow but physiologically relevant because the high concentration (0.1 mg/ml) of prothrombin in the blood provides a reservoir for continuous thrombin generation by autoactivation. It is also possible that autoactivation in vivo unravels under a much shorter time scale because of conditions favoring sequestration of prothrombin by histone H4 or the presence of other cofactors yet to be identified. Other zymogens autoactivate upon interaction with external cofactors. Hyaluronan-binding protein (factor VII activating protease) autoactivates upon binding of histones (8), negatively charged molecules such as heparin and RNA (9, 29, 38 -40), and spermidine (41). Coagulation factors XI and XII autoactivate upon binding of RNA (9). Histone H4 specifically requires the Gla domain to promote autoactivation of prothrombin and likely induces exposure of Arg-320 (Arg-15) to solvent via a long range allosteric effect that propagates from the Gla domain to the activation domain situated nearly 80 Å away (5). The effect is mechanistically relevant and draws attention to the presence of long range communication within the prothrombin molecule mediated by the extreme flexibility of its structure (5).
Fluorescence and LRET measurements show that histone H4 binds selectively to the collapsed conformation of the zymogen with an affinity in the low nM range and induces a new conformation where the distance between Ser-101 in kringle-1 and Ser-210 in kringle-2 increases from Յ34 to 47 Å. The new conformation is distinct from the fully extended form of prothrombin where the same distance is 54 Å (5). Histone H4 binds to prothrombin according to a kinetic mechanism that includes conformational selection and induced fit (Fig. 4), a highly versatile scheme whose properties span the entire landscape allowed for independent relaxations (23). The conformational selection component of the kinetic mechanism is supported by stopped flow measurements where histone H4 binds selectively to one of the two possible pre-existing conformations of prothrombin. The result is mechanistically important because it reinforces the notion that trypsin-like zymogens exist in multiple conformations as probed by binding to the active site (16) or auxiliary domains such as the Gla domain reported here.
Whether a linkage exists in prothrombin between the E*-E equilibrium affecting the active site region (7,37,42) and the equilibrium between the collapsed and fully extended conformations made possible by the flexibility of the linker between kringle-1 and kringle-2 (5) remains to be established. The possibility is intriguing as it suggests a long range communication between the catalytic domain of the zymogen and its auxiliary domains, thereby offering a much needed structural perspective on the mechanism of prothrombin activation.
Because histone H4 promotes autoactivation, we speculate that the new conformation produced by induced fit from the original collapsed form exposes Arg-320 (Arg-15) to solvent for proteolytic attack and stabilizes the active site in the E form. In this context, it becomes of interest to ponder what conformation of prothrombin is stabilized upon interaction with the prothrombinase complex and whether the selection depends on the environment in which prothrombin activation takes place. Based on the results reported here, the fully extended conformation of prothrombin is not prone to autoactivation and may be the one stabilized by assembly of the prothrombinase complex on the surface of platelets, thereby making cleavage at Arg-271 preferred over cleavage at Arg-320 under conditions relevant to physiology (3,4). On the other hand, conditions where factor Xa and cofactor Va induce conformational transitions in prothrombin similar to those induced by histone H4 may direct cleavage at Arg-320 and promote thrombin generation along the meizothrombin pathway, where cleavage at Arg-320 precedes cleavage at Arg-271 (1). In this scenario, selection between the two sites of cleavage at Arg-271 or Arg-320 would depend on their solvent accessibility and on the conformation of the linker domain between kringle-1 and kringle-2.
The induced fit component of the kinetic mechanism of histone H4 recognition also deserves attention. Stopped flow data do not detect such component, but that may be due to a lack of spectroscopic signal associated with it. Alternatively, the induced fit component may be a simplification of a more general mechanism where the new conformation stabilized by histone H4 binding to the collapsed form actually pre-exists in solution but in minuscule amounts. A mechanism where the pre-existing equilibrium includes such conformation present only as 1% of the total population would fit adequately the LRET data in Fig. 4 and the stopped flow data in Fig. 3, thereby obviating the need for induced fit. This argument is in line with the recent conclusion that conformational selection is always sufficient and induced fit is never necessary as a mechanism of ligand binding (23). Conclusive assessment of the role of induced fit in prothrombin function will require measurements of the underlying conformational transitions at the single molecule level.
The results reported here broaden our understanding of the prothrombotic activity of histones in the context of the well established link between inflammation and thrombosis (28). Infusion of intravenous histones into mice causes sepsis, thrombosis, and death (11). The concentration of histones in the blood reaches 20 M under pathological conditions (43) and is sufficient to saturate prothrombin and promote autoactivation leading to thrombin generation within few hours. Pro-longed exposure to histone H4 is equivalent to a slow infusion of small amounts of thrombin in the circulation, which eventually culminates in prothrombotic and proinflammatory effects (28). Prothrombin autoactivation induced by endogenous cofactors such as histone H4 or other mediators of inflammation yet to be identified demonstrates that thrombin can be generated by a mechanism that bypasses activation of the blood coagulation cascade.