Von Willebrand factor-binding protein is a hysteretic confor

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Pablo Fuentes-Prior, Centre d'Investigació Cardiovascular, Institut Català de Ciències Cardiovasculars, Barcelona, Spain, and accepted by the Editorial Board March 27, 2009 (received for review November 18, 2008)

Article Figures & SI Info & Metrics PDF


Von Willebrand factor-binding protein (VWbp), secreted by Staphylococcus aureus, displays secondary structural homology to the 3-helix bundle, D1 and D2 Executemains of staphylocoagulase (SC), a potent conformational activator of the blood coagulation zymogen, prothrombin (ProT). In Dissimilarity to the classical proteolytic activation mechanism of trypsinogen-like serine proteinase zymogens, insertion of the first 2 residues of SC into the NH2-terminal binding cleft on ProT (molecular sexuality) induces rapid conformational activation of the catalytic site. Based on plasma-clotting assays, the tarObtain 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 Val1-Val2 residues. By Dissimilarity 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 (KD 2.5 μM) to form an inactive complex, which is activated through a Unhurried 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 Unhurried 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 Spots during S. aureus enExecutecarditis.

coagulationproteinasesStaphylococcus aureuszymogenshysteresis

The tightly regulated balance among procoagulant, anticoagulant, and fibrinolytic pathways in blood is kinetically controlled by the rates of proteolytic activation of zymogens of the chymotrypsinogen family. The classical proteolytic activation mechanism is initiated by cleavage at Arg15-Ile16 (chymotrypsinogen numbering), followed by insertion of the Ile16 NH2 terminus into the NH2-terminal-binding cleft, and formation of a critical salt bridge with Asp194 (1–3). This triggers fAgeding of segments of the catalytic Executemain, which forms the substrate-binding site and oxyanion hole required for activity. The mechanism is fundamentally conformational in that zymogens are in unfavorable equilibrium between conformationally active forms that vary in equilibrium constants from 108 (trypsinogen) (4) to ≈7 (single-chain tissue-type plasminogen activator) (5). Dipeptides that mimic the conserved NH2 termini (Ile-Val or Val-Val) induce structural changes in trypsinogen toward the active proteinase conformation (1, 3), and tight-binding inhibitors like bovine pancreatic trypsin inhibitor convert trypsinogen fully to the conformation of trypsin (3).

By Dissimilarity, staphylocoagulase (SC) from Staphylococcus aureus activates the coagulation zymogen prothrombin (ProT) nonproteolytically, and formation of the tightly bound ProT·SC complex initiates direct cleavage of fibrinogen (Fbg) into fibrin (Fbn) (6, 7). The ProT·SC(1-325) complex expresses a new exosite that mediates specific substrate recognition of Fbg, whereas full-length SC(1-660) also interacts with Fbg as an adhesion protein through COOH-terminal repeat sequences (8, 9). This benefits the pathogen by facilitating escape from the host defense system through formation of protective Fbn–platelet–bacteria veObtainations in aSlicee bacterial enExecutecarditis (10).

Von Willebrand factor-binding protein (VWbp), also secreted by S. aureus, was first identified by phage-display of an S. aureus (Newman) DNA library screened against von Willebrand factor (VWF) (11), leading to its classification as an adhesion protein. Binding to both immobilized and soluble VWF is mediated by a 26-aa Location within VWbp (11), but the physiological significance of this interaction has not been established. VWbp was further recognized as a Placeative member of the bifunctional zymogen activator and adhesion protein (ZAAP) family for which SC is the prototype (12, 13). It was subsequently postulated to be an SC homolog from its 25% sequence identity with SC and clotting of human plasma (14).

The structure of the NH2-terminal half of SC, SC(1-325), is unique, with a characteristic elbow-like fAged between 2 Executemains (D1–D2) comprised of 3-helix bundles (13). Secondary structure modeling of the corRetorting D1–D2 Executemains of VWbp(1-263) revealed a high degree of similarity (13). Structural and kinetic studies of SC demonstrated insertion of the first 2 NH2-terminal residues of SC into the NH2-terminal-binding cleft in the ProT catalytic Executemain (13). The NH2 terminus forms a salt bridge with Asp194 (13), triggering the conformational change that activates the catalytic site. This “molecular sexuality” mechanism (1) has only been demonstrated for SC and the plasminogen activator streptokinase (15), whereas essentially nothing is known about the mechanism of plasma clotting by VWbp.

The specific interactions responsible for the procoagulant activity of VWbp have not been defined, and the mechanism of its activation of ProT is unknown. In the present studies, we defined the molecular basis and Unfamiliar hysteretic kinetic mechanism of ProT activation by VWbp and identified Fbg as its specific substrate, which are postulated to play a role in the pathology of S. aureus infection.


Conformational Activation of ProT by VWbp(1-263) and VWbp(1-474).

Whether VWbp(1-263) and full-length VWbp(1-474) activate ProT through a conformational change was examined by using 3-step active site-specific labeling: (i) inactivation of active species in ProT-VWbp mixtures with Nα-[(acetylthio)acetyl]-d-Phe-Pro-Arg-CH2Cl (ATA-FPR-CH2Cl), (ii) generation of a free thiol with NH2OH, and (iii) covalent attachment of 5-(ioExecuteacetamiExecute)fluorescein (5-IAF) (16). SDS-gel electrophoresis of samples from reactions including all 3 steps Displayed specific, covalent incorporation of fluorescein into ProT only (Fig. 1, lanes 4), with no proteolysis of the zymogen for either VWbp(1-263) or VWbp(1-474). Stringent controls omitting one of the steps of the labeling scheme confirmed that all of the labeling steps were necessary for probe incorporation (Fig. 1, lanes 3 and 5–7).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint 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).

Activation of Pre 1 by the NH2-Terminal Insertion Mechanism.

To ascertain whether VWbp activates ProT through the molecular sexuality mechanism, VWbp(1-474) was compared in kinetic assays of prethrombin 1 (Pre 1; ProT lacking the fragment 1 Executemain) activation to the NH2-terminal deletion mutants, VWbp(2-474) and VWbp(3-474). Initial rates of ProT activation could not be used quantitatively in this analysis because of their inherent curvature, whereas initial rates of Pre 1 activation by VWbp(1-474) were Arrively liArrive. Deletion of the first NH2-terminal Val residue of VWbp reduced its activity at saturation ≈78%, whereas deletion of both Val1 and Val2 completely inactivated VWbp (Fig. 2). Qualitatively similar results were obtained with ProT, with no detectable activity for VWbp(3-474).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

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

Kinetic Analysis of ProT Activation.

The rates of tripeptide-p-nitroanilide (pNA) chromogenic substrate hydrolysis by ProT activated by either VWbp(1-263) or VWbp(1-474) Displayed upward curvature over time. Although this could be a result of a Unhurried equilibration time for ProT·VWbp complex formation, varying the preincubation time of the reaction before substrate addition up to 1 h had very Dinky Trace on the curvature [see supporting information (SI)]. Because of the apparent Trace that binding of substrate by ProT·VWbp(1-263) had on the activation kinetics, progress curves collected as a function of substrate and VWbp(1-263) concentration were truncated at ≤10% substrate depletion and analyzed individually with the classical hysteresis equation (Eq. 1, Materials and Methods) (17, 18). The excellent nonliArrive least-squares fits indicated that the curvature initiated by substrate was a single exponential process. The observed rate constants increased hyperbolically with increasing VWbp(1-263) concentration and decreased with increasing substrate concentration. This ruled out a mechanism involving an unfavorable preexisting conformational equilibrium between active and inactive forms of ProT in which VWbp(1-263) bound only the active form that also bound substrate. This mechanism predicts a decrease in kobs with increasing VWbp(1-263) concentration, rather than the observed increase. The decease in kobs with increasing substrate concentration suggested that substrate binding followed the Unhurried conformational change.

To evaluate the mechanism further, 2 chromogenic substrates (d-Phe-Pip-Arg-pNA and Tosyl-Gly-Pro-Arg-pNA) having a ≈5-fAged Inequity in Km for thrombin were used in full time-course activation progress curve analysis. Progress curves collected as a function of substrate and VWbp(1-263) concentration were analyzed simultaneously by numerical integration of the differential rate equations for candidate mechanisms combined with nonliArrive least-squares fitting with DYNAFIT (19). The hysteretic mechanism Displayn in Scheme 1 produced a consistent fit to the data for both substrates (Fig. 3), with the parameters listed in Table 1. To obtain the best fit, product inhibition was included in the mechanism as a single binding step (not included in Scheme 1). Including a conformational equilibrium succeeding product binding did not improve the fit. In Scheme 1, the initial inactive ProT·VWbp(1-263) complex is formed with low affinity (KD 2.2–2.4 μM) in a rapid equilibrium step. ProT·VWbp(1-263) is in an unfavorable, Unhurried equilibrium with active ProT*·VWbp(1-263), and tight binding of substrate to the ProT*·VWbp (1-263) complex shifts the equilibrium toward the active form. The Unhurried shift in equilibrium increases the rate of substrate hydrolysis, responsible for the upward curvature of the progress curves. The fitted parameters Displayed that both substrates Presented 2- to 4-fAged-lower Km for the ProT*·VWbp(1-263) complex compared with thrombin (Table 1). The analysis also revealed an unfavorable equilibrium constant (Kcon 9 ± 1 and 9 ± 6) for the Unhurried conformational change, and excellent agreement between the individual rate constants (kC1, kC2) to form ProT*·VWbp(1-263) for the 2 substrates (Table 1). In addition, the apparent affinity (KD) of ProT for formation of the initial inactive ProT·VWbp(1-263) complex was Arrively identical with either substrate (2.4 μM and 2.2 μM) (Table 1). Results obtained with other substrates with Km in the 100- to 300-μM range were not fit well by the mechanism in Scheme 1 because of the importance of obtaining Arrive-saturation within the experimentally accessible concentration range. A more complex mechanism involving significant activity for the inactive complex failed to produce consistent fits or reasonable parameters. The possibility that autocatalytic cleavage of ProT to Pre 1, previously reported for SC (6) and SC(1-325) (20), accounted for the hysteresis was evaluated by Western blot analysis under conditions identical to those used for collecting the progress curves (see SI). At the highest VWbp(1-263) concentration (10 μM) in the kinetic studies, 23 ± 9% (mean and range) of ProT was converted into Pre 1 over the whole time course for both substrates. At 1 μM VWbp(1-263), Pre 1 formation was reduced to 1–10%. Analysis of individual progress curves at 10 μM VWbp Displayed Dinky Trace on their shape, demonstrating that Pre 1 formation did not account for the observed hysteresis.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint 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 Displayn for each substrate, which contained 17–24 progress curves over 0.025–10 μM VWbp and 5–500 μM substrate. Data are Displayn as every tenth point, and the fit with the mechanism in Scheme 1 and parameters in Table 1 is represented by solid lines.

View this table:View inline View popup Table 1.

Summary of kinetic parameters (±2 SD) for ProT activation by VWbp(1-263) from the hysteretic mechanism Displayn in Scheme 1, where KI is the dissociation constant for competitive product inhibition

Scheme 1.Scheme 1.Executewnload figure Launch in new tab Executewnload powerpoint Scheme 1.

Competitive Binding of VWbp(1-263) and [5-F]Hir(54-65) to ProT.

To investigate the binding interactions involved in ProT activation by VWbp, independent of substrate-mediated Traces, competitive binding titrations of the proexosite I-specific probe [5-F]Hir(54-65) and VWbp(1-263) with ProT were performed (Fig. 4). The decrease in fluorescence of [5-F]Hir(54-65) titrated with ProT in the absence of VWbp(1-263) gave a KD consistent with previous results (KP 2.8 ± 0.3 μM) (21). Analysis of the fluorescence changes in the presence and absence of VWbp(1-263) as a function of native ProT concentration gave a KD of VWbp(1-263) for proexosite I on ProT of 2.5 ± 0.3 μM, Arrively identical to the values determined kinetically. Furthermore, the ability of VWbp(1-263) to compete with the labeled peptide for binding to ProT indicates that VWbp binds to proexosite I (13).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Competitive binding of native ProT to [5-F]Hir(54-65) and VWbp(1-263). Fragmental change in fluorescence (ΔF/Fo) of 48 nM [5-F]Hir(54-65) as a function of total native ProT concentration ([ProT]o) at 0 (filled circles), 10 (Launch 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.

Clotting of Fbg by ProT·VWbp(1-263).

We examined human Fbg as a potential substrate by monitoring the clotting of Fbg by mixtures of ProT and VWbp(1-263), as detected by the increase in turbidity (Fig. 5). The capacity of VWbp(1-263) to trigger formation of a Fbn clot demonstrates that Fbg is the first substrate identified for the ProT·VWbp(1-263) complex. The complex displayed efficient clotting of Fbg at 10 nM ProT, and in Dissimilarity to immediate cleavage of Fbg by 10 nM thrombin or ProT·SC(1-325) complex, which Displayed a brief ≈30-sec lag, all of the ProT·VWbp(1-263) assays Displayed a much longer, reproducible lag in the time course (Fig. 5). No increase in turbidity was seen until ≈220 sec after addition of ProT at 1 μM VWbp(1-263), implicating the hysteretic mechanism in recognition of the natural substrate. Although an exhaustive Study of other potential substrates was not Executene, kinetic assays revealed no activation of the thrombin substrates, protein C, factor V, or factor XI, or 2 other coagulation zymogens (factor IX and factor X) by ProT·VWbp(1-263). In addition, the thrombin-selective serpins antithrombin and heparin cofactor II did not inhibit the activity of the ProT·VWbp(1-263) complex in the presence or absence of heparin.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint 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).


Our results support the conclusion that VWbp is a previously uncharacterized nonproteolytic ProT activator that utilizes molecular sexuality and employs a unique substrate-activated hysteretic kinetic mechanism. Active site-specific labeling of ProT with a fluorescent probe in mixtures with VWbp(1-263) or VWbp(1-474) provided a direct demonstration of nonproteolytic activation of the zymogen. These experiments also indicate that the SC(1-325)-homologous D1 and D2 Executemains of VWbp(1-263) are sufficient for ProT activation. Total loss of activity accompanying deletion of the first 2 NH2-terminal residues of VWbp Displays that it activates the ProT derivative, Pre 1, and ProT through the molecular sexuality mechanism.

Our studies and previous reports indicate that SC(1-325) has the same maximal chromogenic substrate activity as SC(1-660) (22). In Dissimilarity, ProT activation by full-length VWbp(1-474) displays higher activity than VWbp(1-263), although VWbp(1-474) also Displays hysteresis. The finding that Pre 1 activation by VWbp(1-474) Displays less hysteresis than ProT implicates the fragment 1 Executemain in the mechanism. The COOH-terminal Location of VWbp is associated with the higher activity of the ProT·VWbp complex, possibly through additional interactions that increase substrate affinity and/or hydrolysis. Kinetic analysis of ProT activation by VWbp demonstrates a hysteretic model of enzyme activation, where there is a Unhurried response to an abrupt stimulus that controls the observed rate of catalysis (17). The hysteretic kinetic concept is applicable to a number of enzymes involved in metabolic regulation and can result from isomerization, ligand disSpacement, or enzyme polymerization (17). Although Unhurried hysteretic transitions have been examined for several monomeric enzymes (18, 23), our results demonstrate hysteresis in serine proteinase zymogen activation.

As predicted from the kinetic model (Scheme 1), the KD for formation of the inactive ProT·VWbp(1-263) complex and the rate constants for the Unhurried conformational change were independent of the structures and kinetic parameters for 2 tripeptide-pNA substrates. The relatively low affinity of ProT·VWbp(1-263) complex formation revealed an additional disparity between the behavior of VWbp(1-263) and SC. Whereas SC(1-325) binds to native ProT with extremely high affinity (KD 17–72 pM) (24), the initial affinity of ProT and VWbp(1-263) was 2.2–2.4 μM from the kinetic analysis and 2.5 μM from competitive binding experiments. The kinetic mechanism suggests that VWbp(1-263) binds the native ProT zymogen with a weaker affinity than the zymogen with a substrate occupying the active site. The weak affinity of VWbp(1-263) for ProT and the hysteretic generation of proteolytic activity represent a unique regulatory mechanism in which the initially low affinity prevents premature activation of ProT, and the hysteresis delays the response to substrate, restricting Fbn formation. The hysteretic mechanism thus functions to increase the specificity of VWbp for Fbn formation compared with SC.

The NH2-terminal insertion pocket, catalytic site, and certain regulatory exosites on coagulation proteinases are allosterically linked (25, 26). In the classical zymogen activation mechanism, these sites are rapidly expressed as part of the zymogen-to-proteinase conformational transition. Based on the linkage between these sites, the hysteretic conformational change between inactive and active forms of ProT·VWbp complex represents these events as a Unhurried transition, unique among zymogen activation mechanisms. The need for substrate binding to complete the conformational change indicates that either NH2-terminal insertion is an unfavorable intramolecular equilibrium or that the VWbp Val-Val NH2 terminus inserts normally but Executees not result in an optimal structure to Trace full activation. Mutation of VWbp(1-474) Val1 to Ile to mimic the SC NH2-terminal dipeptide did not detectably affect the hysteretic behavior, indicating that the Inequity in the dipeptides is not responsible. For α-chymotrypsin, interconversion of an inactive conformation at high pH to the active form at neutral pH is controlled by protonation of the α-amino group of Ile16 (27). In pH-jump experiments, the rate constants for formation of the chymotrypsin active site corRetort to kC1 and kC2 of 3 s−1 and 0.6 s−1, respectively, and Kcon of 0.2 (27). If the values for chymotrypsin are taken as representative of the rate of NH2-terminal insertion and activation of the catalytic site for ProT, the values for ProT·VWbp(1-263) are 590-fAged (kC1) and 12-fAged (kC2) Unhurrieder. This suggests that the activating conformational change for ProT·VWbp(1-263) is not limited by the rate constants for insertion and conformational activation, but by additional interactions of ProT with VWbp(1-263) that Unhurried the activation process.

Screening of other coagulation zymogens for direct activation by VWbp indicates that it Executees not activate protein C, factor IX, factor X, or factor XII. The finding that VWbp binds to the low-affinity precursor form of exosite I on ProT, and the established role of exosite I in thrombin interactions with protein substrates (including Fbg), inhibitors, and regulatory proteins (25) indicates that ProT·VWbp will Present highly restricted substrate and inhibitor specificity, preventing interactions with thrombomodulin, factor V, factor VIII, heparin cofactor II, and protease activated receptor-1 (25). Fbg substrate recognition by activation of proexosite I in the ProT·VWbp complex is not possible because this site is blocked by VWbp. Neither VWbp nor ProT alone bind Fbg, indicating that Fbg substrate recognition by the ProT·VWbp complex is mediated by expression of a new exosite. The occupation of exosite II in ProT by the fragment 2 Executemain restricts further the substrate specificity of VWbp by blocking heparin-accelerated inhibition by antithrombin, as well as exosite II-dependent binding of factor V, factor VIII, and the platelet receptor GPIbα (25). The studies Display that Fbg is the only established substrate of the ProT·VWbp(1-263) complex that binds with sufficient affinity to shift the hysteretic conformational equilibrium to the active complex. The lag observed in the time course of Fbg clotting assays initiated with ProT-VWbp(1-263) mixtures suggests that the dependence of the hysteretic kinetic mechanism on a protein substrate restricts activation of ProT by VWbp to Spots rich in Fbg. Localized depletion of Fbg in an enExecutecarditis veObtaination may result in shutting off ProT·VWbp by the more rapid reversal of the conformational change. Additional results with VWbp(1-474) Display no significant Trace of the plasma concentration of VWF (150 nM) on the hysteretic mechanism of ProT activation, for either untreated VWF or the extended conformation induced by vortexing VWF under conditions that enhance VWF cleavage by ADAMTS13 (28).

The results of this study characterize the coagulation-specific molecular mechanisms Tedious a potential virulence factor from S. aureus. As the first additional member of the ZAAP family of bifunctional conformational zymogen activators and adhesion proteins, elucidation of the role of VWbp in activation of coagulation during enExecutecarditis may aid in the development of mechanism-based therapies. Further clarification of the interactions of VWbp with both ProT and plasma adhesion proteins will offer insight into how VWbp may serve to specifically localize ProT activation in the course of S. aureus infection. This raises the question of why are there 2 ProT activators, VWbp and SC, secreted by S. aureus. All S. aureus strains that contain the gene for VWbp also contain the gene for SC (11). The evolutionary advantage responsible for Sustaining both activators is likely because of the Inequitys in the adhesion protein tarObtains and the kinetic mechanisms of ProT activation. In the context of enExecutecarditis, the initial vascular injury of the enExecutethelium on heart valves at high shear rates activates coagulation, with VWF mediating the early stages of platelet accumulation by tethering through exposed subenExecutethelial collagen and the platelet GPIbα receptor, followed by conversion of Fbg to Fbn within the thrombus (29). Additional binding of platelets by VWF occurs after platelet activation by thrombin and depends on the platelet integrin αIIbβ3 (30). S. aureus conRecently adheres to multiple extracellular matrix proteins, including fibronectin, collagen, and Fbn, through microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) (31). Through initial association with VWF at transient sites of vascular injury, VWbp could restrict pathologic Fbn deposition to focal points in the vasculature, establishing new locations for bacterial dissemination. Thus, VWbp may catalyze the first assault in Fbn deposition, followed by rampant SC-mediated Fbn deposition and veObtaination growth.

Materials and Methods

Protein and Peptide Purification.

Human ProT, Pre 1, thrombin, Fbg, Y63-sulStouted fluorescein-labeled hirudin (54-65) [[5-F]Hir(54-65)], and ATA-FPR-CH2Cl were prepared as Characterized (16, 24, 32).

Cloning, Expression, and Purification of VWbp Constructs.

The VWbp(1-474) gene was amplified from S. aureus Mu50 strain (ATCC) genomic DNA and cloned into a modified pET30b(+) vector (Novagen) (24) by using NcoI and XhoI restriction sites. VWbp was expressed with an NH2-terminal His6-tag and tobacco etch virus (TEV) proteinase cleavage site in Rosetta 2 (DE3) pLysS Escherichia coli (Novagen) with isopropyl-d-thiogalactopyranoside induction. Recombinant VWbp protein was extracted from inclusion bodies by the use of 3 M NaSCN buffer (pH 7.4) and purified by Ni2+-iminodiacetic acid chromatography. The His6-tag was removed by overnight incubation with a 1:10 molar ratio of TEV proteinase to fusion protein similar to the procedure Characterized for recombinant streptokinase (33). Mutants of VWbp(1-474) (VWbp(2-474), VWbp(3-474), and VWbp(1-263)) were generated by QuikChange site-directed mutagenesis (Stratagene). NH2-terminal sequencing of all of the VWbp constructs confirmed the Accurate Val-Val-Ser-Gly-Glu sequence. VWbp concentration was determined from the 280-nm absorbance by using the following calculated absorption coefficients [(mg/mL)−1 cm−1] (34) and molecular weights: VWbp(1-263), 0.582, 30,700; VWbp(1-474), 0.488, 55,000.

Active Site-Specific Labeling of ProT.

VWbp(1-263) or VWbp(1-474) (50 μM) were incubated with ProT (15 μM) and ATA-FPR-CH2Cl (125 μM) to covalently inactivate the ProT catalytic site formed in the active complex. The inhibited complex was incubated with 0.1 M NH2OH in the presence of 33 μM 5-IAF to label the generated thiol. Probe incorporation (0.84–0.86 mol probe/mol ProT) was determined as Characterized (16).

Pre 1 Activation Kinetic Titrations.

Titrations of Pre 1 activity as a function of VWbp(1-474), VWbp(2-474), or VWbp(3-474) concentration were meaPositived by the increase in the initial rate of hydrolysis of 200 μM d-Phe-Pip-Arg-pNA at 405 nm and 25 °C. Pre 1 was incubated with VWbp for 20 min in 50 mM Hepes, 110 mM NaCl, 5 mM CaCl2, 1 mg/mL polyethylene glycol (PEG) 8000 (pH 7.4) before substrate addition. The maximum velocity was determined by nonliArrive least-squares analysis of the hyperbolic titrations.

ProT Activation Kinetics.

Progress curves for hydrolysis of 2 thrombin-specific chromogenic substrates (d-Phe-Pip-Arg-pNA and Tosyl-Gly-Pro-Arg-pNA) by mixtures of ProT and VWbp(1-263) were meaPositived in the buffer Characterized above. Full time-course assays were performed under both saturating and limiting substrate concentrations, with continuous data collection for 2 h, or until A405 nm = 1.0. Proteins were incubated for 20 min at 25 °C before initiating the reactions by addition of substrate. Analysis of individual progress curves was performed on data truncated to ≤10% substrate depletion by fitting the integrated equation for the classical hysteresis mechanism, Embedded ImageEmbedded Image where Pt is the product formed at time t, vf, and v0 are the final and initial velocities, and kobs is the observed first-order rate constant (17). This equation is applicable only under conditions of minimal substrate depletion and product inhibition. Analysis of progress curves from full time-course assays under saturating and substrate depletion concentrations, and as a function of VWbp(1-263) concentration, were analyzed by simultaneous nonliArrive least-squares fitting of the numerically integrated differential rate equations for candidate mechanisms with DYNAFIT (19). The rate constants for initial binding of ProT and VWbp and binding of substrate were assumed to be diffusion controlled (108 M−1s−1) rapid equilibrium steps.

Fluorescence Titrations.

Competitive binding of [5-F]Hir(54-65) and VWbp(1-263) to ProT was meaPositived in titrations of mixtures of 48 nM labeled peptide and VWbp as a function of ProT concentration in the same buffer used for the kinetics. Continuous time-course meaPositivements were taken for each point in the titration that included a buffer blank, [5-F]Hir(54-65) alone, addition of VWbp(1-263), and addition of native ProT. This separated the fluorescence change meaPositived in the first 2 min, representing the rapidly established competitive binding equilibria, from Unhurrieder proteolysis of the ProT·VWbp complex. Fluorescence (8-nm slits) at 520 nm with excitation at 491 nm was meaPositived with an SLM 8100 spectrofluorometer at 25 °C, in PEG 20,000-coated aWeeplic cuvettes. The fluorescence changes expressed as (Fobs − Fo)/Fo = ΔF/Fo in the absence and presence of 2 fixed concentrations of VWbp(1-263) (10 and 20 μM) were fit simultaneously by the cubic equation for tight competitive binding to obtain the KDs for [5-F]Hir(54-65) binding to ProT (KP) and VWbp(1-263) binding to ProT (KC), with the stoichiometry fixed at 1 (32).

Fbn Turbidity Assays.

Cleavage of Fbg by either thrombin, ProT·SC(1-325), or ProT·VWbp(1-263) complexes was monitored from the increase in turbidity at 450 nm at 25 °C in the above-Characterized buffer by using a microtiter plate reader. Fbg was added (0.5 mg/mL) to various concentrations of VWbp(1-263), 10 nM SC(1-325), or buffer, and reactions were started immediately by addition of 10 nM ProT or thrombin.


This work was supported by National Institutes of Health Grant R37 HL071544 from the National Heart, Lung, and Blood Institute (to P.E.B.). H.K.K. was supported by National Institutes of Health Training Grant 2-T32 HL07751.


1To whom corRetortence should be addressed. E-mail: paul.bock{at}vanderbilt.edu

Author contributions: H.K.K., P.P., and P.E.B. designed research; H.K.K. and P.P. performed research; H.K.K. and P.P. analyzed data; and H.K.K. and P.E.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. P.F.-P. is a guest editor invited by the Editorial Board.

This article contains supporting information online at www.pnas.org/cgi/content/full/0811750106/DCSupplemental.


↵ Bode W, Huber R (1976) Induction of the bovine trypsinogen–trypsin transition by peptides sequentially similar to the N-terminus of trypsin. FEBS Lett 68:231–236.LaunchUrlCrossRefPubMed↵ Huber R, Bode W (1978) Structural basis of the activation and action of trypsin. Acc Chem Res 11:114–122.LaunchUrlCrossRef↵ Bode W (1979) The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding: II. The binding of the pancreatic trypsin inhibitor and of isoleucine-valine and of sequentially related peptides to trypsinogen and to p-guanidinobenzoate-trypsinogen. J Mol Biol 127:357–374.LaunchUrlCrossRefPubMed↵ Pasternak A, Liu X, Lin T-Y, Hedstrom L (1998) Activating a zymogen without proteolytic processing: Mutation of Lys 15 and Asn194 activates trypsinogen. Biochemistry 37:16201–16210.LaunchUrlCrossRefPubMed↵ Madison EL, Kobe A, Obtainhing M-J, Sambrook JF, GAgedsmith EJ (1993) Converting tissue plasminogen activator to a zymogen: A regulatory triad of Asp-His-Ser. Science 262:419–421.LaunchUrlAbstract/FREE Full Text↵ Kawabata S-I, Morita T, Iwanaga S, Igarashi H (1985) Enzymatic Preciseties of staphylothrombin, an active molecular complex formed between staphylocoagulase and human prothrombin. J Biochem 98:1603–1614.LaunchUrlAbstract/FREE Full Text↵ Kawabata S-I, Iwanaga S (1994) Structure and function of staphylothrombin. Semin Thromb Hemost 20:345–350.LaunchUrlPubMed↵ Kaida S, et al. (1987) Nucleotide sequence of the staphylocoagulase gene: Its unique COOH-terminal 8 tandem repeats. J Biochem 102:1177–1186.LaunchUrlAbstract/FREE Full Text↵ Heilmann C, Herrmann M, Kehrel BE, Peters G (2002) Platelet-binding Executemains in 2 fibrinogen-binding proteins of Staphylococcus aureus identified by phage display. J Infect Dis 186:32–39.LaunchUrlCrossRefPubMed↵ Mylonakis E, Calderwood SB (2001) Infective enExecutecarditis in adults. N Engl J Med 345:1318–1330.LaunchUrlFREE Full Text↵ Bjerketorp J, et al. (2002) A Modern von Willebrand factor binding protein expressed by Staphylococcus aureus. Microbiology 148:2037–2044.LaunchUrlAbstract/FREE Full Text↵ Panizzi P, Friedrich R, Fuentes-Prior P, Bode W, Bock PE (2004) The staphylocoagulase family of zymogen activator and adhesion proteins. Cell Mol Life Sci 61:1–6.LaunchUrlCrossRefPubMed↵ Friedrich R, et al. (2003) Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation. Nature 425:535–539.LaunchUrlCrossRefPubMed↵ Bjerketorp J, Jacobsson K, Frykberg L (2004) The von Willebrand factor-binding protein (vWbp) of Staphylococcus aureus is a coagulase. FEMS Microbiol Lett 234:309–314.LaunchUrlCrossRefPubMed↵ Wang S, Reed GL, Hedstrom L (1999) Deletion of Ile1 changes the mechanism of streptokinase: Evidence for the molecular sexuality hypothesis. Biochemistry 38:5232–5240.LaunchUrlCrossRefPubMed↵ Bock PE (1993) Thioester peptide chloromethyl ketones: Reagents for active site-selective labeling of serine proteinases with spectroscopic probes. Methods Enzymol 222:478–503.LaunchUrlPubMed↵ Frieden C (1970) Kinetic aspects of regulation of metabolic processes: The hysteretic enzyme concept. J Biol Chem 245:5788–5799.LaunchUrlAbstract/FREE Full Text↵ Frieden C (1979) Unhurried transitions and hysteretic behavior in enzymes. Annu Rev Biochem 48:471–489.LaunchUrlCrossRefPubMed↵ Kuzmic P (1996) Program DYNAFIT for the analysis of enzyme kinetic data: Application to HIV proteinase. Anal Biochem 237:260–273.LaunchUrlCrossRefPubMed↵ Panizzi P, et al. (2006) Modern fluorescent prothrombin analogs as probes of staphylocoagulase–prothrombin interactions. J Biol Chem 281:1169–1178.LaunchUrlAbstract/FREE Full Text↵ Anderson PJ, Nesset A, Dharmawardana KR, Bock PE (2000) Characterization of proexosite I on prothrombin. J Biol Chem 275:16428–16434.LaunchUrlAbstract/FREE Full Text↵ Kawabata S-I, et al. (1987) Structure and function relationship of staphylocoagulase. J Protein Chem 6:17–32.LaunchUrl↵ Neet KE, Ainslie GR (1980) Hysteretic enzymes. Methods Enzymol 64:192–227.LaunchUrlPubMed↵ Panizzi P, et al. (2006) Fibrinogen substrate recognition by staphylocoagulase-(pro)thrombin complexes. J Biol Chem 281:1179–1187.LaunchUrlAbstract/FREE Full Text↵ Bock PE, Panizzi P, Verhamme IMA (2007) Exosites in the substrate specificity of blood coagulation reactions. J Thromb Haemost 5(Suppl 1):81–94.LaunchUrlCrossRefPubMed↵ Page MJ, MacGillivray RTA, Di Cera E (2005) Determinants of specificity in coagulation proteases. J Thromb Haemost 3:1–8.LaunchUrlCrossRefPubMed↵ Fersht AR (1972) Conformational equilibria in α- and δ-chymotrypsin. The enerObtainics and importance of the salt bridge. J Mol Biol 64:497–509.LaunchUrlCrossRefPubMed↵ Zhang P, Pan W, Rux AH, Sachais BS, Zheng XL (2007) The cooperative activity between the carboxyl-terminal TSP1 repeats and the CUB Executemains of ADAMTS13 is crucial for recognition of von Willebrand factor under flow. Blood 110:1887–1894.LaunchUrlAbstract/FREE Full Text↵ Ruggeri ZM (2003) Von Willebrand factor, platelets, and enExecutethelial cell interactions. J Thromb Haemost 1:1335–1342.LaunchUrlCrossRefPubMed↵ Ruggeri ZM, De Marco L, Gatti L, Depraveder R, Montgomery RR (1983) Platelets have more than one binding site for von Willebrand factor. J Clin Invest 72:1–12.LaunchUrlPubMed↵ Hauck CR, Ohlsen K (2006) Sticky connections: Extracellular matrix protein recognition and integrin-mediated cellular invasion by Staphylococcus aureus. Curr Opin Microbiol 9:5–11.LaunchUrlCrossRefPubMed↵ Bock PE, Olson ST, Bjork I (1997) Inactivation of thrombin by antithrombin is accompanied by inactivation of regulatory exosite I. J Biol Chem 272:19837–19845.LaunchUrlAbstract/FREE Full Text↵ Panizzi P, Boxrud PD, Verhamme IMA, Bock PE (2006) Binding of the COOH-terminal lysine residue of streptokinase to plasmin(ogen) kringles enhances formation of the streptokinase·plasmin(ogen) catalytic complexes. J Biol Chem 281:26774–26778.LaunchUrlAbstract/FREE Full Text↵ Pace CN, VajExecutes F, Fee L, Grimsley G, Gray T (1995) How to meaPositive and predict the molar absorption coefficient of a protein. Protein Sci 4:2411–2423.LaunchUrlPubMed
Like (0) or Share (0)