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Originally published In Press as doi:10.1074/jbc.M705871200 on February 8, 2008

J. Biol. Chem., Vol. 283, Issue 15, 9531-9542, April 11, 2008
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Degradation of Circulating von Willebrand Factor and Its Regulator ADAMTS13 Implicates Secreted Bacillus anthracis Metalloproteases in Anthrax Consumptive Coagulopathy*

Myung-Chul Chung, Taissia G. Popova, Shelley C. Jorgensen, Li Dong, Vikas Chandhoke, Charles L. Bailey, and Serguei G. Popov1

From the National Center for Biodefense and Infectious Diseases, College of Sciences, George Mason University, Manassas, Virginia 20110

Received for publication, July 17, 2007 , and in revised form, January 29, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Pathology data from the anthrax animal models show evidence of significant increases in vascular permeability coincident with hemostatic imbalances manifested by thrombocytopenia, transient leucopenia, and aggressive disseminated intravascular coagulation. In this study we hypothesized that anthrax infection modulates the activity of von Willebrand factor (VWF) and its endogenous regulator ADAMTS13, which play important roles in hemostasis and thrombosis, including interaction of endothelial cells with platelets. We previously demonstrated that purified anthrax neutral metalloproteases Npr599 and InhA are capable of cleaving a variety of host structural and regulatory proteins. Incubation of human plasma with these proteases at 37 °C in the presence of urea as a mild denaturant results in proteolysis of VWF. Also in these conditions, InhA directly cleaves plasma ADAMTS13 protein. Npr599 and InhA digest synthetic VWF substrate FRETS-VWF73. Amino acid sequencing of VWF fragments produced by InhA suggests that one of the cleavage sites of VWF is located at domain A2, the target domain of ADAMTS13. Proteolysis of VWF by InhA impairs its collagen binding activity (VWF:CBA) and ristocetin-induced platelet aggregation activity. In plasma from anthrax spore-challenged DBA/2 mice, VWF antigen levels increase up to 2-fold at day 3 post-infection with toxigenic Sterne 34F2 strain, whereas VWF:CBA levels drop in a time-dependent manner, suggesting dysfunction of VWF instead of its quantitative deficiency. This conclusion is further supported by significant reduction in the amount of VWF circulating in blood in the ultra-large forms. In addition, Western blot analysis shows proteolytic depletion of ADAMTS13 from plasma of spore-challenged mice despite its increased expression in the liver. Our results suggest a new mechanism of anthrax coagulopathy affecting the levels and functional activities of both VWF and its natural regulator ADAMTS13. This mechanism may contribute to hemorrhage and thrombosis typical in anthrax.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacillus anthracis, the causative agent of anthrax, is a zoonotic, Gram-positive, spore-forming Bacillus. It is capable of causing a highly lethal systemic infection in people and experimental animals upon inhalation of the spores. The pathological mechanisms underlying high mortality of inhalation anthrax infection are complex and remain insufficiently understood. The major virulence factors, lethal toxin (LT)2 and edema toxin (ET) produced by B. anthracis can cause death of experimental animals with symptoms resembling those of anthrax infection. Therefore, many studies have been focused on the cytotoxic effects of LT, including the cells of monocyte lineage (peripheral blood monocytes, macrophages, and dendritic cells) (1) and epithelial cells (2). It has also been reported that the LT-induced cell death could cause a massive release of the pro-inflammatory cytokines, such as the tumor necrosis factor {alpha} (TNF{alpha}), which may result in lethality (3), but recent data clearly indicate that the intoxication mediated by LT in mice and rats is independent of either the macrophage susceptibility or the TNF{alpha} release (1). The animals die from a hypoxic liver failure and a circulatory collapse, although the mediators of death remain unknown. Other secreted bacterial factors including proteases and hemolysins have also been reported as substantial contributors to anthrax pathology and mortality (4, 5).

In the effort to investigate anthrax toxicity, our attention was attracted to the mechanisms, which are not cytotoxic per se but could potentially result in the lethal imbalance of the host homeostasis and failure of vital organs. Relevant to this hypothesis, blood vessel leakage, hemorrhage, and disseminated intravascular coagulopathy (DIC), also referred to as consumptive coagulopathy, are well known pathologic features in human anthrax cases (6, 7). DIC is often associated with intravascular thrombosis formation accompanied by a decrease in the amount of circulating thrombocytes (thrombocytopenia (TP)), tissue hypoxia, activation of the fibrinolytic pathways, depletion of coagulation factors, inability to maintain hemostasis, and overt bleeding. Clinical manifestations range from asymptomatic compensated DIC to shock and death. In the nonhuman primate model the infection with a toxigenic B. anthracis Sterne 34F2 also results in changes of vascular permeability, DIC, and systemic inflammation (8).

Similar to the infectious process, in the animal models of anthrax toxemia, LT and ET have been implicated in vascular leakage (9, 10) and TP. Mice and rats administered LT developed TP, which was stronger in non-survivors relative to survivors. Culley et al. (11) reported that TP in mice was invariably associated with death. Other pathophysiological changes included hypofibrinogenemia, elevated prothrombin time, and elevated activated partial thromboplastin time, indicating DIC and/or circulatory shock as underlying mechanisms. Both ET and LT suppressed platelet aggregation and increased in vivo bleeding time albeit by apparently different mechanisms (12, 13). ET abrogated aggregation in vitro induced by thrombin or ADP, whereas LT was active against the arachidonic acid-induced but not the thrombi-induced platelet aggregation. TP in mice was associated with the decreased capacity of the circulating platelets to adhere to fibrinogen.

In addition to LT and ET, other pathogenic factors may also interfere with the host hemostasis. We recently demonstrated that culture supernatant of non-toxigenic B. anthracis delta Ames strain caused hemorrhages in a murine skin test and was lethally toxic to mice upon intratracheal administration (4). Specific antibodies against secreted proteases present in the supernatant reduced its hemorrhagic activity and showed a protective effect in spore-challenged mice. We also found that LT and B. anthracis hemolytic proteins can increase permeability of the epithelial barriers in the process which is accompanied by the accelerated shedding of cell surface proteoglycans (namely syndecans-1 and -4) and E-cadherin through the induction of host cell sheddase (14). Our data showed that anthrax proteases other than LT can also participate in the release of soluble syndecans (15). Taking into account that shed cell surface proteoglycans share considerable structural similarity with heparin, a widely used anticoagulant, we suggested that the ectodomain shedding may contribute to hemostatic abnormalities during anthrax infection.

We recently purified two major B. anthracis metalloproteases, Npr599 and InhA from culture supernatants of B. anthracis delta Ames and identified their activity toward plasma proteins and extracellular matrix molecules (15). Specifically, the proteases have been found to degrade plasminogen, fibrinogen, {alpha}2-macroglobulin, {alpha}2-antiplasmin, and {alpha}1-antitrypsin, indicating their potential role in modulation of blood hemostasis and thrombosis. In addition, the property of these proteases to cleave extracellular matrix proteins such as collagens, fibronectin, laminin, and syndecan-1, which are important for integrity of cell-to-cell and cell-to-extracellular matrix contacts, implicated them as pathogenic factors that are likely involved in the increase of endothelial barrier permeability and hemorrhage.

Among plasma proteins, von Willebrand factor (VWF) is a multimeric glycoprotein that contributes to the recruitment of platelets to the injured vessel wall (16). Increased proteolysis of ULVWF in normal plasma results in bleeding disorders, which is a general manifestation found in von Willebrand disease, a common genetic bleeding disorder of humans caused by mutations of VWF gene (for review, see Ref. 17). VWF is secreted constitutively from endothelial cells and megakaryocytes/platelets predominantly as "ultra-large" multimers (ULVWF) and tethers to platelets as well as collagens of basement matrix at sites of vascular damage to form a primary platelet plug (18). The tethering activity of VWF depends on its molecular size. ULVWF multimers not only bind to circulating platelets more readily than smaller forms but also undergo marked conformational changes under high shear stress. This multimeric adhesiveness depends on the activity of zinc metalloprotease ADAMTS13, which cleaves VWF at the peptide bond between residues Tyr1605 and Met1606 of the A2 domain (19, 20). We, therefore, suggested to test if VWF and its natural regulator ADAMTS13 are targets of B. anthracis proteolytic enzymes.

In several pathological conditions such as von Willebrand disease type 2B, thrombotic thrombocytopenic purpura, hemolysis-elevated liver enzymes-low platelets, and antiphospholipid syndrome, ADAMTS13 activities or levels are significantly reduced (for review, see Ref. 21). Its absence or reduced activity results in TP, renal dysfunction, fluctuating neurological symptoms, microangiopathic hemolytic anemia, and fever. Other pathological conditions associated with the increase in the level of circulating ULVWF and/or ADAMTS13 deficiency include infections such as parasitic malaria (22), hemolytic uremic syndrome caused by Escherichia coli O157:H7 (23–25), and Rocky Mountain spotted fever caused by Rickettsia rickettsii (26, 27). However VWF and ADAMTS13 pathologies in anthrax have not been studied.

We demonstrate that during anthrax infection, activities and protein levels of both VWF and ADAMTS13 are modulated in plasma of spore-challenged mice. The in vitro experiments suggest that secreted proteases Npr599 and InhA turn off the natural regulation of VWF cleavage by ADAMTS13 and generate proteolytically cleaved VWF defective in collagen binding and platelet aggregation activity. The results implicate secreted metalloproteases as a likely cause of hemorrhage and thrombosis in anthrax pathogenesis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Recombinant B. anthracis Proteins—B. anthracis non-encapsulated Sterne strain 34F2 [pXO1+, pXO2–] was obtained from the Colorado Serum Co. The non-toxigenic delta Sterne strain [pXO1–, pXO2–] was from the collection of the National Center for Biodefense and Infectious Diseases (George Mason University, VA). The non-toxigenic delta Ames strain [pXO1–, pXO2–] was a kind gift from Dr. S. Leppla, National Institutes of Health. For all strains the absence or presence of plasmids was confirmed by PCR with specific primers. Lethal factor (LF) was purchased from List Biologicals Laboratories. Anthralysin O (AnlO) was expressed in E. coli and isolated as described (28).

Proteolysis of VWF and ADAMTS13 in Human Plasma—Human normal plasma obtained from Sigma (1:10 dilution) was incubated with culture supernatants or purified proteases Npr599 and InhA of B. anthracis delta Ames in the presence or absence of urea (final 1.5 M). Culture supernatants were prepared from overnight LB cultures in the presence and absence of 0.5% glucose. Proteolytic activity of culture supernatants against casein was nullified by the presence of 0.5% glucose in LB. Purification of Npr599 and InhA from culture supernatants was performed as described (15). After overnight incubation at room temperature (arbitrary 21 °C) or 37 °C, the resulting products in the non-reducing native SDS sample buffer (Invitrogen) were separated by 1% agarose-SDS-Tris-glycine gel for multimer analysis and by 6% SDS-PAGE gel under reducing conditions (32 mM dithiothreitol) for monomer analysis. The Western blots were performed with HRP-conjugated rabbit anti-human VWF antibody P226 (Dako) in a 1:2000 dilution after protein transfer onto a PVDF membrane. For detection of the InhA-treated VWF fragments, three more antibodies, namely 3E2D10 (against amino acids 845–949), H-300 (against amino acid 2514–2813), and 14XX2 (against A2 domain) from Santa Cruz Biotechnology were used as primary antibodies. For proteolysis of ADAMTS13, 50 µl of human plasma (1:10 dilution) were incubated with 0.2 µg of Npr599, InhA, or LF in 5 mM Tris-HCl, pH 8.0, at 37 °C overnight. The samples were analyzed by Western blot using anti-ADAMTS13 antibody BL154G (Bethyl Laboratories).

Proteolysis of a Synthetic Peptide FRETS-VWF73—FRETS-VWF73 (Peptides International, final 2 µM) was incubated with plasma (4 µl), purified Npr599, or InhA (2 µM each) in 100 µlof reaction buffer (5 mM Tris-HCl, 25 mM CaCl2, and 0.005% Tween 20, pH 6.0). The reaction was incubated at 37 °C, and change of fluorescence over time was monitored (excitation at 355 nm, emission at 460 nm). Initial velocities were obtained from plots corresponding to less than 20 and 70% full hydrolysis of FRETS-VWF73 against Npr599 and InhA, respectively. The slopes of these plots were divided by the fluorescence change corresponding to complete hydrolysis and then multiplied by the substrate concentrations to obtain initial velocity in units of µM·min–1.

N-terminal Sequencing of VWF Fragments—Purified native human VWF (Sigma) was incubated with InhA in a buffer consisting of 5 mM Tris-HCl, pH 7.5, 1 mM CaCl2, and 1.5 M urea at 37 °C for 2 h. The products were loaded onto 10% NuPAGE MES gel (Invitrogen) and separated under reducing conditions (32 mM dithiothreitol). After transferring the proteins onto PVDF membranes, automated N-terminal sequencing was performed using an automated Edman degradation sequencer (Applied Biosystems) at the Biosynthesis (Lewisville, TX).

Collagen Binding Assay—Acid-soluble collagen type III from human placenta (Sigma) was dissolved in 3% acetic acid. The collagen solution (100 µl) was rapidly diluted to a final concentration of 20 µg/ml in phosphate-buffered saline (PBS) and then immobilized on a MaxSorp microplate (Nunc) for 1 h at room temperature. After coating, the plate was blocked with 5% BSA in PBS. Plasma (1:80) was incubated with proteases (10, 1, 0.1, 0.01 µg/ml) in the absence or the presence of 1.5 M urea at 37 °C for 3 h, and the reaction was stopped by adding 5 mM EDTA. For assaying the VWF collagen binding activity (VWF: CBA) in mouse plasma, the total protein content in each plasma sample was measured with Bradford reagent (Bio-Rad) and BSA as the standard. The amounts of plasma corresponding to 10 µg of total protein from each sample were dissolved in 100 µl of PBS and loaded on the collagen-coated plate for 1 h at room temperature. After the plate was washed 5 times with PBS containing 0.05% Tween 20 (PBST), HRP-conjugated VWF antibody in dilution 1:2000 was added and incubated for 1 h at room temperature. After washing with PBST, the reaction was visualized by the addition of tetramethyl bezidine solution (100 µl) followed by stop reaction with the addition of 2 M H2SO4. The absorbance was read at 450 nm on a microplate reader. Experiments using o-phenanthroline were performed similarly, except that proteases were preincubated for 5 min with 5 mM o-phenanthroline. As a negative control, BSA was used instead of collagen, and the optical density of the sample was subtracted by the optical density detected in the BSA-coated well.

VWF Overlay Assay—Proteases were run on a 10% SDS-PAGE gel and then electrophoretically transferred onto a PVDF membrane. The membrane was soaked into PBST overnight at 4 °C to renature the proteins and then was incubated with normal human plasma (diluted 1:20 with PBST) for 1.5 h at room temperature. The plasma was washed out with PBST 5 times for 4 min, and the membrane was incubated with HRP-conjugated anti-VWF antibody (1:2000) for 1 h followed by the HRP reaction.

Ristocetin Cofactor-induced Platelet Aggregation (RIPA) Assay—RIPA assay was performed using a Helena ristocetin cofactor assay kit (Helena Laboratories). 2-Fold diluted plasma (100 µl) was incubated with 0.2 µg of proteases in 1.5 M urea at 37 °C overnight, and EDTA (5 mM) was added to stop the reaction. 10 µl of protease-treated plasma and 10 µl of ristocetin (10 mg/ml) were added to 80 µl of platelet suspension (~3 x 105/ml). The mixture was incubated for 10 min with vigorous orbital shaking. To assess microscopic analysis, the aggregates were fixed with 1% paraformaldehyde for 10 min and examined under the microscope. Quantification of platelet aggregation was assessed by expression of platelet marker CD41 using flow cytometry. Ristocetin cofactor-induced platelet aggregates were incubated with phosphatidylethanolamine-labeled anti-CD41 antibody (eBioscience) for 10 min. A fixing step was followed by adding 2% paraformaldehyde for 10 min, and the aggregates were diluted with 1 ml of PBS for 2 h at room temperature. Platelet aggregation was calculated by counting the number of free platelets (<5 µm) shifted to a bitmap representing platelet aggregates (>5 µm) as described by Jy et al. (29).

Analysis of VWF Released from Human Umbilical Vein Endothelial Cells (HUVEC)—HUVEC from Clonetics Corp. were maintained in EBM medium (Clonetics) at 37 °C in an atmosphere of 95% air and 5% CO2. Cells were incubated with AnlO (0.01 and 0.1 µg/ml), and culture supernatants (diluted 100- and 10-fold) of B. anthracis delta Ames were prepared as described (15). After incubation for 15 min (AnlO) and 60 min (culture supernatants), 500 µl of the conditioned media were precipitated with cold EtOH (1 ml), and the precipitates were dissolved in 15 µl of 2x native sample buffer (Invitrogen). ULVWF multimers were visualized in 1% agarose-SDS-Tris-glycine gel followed by Western blot on a PVDF membrane with anti-VWF-HRP (1:2000) as described above.

Plasma and Serum Preparation from Spore-challenged Mice—The 9-week-old mice (DBA/2 from The Jackson Laboratory) were challenged intraperitoneally with 1 x 107 spores of B. anthracis non-encapsulated Sterne strain 34F2 [pXO1+, pXO2–] and non-toxigenic delta Sterne strain [pXO1–, pXO2–]. The LD50 of 3 x 106 spores (LD50) for Sterne strain by the intraperitoneal route was established earlier (14). The delta Sterne strain challenge in the same dose resulted in no lethality. The protocol of experiments has been approved by the Animal Care and Use Committees of George Mason University, Manassas, VA and the United States Department of Defense. Every 24 h post-challenge, blood from 5 mice was individually drawn via orbital sinus into solution of EDTA (final concentration of 0.1 M) to inhibit coagulation and proteolysis (15) and was centrifuged at 900 x g for 15 min to obtain platelet-poor plasma. Serum was prepared by centrifugation of blood coagulated at room temperature. For the ELISA assay of VWF antigen (VWF: Ag), plasma from each mouse (10 µg of total protein) was diluted in 100 µl of PBS and used to coat wells of the Nunc Maxisorp plates (eBiosciences) overnight at 4 °C. After incubation, the plates were washed 3 times with 100 µl per well of PBS, 0.1% Tween 20 and blocked for 1 h at 4 °C with 100 µl per well of PBS plus 5% BSA (Sigma). Plates were incubated in 100 µl per well of fresh blocking solution plus 1:2000 dilution of HRP-conjugated anti-human VWF antibody P226 (Dako) for 1 h at room temperature, washed 7 times with 200 µl per well of PBS, 0.1% Tween 20, and then developed using tetramethyl benzidine reagent (Beckman Coulter) added to all wells and incubated at room temperature for 30 min. Absorbance was detected at 450/570 nm using µQuant plate reader (Bio-Tek Instruments). The average absorbance calculated for each treatment group was corrected for the average absorbance of the control wells without plasma, and the results were presented as a percent change relative to the untreated control group. The assay was validated using serial dilutions of control plasma and purified VWF (not shown).

Reverse Transcriptase-PCR—Liver samples from spore-challenged mice were collected and immediately frozen in liquid nitrogen and stored frozen at –80 °C. Total RNA from liver was isolated using the Qiagen RNA isolation kit according to the manufacturer's recommendation. Isolated RNA was converted to cDNA using the SuperScript II reverse transcriptase-PCR reverse transcription kit (Invitrogen). For determination of ADAMTS13 mRNA expression, a conventional PCR was performed using a Platinum PCR premix (Invitrogen) with specific primers, the exon 21/22-specific sense primer (5'-TTGTGGGAGAGGTCTGAAGGAACT) and the pseudoexon 24-specific antisense primer (5'-TCAGCGCCATCTTGTGACGGCGAA). After amplification, PCR products were separated on a 1.0% agarose gel and stained with ethidium bromide. Photographs were scanned and analyzed by densitometry. The expression levels were normalized by β-actin mRNA levels.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
ULVWF Multimers Are Susceptible to Anthrax Proteolytic Enzymes—The specific cleavage of ULVWF multimers in physiological conditions by ADAMTS13 depends on the conditions of blood flow, suggesting that conformational changes in VWF upon fluid shear stress are necessary to expose the domain A2 cleavage site or the ADAMTS13-binding sites on the VWF molecule (23, 30). Mild denaturation of VWF in plasma with urea or guanidine hydrochloride in static conditions in vitro also stimulates cleavage (31), which takes place faster at subphysiological temperatures (e.g. at 21 °C) relative to 37 °C (32). To investigate if anthrax factors can facilitate VWF proteolysis in vitro, human normal plasma was incubated with supernatants of atoxigenic B. anthracis cultures or the purified proteases Npr599 and InhA in the absence or presence of urea at room temperature or 37 °C. Without urea in plasma, ULVWF was resistant to overall degradation (Fig. 1, A and B) at either temperature, although some sensitivity was detected in the presence of InhA and Nrp599 (Fig. 1A). In the presence of urea at room temperature, ULVWF was effectively cleaved by endogenous ADAMTS13 to a main product of ~200 kDa corresponding to a dimer of cleaved subunits (Fig. 1A, lane 13). Under these conditions, the supernatant of B. anthracis culture in LB media (Fig. 1A, lane 8), InhA (Fig. 1A, lane 11), and to a smaller extent Npr599 further enhanced ULVWF cleavage. However, at 37 °C even in the presence of urea, ULVWF cleavage was detectable only in the case of anthrax culture supernatants and isolated proteases. LF did not cleave VWF, and EDTA completely inhibited all metalloprotease activity in agreement with our previous observations (15).


Figure 1
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  		FIGURE 1.
Secreted anthrax proteases Npr599 and InhA degrade ULVWF in human plasma. Plasma was incubated with proteases at 21 °C (A) and 37 °C (B) overnight in the presence (+) or absence (–) of 1.5 M urea. The resulting products were separated by 1% agarose-SDS-Tris-glycine gel without reducing agents. After transferring onto a PVDF membrane, the products were visualized with HRP-conjugated VWF antibody. BaCS (LB) and BaCS (LBG) represent supernatants of B. anthracis culture in LB medium and in LB medium containing 0.5% glucose, respectively. Blots are representative of three individual experiments.

 


Figure 2
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  		FIGURE 2.
Western blot analysis of VWF monomers and ADAMTS13 in protease-treated human plasma. Plasma was incubated with anthrax proteases at 21 °C (A), 37 °C (B), and 37 °C with EDTA (C) overnight, separated by 6% SDS-PAGE gel under indicated conditions, and probed with HRP-conjugated VWF antibody on a PVDF membrane. ADAMTS13 protein in plasma was detected using anti-ADAMTS13 antibody under reducing conditions at 37 °C (D). Arrowheads indicate proteolytic products of VWF. Blots are representative of duplicate experiments.

 
The ULVWF cleavage products in the presence of urea were also analyzed in 6% SDS-PAGE gel under reducing conditions (Fig. 2). In agreement with Fig. 1, a higher degree of cleavage was detectable at room temperature, with InhA being the most active. At 37 °C, the intermediate degradation products were also detectable. Overall, the above results suggested that anthrax proteases were likely to display their effect on VWF in physiological conditions mimicked by the presence of urea at room temperature, when the conformation of VWF is susceptible to the endogenous ADAMTS13.


Figure 3
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  		FIGURE 3.
Kinetic analysis of FRETS-VWF73 proteolysis by Npr599 and InhA. Npr599 or InhA (2 µM each) were incubated with substrate in a reaction buffer at 37 °C. Extent of proteolysis was measured using fluorescence (excitation at 355 nm, emission at 460 nm). Average initial velocity (Vo) was obtained from the change of fluorescence within 2 min (n = 3).

 
To investigate the susceptibility of ADAMTS13 to the proteases, normal plasma was incubated with proteases in the absence of a denaturant. Western blot analysis with antibodies against ADAMTS13 showed that InhA effectively degraded ADAMTS13 (Fig. 2D), which is consistent with the absence of ADAMTS13-specific products in the presence of InhA (Fig. 2, A and B). These results can be explained by a pathogenic mechanism in which anthrax proteases may take complete control over proteolysis of VWF by endogenous ADAMTS13 playing an important role in primary homeostasis.

Npr599 and InhA Cleave a Synthetic VWF Substrate FRETS-VWF73, and InhA Degrades Native VWF—Recently, a minimal synthetic ADAMTS13 substrate FRETS-VWF73, which contains the cleavage site of VWF A2 domain, was developed for the fluorescence resonance energy transfer (FRET) assay. The peptide substrate corresponds to residues Asp1596-Arg1668 and contains the Tyr1605-Met1606 bond that is cleaved by ADAMTS13 (33, 34). Fig. 3 shows that FRETS-VWF73 can serve as a substrate for proteolysis by Npr599 and InhA in a concentration-dependent manner. The data suggest that with this substrate, InhA is a stronger protease than Npr599.

To further determine the cleavage sites of native VWF by Npr599 and InhA, we treated native VWF with the proteases and sequenced the N-terminal amino acids of its fragmented products. As expected, under mild denaturing conditions (1.5 M urea, room temperature) the VWF was readily degraded by InhA, which produced 4 major fragments (F1–F4) detected with Coomassie Blue dye (Fig. 4A). Using patterns obtained with domain-specific antibodies, the fragments were assigned as shown in Fig. 4D. The N-terminal sequences of F1 and F4 were identified as SLS(C)RP and VLQR(C), respectively. Because cysteine residues cannot be detected in automated Edman degradation, the fourth amino acid of fragment F1 and the fifth amino acid of fragment F4 are assumed to be cysteines according to the VWF protein sequence.


Figure 4
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  		FIGURE 4.
Identification of InhA cleavage sites in VWF. Purified VWF was incubated with InhA at 37 °C for 2 h. The samples were separated by 10% PAGE gel and transferred onto a PVDF membrane. The membrane was stained with Coomassie blue (A) and analyzed by Western blot with domain specific antibodies (B). Each band was subjected to an automatic N-terminal sequencing (C). Parentheses indicate the undetected or ambiguously detected amino acids in VWF sequence. Each fragment was assigned as shown in D. Sequence of FRETS-VWF73 and predicted cleavage of VWF by InhA identified by N-terminal sequencing are shown in D. Nma (N-β-[2-(methylamino)benzoyl]-2,3-diaminopropionic acid) and Dnp (N-β-[2,4-dinitrophenyl]-2,3-diaminopropionic acid) for FRET assay were substituted for glutamine and asparagine, respectively. Vertical arrows indicate cleavage sites for ADAMTS13 and InhA, respectively. The horizontal arrow indicates FRET between Nma and Dnp sensitive to peptide cleavage.

 


Figure 5
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  		FIGURE 5.
Detection of VWF proteolysis by Npr599 and InhA using the collagen binding assay. Plasma was incubated with different concentrations of proteases in the absence and presence of inhibitor o-phenanthroline and was loaded on the collagen-coated plate for 1 h at room temperature. Collagen-bound VWF was detected with HRP-conjugated anti-VWF antibody P226. The amount of antibody epitope bound to collagen (% collagen binding) was calculated relative to control without protease and inhibitor after background subtraction (n = 4, p < 0.001 to protease-treated control without inhibitor).

 
The Leu1664-Val1665 bond cleaved by InhA is located within the A2 domain. A similar sequence 1601PNLVYM1606 within the FRET region of FRETS-VWF73 is a likely second InhA cleavage site. It overlaps with the ADAMTS13 cleavage site 1603LVYMVT1608, and its presence explains the sensitivity of the substrate fluorescence to InhA. Minor fragments such as Fn and Fc were also detected, but it has to be further studied if they originate from the activity of InhA or possible impurities in the enzyme preparation. The N-terminal sequences of F2 and F3 could not be determined due to complexity of peptide mixtures. Fig. 4 summarizes the localization of cleavage sites of VWF by InhA. Overall, the cleavage data demonstrate that anthrax protease InhA mimics ADAMTS13 with regard to generation of the N-terminal fragment of mature VWF different only in one amino acid from the endogenously produced fragment, but in contrast to ADAMTS13, it further degrades the C-terminal fragment.

Modification of Collagen Binding Assay to Detect VWF Degradation by Npr599 and InhA—Binding of VWF to the subendothelial collagen is the first step in the initiation of primary hemostasis (35). Native conformation of VWF is essential for the interaction with collagen, but a limited reduction of the ULVWF multimers to the VWF dimers results in a decreased binding (36). However, the quantitative assessment of the VWF proteolysis by bacterial proteases is complicated due to the presence of multiple cleavage sites. We suggested that the collagen binding assay could be used as a sensitive measure of VWF proteolytic degradation. In the case when a specific N-terminal antibody is used for the detection of proteolyzed VWF after its binding to collagen, the cleavage events destroying either the antibody epitope or the A3 collagen binding domain or the functionally important A1, A2 domains between the antibody epitope and A3 domain would result in the loss of signal. We performed the assay using normal human plasma and human placenta type III collagen. In the absence of urea, no significant inhibition of antibody-generated signal by treatment of VWF with InhA was observed at different concentrations, indicating that the collagen-binding site of VWF was available for interaction and that in agreement with data presented in Fig. 1B, the exposed regions of the protein were not susceptible to the proteases in static conditions (data not shown). However, in the presence of 1.5 M urea at room temperature, Npr599 and InhA strongly increased VWF proteolysis in a concentration-dependent manner (Fig. 5). LF did not show any increase in proteolysis at different concentrations (0.01–10 µg/ml). Incubation of proteases with o-phenanthroline resulted in a complete recovery of VWF collagen binding (Fig. 5). This suggests that Npr599 and InhA may be able to modulate the VWF function by proteolysis in the conditions of shear stress in vivo after unfolding of the VWF conformation into to a protease-susceptible state.

Npr599 and InhA Bind to VWF and Inhibit Ristocetin-induced Platelet Aggregation—With regard to inhibition of VWF binding to collagen, we tested if the proteases can associate with VWF similar to ADMATS13, which forms a relatively stable complex with the VWF spacer domain (37). The proteases were transferred onto the PVDF membrane and visualized with naphthol blue (Fig. 6A), and the membrane was used in the overlay assay with VWF (Fig. 6B). The VWF binding to Npr599 was readily detectable, whereas the InhA binding to VWF was weaker and took place preferentially with the C-terminal-truncated fragment (18 kDa) compared with the catalytic domain-containing fragment (46 kDa) (15). The latter fragment is likely involved in the dynamic interaction with the substrate and may retain catalytic activity in the above assay conditions. Therefore, at least two regions of InhA can associate with VWF, and it was interesting to test if the protease binding to VWF could modulate the VWF property to induce platelet aggregation.


Figure 6
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  		FIGURE 6.
Npr599 and InhA bind to VWF. Proteases were run on 6% SDS-PAGE gel, electrophoretically transferred onto a PVDF membrane, and visualized with Naphtol Blue (A). The membrane was soaked in PBST overnight at 4 °C to renature the proteins. After overlaying with human normal plasma, protease-interacting VWF was detected with HRP-conjugated VWF antibody (B). Blots are representative of three individual experiments.

 
The RIPA is a sensitive test of interaction between VWF and its platelet receptor glycoprotein-Ib{alpha} (38). Cleavage of VWF by ADAMTS13 negatively regulates the interaction (39). To investigate the effect of Npr599 and InhA on RIPA, plasma VWF was preincubated with the proteases in the presence of urea, and then formalin-fixed platelets and ristocetin were added to induce platelet aggregation. Platelet aggregation by different concentrations of plasma was examined under the microscope. The Npr599-treated plasma showed similar RIPA activity as control plasma; however, InhA completely inhibited platelet aggregation (Fig. 7). Quantification of RIPA was performed by flow cytometry using anti-CD41 antibody to detect free platelets. This method is more sensitive and quantitative than conventional aggregometry in detecting the early stages of platelet aggregation (29). As shown in Fig. 7, preincubation of plasma with Npr599 and InhA inhibited RIPA up to 32 and 84%, respectively, compared with the untreated control. As a negative control, LF exhibited no significant decrease in RIPA. A preincubation of InhA with 5 mM o-phenanthroline, known to inhibit its enzymatic activity (15), blocked the inhibitory effect of InhA on RIPA (Fig. 7D). This suggests that the inhibition of platelet aggregation by InhA-treated VWF requires the InhA enzymatic activity and that the InhA binding to VWF demonstrated above (Fig. 6) does not play a regulatory role, although it may be important for the effective substrate cleavage by InhA.

Anthrax Proteases Are Capable of Inducing and Then Degrading VWF Multimers from HUVECs—It appears that the property of InhA to degrade ULVWF and, thus, to interfere with VWF-mediated host responses such as platelet aggregation may contribute to anthrax virulence through prolongation of the bleeding time and induction of hemorrhage similar to conditions associated with the mutant forms of VWF (17). A release of ULVWF by endothelial cells is an important component of host response to stress, injury, and inflammation aimed to preserve the integrity of endothelial barriers in the variety of pathophysiological conditions. To investigate whether anthrax pathogenic factors can stimulate secretion of ULVWF multimers, we chose to employ HUVECs known to secrete ULVWF multimeric strings after stimulation with histamine, cytokines, or Shiga toxins of E. coli O157:H7 (40). HUVECs were stimulated with B. anthracis delta Sterne culture supernatants containing secreted proteases as major secreted products (15) as well as the isolated B. anthracis hemolysin, AnlO, previously found to be involved in the host cell stress response (14). Secreted factors from B. anthracis culture supernatant caused a strong concentration-dependent release of VWF into the HUVEC culture media evaluated with a specific ELISA (data not shown).

Analysis of VWF polymerization after stimulation of HUVECs with B. anthracis culture supernatant in comparison with human normal plasma (Fig. 8) showed that VWF was present in a cleaved, dimeric form, in agreement with the presence of InhA and other proteolytic enzymes among B. anthracis-secreted proteins (41, 42). Consistent with the fact that AnlO is a pore-forming toxin but not a protease, the AnlO-induced VWF was highly aggregated (Fig. 8A). Under reducing conditions, the products of VWF cleavage by secreted bacterial proteases were undetectable in a Western blot using a VWF-specific antibody, indicating a proteolytic degradation of the protein antibody epitope(s) (Fig. 8B). In contrast, a band corresponding to a VWF monomer in normal plasma was presented in Western blot of the AnlO-treated cells (Fig. 8B). These data show that endothelial cells can respond to anthrax pathogenic factors by VWF secretion and demonstrate the capacity of secreted proteases to degrade the ULVWF multimers secreted from HUVECs.


Figure 7
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  		FIGURE 7.
RIPA activity of VWF is inhibited by its proteolysis with InhA. Platelets were incubated with ristocetin and protease-treated plasma as described under "Experimental Procedures." After incubation for 10 min, aggregated platelets were fixed with 2% paraformaldehyde followed by incubation at room temperature for 2 h. Platelet aggregation was quantified by using flow cytometry with phosphatidylethanolamine (PE)-labeled anti-CD41 antibody. Diagrams show free platelets in the aggregation assay in control plasma (A), in Npr599-treated plasma (B), and in InhA-treated plasma (C). Pictures of platelet aggregation were taken under a light microscope (inset in each panel). Free platelet content was used for calculation of aggregation relative to control (n = 3) (D). Platelet aggregations of 0 and 100% represent free platelets in conditions without plasma and with normal plasma, respectively.

 
ADAMTS13 and ULVWF Are Depleted in Spore-challenged Plasma—The in vitro data presented above prompted us to test if stimulation of VWF release suggested by the HUVEC cell model and concomitant degradation of both VWF and ADAMTS13 by secreted proteases reflected the features of anthrax infection in vivo. Mice (DBA/2) were challenged with B. anthracis spores of either the toxigenic Sterne 34F2 strain [pXO1+, pXO2–] or the atoxigenic delta Sterne strain [pXO1–, pXO2–]. Blood samples were drawn every 24 h post-challenge. Under the conditions of our experiments, the delta Sterne strain caused no clinical signs of infection or detectable bacteremia, whereas about 50% of Sterne-challenged mice died at day 3 post-infection with massive proliferation of bacteria in the spleen and liver (Refs. 43 and 44 and data not shown). The proteolytic activities of culture supernatants for both strains tested with casein as substrate were similar (±10%) (data not shown).

ADAMTS13 was significantly depleted from serum during infection at day 3 post-Sterne spore challenge and at day 2 post-delta Sterne spore challenge. All Sterne-challenged animals died by day 5, but all delta Sterne-challenged mice survived after they recovered the level of ADAMTS13 at day 3 post-challenge (Fig. 9A) consistent with the non-virulent nature of the delta Sterne strain. In both challenge groups the animals responded to infection with the activation of ADAMTS13 gene transcription in the liver, where the protein is synthesized. The expression of the gene returned to normal upon recovery in the case of the delta Sterne-challenged mice (Fig. 9B). In contrast, the aggravating condition of the Sterne-challenged mice was accompanied by a steady increase in the ADAMTS13 gene transcription (up to ~2.8-fold at day 3 post-challenge). The profile of the ADAMTS13 transcription was mirrored by the amount of VWF antigen recognized by a specific antibody in the plasma of infected mice (Fig. 11B), perhaps indicating an increased physiological demand of the host to maintain hemostasis in response to ADAMTS13 depletion and the elevated levels of VWF.

Taking into account the capacity of secreted proteases to degrade ADAMTS13 and VWF, we suggested that the multimerization state of VWF in the plasma of challenged animals would be affected by the infectious process. When plasma samples from spore-challenged mice were analyzed on a SDS-agarose gel, Sterne spore-challenged mice displayed significant reduction of high MW multimers starting at day 3 (Fig. 10). This indicated the onset of intensive proteolysis of VWF multimers before death. The plasma from delta Sterne-challenged mice showed a similar trend (data not shown). However, some variability noticed in the VWF multimerization between independent experiments precluded a direct visual comparison of the results. To account for possible differences due to sample handling, we prepared and tested all plasma samples for collagen binding in a single experiment. The results are presented in Fig. 11. As expected, the Sterne-challenged mice demonstrated early dramatic loss of VWF recognized by the specific antibody (P226) in the collagen binding assay (Fig. 11A) despite the increase in the total amount of circulating VWF antigen (Fig. 11B) detected by the ELISA. Although the ELISA sensitivity may be influenced by the degradation of the antibody epitope, the lethal infection process seems to stimulate a continuous production of VWF. In the case of delta Sterne challenge, there was a partial loss of the antibody-detectable, collagen-bound VWF with a trend to recover a normal function of VWF (Fig. 11A). Overall, VWF antigen levels showed a reduction of collagen binding activity in the plasma for both B. anthracis strains (Fig. 11C), which correlated with the accumulation of functionally defective VWF during lethal Sterne infection (R2 = 0.72). The above observations in challenged mice are consistent with the in vitro data obtained with purified enzymes.


Figure 8
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  		FIGURE 8.
Culture supernatants of B. anthracis induce but degrade ULVWF in HUVECs. HUVECs were incubated with AnlO for 15 min and with culture supernatants of B. anthracis delta Ames in LB medium for 60 min. Ethanol precipitates of cell culture supernatants were employed for ULVWF analysis in 1% agarose-SDS-Tris-glycine gel followed by immunoblot analysis with anti-VWF antibody (A). Monomers of VWF were analyzed in 6% SDS-PAGE under a reducing condition (B).

 


Figure 9
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  		FIGURE 9.
ADAMTS13 depletion in sera of B. anthracis spore-challenged mice. Pooled sera (n = 5) taken from spore-challenged mice at 6 h, day 1, day 2, and day 3 were transferred onto a nitrocellulose membrane, and the membrane was probed with anti-ADAMTS13 antibody (A). ADAMTS13 expression in liver was analyzed by reverse transcriptase-PCR with cDNA prepared by a conventional reverse transcription of RNA from livers of spore-challenged mice. Amplified products were estimated by densitometry and normalized by β-actin mRNA levels (B)(n = 3).

 


Figure 10
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  		FIGURE 10.
ULVWF multimers are degraded in plasma of Sterne spore-challenged mice. After incubation at room temperature overnight, plasma (n = 3) taken from spore-challenged mice was subjected to ULVWF analysis in a 1% agarose-SDS gel, as described under "Experimental Procedures." The blot was developed with a long (A) or a short (B) exposure time.

 


Figure 11
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  		FIGURE 11.
Anthrax infection results in the increased amount of circulating proteolytically degraded VWF. The N terminus-specific antibody P226 was used to detect the amount of VWF epitope in circulation (VWF:Ag) and after binding to collagen (VWF:CBA). Plasma (10 µg of protein per well) in PBS was added to a collagen-coated Maxisorp microplate for 1 h. After washing the plate with PBS, collagen-bound VWF was detected colorimetrically by probing with HRP-conjugated VWF antibody (A). VWF antigen levels were determined by a conventional ELISA using P226 as a primary antibody. Plasma (10 µg) was coated onto a microplate overnight. Total VWF antigen was determined colorimetrically as above (B). Error bars indicate S.D. (n = 10 for control and n = 5 for spore-challenged samples). Correlation between VWF:CBA and VWF:Ag uses the data presented in panels A and B (C). Open circles, squares, triangles, and diamonds represent control, day 1, day 2, and day 3 post-spore challenge, respectively. Black and gray distinguish between Sterne and delta Sterne challenge, respectively. Solid and dotted lines represent best-fit lines of linear regression between VWF:CBA and VWF:Ag for Sterne and delta Sterne challenges, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Secreted metalloproteases Npr599 and InhA are candidate B. anthracis virulence factors capable of degrading a variety of biologically important substrates (15). In this report we extended analyses of these proteases to cleavage of VWF and its natural regulator ADAMTS13. The choice of these proteins was determined by their important roles in hemostasis along with the observations on severe DIC-like coagulopathy in anthrax infection (6, 8, 45). For example, VWF-deficient mice develop spontaneous bleeding in intra-abdomen and massive hemorrhage in the neck and head (46). On the other hand, accumulation of ULVWF multimers in ADAMTS13 knock-out mice results in a prothrombotic state (47). In humans the ADAMTS13 deficiency may lead to a condition known as thrombotic thrombocytopenic purpura. As a result of the abnormal processing of ULVWF, most patients with thrombotic thrombocytopenic purpura develop platelet-rich thrombi in the microvasculature, renal failure, and neurologic dysfunction, and 90% of them ultimately die (48). Recently, Ono et al. (49) demonstrated that sepsis-induced DIC was associated with a severe secondary ADAMTS13 deficiency. In these patients the amount of antigen and enzymatic activity of ADAMTS13 were decreased to less than 20% that of their normal levels and were accompanied by increased multimerization of VWF.

Currently, DIC in anthrax has no mechanistic explanation. A previous study in LT-injected mice suggested that blood vessel leakage and hemorrhage led to DIC and circulatory shock as underlying pathophysiological mechanisms (11). However, there were no reports related to ADAMTS13 and/or VWF pathologies in anthrax. Our findings point toward possible roles of metalloproteases Npr599 and InhA in a life-threatening anthrax coagulopathy. We demonstrate that these proteases are capable of cleaving VWF in vitro. Specifically, InhA cleaves VWF at least at three sites, two of which are located within the A2 domain of the molecule, including the one in the immediate proximity to the endogenous ADAMTS13 cleavage site. The cleavage pattern suggests that InhA could be more potent than ADAMTS13 and, therefore, could cause a significant reduction of ULVWF multimers in the blood. Normally, VWF is cleaved rapidly by ADAMTS13 in the conditions of high fluid shear stress but is resistant to cleavage under static conditions (30). A pathological increase in shear stress-induced proteolysis of VWF appears to account for the association of severe aortic stenosis and gastrointestinal bleeding known as Heyde syndrome (50). Fig. 1 shows that InhA is capable of cleaving VWF in conditions mimicking sheer stress when the protein is partially unfolded by urea. Hemorrhagic lymphadenitis and meningitis are common pathologic features of inhalation anthrax (6, 51, 52), and it seems plausible that during anthrax pathogenesis the InhA may contribute to hemorrhage by cleaving VWF and, therefore, reducing the capacity of the host to repair vascular damage.

One of the VWF functions is to bridge platelets to exposed reactive surfaces at sites of vascular injury. In this process the collagen binding domain is involved in anchoring of VWF to damaged vessels. We found that the InhA-mediated degradation was associated with a strong reduction in the amount of intact VWF capable of binding to collagen (Fig. 7). In addition, binding of VWF to platelets in the presence of ristocetin was strongly inhibited by InhA, but not by Npr599 or LF, the proteolytic component of LT.

We also found that in human plasma, InhA effectively degraded not only VWF but ADAMTS13. Various proteases have been shown to degrade ADAMTS13 in vitro. Thrombin, plasmin, and granulocyte elastase generated during DIC can cause inactivation of ADAMTS13 (49, 53), and InhA is the first bacterial protease implicated in this process. In addition, in the experiments with HUVECs we found that InhA and hemolytic protein AnlO as well as B. anthracis proteins secreted in the culture media induced a strong release of VWF accompanied by its concomitant cleavage (Fig. 8). When microvascular endothelial cell layers are damaged by pathogen infection or trauma, ULVWF multimers secreted from endothelial cells bind to the extracellular matrix components including the collagen. Glycoprotein-1b{alpha} receptor of platelets interacts with ULVWF multimers resulting in subsequent growth of hemostatic plug formation. In these circumstances the ADAMTS13 deficiency is expected to be pro-thrombotic because it increases the VWF multimerization. In contrast, VWF deficiency is associated with spontaneous bleeding (17, 46). When both ADAMTS13 and VWF can be degraded by InhA, the hemostatic consequences of its proteolytic activity are difficult to predict based only on the in vitro results, and the balance between pro- and anti-thrombotic factors may depend on a specific biological setting. Experiments with human plasma (Fig. 2B) show that in the presence of InhA the pattern of VWF cleavage does not contain bands typical for ADAMTS13 activity and, therefore, suggests that InhA can cause a fast preferential degradation of ADAMTS13. This, however, may not correctly reflect the situation in vivo because a balance between pro- and anti-thrombotic VWF-associated processes in anthrax infection may depend on a number of factors, such as individual host susceptibility, differential expression, and distribution of pathogenic proteins within the infected host, blood flow hydrodynamics in the vessels of different size, etc. This consideration may at least in part explain variability of hemostatic abnormalities often observed between patients (7) and even inbred experimental animals (8) as well as the paradoxical simultaneous presence of hemorrhage and thrombosis in the same patient.

To confirm that our in vitro observations were relevant to situation in vivo, we investigated the role of VWF and ADAMTS13 in anthrax murine model during systemic infection. Available data demonstrate that InhA (encoded by BA0672 and BA1295 genes) can be detected in the sera of infected animals as active protein (54, 55). In mice challenged with a lethal dose of Sterne spores, the protein levels of ADAMTS13 were significantly reduced on day 3, although mRNA levels remained steadily elevated until death (>50% of animals died by day 3) (Fig. 9). Reduction in both the ULVWF amount and the level of intact VWF capable of binding to collagen coincided with the depletion of ADAMTS13 and remained low through the entire course of infection with Sterne spores, whereas the antigen level of VWF continued to grow (Fig. 11). Overall, the results of the virulent Sterne challenge indicated the onset of the overwhelming proteolysis of circulating ADAMTS13 and ULVWF before death. In comparison, all animals challenged with the same dose of non-virulent delta Sterne spores experienced similar but less severe symptoms, demonstrated recovery from disease, and ultimately survived. In the case of both strain challenges the deficiency of ADMATS13 did not induce an increase of ULVWF levels in anthrax plasma. Furthermore, a decrease of ADAMTS13 showed no correlation with either degradation of ULVWF or antigen level. It is, therefore, plausible that, similar to experiments with human plasma in vitro, during infection the secreted proteolytic factors of B. anthracis quickly deplete ADAMTS13 and take complete control over the fate of circulating VWF. Fig. 11C shows that in the case of steadily progressing Sterne infection, gradual reduction in the amount of collagen-bound VWF antigen correlates with increased total amount of VWF antigen in plasma. It may reflect systemic endothelial injury and consequent activation. Before death, the animals demonstrate almost a complete loss of intact VWF, which therefore, might be considered as a marker of lethality.

Currently, it seems to be firmly established that systemic anthrax is associated with overt or probable DIC in human patients (6, 45, 52) as well as in the recently developed baboon model (8) showing a significant increase in vascular permeability coincident with hemostatic imbalances manifested by TP, transient leucopenia, and microthrombosis. The latter is a typical feature of anthrax (56), but the mechanism of its formation is unknown. Thrombosis is commonly attributed to the increase in ULVWF but not the VWF fragmentation found in our experiments. Nevertheless, increased fragmentation of VWF has been reported during the acute disease phase in patients with hemolytic uremic syndrome and thrombotic thrombocytopenic purpura in association with thrombosis, suggesting a permissive role of fragmented VWF in the formation of microvascular thrombi (25). A recent comparison study with normal and VWF–/– mice strongly suggested the existence of a ligand for platelet glycoprotein-Ib{alpha} other than VWF during thrombus formation in the injured vessels (57). Furthermore, thrombus formation observed in fibrinogen-deficient mice and in mice doubly deficient for fibrinogen and VWF showed the existence of a novel platelet aggregation pathway independent of both fibrinogen and VWF (58, 59). These results explain the formation of microvascular thrombi under VWF fragmentation in anthrax, but the precise role of VWF in anthrax thrombosis requires further studies.

In summary, our results demonstrate a new proteolytic mechanism of hemostatic imbalance in anthrax, which affects both the levels and activities of VWF and its natural regulator ADAMTS13 in plasma of spore-challenged mice, correlates with the severity of infectious process, and could contribute to hemorrhage and thrombosis typical in challenged animals and anthrax patients. The experiments with human plasma and purified anthrax proteases indicate that InhA and Npr599, but not LT, could be considered as pathogenic factors effectively compromising functions of VWF and ADAMTS13 in the repair of vascular damage and regulation of platelet recruitment to injured endothelium.


   FOOTNOTES
 
* This work was supported by United States Department of Defense Grant DAMD 17-03-C-0122. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: National Center for Biodefense and Infectious Diseases, George Mason University, 10900 University Blvd., Manassas, VA 20110. Tel.: 703-993-4713; Fax: 703-993-4288; E-mail: spopov{at}gmu.edu.

2 The abbreviations used are: LT, lethal toxin; ADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; AnlO, anthralysin O; DIC, disseminated intravascular coagulopathy; InhA, immune inhibitor A metalloprotease; LF, lethal factor; Npr, neutral protease; RIPA, ristocetin-induced platelet aggregation; VWF, von Willebrand factor; ULVWF, ultra-large VWF; VWF·CBA, collagen binding activity of VWF detected with antibody P226; VWF·Ag, level of VWF antigens; ET, edema toxin; TP, thrombocytopenia; HRP, horseradish peroxidase; PVDF, polyvinylidene difluoride; MES, 4-morpholineethanesulfonic acid; BSA, bovine serum albumin; PBS, phosphate-buffered saline; HUVEC, human umbilical vein endothelial cells; ELISA, enzyme-linked immunosorbent assay; FRET, fluorescence resonance energy transfer. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Weidong Zhou, Bryan Millis, and Thomas Huff (George Mason University) and Edward Cedrone (American Type Culture Collection) for technical assistance.



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