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J. Biol. Chem., Vol. 278, Issue 32, 30136-30141, August 8, 2003
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From the
Department of Pathology and Immunology,
¶Howard Hughes Medical Institute, and
||Department of Medicine, Washington University
School of Medicine, St. Louis, Missouri 63110
Received for publication, May 21, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The VWF-cleaving protease was recently purified and identified as a new member of the ADAMTS family of metalloproteases (10, 11), so named for the combination of a disintegrin-like and metalloprotease (reprolysin type), with thrombospondin type 1 motifs (12). The primary structure of the ADAMTS13 precursor was determined by cDNA cloning (13, 14) and by positional cloning in families with inherited ADAMTS13 deficiency (15). It consists of 1427 amino acid residues comprising a signal peptide, a short propeptide ending in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrin domain, a thrombospondin-1 repeat (TSP1), a cysteine-rich domain and spacer characteristic of the ADAMTS family, seven additional TSP1 repeats, and two CUB domains (13–15). Several alternatively spliced mRNA species have been identified that could encode truncated forms of ADAMTS13 lacking various C-terminal domains (13–16).
The complex multidomain structure of ADAMTS13 is conserved across vertebrates as diverse as mammals, birds, and fish (17),2 suggesting that motifs outside the metalloprotease domain are required for its biological function. This concept is supported by the finding that missense mutations in domains far from the metalloprotease domain cause inherited ADAMTS13 deficiency and thrombotic microangiopathy (15, 18, 19). The specificity of ADAMTS13 for a single bond in VWF and the remarkable regulation of cleavage by tensile stress on the substrate suggest that accessory domains are critical for substrate recognition. However, the structural requirements for ADAMTS13 activity have not been determined. To address this question, ADAMTS13 constructs with nested C-terminal deletions were characterized. The results suggest that the spacer domain is required to recognize and cleave VWF. More distal domains may contribute to activity but do not appear to be necessary in vitro.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Using ADAMTS13 constructs in vector pcDNA3.1/V5-His-TOPO as the template, recombination sites attB1 and attB2 were introduced at the 5'- and 3'-ends of the insert by PCR with oligonucleotides 5'-ggg gac aag ttt gta caa aaa agc agg ctt cac cAT GCA CCA GCG TCA CCC CCG GGC AAG-3' (attB1 site in lowercase) and 5'-ggg gac cac ttt gta caa gaa agc tgg gtc TCA ATG GTG ATG GTG ATG ATG ACC G-3' (attB2 site in lowercase). PCR products were recombined with plasmid pDONR201 and BP Clonase to prepare entry clones according to the manufacturer's instructions (Invitrogen). Entry clones were sequenced to verify the accuracy of the PCR and recombination reactions. Bacmid expression vectors were prepared by recombination of each entry clone with vector pDEST8 and LR Clonase (Invitrogen). Baculovirus stocks were prepared for each bacmid by transfection of Spodoptera frugiperda Sf9 cells and repeated amplification of the resultant virus.
Recombinant Protein Expression—COS-1 or COS-7 cells (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin, and streptomycin. Cells were seeded on 6-well tissue culture plates at 45% confluence 12 h before transfection in serum-free Opti-MEM I (Invitrogen) containing 6 µl of LipofectAMINE 2000 (Invitrogen) per 1 µg of plasmid DNA. After 4–5 h, the transfection mixture was replaced with 2 ml of fresh serum-free Opti-MEM I. The medium was collected 48 h after transfection, and phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM. Cell debris was removed by centrifugation, and the conditioned medium was stored at –70 °C.
Sf9 cells were cultured in 1-liter bottles at 27 °C with shaking in 100 ml of protein-free Sf-900 II SFM medium (Invitrogen) to a density of 2 x 106/ml. Baculovirus stocks were added at a multiplicity of infection of 0.1, and cultures were incubated for an additional 96 h. Media were dialyzed against phosphate-buffered saline (PBS), concentrated by ultrafiltration, and stored at –20 °C.
Recombinant proteins were visualized by SDS-PAGE and Western blotting with monoclonal anti-V5 antibody (Invitrogen), peroxidaseconjugated goat anti-mouse IgG (DAKO Corp., Carpinteria, CA), and the chemiluminescent ECL detection system (Amersham Biosciences) as described previously (20). The luminograms were scanned, and the relative amount of proteins detected was estimated by densitometry using NIH Image 1.62 (developed at the National Institutes of Health and available on the Internet at rsb.info.nih.gov/nih-image/). The absolute concentration of V5-tagged ADAMTS13 constructs was determined by standardization with the identically tagged Positope reference protein (Invitrogen).
ADAMTS13 Assays—ADAMTS13 protease activity was assayed by a collagen-binding method (21) with minor modifications (22). The multimer distribution of VWF after incubation with ADAMTS13 variants was assessed also by SDS-agarose gel electrophoresis and Western blotting with peroxidase-conjugated polyclonal anti-VWF antibody (catalog no. P0226, DAKO) as described previously (23).
The specific cleavage of VWF subunits by ADAMTS13 was confirmed by SDS-PAGE
and Western blotting as described above, but samples were reduced with 2%
-mercaptoethanol, and proteins were detected with polyclonal anti-VWF
antibody (23).
Immunofluorescence Microscopy—COS-7 cells were grown to 80% confluence in 4-well Lab-Tek glass chamber slides (Nalge Nunc Int., Naperville, IL) in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were washed once with PBS and transfected with plasmid DNA mixed with LipofectAMINE 2000 (1:6, w/v) in serum-free Opti-MEM I (Invitrogen). After 5 h, the medium was removed and replaced with fresh Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. After 48 h, the cells were washed with ice-cold PBS and fixed for 10 min at –20 °C with ethanol:acetic acid (9:1) or at room temperature with 2% paraformaldehyde in PBS. The cells were washed three times with PBS, and nonspecific antibody binding sites were blocked with 1.5% bovine serum albumin in PBS for 30 min. The cells were incubated sequentially for 1 h each at 20 °C with anti-V5 IgG (1:1000) followed by Cy3-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) in PBS containing 1.5% bovine serum albumin. The cells were washed three times with PBS between the incubations with antibodies. For nuclear staining, cells fixed with paraformaldehyde were permeabilized with 0.075% saponin for 3 min and stained with Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA).
Extracellular Matrix Localization—RFL-6 primary rat lung
fibroblasts (CCL-192, ATCC) were maintained in Kaighn's modification of Ham's
F-12 medium (ICN Biomedicals, Irvine, CA) with 20% fetal bovine serum, 2
mM L-glutamine, 0.1 mM non-essential amino acids, 1
mM sodium pyruvate, 100 units/ml penicillin, and 100 µg/ml
streptomycin. Cells at 50% confluence in T25 flasks were transfected with 2
µg of pcADAMTS13-V5-His, 12 µl of LipofectAMINE, and 8 µl of Plus
reagent (Invitrogen) in 2 ml of Opti-MEM I. At 48 h, medium was collected, and
cells were washed extensively with ice-cold PBS. The cells were lysed on ice
for 30 min in 2 ml of water containing 0.1% protease inhibitor mixture (Sigma)
and 1 mM phenylmethylsulfonyl fluoride. The lysate was removed, and
remaining cell membranes and matrix were washed extensively with ice-cold
water. The extracellular matrix was solubilized by scraping into 100 µl of
SDS-PAGE sample buffer (15 mM Tris-HCl, pH 6.8, 2.5% glycerol, 0.5%
SDS, 178 mM -mercaptoethanol, and 0.25% bromphenol blue).
Equal percentages of lysate, medium, and matrix solutions were incubated at
100 °C for 5 min, then diluted 2.5-fold in 50 mM sodium
citrate, pH 5.5, with or without 500 units of recombinant Streptomyces
plicatus endoglycosidase H (New England Biolabs, Inc., Beverly, MA) and
incubated at 37 °C for 2 h. Products were analyzed by SDS-PAGE and Western
blotting with anti-V5 antibody or monoclonal anti-human fibronectin antibody
(clone IST-3, catalog no. F0791, Sigma).
Cell Surface Biotinylation—Transfected cells in 6-well
plates were washed three times with ice-cold PBS and incubated at 4 °C for
30 min with 1 ml of PBS containing 1.5 mg/ml
sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (NHS-SS-Biotin,
Pierce) (24). Reaction was
stopped by washing with ice-cold PBS, and cells were lysed in 1.5 ml of PBS
containing 1% Triton X-100 and 1% protease inhibitor mixture (Sigma). The
lysate was clarified by centrifugation and split into two equal samples.
ADAMTS13 proteins in one sample were concentrated by adsorption onto 100 µl
of TALON metal affinity beads (BD Biosciences Clontech). Biotin-labeled
proteins from the second sample were adsorbed onto 100 µl of
streptavidin-agarose (Pierce) at room temperature for 2 h, and unbound
ADAMTS13 in the flow-through fraction was concentrated by adsorption onto 100
µl of TALON beads. After washing four times with PBS, proteins were eluted
from TALON or streptavidin beads with 40 µl of 0.4% SDS containing 5%
-mercaptoethanol. Samples (30 µl) were diluted with 30 µl of water
and incubated at 37 °C for 16 h in a total volume of 50 µl containing
100 mM sodium citrate phosphate, pH 5.5, 2% Triton X-100, 4%
-mercaptoethanol, and 0.02% sodium azide without (control) or with 2
milliunits of recombinant S. plicatus endoglycosidase H (Oxford
Glycosciences, Abingdon, UK)
(24). Products were analyzed
by SDS-PAGE and Western blotting with anti-V5 antibody.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2-fold in the amount of secreted protein detected by Western blotting with
anti-V5 antibody.
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The cellular localization of ADAMTS13 proteins was assessed by immunofluorescence microscopy (Fig. 2). The variant containing only the metalloprotease domain (del6) exhibited a perinuclear distribution consistent with localization in the Golgi apparatus. Larger constructs exhibited a more extensively diffuse plus granular pattern consistent with transient localization in the endoplasmic reticulum during biosynthesis and secretion. Staining of nonpermeabilized cells, either in culture at 4 °C or after fixation with paraformaldehyde, did not identify any ADAMTS13 variant on the cell surface. The absence of cell-surface ADAMTS13 was confirmed for full-length ADAMTS13 by a chemical modification approach (Fig. 3). Cell surface proteins were biotinylated and recovered from cell lysates by affinity chromatography on avidin-agarose. Contamination of the biotinylated fraction by intracellular proteins was assessed by digestion with endoglycosidase H, which does not act on complex oligosaccharides of secreted ADAMTS13 in conditioned medium. Almost all of the ADAMTS13 was not biotinylated and had endoglycosidase H-sensitive oligosaccharides consistent with localization in the endoplasmic reticulum. Trace amounts of biotinylated ADAMTS13 had endoglycosidase H-sensitive oligosaccharides and probably represent intracellular protein derived from broken cells. Therefore, secreted ADAMTS13 does not bind detectably to COS-7 cells.
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Because some ADAMTS proteases interact with extracellular matrix components, the association of ADAMTS13 with extracellular matrix was examined. Recombinant full-length ADAMTS13 was not detected in the extracellular matrix of transfected COS-1 cells by immunofluorescence or by Western blotting (data not shown), but COS-1 cells produce a relatively sparse matrix in culture. Therefore, similar experiments were performed in primary rat lung fibroblasts, which produce a more substantial matrix and have been used extensively in studies of extracellular matrix localization (25). In RFL-6 cells, the oligosaccharides of intracellular ADAMTS13 were endoglycosidase H-sensitive, whereas oligosaccharides of secreted ADAMTS13 were endoglycosidase H-resistant (Fig. 4). Trace amounts of endoglycosidase H-sensitive ADAMTS13 were recovered in solubilized extracellular matrix (Fig. 4), although endoglycosidase H-resistant fibronectin was abundant (data not shown). Overexposure did not demonstrate any ADAMTS13 in the matrix fraction that was resistant to endoglycosidase H. Similar results were obtained by immunofluorescence: fibronectin was detected readily in RFL-6 extracellular matrix, but ADAMTS13 was not. Thus, secreted ADAMTS13 does not appear to bind to the extracellular matrix of cultured RFL-6 or COS-1 cells.
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Proteolytic Activity of ADAMTS13 Variants—Recombinant
ADAMTS13 proteins were assayed under conditions previously established for
plasma ADAMTS13. ADAMTS13 does not cleave VWF at a significant rate under
conditions of physiological ionic strength and in the absence of fluid shear
stress. However, reaction occurs readily in buffers of low ionic strength that
are supplemented with low concentrations of urea or guanidine
(6,
7). The relative concentration
of recombinant ADAMTS13 variants was determined by Western blotting and
densitometry (Fig.
5A), and equal amounts were incubated with purified
plasma VWF in the presence of 1.5 M urea. The affinity of VWF for
collagen varies directly with multimer length. Therefore, cleavage of VWF
multimers can be assayed as a decrease in collagen binding activity
(21)
(Fig. 5B). At the
concentration tested, the activity of full-length recombinant ADAMTS13 was
similar to that in an equal volume of plasma. As described for plasma
ADAMTS13, the activity of recombinant ADAMTS13 was inhibited by chelation of
divalent metal ions or by autoantibodies to ADAMTS13 from a patient with
idiopathic TTP (22) confirming
the specificity of the reaction. Construct del1, which lacks both CUB domains,
had normal activity toward VWF. Further deletion of TSP1 domains 2–8 had
a variable effect. Among four experiments, the average activity of construct
del2 was 70% relative to the full-length protein, but the standard error
was large, and the activity of del2 was not significantly different from that
of del1 or full-length ADAMTS13. Deletion of the spacer domain in construct
del3 abolished activity, and the shorter constructs del4, del5, and del6 also
were inactive. The results obtained in the collagen-binding assay were
confirmed by direct visualization of VWF multimers. Full-length ADAMTS13 and
truncation mutants del1 and del2 digested VWF rapidly into small multimers,
whereas constructs del3, del4, del5, and del6 had no discernable effect on the
VWF multimer distribution (data not shown).
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Plasma ADAMTS13 (6, 7) and recombinant full-length ADAMTS13 (26) cleave the Tyr1605-Met1606 bond of the VWF subunit, generating characteristic fragments of 140 kDa and 176 kDa that also are produced during the normal catabolism of VWF in vivo (27). Active recombinant ADAMTS13 variants lacking the CUB domains (del1) or further lacking TSP1 repeats 2–8 (del2) produced VWF fragments similar to those generated by full-length recombinant or plasma ADAMTS13 (Fig. 6).
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The results obtained with recombinant ADAMTS13 expressed in mammalian COS-7
cells were confirmed with selected variants expressed in baculovirus-infected
Sf9 cells (Fig. 7). Proteins
expressed in Sf9 cells had slightly lower apparent mass than their
counterparts expressed in COS-7 cells, suggesting that Sf9 cells may add
smaller oligosaccharides. Full-length ADAMTS13 expressed in both cell types
gave rise to a similar C-terminal degradation product of 45 kDa
(Fig. 7), suggesting that a
site between TSP1 repeats 6 and 8 may be particularly susceptible to
proteolysis.
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In COS-7 cells, all variants were expressed at a level of 9–13
nmol/liter conditioned medium. Surprisingly, expression in Sf9 cells was not
markedly better. All species were expressed at 6–13 nmol/liter
conditioned medium. Optimal expression required a low multiplicity of
infection of 0.1, and almost no ADAMTS13 protein was recovered at a more
typical multiplicity of infection of 1 or 5, suggesting that active ADAMTS13
may be toxic to insect cells.
Whether expressed in COS-7 cells or Sf9 cells, full-length ADAMTS13 and the
variant truncated after the spacer domain (del2) had similar specific
activities of 80–110 units/nmol
(Fig. 7), confirming that
domains C-terminal to the spacer domain are not required for proteolytic
activity in vitro. If full-length recombinant and plasma ADAMTS13
(170 kDa) have similar specific activity, the results suggest that the
plasma concentration of active ADAMTS13 is 1.6 µg/ml, which is similar
to the value of 1 µg/ml based upon the recovery of ADAMTS13 during
purification from plasma
(11).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Although several ADAMTS proteases associate with extracellular matrix or cell surfaces, ADAMTS13 does not appear to fit this pattern. Full-length ADAMTS13 did not bind detectably to the surface of COS-7 cells (Fig. 3) and was not incorporated into the extracellular matrix of COS-1 or RFL-6 cells (Fig. 4). If ADAMTS13 binds cell surfaces or matrix in vivo, such interactions could depend on binding partners that are not expressed in these cultured cell lines.
The properties of truncated ADAMTS13 proteins suggest that substrate recognition requires the participation of several domains. Full-length recombinant ADAMTS13 was able to cleave VWF with apparently normal specificity, but truncation mutants lacking the spacer domain could not (Figs. 5, 6, 7). Therefore, TSP1 repeats 2–8 and the CUB domains are not required for VWF cleavage in vitro. Whether they are important under fluid shear stress or under more physiological conditions in vivo requires further study. If these C-terminal domains were necessary in vivo, one might predict the occurrence of patients with inherited TTP caused by mutations in the ADAMTS13 gene but with normal plasma ADAMTS13 activity upon laboratory testing. To date, however, all reported mutations distal to the spacer domain have been associated with severe ADAMTS13 deficiency (15, 19, 34).
Deletion of the ADAMTS13 spacer domain abolished the activity of construct
del3 toward VWF (Figs. 5,
6,
7), suggesting that the spacer
domain participates in substrate recognition. This conclusion would be
strengthened if construct del3 and smaller variants were shown to have
catalytic activity toward another substrate, to react with an active site
inhibitor, or to have native conformation by another method. Unfortunately, no
ADAMTS13 substrates other than VWF and certain fragments thereof have been
reported. A variety of other proteins and small synthetic substrates have been
tested, but none were cleaved detectably; also, with the exception of
metal-ion chelators (6,
7), no synthetic or natural
active site inhibitor including
2-macroglobulin4
has been shown to react with ADAMTS13. The efficient synthesis and secretion
of small inactive ADAMTS13 constructs (Figs.
2 and
7) suggest they are folded
properly, but direct evidence for structural integrity will require a
different experimental approach.
Although not addressed in this study, the more proximal disintegrin and Cys-rich domains and the first TSP1 repeat may also participate in substrate recognition. For example, the mutation P475S in the ADAMTS13 Cys-rich domain was identified in a Japanese patient with inherited TTP; the corresponding recombinant mutant ADAMTS13 was secreted efficiently but had extremely low activity toward VWF (18). Other missense mutations that cause inherited ADAMTS13 deficiency have been found in the disintegrin, Cys-rich, and first TSP1 domains (15, 19), but protein expression studies have not yet been reported for them.
Proteolytic processing can generate distinct forms of several ADAMTS proteases, and this variation may control tissue localization or substrate specificity. For example, ADAMTS4 is autoproteolytically cleaved into products that lack variable portions of the spacer and Cys-rich domains, and the truncated forms have reduced affinity for sulfated glycosaminoglycans (32). Some cells remove the C-terminal TSP1 repeats from ADAMTS1, which reduces its affinity for heparin and endothelial cells (29). At least one site within the C-terminal TSP1 repeats of recombinant full-length ADAMTS13 appears to be sensitive to cleavage (Fig. 7), suggesting that it could be subject to proteolytic processing in vivo. Such processing could occur intracellularly as indicated by the sensitivity of the C-terminal fragment to digestion with endoglycosidase H (Fig. 3). Additional structural heterogeneity for several ADAMTS proteases may be produced by alternative mRNA splicing (16, 35, 36), although the biological significance of the predicted products has not been established. In the case of ADAMTS13, alternatively spliced forms have been cloned that encode variants truncated after the metalloprotease domain, the spacer domain, the second TSP1 repeat, or the first CUB domain (13–15). Similar deletion mutants (Fig. 1) were secreted efficiently by COS-7 cells and Sf9 cells, and ADAMTS13 purified from plasma was shown to contain species of 110, 130, 140, and 150 kDa that had the same N-terminal amino acid sequence (11). Thus, truncated forms of ADAMTS13 may circulate in vivo and could potentially be derived from alternatively spliced transcripts. Based on the properties of recombinant ADAMTS13 mutants (Figs. 5, 6, 7), such variation in the complement of C-terminal domains would dramatically affect ADAMTS13 substrate specificity.
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FOOTNOTES |
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Present address: Dept. of Pathology and Laboratory Medicine, The Children's
Hospital of Philadelphia, University of Pennsylvania School of Medicine, 34th
St. and Civic Center Blvd., Philadelphia, PA 19104.
** To whom correspondence should be addressed: Howard Hughes Medical Inst., Washington University School of Medicine, 660 S. Euclid Ave., Box 8022, St. Louis, MO 63110. Tel.: 314-362-9029; Fax: 314-454-3012; E-mail: esadler{at}im.wustl.edu.
1 The abbreviations used are: TTP, thrombotic thrombocytopenic purpura; PBS,
phosphate-buffered saline; TSP1, thrombospondin type 1; VWF, von Willebrand
factor.
2 J. E. Sadler, unpublished observations.
3 X. Zheng, K. Nishio, E. M. Majerus, and J. E. Sadler, unpublished
observations.
4 X. Zheng and J. E. Sadler, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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5 µl of concentrated
conditioned medium) were incubated for 16 h with VWF substrate in buffer
containing 5 mM Tris-HCl, pH 8.0, 3 mM BaCl2,
and 1.5 M urea, and the integrity of the remaining VWF multimers
was assessed in a collagen binding assay. The results were normalized to the
values obtained with full-length ADAMTS13 (FL). The bars
represent the S.E. of four independent assays.
