|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
PLENARY PAPER
From the Central Hematology Laboratory, University
Hospital, Inselspital, Bern, Switzerland.
von Willebrand factor-cleaving protease (vWF-cp) is responsible
for the continuous degradation of plasma vWF multimers released from endothelial cells. It is deficient in patients with thrombotic thrombocytopenic purpura, who show unusually large vWF multimers in
plasma. Purified vWF-cp may be useful for replacement in these patients, who are now treated by plasma therapy. In this study, vWF-cp
was purified from normal human plasma by affinity chromatography on the
IgG fraction from a patient with autoantibodies to vWF-cp and by a
series of further chromatographic procedures, including affinity
chromatography on Protein G, Ig-TheraSorb, lentil lectin, and heparin.
Four single-chain protein bands, separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, showed Mr of 150, 140, 130, and 110 kd and were
found to share the same N-terminal amino acid sequence, suggesting that they were derived from the same polypeptide chain that had been partially degraded at the carboxy-terminal end. A hydrophobic sequence
(Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val-Gly) of the first 15 residues was established. The protease migrates in gel
filtration as a high-molecular-weight complex with clusterin, a 70-kd
protein with chaperonelike activity. vWF-cp bound to clusterin is
dissociated by the use of concentrated chaotropic salts. vWF-cp in
normal human plasma or serum is not associated with clusterin, suggesting that the observed complex is due to vWF-cp denaturation during the purification procedure. Activity of vWF-cp is unusually stable during incubation at 37°C; its in vitro half-life in citrated human plasma, heparin plasma, or serum is longer than 1 week. There was
even a temporary increase in protease activity during the first 3 days
of incubation.
(Blood. 2001;98:1654-1661) von Willebrand factor (vWF) is a blood glycoprotein
that is required for normal hemostasis. It is synthesized in
endothelial cells and megakaryocytes from identical 250-kd subunits
into disulfide-linked multimers ranging in size from about 500 to
20 000 kd. Each subunit contains binding sites for several fibrillar
and nonfibrillar collagen types, extracellular matrix, platelet
glycoproteins GPIb and GPIIb/IIIa, coagulation factor VIII, heparin,
sulfatides, ristocetin, and botrocetin.1 Multiple
interactions of repeating binding sites in vWF multimers with proteins
of the subendothelium and with receptors on the platelet surface lead
to adhesion of platelets to the exposed subendothelium, which resist
remarkable shear forces encountered in the circulating blood,
particularly in small vessels and stenosed arteries. Deficiency or
molecular abnormality of vWF leads to von Willebrand disease, the most
common inherited bleeding disorder. On the other hand, increased levels of vWF, which may be the result of chronic endothelial cell injury, have been associated with atherogenesis,2 deep vein
thrombosis,3 myocardial infarction,4,5 and
ischemic stroke.5,6 Therefore, vWF has been proposed as a
target for antithrombotic treatment.7,8
After secretion into plasma, vWF multimers undergo proteolytic
degradation; the 250-kd polypeptide subunits are degraded to fragments
of 140, 176, and 189 kd9 by proteolytic cleavage of the
peptide bond Tyr842-Met843.10 A specific vWF-cleaving protease (vWF-cp), responsible for in vivo degradation of vWF, has been
partially purified and characterized.11,12 This protease seems to be different from all other described
proteases,11 although some similarity with matrix
metalloproteinases (MMPs) has been reported.13 vWF-cp
showed an uncommonly high molecular weight11,12 and an
unusually long biologic half-life.14
The largest multimeric forms of vWF in the flowing blood are
hemostatically most active. In healthy persons, they appear not to
interact with circulating platelets. Unusually large molecular forms of
vWF were found in patients suffering from relapsing thrombotic thrombocytopenic purpura (TTP).15 These very large
multimers showed increased binding to platelets under high levels of
shear stress.16 Moake et al15 suggested that
the presence of unusually large vWF multimers may be due to either
excessive secretion of endothelial vWF or impaired degradation of the
highest multimers because of deficiency of a disulfide bond reductase
or a protease. Recent studies indeed showed complete constitutional
deficiency of vWF-cp activity in patients with familial
TTP17-19 and the presence of autoantibodies inhibiting
vWF-cp in patients with nonfamilial TTP.18,20,21
Plasma infusion or exchange is considered to be the therapy of choice
in patients with TTP. Until the early 1960s, less than 3% of patients
with TTP survived.22 During the next decades, the
treatment of TTP with plasma infusion and exchange resulted in better
than 80% survival. The effectiveness of treatment of TTP by plasma
infusion and exchange has been ascribed to replacement of the missing
vWF-cp activity and removal of autoantibody.17,23 The
dependence on plasma exchange set off a massive consumption of plasma
for therapy of TTP patients, as reported by the Canadian Apheresis
Group.24 In 1997, TTP was the most frequent indication for
plasma exchange, accounting for about 40% of all such procedures. Obviously, a plasma product enriched in vWF-cp or a recombinant vWF-cp
would represent an important advantage in therapy of TTP. Furthermore,
vWF-cp may be considered as a potential agent affecting vWF binding to
subendothelial components and platelets, and thus impeding thrombotic
vascular occlusion. The development of plasma or recombinant vWF-cp
preparations depends on information concerning the biochemical behavior
and structural properties of vWF-cp.
The present study concerns the isolation of vWF-cp from normal human
plasma by several chromatographic methods, including immunoadsorption
on autoantibodies from a patient with acquired TTP. In addition to a
short N-terminal amino acid sequence, the binding to clustering during
purification and the stability of vWF-cp are described.
Materials
Purification of vWF-cp
First step.
A pool of platelet-poor plasma from 4 healthy volunteers was
anticoagulated by citrate-phosphate-dextrose and stored at Second step.
Protein fractions from 8 runs of the first step, containing vWF-cp
activity, were pooled and diluted 1:2 with distilled water to lower the
ionic strength. The diluted sample passed through a column of
Ig-TheraSorb (1.6 × 25 cm), kindly provided by Dr R. Koll
(PlasmaSelect, Teterow, Germany), consisting of Sepharose-CL-4B coupled
with sheep antibodies to human IgG, IgM, IgA, and IgE. The Ig-TheraSorb
column was equilibrated and eluted with 10 mM Tris, 75 mM NaCl, pH 7.4. Subsequently the unadsorbed proteins, in a total volume of about 1600 mL, were applied to an anionic exchange column (High Q Support;
1.6 × 2.5 cm) equilibrated with the latter buffer at a flow rate of
90 mL/h. The proteins bound to High Q Support were eluted with 20 mM
Tris, 0.5 M NaCl, 1 mM MnCl2, pH 7.4, and led directly onto
a column of lentil lectin-Sepharose (1.6 × 12.5 cm) equilibrated
with the same buffer. Chromatography was performed at a flow rate of 60 mL/h, and the proteins were eluted in 2 steps by 20 mM Tris, 0.5 M
NaCl, pH 7.4, containing 30 mM and 0.3 M Third step.
The protein fractions eluted with 0.3 M Anti- Anticlusterin immunoaffinity chromatography One milliliter of the peak fraction eluted from the initial immunoaffinity column (first step) or 1 mL normal human plasma was applied on a column of anticlusterin-Sepharose (2 mL) that had been prepared by coupling a murine monoclonal anti-human clusterin antibody to CNBr-activated Sepharose-4B. The column had a ligand density of 0.4 mg IgG/mL and was equilibrated with TBS. After application of the sample, flow through the column was stopped for 30 minutes. The column was then washed with TBS, and the bound proteins were eluted by 3.0 M NaSCN in TBS. The chromatography was performed at a flow rate of 12 mL/h, and the collected 1-mL fractions were immediately dialyzed against TBS.In another experiment, a 1-mL sample of partially purified vWF-cp from the initial immunoaffinity step was applied on the anticlusterin-Sepharose column, and proteins were eluted stepwise by the following: (1) 5 mM Tris, 1 mM CaCl2, pH 8.0; (2) 5 mM Tris, 1.0 M urea, 1 mM CaCl2, pH 8.0; (3) 10 mM Tris, 0.5 M NaCl, pH 7.4; and (4) 3.0 M NaSCN in TBS. Activity of vWF-cp Assay of vWF-cp was performed as described previously using sodium dodecyl sulfate (SDS)-agarose gel electrophoresis on 1.4% agarose and immunoblotting with anti-vWF antibody for analysis of digested vWF.11Stability of vWF-cp Blood samples were collected from the same healthy subject as citrated blood (1 volume 0.106 M Na3-citrate per 9 volumes blood), heparin blood, or native blood allowed to clot completely before centrifugation. An aliquot of the serum sample was dialyzed for 4 hours at 37°C against 1 mM CaCl2, 5 mM Tris, pH 8.0, and subsequently incubated for 2 weeks at 37°C. In addition, a fraction of partially purified vWF-cp, obtained by affinity chromatography of normal plasma on patient IgG-AffiGel column (first step of vWF-cp purification; see above), was also incubated for 2 weeks at 37°C in 0.15 M NaCl, 10 mM Tris, 1 mM Na3-citrate, pH 7.4. After incubation, the samples were submitted to collagen binding assay of vWF-cp.25SDS-polyacrylamide gel electrophoresis and silver staining SDS-polyacrylamide slab gels with a continuous gradient from 4% to 12% polyacrylamide and a thickness of 3 mm were made according to Laemmli.26 Proteins were reduced with 65 mM dithiothreitol for 20 minutes at 60°C. Electrophoresis was performed for 16 hours at 60 V. The proteins in the gel were then fixed by a mixture of 25% methanol, 7.5% acetic acid, 1.74% glycerol, and 65.8% distilled H2O (fixing solution) for at least 3 hours. After washing 4 times for 1 hour with distilled H2O, the gel was incubated for 5 minutes in 0.02% Na2S2O3, rinsed twice for 1 minute with H2O, and incubated twice for 20 minutes with a solution of 0.1% Ag nitrate. The gels were then washed again twice for 5 minutes with H2O, and the staining was continued by incubation in 2.5% Na2-carbonate and 0.015% formaldehyde, and terminated using the fixing solution.Immunoblotting of reduced vWF Proteins were electrotransferred from the SDS-polyacrylamide slab gel to a nitrocellulose membrane during 3 hours at 26 V in a buffer containing 0.04% SDS and 50 mM NaH2PO4, pH 7.4. The membrane was blocked with 2.5% BSA in TBS and incubated with rabbit anti-human vWF and goat anti-rabbit IgG antibody labeled with alkaline phosphatase. The vWF subunit and its fragments were visualized by incubation with 0.01% NBT, 0.006% BCIP, and 4 mM MgCl2 in 0.1 M ethanolamine, pH 9.8.Dot blots Samples eluted from anticlusterin-Sepharose were diluted in TBS, and 3-µL aliquots were applied to a nitrocellulose membrane. After blocking the membrane with 2.5% BSA in TBS, pH 7.4, for 30 minutes at 37°C, clusterin was detected following overnight incubation with a monoclonal mouse anti-human clusterin antibody (dilution 1:2500 in TBS containing 2.5% BSA) and a rabbit anti-mouse IgG antibody conjugated with horseradish peroxidase (dilution 1:1000, 2 hours), followed by staining with a solution of 0.05% diaminobenzidine, 0.007 N NaOH, and 0.012% H2O2.Amino acid sequencing of vWF-cp The final protein preparation from the third step of isolation was subjected to electrophoresis on a 1.5-mm-thick SDS-polyacrylamide gel according to Laemmli.26 A gradient of 4% to 12% polyacrylamide was used for fractionation of high-molecular-weight proteins, and a gradient of 8% to 12% polyacrylamide was used for low-molecular-weight proteins. After electrophoresis under nonreducing or reducing conditions (final concentration 65 mM dithiothreitol), the proteins were blotted onto PVDF membranes and stained for 2 minutes with 0.25% Coomassie Blue in 45% methanol, 9% acetic acid, and 46% H2O. After rinsing with a mixture of 50% methanol, 10% acetic acid, and 40% H2O, the visible protein bands were cut out and analyzed on a Procise-cLC Sequencer (Foster City, CA) at the Chemical Institute of the University of Bern.Amino acid analysis Analysis of the composition of the amino acids was performed at the Chemical Institute of the University of Bern from the same sample as used for amino acid sequencing. The protein bands were hydrolyzed in the gas phase over 6 N HCl for 22 hours at 110°C, and the amino acids were determined by high-performance liquid chromatography.
Isolation and characterization of vWF-cp First step.
Affinity chromatography of 100 mL normal human plasma on AffiGel
Hz with the covalently linked IgG fraction of plasma from a patient
with autoantibodies against vWF-cp was used for initial purification of
vWF-cp (Figure 1, first step). The
capacity of the column was sufficient to completely bind vWF-cp from
0.5 mL normal plasma per 1 mL immunoadsorbent. The column was washed overnight with TBS containing 1 mM Na3-citrate and 0.02%
NaN3, and the bound proteins were dissociated from the
antibodies using 3 M NaSCN, which was in turn separated from vWF-cp by
gel filtration on BioGel P-6DG. Because the immunoaffinity column also
retained major amounts of IgG from normal human plasma, the eluted
fractions were filtered on a tandem column of Protein G-Sepharose. The
total amount of protein in the pooled fractions containing vWF-cp
activity corresponded to about 0.2% of initial, as estimated from the
number of absorbance units (AU) measured at 280 nm (Table
1). The loss of about 50% of initial
vWF-cp activity was primarily due to inactivation, as judged from our
preliminary experiments on protease stability in 3 M NaSCN.
SDS-polyacrylamide gel electrophoresis (PAGE); of these
fractions revealed a variety of proteins in Mr range
between 30 and 1000 kd under nonreducing conditions (data not shown). These proteins included IgA, IgM, fibronectin,
Second step.
The eluted protein fractions containing vWF-cp activity from 8 batches
of the first step were pooled and filtered through a column of
Ig-TheraSorb to remove IgA, IgM, and residual IgG. Subsequently, the
proteins from a total volume of 1600 mL were adsorbed on a 5-mL column
of High Q Support, and again eluted with 0.5 M NaCl, 1 mM
MnCl2, in 20 mM Tris buffer, pH 7.4. The resulting solution
was further subjected to chromatography on lentil lectin-Sepharose.
While the major part of vWF-cp activity (about 80%) was found in the
fractions eluted by 0.3 M Third step. Fibronectin was removed by chromatography on heparin-Sepharose. Preliminary experiments showed that vWF-cp activity did not bind to heparin-Sepharose under conditions required for complete adsorption of contaminating fibronectin. The volume of the collected active fractions was again reduced on a small tandem column of High Q Support. The proteins in the protease preparation were further fractionated by gel filtration on Sephacryl S-300 HR. As shown in Figure 1 (third step), the sample was introduced into the Sephacryl column during collection of fraction 28. The activity assay indicated that the protease was eluted from the Sephacryl column in fractions 57 to 61 (Figure 2A). The elution volume of vWF-cp (200-230 mL) was close to the void volume of the Sephacryl column, suggesting that the protease elutes from Sephacryl S-300 HR as a high-molecular-weight protein. SDS-PAGE of the corresponding fractions under nonreducing conditions (Figure 3A) showed a protein band at 70 kd, 4 bands in the range of 110 to 150 kd (110, 130, 140, and 150 kd), and a band at 350 kd, in addition to a smeared band of more than 400 kd. The 350-kd and 70-kd bands disappeared completely after reduction of disulfide bonds (Figure 3B), whereas the unreduced bands within Mr range of 110 to 150 kd appeared to represent single-chain polypeptides because they were also identified in the reduced sample, albeit migrating slightly more slowly (with respective Mr of 120, 160, 170, and 180 kd).
Two-dimensional SDS-PAGE (Figure 4) showed that the 350-kd band produced 2 bands of Mr 170 and 120 kd upon disulfide reduction. N-terminal sequence analysis (Table 2) of the 350-kd protein band (Ser/Asn-Val-Ser-Gly-Lys-Pro-Gln-Tyr-Met-Val) indicated that this band represented  2M. This was confirmed by immunoblotting; the band of 350 kd from the unreduced sample and the band of 170 kd
from the reduced sample reacted with rabbit anti-2M
antibodies (not shown). Affinity chromatography on
anti-2M-Sepharose resulted in complete removal of
2M and full recovery of vWF-cp eluting from the column
in the breakthrough (data not shown). Because the reduced 170-kd
protein band in Figure 3B contained both vWF-cp and
2M, sequence analysis of the reduced bands was performed after SDS-PAGE of 2M-depleted samples. Amino acid
sequences of all 4 polypeptides migrating as bands with Mr
110 to 150 kd and 120 to 180 kd in unreduced and reduced SDS-PAGE,
respectively, indicated an identical N-terminal amino acid sequence
Ala-Ala-Gly-Gly-Ile, suggesting that they represented the same
polypeptide that had been proteolytically cleaved at the
carboxy-terminal portion of the molecule. In addition, all 4 polypeptides had similar amino acid composition (Table
3).
Anticlusterin immunoaffinity chromatography One milliliter of the peak fraction that had been eluted in TBS from the Protein G-Sepharose column at the end of the first purification step was applied to the anticlusterin column consisting of 1 mL Sepharose 4B coupled with murine monoclonal anti-human clusterin antibody. This crude fraction of partially purified vWF-cp contained a mixture of proteins of molecular mass range 30 to 1000 kd, with a total absorbance of 0.2 at 280 nm. Virtually all proteins were adsorbed to the anticlusterin column. The results of the protease assay are shown in Figure 5A. The activity of the initial protease preparation (a) is completely depleted in the breakthrough elution fraction (b). The column was then thoroughly washed with TBS and the protease was eluted with 3 M NaSCN (c). The latter fraction was dialyzed against TBS before analysis. There was no clusterin present in the breakthrough volume (b), as shown by dot blotting with monoclonal anticlusterin antibodies (Figure 5B). There were also no other proteins detectable in the fraction (b) by SDS-PAGE and silver staining (data not shown). Most proteins of the initial protease preparation, including clusterin, were eluted by 3.0 M NaSCN. It is obvious that the protease was retained by complex formation with clusterin trapped within the anticlusterin column. To examine the binding affinity of vWF-cp for clusterin, we performed a stepwise elution using low ionic strength (5 mM Tris, 1 mM CaCl2, pH 8.0), 1 M urea (in 5 mM Tris, 1 mM CaCl2, pH 8.0), and 0.5 M NaCl (in 10 mM Tris, pH 7.4). There was no vWF-cp activity and no clusterin eluted by any of these 3 buffers, but complete release of protease and clusterin was achieved with 3 M NaSCN (data not shown).
Affinity chromatography on an anticlusterin column was also carried out
with 1 mL citrated normal human plasma. Results shown in Figure
6A indicate that the protease activity of
the normal plasma sample (a) appeared in the breakthrough volume
(b1-b5, representing elution fractions 2-6, with volumes of 1 mL each). After washing the column with TBS, the elution was continued after fraction 25 with 3 M NaSCN. Less than 1% of total protease activity appeared in 1-mL fractions 27 and 28, corresponding to lanes c1 and c2,
respectively. Dot blot analysis showed absence of clusterin in the
breakthrough fractions containing vWF-cp, whereas clusterin was eluted
with 3 M NaSCN (Figure 6B). To exclude the influence of chelating
citrate ions, we repeated the latter experiment with 1 mL normal human
serum. The results were identical (data not shown) to those observed
with citrated human plasma.
Stability of vWF-cp Activity of vWF-cp during incubation of citrated plasma, heparin plasma, and serum at 37°C for 2 weeks is shown in Figure 7. The initial activity in the serum and in the heparin plasma was about 20% higher than in citrated plasma that had been diluted by addition of 1/9 blood volume of Na3-citrate solution. There was a steady but slow decrease of protease activity in the citrated plasma (half-life longer than 1 week), whereas the vWF-cp was extremely stable in heparin plasma and in serum. In fact, there was even a temporary increase in protease activity within the first 3 days of incubation. Similar stability was observed in the serum sample that had been dialyzed against 1 mM CaCl2, 5 mM Tris, pH 8.0 before incubation (data not shown). In contrast to vWF-cp activity in plasma or serum, a partially purified protease preparation obtained by affinity chromatography on patient IgG as a complex with clusterin, containing only 0.2% of initial total protein concentration, showed an impaired stability. Its in vitro half-life was about 5 days (not shown).
In response to endothelial cell activation or injury, unusually large and biologically potent vWF multimers are released from the endothelial cell-specific organelles called Weibel-Palade bodies.27 These extremely large vWF multimers have a higher binding affinity for platelet receptors GPIb and/or GPIIb/IIIa than the largest vWF forms present in normal plasma and can induce platelet aggregation under conditions of elevated fluid shear stress.28 Unusually large vWF multimers were detected in patients with TTP,15,17 and increased vWF binding to platelets was demonstrated in TTP patients.16 In 1996, 2 groups of investigators independently identified and partially purified a protease from normal human plasma that was shown to be responsible for in vivo degradation of vWF multimers.11,12 Severe congenital deficiency of this protease was established in patients with familial TTP,17-19 and the presence of vWF-cp-inhibiting autoantibodies was observed in patients with nonfamilial TTP.18,20,21 Another attempt to purify the vWF-cp is described in the present report. The initial purification step consisted of affinity chromatography of normal human plasma on the IgG fraction obtained from a patient with an autoantibody to vWF-cp. The protease was eluted from the immunoadsorbent using 3 M NaSCN and was immediately separated from the chaotropic agent. The resulting fraction contained about 0.2% of initial protein but still contained many contaminating proteins. Of these, IgGs were removed by affinity chromatography on Protein G-Sepharose and on sheep antibodies to human IgG, IgM, IgA, and IgE. Affinity chromatography on lentil lectin-Sepharose resulted in removal of more than 95% contaminating proteins but was also accompanied by a considerable loss of the protease activity. The recovered vWF-cp fraction still contained significant amounts of fibronectin, which had been previously shown to contain latent matrix-degrading protease activities that were generated by cleavage of purified fibronectin with cathepsin D29 and other cathepsins.30 Furthermore, Kempfer et al31 showed that the proteolytic degradation products of fibronectin induced the loss of large vWF multimers at high shear stress. Our results indicate that vWF-cp is not associated with fibronectin because the protease activity was not removed from normal human plasma on gelatin-Sepharose or heparin-Sepharose (data not shown), 2 materials with strong binding affinities for fibronectin. vWF-cp was eluted from Sephacryl S-300 HR in the early fractions after the void volume, suggesting an unusually high molecular weight for a protease, as observed earlier.11 SDS-PAGE of the unreduced material showed 4 bands migrating with Mr of 150, 140, 130, and 110 kd. All 4 polypeptides showed the same N-terminal amino acid sequence (Ala-Ala-Gly-Gly-Ile). The sequence of the first 15 amino acids (Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val-Gly) is very hydrophobic; it contains 4 Leu, 1 Ile, and 2 Val residues. Proteolytic degradation apparently led to cleavage of vWF-cp at multiple sites in the carboxy-terminal portion of the molecule. The amino acid composition showed remarkable proportions of Arg and very low content of Lys. Human proteins showing the best similarity in amino acid composition (ie, fibulin, pregnancy zone protein) were found to lack the N-terminal amino acid sequence of vWF-cp. From the recovery of vWF-cp in the final fraction and taking into
consideration the relative intensities of silver-stained bands of
vWF-cp, The protease was eluted by gel filtration from the Sephacryl S-300 HR
column together with It is known that The identity of the 70-kd protein contaminating the purified vWF-cp
preparation was deduced from the amino acid sequences of the SDS-PAGE
bands. Clusterin (apolipoprotein J) is a carbohydrate-rich 2-chain
protein consisting of a 222-residue Our results indicate that clusterin, vWF-cp, and all contaminating proteins that were eluted with 3 M NaSCN from the patient IgG-AffiGel column were retained on anticlusterin-Sepharose. vWF-cp could not be dissociated from clusterin under conditions enhancing the proteolytic digestion of vWF (5 mM Tris, 1 mM CaCl2, pH 8.0; 1.0 M urea). No detectable ligand was dissociated from the anticlusterin column with 0.5 M NaCl, whereas clusterin, vWF-cp, and all bound contaminating proteins were eluted with 3 M NaSCN, thus explaining the high apparent molecular weight of vWF-cp and clusterin, as judged from gel filtration on Sephacryl S-300 HR. Affinity chromatography of normal human plasma and of normal human serum on anticlusterin-Sepharose showed that the vWF-cp passed unretarded through the column, whereas the clusterin was firmly bound and was again dissociated with 3 M NaSCN. These results indicate that vWF-cp in vivo is not associated with clusterin and that the observed complex formation is due to vWF-cp denaturation during the first step of the purification procedure. Our data show that the protease activity in citrated plasma is inactivated very slowly at 37°C (half-life longer than 1 week) and that the activity even increases initially in heparin plasma or in serum, both containing about 1 mM of free calcium ions. It remains to be investigated whether vWF-cp is present in the circulation as an inactive precursor that is slowly but continuously activated in the presence of calcium ions. There is a practical benefit of these observations for the clinical laboratory. Because of full recovery of vWF-cp in serum and heparin plasma, both forms of patient samples are suitable, in addition to citrated plasma, for detection of vWF-cp deficiency in patients with TTP/hemolytic-uremic syndrome, even if the samples have been stored for longer intervals at room temperature. It must be added that EDTA is not appropriate as a blood anticoagulant because it completely and apparently irreversibly inactivates vWF-cp.25
The reader is referred to the accompanying paper, Fujikawa et al,39 page 1662, characterizing vWF-cp as a metalloprotease.
Submitted March 29, 2001; accepted July 3, 2001.
Supported by grants from the Swiss National Science Foundation (32-47033.96); from the Malcolm Hewitt Wiener Foundation, New York, NY; from the Central Laboratory, Blood Transfusion Service, Swiss Red Cross, Bern, Switzerland; and from Baxter Immuno, Vienna, Austria.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Bernhard Lämmle, Central Hematology Laboratory, University Hospital, Inselspital, CH-3010 Bern, Switzerland; e-mail: bernhard.laemmle{at}insel.ch.
1. Sadler JE. Biochemistry and genetics of von Willebrand factor. Annu Rev Biochem. 1998;67:395-424[CrossRef][Medline] [Order article via Infotrieve]. 2. Badimon L, Badimon JJ, Chesbro JH, Fuster V. von Willebrand factor and cardiovascular disease. Thromb Haemost. 1993;70:111-118[Medline] [Order article via Infotrieve]. 3. Koster T, Blann AD, Briet E, Vandenbroucke JP, Rosendaal FR. Role of clotting factor VIII in effect of von Willebrand factor on occurrence of deep-vein thrombosis. Lancet. 1995;345:152-155[CrossRef][Medline] [Order article via Infotrieve]. 4. Jansson J-H, Nilsson TK, Johnson O. von Willebrand factor in plasma: a novel risk factor for recurrent myocardial infarction. Br Heart J. 1991;66:678-682.
5.
Cortellaro M, Boschetti C, Cofrancesco E, et al.
The PLAT Study: hemostatic function in relation to atherothrombotic ischemic events in vascular disease patients. Principal results.
Arterioscler Thromb.
1992;12:1063-1070
6.
Qizilbash N, Duffy S, Prentice CRM, Boothby M, Warlow C.
von Willebrand factor and risk of ischemic stroke.
Neurology.
1997;49:1552-1556 7. Ruggeri ZM. von Willebrand factor as a target for antithrombotic intervention. Circulation. 1992;86 (suppl 6):III26-III29. 8. Meyer D. von Willebrand factor: a target for inhibition of thrombosis. Nouv Rev Fr Hematol. 1993;35:255-257. 9. Berkowitz SD, Dent J, Roberts J, et al. Epitope mapping of the von Willebrand factor subunit distinguishes fragments present in normal and type IIA von Willebrand disease from those generated by plasmin. J Clin Invest. 1987;79:524-531.
10.
Dent JA, Berkowitz SD, Ware J, Kasper CK, Ruggeri ZM.
Identification of a cleavage site directing the immunochemical detection of molecular abnormalities in type IIA von Willebrand factor.
Proc Natl Acad Sci U S A.
1990;87:6306-6310
11.
Furlan M, Robles R, Lämmle B.
Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis.
Blood.
1996;87:4223-4234
12.
Tsai H-M.
Physiologic cleavage of von Willebrand factor by a plasma protease is dependent on its conformation and requires calcium ion.
Blood.
1996;87:4235-4244
13.
Tsai H-M, Sussman II, Ginsburg D, Lankhof H, Sixma JJ, Nagel RL.
Proteolytic cleavage of recombinant type 2A von Willebrand factor mutants R834W and R834Q: inhibition by doxycycline and by monoclonal antibody VP-1.
Blood.
1997;89:1954-1962 14. Furlan M, Robles R, Morselli B, Sandoz P, Lämmle B. Recovery and half-life of von Willebrand factor-cleaving protease after plasma therapy in patients with thrombotic thrombocytopenic purpura. Thromb Haemost. 1999;81:8-13[Medline] [Order article via Infotrieve]. 15. Moake JL, Rudy CK, Troll JH, et al. Unusually large plasma factor VIII: von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. N Engl J Med. 1982;307:1432-1435[Medline] [Order article via Infotrieve]. 16. Chow TW, Turner NA, Chintagumpala M, et al. Increased von Willebrand factor binding to platelets in single episode and recurrent types of thrombotic thrombocytopenic purpura. Am J Hematol. 1998;57:293-302[CrossRef][Medline] [Order article via Infotrieve].
17.
Furlan M, Robles R, Solenthaler M, Wassmer M, Sandoz P, Lämmle B.
Deficient activity of von Willebrand factor-cleaving protease in chronic relapsing thrombotic thrombocytopenic purpura.
Blood.
1997;89:3097-3103
18.
Furlan M, Robles R, Galbusera M, et al.
Von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome.
N Engl J Med.
1998;339:1578-1584 19. Furlan M, Lämmle B. Aetiology and pathogenesis of thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome: the role of von Willebrand factor-cleaving protease. Baillières Clin Haematol. 2001;14:437-454.
20.
Furlan M, Robles R, Solenthaler M, Lämmle B.
Acquired deficiency of von Willebrand factor-cleaving protease in a patient with thrombotic thrombocytopenic purpura.
Blood.
1998;91:2839-2846
21.
Tsai H-M, Lian EC-Y.
Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura.
N Engl J Med.
1998;339:1585-1593 22. Shepard KV, Bukowski RM. The treatment of thrombotic thrombocytopenic purpura with exchange transfusions, plasma infusions, and plasma exchange. Semin Hematol. 1987;24:178-193[Medline] [Order article via Infotrieve]. 23. Moake JL, Chow TW. Thrombotic thrombocytopenic purpura: understanding a disease no longer rare. Am J Med Sci. 1998;316:105-119[CrossRef][Medline] [Order article via Infotrieve].
24.
Clark WF, Rock GA, Buskard N, et al.
Therapeutic plasma exchange: an update from the Canadian Apheresis Group.
Ann Intern Med.
1999;131:453-462 25. Gerritsen HE, Turecek PL, Schwarz HP, Lämmle B, Furlan M. Assay of von Willebrand factor (vWF)-cleaving protease based on decreased collagen binding affinity of degraded vWF. A tool for the diagnosis of thrombotic thrombocytopenic purpura (TTP). Thromb Haemost. 1999;82:1386-1389[Medline] [Order article via Infotrieve]. 26. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685[CrossRef][Medline] [Order article via Infotrieve]. 27. Sporn LA, Marder VJ, Wagner DD. Inducible secretion of large, biologically potent von Willebrand factor multimers. Cell. 1986;46:185-190[CrossRef][Medline] [Order article via Infotrieve]. 28. Moake JL, Turner NA, Stathopoulos NA, Nolasco LH, Hellums JD. Involvement of large plasma von Willebrand factor (vWF) multimers and unusually large vWF forms derived from endothelial cells in shear stress-induced platelet aggregation. J Clin Invest. 1986;78:1456-1461.
29.
Keil-Dlouha V, Planchenault T.
Potential proteolytic activity of human plasma fibronectin.
Proc Natl Acad Sci U S A.
1986;83:5377-5381 30. Blondeau X, Lambert Vidmar S, Emod I, Turk V, Keil-Dlouha V. Generation of matrix-degrading proteolytic system from fibronectin by cathepsins B, G, H and L. Biol Chem Hoppe Seyler. 1993;374:651-656[Medline] [Order article via Infotrieve]. 31. Kempfer AC, Farias CE, Silaf MR, Carballo GA, Lazzari MA. Degradation products of partially purified fibronectin induced the loss of large von Willebrand factor multimers at high shear rate [abstract]. Thromb Haemost. 1999;82(suppl):100[Medline] [Order article via Infotrieve].
32.
Harpel PC, Mosesson MW.
Degradation of human fibrinogen by plasma
33.
Switzer MEP, Gordon HJ, McKee PA.
Proteolytic activity of
34.
Gonias SL, Pizzo SV.
Reaction of human 35. Wilson MR, Easterbrook-Smith SB. Clusterin is a secreted mammalian chaperone. Trends Biochem Sci. 2000;25:95-98[CrossRef][Medline] [Order article via Infotrieve].
36.
Humphreys DT, Carver JA, Easterbrook-Smith SB, Wilson MR.
Clusterin has chaperone-like activity similar to that of small heat shock proteins.
J Biol Chem.
1999;274:6875-6881 37. Pankhurst GJ, Bennett CA, Easterbrook-Smith SB. Characterization of the heparin-binding properties of human clusterin. Biochemistry. 1998;37:4823-4830[CrossRef][Medline] [Order article via Infotrieve]. 38. Hochgrebe T, Pankhurst GJ, Wilce J, Easterbrook-Smith SB. pH-dependent changes in the in vitro ligand-binding properties and structure of human clusterin. Biochemistry. 2000;39:1411-1419[CrossRef][Medline] [Order article via Infotrieve].
39.
Fujikawa K, Suzuki H, McMullen B, Chung D.
Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family.
Blood.
2001;98:1662-1666
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  S. F. De Meyer, H. Deckmyn, and K. Vanhoorelbeke von Willebrand factor to the rescue Blood, May 21, 2009; 113(21): 5049 - 5057. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Garagiola, C. Valsecchi, S. Lavoretano, H. Oren, M. Bohm, and F. Peyvandi Nonsense-mediated mRNA decay in the ADAMTS13 gene caused by a 29-nucleotide deletion Haematologica, November 1, 2008; 93(11): 1678 - 1685. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Sadler Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura Blood, July 1, 2008; 112(1): 11 - 18. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Lammle, J. A. Kremer Hovinga, and J. N. George Acquired thrombotic thrombocytopenic purpura: ADAMTS13 activity, anti-ADAMTS13 autoantibodies and risk of recurrent disease Haematologica, February 1, 2008; 93(2): 172 - 177. [Full Text] [PDF]
|
|
|
|
|
|
F. Peyvandi, S. Lavoretano, R. Palla, H. B. Feys, K. Vanhoorelbeke, T. Battaglioli, C. Valsecchi, M. T. Canciani, F. Fabris, S. Zver, et al. ADAMTS13 and anti-ADAMTS13 antibodies as markers for recurrence of acquired thrombotic thrombocytopenic purpura during remission Haematologica, February 1, 2008; 93(2): 232 - 239. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Anderson, D. Gailani, H. B. Feys, W. Gao, E. M. Majerus, K. Vanhoorelbeke, and J. E. Sadler Factor XI/ADAMTS13 complexes are quantitatively insignificant in human plasma Haematologica, October 1, 2007; 92(10): 1419 - 1422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. C. Desch and D. G. Motto Thrombotic Thrombocytopenic Purpura in Humans and Mice Arterioscler Thromb Vasc Biol, September 1, 2007; 27(9): 1901 - 1908. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. K. Swisher, J. T. Doan, S. K. Vesely, H. C. Kwaan, B. Kim, B. Lammle, J. A. Kremer Hovinga, and J. N. George Pancreatitis preceding acute episodes of thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: report of five patients with a systematic review of published reports Haematologica, July 1, 2007; 92(7): 936 - 943. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.J. Hunt, S. Tueger, J. Pattison, J. Cavenagh, and D.P. D'Cruz Microangiopathic haemolytic anaemia secondary to lupus nephritis: an important differential diagnosis of thrombotic thrombocytopenic purpura Lupus, May 1, 2007; 16(5): 358 - 362. [Abstract] [PDF]
|
|
|
|
 |
|
 A. Casonato, F. Fabris, E. Pontara, M. G. Cattini, N. Zocca, L. Gallinaro, A. Girolami, and A. Pagnan Diagnosis and Follow-up of Thrombotic Thrombocytopenic Purpura by Means of von Willebrand Factor Collagen Binding Assay Clinical and Applied Thrombosis/Hemostasis, July 1, 2006; 12(3): 296 - 304. [Abstract] [PDF]
|
|
|
|
|
|
W. A. Hassenpflug, U. Budde, T. Obser, D. Angerhaus, E. Drewke, S. Schneppenheim, and R. Schneppenheim Impact of mutations in the von Willebrand factor A2 domain on ADAMTS13-dependent proteolysis Blood, March 15, 2006; 107(6): 2339 - 2345. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ono, J. Mimuro, S. Madoiwa, K. Soejima, Y. Kashiwakura, A. Ishiwata, K. Takano, T. Ohmori, and Y. Sakata Severe secondary deficiency of von Willebrand factor-cleaving protease (ADAMTS13) in patients with sepsis-induced disseminated intravascular coagulation: its correlation with development of renal failure Blood, January 15, 2006; 107(2): 528 - 534. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Soejima, H. Nakamura, M. Hirashima, W. Morikawa, C. Nozaki, and T. Nakagaki Analysis on the Molecular Species and Concentration of Circulating ADAMTS13 in Blood J. Biochem., January 1, 2006; 139(1): 147 - 154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Sadler Thrombotic Thrombocytopenic Purpura: A Moving Target Hematology, January 1, 2006; 2006(1): 415 - 420. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Plaimauer, J. Fuhrmann, G. Mohr, W. Wernhart, K. Bruno, S. Ferrari, C. Konetschny, G. Antoine, M. Rieger, and F. Scheiflinger Modulation of ADAMTS13 secretion and specific activity by a combination of common amino acid polymorphisms and a missense mutation Blood, January 1, 2006; 107(1): 118 - 125. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. J. Hulstein, P. G. de Groot, K. Silence, A. Veyradier, R. Fijnheer, and P. J. Lenting A novel nanobody that detects the gain-of-function phenotype of von Willebrand factor in ADAMTS13 deficiency and von Willebrand disease type 2B Blood, November 1, 2005; 106(9): 3035 - 3042. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Ai, P. Smith, S. Wang, P. Zhang, and X. L. Zheng The Proximal Carboxyl-terminal Domains of ADAMTS13 Determine Substrate Specificity and Are All Required for Cleavage of von Willebrand Factor J. Biol. Chem., August 19, 2005; 280(33): 29428 - 29434. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. G. Levy, D. G. Motto, and D. Ginsburg ADAMTS13 turns 3 Blood, July 1, 2005; 106(1): 11 - 17. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Tao, Y. Wang, H. Choi, A. Bernardo, K. Nishio, J. E. Sadler, J. A. Lopez, and J.-f. Dong Cleavage of ultralarge multimers of von Willebrand factor by C-terminal-truncated mutants of ADAMTS-13 under flow Blood, July 1, 2005; 106(1): 141 - 143. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. Majerus, P. J. Anderson, and J. E. Sadler Binding of ADAMTS13 to von Willebrand Factor J. Biol. Chem., June 10, 2005; 280(23): 21773 - 21778. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. B. Crawley, J. K. Lam, J. B. Rance, L. R. Mollica, J. S. O'Donnell, and D. A. Lane Proteolytic inactivation of ADAMTS13 by thrombin and plasmin Blood, February 1, 2005; 105(3): 1085 - 1093. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Uchida, H. Wada, M. Mizutani, M. Iwashita, H. Ishihara, T. Shibano, M. Suzuki, Y. Matsubara, K. Soejima, M. Matsumoto, et al. Identification of novel mutations in ADAMTS13 in an adult patient with congenital thrombotic thrombocytopenic purpura Blood, October 1, 2004; 104(7): 2081 - 2083. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. P. T. Somerville, J.-M. Longpre, E. D. Apel, R. M. Lewis, L. W. Wang, J. R. Sanes, R. Leduc, and S. S. Apte ADAMTS7B, the Full-length Product of the ADAMTS7 Gene, Is a Chondroitin Sulfate Proteoglycan Containing a Mucin Domain J. Biol. Chem., August 20, 2004; 279(34): 35159 - 35175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Banno, K. Kaminaka, K. Soejima, K. Kokame, and T. Miyata Identification of Strain-specific Variants of Mouse Adamts13 Gene Encoding von Willebrand Factor-cleaving Protease J. Biol. Chem., July 16, 2004; 279(29): 30896 - 30903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Klaus, B. Plaimauer, J.-D. Studt, F. Dorner, B. Lammle, P. M. Mannucci, and F. Scheiflinger Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura Blood, June 15, 2004; 103(12): 4514 - 4519. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. L. Zheng, R. M. Kaufman, L. T. Goodnough, and J. E. Sadler Effect of plasma exchange on plasma ADAMTS13 metalloprotease activity, inhibitor level, and clinical outcome in patients with idiopathic and nonidiopathic thrombotic thrombocytopenic purpura Blood, June 1, 2004; 103(11): 4043 - 4049. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-D. Studt, J. A. K. Hovinga, R. Radonic, V. Gasparovic, D. Ivanovic, M. Merkler, U. Wirthmueller, C. Dahinden, M. Furlan, and B. Lammle Familial acquired thrombotic thrombocytopenic purpura: ADAMTS13 inhibitory autoantibodies in identical twins Blood, June 1, 2004; 103(11): 4195 - 4197. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Padilla, J. L. Moake, A. Bernardo, C. Ball, Y. Wang, M. Arya, L. Nolasco, N. Turner, M. C. Berndt, B. Anvari, et al. P-selectin anchors newly released ultralarge von Willebrand factor multimers to the endothelial cell surface Blood, March 15, 2004; 103(6): 2150 - 2156. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Matsumoto, K. Kokame, K. Soejima, M. Miura, S. Hayashi, Y. Fujii, A. Iwai, E. Ito, Y. Tsuji, M. Takeda-Shitaka, et al. Molecular characterization of ADAMTS13 gene mutations in Japanese patients with Upshaw-Schulman syndrome Blood, February 15, 2004; 103(4): 1305 - 1310. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kokame, M. Matsumoto, Y. Fujimura, and T. Miyata VWF73, a region from D1596 to R1668 of von Willebrand factor, provides a minimal substrate for ADAMTS-13 Blood, January 15, 2004; 103(2): 607 - 612. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. Majerus, X. Zheng, E. A. Tuley, and J. E. Sadler Cleavage of the ADAMTS13 Propeptide Is Not Required for Protease Activity J. Biol. Chem., November 21, 2003; 278(47): 46643 - 46648. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Soejima, M. Matsumoto, K. Kokame, H. Yagi, H. Ishizashi, H. Maeda, C. Nozaki, T. Miyata, Y. Fujimura, and T. Nakagaki ADAMTS-13 cysteine-rich/spacer domains are functionally essential for von Willebrand factor cleavage Blood, November 1, 2003; 102(9): 3232 - 3237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Varadi, J. Schreiner, B. Plaimauer, M. Rieger, F. Scheiflinger, P. Knobl, P. L. Turecek, and H. P. Schwarz ADAMTS13 autoantibody detection by quantitative immunoblotting Blood, September 1, 2003; 102(5): 1932 - 1933. [Full Text] [PDF]
|
|
|
|
|
|
J.-f. Dong, J. L. Moake, A. Bernardo, K. Fujikawa, C. Ball, L. Nolasco, J. A. Lopez, and M. A. Cruz ADAMTS-13 Metalloprotease Interacts with the Endothelial Cell-derived Ultra-large von Willebrand Factor J. Biol. Chem., August 8, 2003; 278(32): 29633 - 29639. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zheng, K. Nishio, E. M. Majerus, and J. E. Sadler Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13 J. Biol. Chem., August 8, 2003; 278(32): 30136 - 30141. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Llamazares, S. Cal, V. Quesada, and C. Lopez-Otin Identification and Characterization of ADAMTS-20 Defines a Novel Subfamily of Metalloproteinases-Disintegrins with Multiple Thrombospondin-1 Repeats and a Unique GON Domain J. Biol. Chem., April 4, 2003; 278(15): 13382 - 13389. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-M. Tsai Advances in the Pathogenesis, Diagnosis, and Treatment of Thrombotic Thrombocytopenic Purpura J. Am. Soc. Nephrol., April 1, 2003; 14(4): 1072 - 1081. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Schneppenheim, U. Budde, F. Oyen, D. Angerhaus, V. Aumann, E. Drewke, W. Hassenpflug, J. Haberle, K. Kentouche, E. Kohne, et al. von Willebrand factor cleaving protease and ADAMTS13 mutations in childhood TTP Blood, March 1, 2003; 101(5): 1845 - 1850. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Zheng, A. M. Pallera, L. T. Goodnough, J. E. Sadler, and M. A. Blinder Remission of Chronic Thrombotic Thrombocytopenic Purpura after Treatment with Cyclophosphamide and Rituximab Ann Intern Med, January 21, 2003; 138(2): 105 - 108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-f. Dong, J. L. Moake, L. Nolasco, A. Bernardo, W. Arceneaux, C. N. Shrimpton, A. J. Schade, L. V. McIntire, K. Fujikawa, and J. A. Lopez ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions Blood, December 1, 2002; 100(12): 4033 - 4039. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-M. Tsai, B. Lammle, V. Bianchi, L. Alberio, M. Furlan, G. Remuzzi, M. Galbusera, and P. M. Mannucci Deficiency of ADAMTS13 and thrombotic thrombocytopenic purpura Blood, November 15, 2002; 100(10): 3839 - 3842. [Full Text] [PDF]
|
|
|
|
|
|
B. Plaimauer, K. Zimmermann, D. Volkel, G. Antoine, R. Kerschbaumer, P. Jenab, M. Furlan, H. Gerritsen, B. Lammle, H. P. Schwarz, et al. Cloning, expression, and functional characterization of the von Willebrand factor-cleaving protease (ADAMTS13) Blood, November 15, 2002; 100(10): 3626 - 3632. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. Flannery, W. Zeng, C. Corcoran, L. A. Collins-Racie, P. S. Chockalingam, T. Hebert, S. A. Mackie, T. McDonagh, T. K. Crawford, K. N. Tomkinson, et al. Autocatalytic Cleavage of ADAMTS-4 (Aggrecanase-1) Reveals Multiple Glycosaminoglycan-binding Sites J. Biol. Chem., November 1, 2002; 277(45): 42775 - 42780. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Sadler A new name in thrombosis, ADAMTS13 PNAS, September 3, 2002; 99(18): 11552 - 11554. [Full Text] [PDF]
|
|
|
|
|
|
K. Kokame, M. Matsumoto, K. Soejima, H. Yagi, H. Ishizashi, M. Funato, H. Tamai, M. Konno, K. Kamide, Y. Kawano, et al. Mutations and common polymorphisms in ADAMTS13 gene responsible for von Willebrand factor-cleaving protease activity PNAS, September 3, 2002; 99(18): 11902 - 11907. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Moake Thrombotic Microangiopathies N. Engl. J. Med., August 22, 2002; 347(8): 589 - 600. [Full Text] [PDF]
|
|
|
|
|
|
G. Remuzzi, M. Galbusera, M. Noris, M. T. Canciani, E. Daina, E. Bresin, S. Contaretti, J. Caprioli, S. Gamba, P. Ruggenenti, et al. von Willebrand factor cleaving protease (ADAMTS13) is deficient in recurrent and familial thrombotic thrombocytopenic purpura and hemolytic uremic syndrome Blood, July 18, 2002; 100(3): 778 - 785. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Bianchi, R. Robles, L. Alberio, M. Furlan, and B. Lammle Von Willebrand factor-cleaving protease (ADAMTS13) in thrombocytopenic disorders: a severely deficient activity is specific for thrombotic thrombocytopenic purpura Blood, June 28, 2002; 100(2): 710 - 713. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Arya, B. Anvari, G. M. Romo, M. A. Cruz, J.-F. Dong, L. V. McIntire, J. L. Moake, and J. A. Lopez Ultralarge multimers of von Willebrand factor form spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: studies using optical tweezers Blood, May 13, 2002; 99(11): 3971 - 3977. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. George, J. E. Sadler, and B. Lammle Platelets: Thrombotic Thrombocytopenic Purpura Hematology, January 1, 2002; 2002(1): 315 - 334. [Abstract] [Full Text]
|
|
|
|
|
|
K. Fujikawa, H. Suzuki, B. McMullen, and D. Chung Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family Blood, September 15, 2001; 98(6): 1662 - 1666. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zheng, D. Chung, T. K. Takayama, E. M. Majerus, J. E. Sadler, and K. Fujikawa Structure of von Willebrand Factor-cleaving Protease (ADAMTS13), a Metalloprotease Involved in Thrombotic Thrombocytopenic Purpura J. Biol. Chem., October 26, 2001; 276(44): 41059 - 41063. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
20°C in
50-mL aliquots. After thawing and centrifuging at 1100g for 5 minutes, 100 mL of the pooled plasma was applied onto a column of
AffiGel Hz (2.6 × 40 cm) coupled with the IgG fraction from a
patient with acquired vWF-cp deficiency that had been isolated from
plasma by affinity chromatography on Protein A-Sepharose, using 0.1 M
Na3-citrate, pH 4.0, for elution. Coupling was performed according to the manufacturer's instructions, and the resulting ligand
density was 2.8 mg total IgG per milliliter gel. The IgG-AffiGel column
was equilibrated with 10 mM Tris, 0.15 M NaCl, 1 mM
Na3-citrate, 0.02% NaN3, pH 7.4. The adsorbed
proteins were eluted using 3.0 M Na thiocyanate (NaSCN) in the
equilibration buffer and immediately separated from the chaotropic salt
by gel filtration on a BioGel P-6DG (2.6 × 40 cm) column directly
connected to the immunoaffinity column. The eluted proteins were
further applied to a column with Protein G-Sepharose (1.6 × 5 cm).
During the plasma application, the flow rate was set to 30 mL/h; during
elution from the IgG-AffiGel column, to 100 mL/h; and during
chromatography on Protein G-Sepharose, to 50 mL/h.
represents
fraction with vWF-cp activity.
), heparin plasma (
), and in normal
serum (
).
-subunit (N-terminal sequence
Asp-Gln-Thr-Val-Ser). Clusterin binds to a variety of biologic ligands,
such as IgG, complement components, high-density lipoprotein particles,
lipids, heparin, and others.