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Plenary paper
Partial amino acid sequence of purified von Willebrand factor–cleaving protease
Helena E. Gerritsen, Rodolfo Robles, Bernhard Lämmle, and Miha Furlan
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 sulfatepolyacrylamide 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-LeuGlu-Leu-Leu-Val-Ala-Val-Gly) of the first
15 residues was established. The protease migrates in gel filtration as a highmolecular-weight complex with clusterin,
a 70-kd protein with chaperonelike activity. vWF-cp bound to clusterin is dissoci-
ated 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)
© 2001 by The American Society of Hematology
Introduction
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
From the Central Hematology Laboratory, University Hospital, Inselspital, Bern,
Switzerland.
Reprints: Bernhard Lämmle, Central Hematology Laboratory, University Hospital,
Inselspital, CH-3010 Bern, Switzerland; e-mail: [email protected].
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.
1654
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.
© 2001 by The American Society of Hematology
BLOOD, 15 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 6
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BLOOD, 15 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 6
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 and methods
Materials
Protein A–Sepharose CL-4B, Protein G–Sepharose-4FF, CNBr-activated
Sepharose-4B, heparin–Sepharose HiTrap, lentil lectin–Sepharose-4B, and
Sephacryl S-300 HR were purchased from Amersham-Pharmacia (Uppsala,
Sweden). AffiGel Hz, BioGel P-6DG, and High Q Support were obtained
from Bio-Rad (Richmond, CA). Ig-TheraSorb was from Serag-Wiessner
(Naila, Germany). Bovine serum albumin (BSA) and 4-aminophenylmercuric acetate were purchased from Fluka (Buchs, Switzerland). ␣-Methyl
D-mannoside was from Calbiochem (San Diego, CA). Rabbit anti–human
vWF antibodies (A0082), rabbit anti–human ␣2-macroglobulin antibodies
(A0033), rabbit anti–human IgA antibodies (A0262), rabbit anti–human
IgG antibodies (A0423), rabbit anti–human IgM antibodies (A0425), goat
anti–rabbit IgG antibodies labeled with alkaline phosphatase (D0487), and
peroxidase-conjugated rabbit anti–mouse immunoglobulins (P0260) were
purchased from Dako (Glostrup, Denmark). Rabbit anti–human fibronectin
(F-3648) was from Sigma (St Louis, MO). Human fibronectin was obtained
from the Central Laboratory, Blood Transfusion Service, Swiss Red Cross
(Bern, Switzerland). Rabbit antibodies to human clusterin (sc-8354) were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA), whereas
murine monoclonal anti–human SP40 (clusterin) antibodies were obtained
from Quidel (Mountain View, CA). The Protran nitrocellulose transfer
membrane was purchased from Schleicher & Schuell (Dassel, Germany).
Polyvinylidene difluoride (PVDF) transfer membrane was from Millipore
(Bedford, MA). BCIP (5-bromo-4-chloro-3-indolyl phosphate p-toluidine
salt) and NBT (p-nitro blue tetrazolium chloride) were purchased from
Bio-Rad. Other chemicals were purchased either from Fluka or from Merck
(Darmstadt, Germany). Tris-buffered saline (TBS) contained 10 mM Tris
and 0.15 M NaCl, pH 7.4.
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 ⫺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
PURIFIED vWF-CLEAVING PROTEASE
1655
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.
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 ␣-methyl
mannoside, respectively.
Third step. The protein fractions eluted with 0.3 M ␣-methyl mannoside were dialyzed against 10 mM Tris, 75 mM NaCl, pH 7.4, and passed
through a column of heparin–Sepharose (1.4 ⫻ 3.2 cm) equilibrated in the
same buffer. The unadsorbed protein fractions were again applied on the
High Q Support column and eluted as described above. The proteins were
finally fractionated by gel filtration on a column of Sephacryl S-300 HR
(2.6 ⫻ 100 cm) equilibrated with TBS. Fractions of 7 mL were collected at
a flow rate of 42 mL/h.
Anti–␣2-macroglobulin immunoaffinity chromatography
An aliquot of the pooled fractions of the third step of isolation containing
vWF-cp activity was loaded on a 1-mL column of anti–␣2-macroglobulin
(anti-␣2M)–Sepharose. The latter immunoadsorbent was prepared by
coupling a rabbit anti-␣2M antibody to CNBr-activated Sepharose-4B
according to the manufacturer’s instructions, resulting in a ligand density of
4.9 mg IgG/mL. The column was equilibrated with TBS and the chromatography was performed at a flow rate of 12 mL/h. The bound proteins were
eluted with 3.0 M NaSCN in TBS, and the collected fractions were
immediately dialyzed against TBS.
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 anticlusterinSepharose 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.11
Stability 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
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1656
BLOOD, 15 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 6
GERRITSEN et al
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.25
SDS–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-␮L
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.
Results
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, ␣2M, and several other unidentified protein bands.
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
␣-methyl mannoside, only a minor amount of vWF-cp was eluted
by 30 mM ␣-methyl mannoside, and no activity was found in the
breakthrough fractions of lentil lectin–Sepharose. The protein
content of the active fractions after the second purification step was
reduced to 0.006% of initial AU (Table 1).
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,
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BLOOD, 15 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 6
PURIFIED vWF-CLEAVING PROTEASE
1657
Figure 1. Chromatographic purification of von Willebrand factor–cleaving protease. Eight batches of 100
mL normal human plasma each were first fractionated by
immunoaffinity chromatography on the anti–vWF-cp autoantibody–containing IgG fraction from a patient with
acquired TTP. Elution fractions collected under the last
column of each step were counted from the start of each
step. The fractions containing vWF-cp activity were
pooled and further subjected to chromatography, as
represents fraction with
indicated schematically.
vWF-cp activity.
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
Table 1. Purification of von Willebrand factor–cleaving protease
Protein absorbance units
(280 nm)
Total
Percent of
initial
vWF-cp activity
plasma units*
Total
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 ␣2Mdepleted 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,
Percent of
initial
Initial material: 800 mL
normal plasma
48 000
100
800
100
0.22
400
50
3
0.006
38
4.8
1.1
0.002
18
2.3
First step
IgG-AffiGel
BioGel P-6DG
Protein G–Sepharose
105
Second step
Ig-TheraSorb
High Q Support
Lentil lectin–Sepharose
Third step
Heparin-Sepharose
High Q Support
Sephacryl S-300 HR
*One plasma unit is the activity of vWF-cp in 1 mL normal human plasma.
Figure 2. Activity of vWF-cleaving protease in fractions eluted from Sephacryl
S-300 HR. (A) SDS-agarose gel electrophoresis of unreduced vWF substrate after
incubation with chromatographic fractions eluted from Sephacryl S-300 HR. Disappearance of high-molecular-weight vWF multimers is indicative of vWF-cp activity. (B)
SDS-PAGE of reduced vWF substrate after incubation with citrated normal plasma
diluted 1:20 (lane b) or with a 4-fold concentrated pool of purified vWF-cp preparation
(lane c). The vWF subunit is completely degraded to fragments of 170 and 140 kd.
The protease-free control in lane a contains only the undegraded vWF subunit of
250 kd.
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GERRITSEN et al
Table 2. N-terminal amino acid sequence of polypeptide bands separated by
SDS-PAGE of purified von Willebrand factor–cleaving protease
Mr
Amino acid sequence
350 kd unreduced
Ser-Val-Ser-Gly-Lys-Pro-Gln-Tyr-Met-Val*
150 kd unreduced
Ala-Ala-Gly-Gly-Ile
140 kd unreduced
Ala-Ala-Gly-Gly-Ile
130 kd unreduced
Ala-Ala-Gly-Gly-Ile
110 kd unreduced
Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu
70 kd unreduced
Asp/Ser-Gln/Leu-Thr/Met-Val/Pro-Ser/Phe†
180 kd reduced
Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu
170 kd reduced
Ala-Ala-Gly-Gly-Ile
160 kd reduced
Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val-Gly
120 kd reduced
Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val-Gly
40 kd reduced
Asp-Gln-Thr-Val-Ser†
*Identified as ␣2-macroglobulin.
†Identified as clusterin.
Figure 3. SDS-PAGE of unreduced and disulfide-reduced fractions eluted from
the Sephacryl S-300 HR column. (A) Unreduced fractions; (B) reduced fractions.
Migration rates of unreduced and reduced protein standards are indicated in the
middle of both panels. Proteins were detected by silver staining.
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).
The unreduced polypeptide band of 70 kd migrated as bands of
Mr 30 to 40 kd after reduction (Figure 4). The sequences of both
bands (Table 2) were aligned with the PIR Database (NBRF)
entries and strongly suggested that they represented unreduced and
reduced clusterin (apolipoprotein J), respectively. The unreduced
70-kd electrophoretic band was transferred to nitrocellulose and
was found to bind rabbit antibodies to human clusterin (results
not shown).
The final recovery of vWF-cp was 2.3% of initial protease
activity, and the specific activity of the purified preparation was
increased about 1000-fold (Table 1). The substantial loss of
protease activity may be ascribed primarily to inactivation of
vWF-cp during the purification procedure.
To examine whether the purified vWF-cp preparation degrades
vWF to the same fragments as the protease responsible for
physiologic degradation of vWF in normal human plasma, we
digested purified vWF either with a dilution of normal plasma or
with the purified vWF-cp after activation with BaCl2. Analysis of
degraded vWF by SDS-PAGE under reducing conditions and
immunoblotting (Figure 2B) revealed the same vWF fragments of
170 and 140 kd in both digestion mixtures.
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
Table 3. Amino acid composition of von Willebrand factor–cleaving protease
No. residues/100 residues
Amino acid
Figure 4. Two-dimensional electrophoretic analysis of a pool of active fractions
collected from the Sephacryl S-300 HR column. The slowest migrating unreduced
proteins (Mr 350 kd or greater) produced subunits of 170 and 120 kd after disulfide
reduction, whereas the 70-kd band was reduced to 40 kd. Four protease bands in the
Mr range of 110 to 150 kd are single-chain polypeptide chains.
150 kd
140 kd
130 kd
110 kd
Asx
6.7
7.0
7.4
8.3
Glx
12.2
12.0
12.8
11.8
Ser
8.4
8.9
8.8
9.2
Gly
11.8
12.1
12.1
12.9
His
2.5
2.3
2.5
2.4
Arg
8.3
7.6
8.1
7.2
Thr
5.6
5.5
5.6
5.7
Ala
10.1
9.5
9.6
8.2
Pro
8.9
8.5
8.3
8.1
Tyr
2.1
2.7
2.3
2.4
Val
7.0
6.9
6.7
6.5
Ile
2.7
2.9
2.6
3.0
Leu
10.0
9.9
9.1
9.7
Phe
2.6
2.8
2.6
3.2
Lys
1.0
1.3
1.3
1.4
Four unreduced polypeptide bands from SDS-PAGE of purified vWF-cp with Mr
150, 140, 130, and 110 kd were analyzed.
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BLOOD, 15 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 6
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
PURIFIED vWF-CLEAVING PROTEASE
1659
Figure 6. Affinity chromatography of citrated normal human plasma on
anticlusterin-Sepharose. (A) Protease activities of normal plasma (a), of the first 5
breakthrough fractions (b1-b5), and of the first 2 fractions eluted with NaSCN (c1, c2).
vWF-cp passed undelayed through the anticlusterin column, although clusterin was
completely sequestered, as shown by the dot blot analysis (B). There was no vWF-cp
released from the column together with clusterin during elution with NaSCN.
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).
Discussion
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
Figure 5. Affinity chromatography on anticlusterin-Sepharose of the crude
protease preparation eluted from the IgG-AffiGel column with NaSCN. (A)
vWF-cp activity in the protease preparation before application onto anticlusterin
column (a), in the breakthrough fraction (b), and in the NaSCN-eluted fraction (c). The
activity was bound in the anticlusterin column and released during elution with
NaSCN. (B) Dot blot analysis of the same 3 samples, using dilutions (dil) 1:2 to 1:32,
shows complete clusterin removal on anticlusterin-Sepharose and its dissociation in
the chaotropic medium.
Figure 7. Stability of vWF-cp during incubation at 37°C. The activity of vWF-cp
was analyzed by collagen-binding assay in citrated normal plasma (F), heparin
plasma (Œ), and in normal serum (f).
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1660
GERRITSEN et al
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 matrixdegrading 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-HisLeu-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, ␣2M, and clusterin in the electrophoretic gels, it may be
roughly estimated that the concentration of vWF-cp in normal
human plasma is about 1 ␮g/mL. Our first attempt11 to isolate
vWF-cp resulted in a 10-fold better purification (approximately
10 000-fold) than that observed in the present study (approximately
1000-fold). Both figures were derived from the relative amounts of
the protease activity divided by protein concentrations (absorbance
at 280 nm) in the initial and final material. This difference may be
explained by an excessive rate of protease inactivation under
BLOOD, 15 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 6
conditions of the present purification method, by activation of the
protease during the previous procedure,11 or both. It should be
pointed out that the present purification method used more
aggressive conditions (eg, 3 M NaSCN) than the previous procedure and that the protease was strongly associated with clusterin
after affinity chromatography on patient IgG. The question remains
open whether the recovered activity represents the total amount of
the protease or whether an unknown fraction of the protease is
present in plasma in an inactive proenzyme form, as might be
hypothesized from our results shown in Figure 7, and became
activated (eg, by calcium ions) in the previous purification method.
The protease was eluted by gel filtration from the Sephacryl
S-300 HR column together with ␣2M (molecular mass 720 kd) and
an unknown protein of 70 kd. Almost simultaneous elution of
vWF-cp with several protein molecules of different sizes suggests
that the protease forms a complex with some other plasma proteins.
Complex formation may also explain the unusually high molecular
mass (200-300 kd) of vWF-cp in both earlier reports on vWF-cp
purification.11,12
It is known that ␣2M interacts with and inhibits virtually any
protease and that covalent complexes of the ␣2M with plasmin32 or
thrombin33 still possess amidolytic but much less proteolytic
activity toward their protein substrates. On the other hand, when
bound to ␣2M, a protease is largely “protected” from inhibition by
other large protease inhibitors. The possibility that vWF-cp may be
firmly associated with ␣2M was tested by immunoaffinity chromatography on anti-␣2M–Sepharose. The activity of the purified
protease fraction from Sephacryl S-300 HR, still containing ␣2M,
appeared unchanged in the void volume of the anti-␣2M–Sepharose
column, suggesting that there was apparently no association
between vWF-cp and ␣2M. This result is supported by the
unusually long biologic half-life of vWF-cp14 because ␣2Mprotease complexes are rapidly cleared from the circulation.34
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 ␣-subunit
(N-terminal sequence Ser-Leu-Met-Pro-Phe) and a 205-residue
␤-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.35 It has a chaperonelike activity similar to that of small
heat-shock proteins36; it appears to bind to hydrophobic regions
of partly unfolded proteins and thus to protect stressed proteins
from precipitation. The clusterin concentration in serum is 50 to
370 ␮g/mL.36 It exists in polymerized forms at physiologic pH
and as monomer occurring preferentially under slightly acidic
conditions, suggesting that pH-induced changes in the structure
of clusterin are responsible for pH-dependent binding of heparin37 or protein ligands.38
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
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BLOOD, 15 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 6
PURIFIED vWF-CLEAVING PROTEASE
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.
1661
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
Note added in proof. The reader is referred to the accompanying paper, Fujikawa et al,39 page 1662, characterizing vWF-cp as a
metalloprotease.
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2001 98: 1654-1661
doi:10.1182/blood.V98.6.1654
Partial amino acid sequence of purified von Willebrand factor−cleaving
protease
Helena E. Gerritsen, Rodolfo Robles, Bernhard Lämmle and Miha Furlan
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