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Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1637-1647
Evidence for Extracellular Processing of Pro-von Willebrand Factor
After Infusion in Animals With and Without Severe von Willebrand
Disease
By
P.L. Turecek,
L. Pichler,
W. Auer,
G. Eder,
K. Varadi,
A. Mitterer,
W. Mundt,
U. Schlokat,
F. Dorner,
L.O. Drouet,
J. Roussi,
J.A. van Mourik, and
H.P. Schwarz
From Baxter Hyland Immuno, Vienna, Austria; CLB, Sanquin Blood Supply
Foundation, Amsterdam, The Netherlands; Hôpital
Lariboisière, Paris, France; and Hôpital Raymond
Poincaré, Garches, France.
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ABSTRACT |
Although proteolytic processing of pro-von Willebrand factor
(pro-vWF) resulting in free propeptide and mature vWF is known to be
initiated intracellularly, vWF released from endothelial cells may
contain a high proportion of incompletely processed pro-vWF. Because
pro-vWF is only rarely detectable in normal human plasma, we
investigated whether extracellular processing of pro-vWF is possible. A
recombinant preparation (rpvWF) containing both pro-vWF and mature vWF
subunits was infused into 2 pigs and 1 dog with severe von Willebrand
disease, 2 mice with a targeted disruption of the vWF gene, and 2 healthy baboons. Total vWF antigen (vWF:Ag), free propeptide, and
pro-vWF were measured using enzyme-linked immunosorbent assay
techniques in blood samples drawn before and after infusion. vWF:Ag
increased promptly. No pro-vWF could be detected when the first
postinfusion sample was drawn after 30 minutes (pigs) or 60 minutes
(mice), but pro-vWF was detectable for short periods when postinfusion
samples were drawn after 15 minutes (dog) or 5 minutes (baboons). In
contrast, free propeptide was increased at the first timepoint
measured, suggesting that it was generated from the pro-vWF in the
rpvWF preparation. vWF multimers were analyzed in the rpvWF preparation
and in plasma samples drawn before and after infusion of rpvWF using
ultra-high resolution 3% agarose gels to allow separation of homo- and
hetero-forms of the vWF polymers. Within 30 minutes after
infusion in the pigs, 1 hour in the dog and the mice, and within 2 hours in the baboons, the multimer pattern had changed to that
typically seen in mature vWF. These data indicate that propeptide
cleavage from unprocessed vWF can occur extracellularly in the
circulation. The enzyme or enzymes responsible for this cleavage in
plasma remain to be identified.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
VON WILLEBRAND FACTOR (vWF) is a large
multimeric adhesive glycoprotein that circulates in plasma and is also
found in platelets, megakaryocytes, endothelial cells, and the
subendothelial matrix.1,2 The molecule has a dual function
in hemostasis because it mediates platelet adhesion at sites of
vascular injury, which is necessary for primary hemostasis, and it
stabilizes factor VIII (FVIII) in the circulation.3 The
pathophysiological significance of the different biological functions
of this protein is demonstrated by von Willebrand disease, in which
severe von Willebrand deficiency results in defective platelet
adhesion, secondary FVIII deficiency, and prolonged bleeding
time.4,5
vWF is synthesized in endothelial cells and megakaryocytes as
pre-pro-vWF, a 2,813-amino acid precursor protein that consists of
a signal peptide of 22 amino acid residues, a 741-amino acid propeptide
(also called von Willebrand antigen II6), and the 2,050-amino acid mature vWF subunit. During biosynthesis, pre-pro-vWF is subjected to a series of intracellular posttranslational
modifications.3,7,8 After cleavage of the signal peptide,
the remaining pro-vWF undergoes glycosylation, C-terminal dimerization,
sulfation and carbohydrate processing, aminoterminal multimerization,
and proteolytic removal of the propeptide from the aminoterminal end of
the vWF subunit. Intracellular propeptide cleavage coincides with
aminoterminal multimerization in the trans-Golgi network.9
This posttranslational endoproteolysis is accomplished by furin
(PACE),10-13 a membrane-associated, calcium-dependent
endoprotease belonging to a family of subtilisin-like serine proteases.
Propeptide cleavage is necessary for the binding of FVIII to
vWF,14-16 but not for multimerization.17,18
When cleaved, the propeptide remains noncovalently associated with the
mature vWF multimers,9 and both are stored in intracellular granules such as the Weibel-Palade bodies in endothelial cells for
release through the regulated pathway.19 However, the
majority of vWF produced by endothelial cells is secreted via the
constitutive pathway. After secretion, the free propeptide circulates
in normal human plasma as a homodimer6 at a concentration
of about 1 µg/mL with a half-life of 2 to 3 hours, while mature vWF
multimers circulate at a concentration of about 10 µg/mL with a
half-life of about 12 hours.20,21
Constitutively secreted vWF may contain a high proportion of
incompletely processed pro-vWF.22,23 However, pro-vWF is
detectable in normal human plasma only on rare occasions, and even then
only trace amounts are observed. In a study of healthy human subjects, administration of 1-deamino-8-d-arginine vasopressin (DDAVP) or endotoxin induced an increase in pro-vWF.21 Even after
stimulation, pro-vWF levels were still approximately 1,000-fold lower
than free propeptide levels. The absence of significant amounts of pro-vWF in plasma might be explained if rapid cleavage of pro-vWF occurred after secretion from endothelial cells. To investigate whether
extracellular processing of pro-vWF can indeed take place in vivo, we
studied the structure and properties of vWF in plasma before and after
infusion of a hetero-multimeric recombinant preparation consisting of
both pro-vWF and mature vWF in canine, porcine, and murine models of
von Willebrand disease and in healthy baboons.
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MATERIALS AND METHODS |
Assays
Total vWF antigen (vWF:Ag), consisting of mature and pro-vWF, was
measured by enzyme-linked immunosorbent assay (ELISA) using polyclonal
rabbit anti-human vWF antibodies (Asserachrom vWF; Boehringer-Mannheim,
Mannheim, Germany). vWF:Ag was expressed in human plasma-equivalent
units per milliliter (U/mL) using the standard preparation from the
test kit. For comparison with propeptide and pro-vWF, 1 U/mL was
assumed to be equivalent to 50 nmol/L.21
Free propeptide was measured as antigen by ELISA with murine antibodies
directed against vWF-propeptide as described by Borchiellini et
al.21 (CLB-Pro 35 for capture; CLB-Pro 14.3 for detection.) A pool of normal human plasma (Reference Plasma 100%; Immuno, Vienna,
Austria) containing 5.5 nmol/L vWF propeptide (calibrated against
purified recombinant propeptide21) served as a reference. Pro-vWF antigen was determined in an ELISA with the same capture antibody directed against propeptide (CLB Pro-35) as in the ELISA for
propeptide, but using a horseradish peroxidase-labeled polyclonal rabbit anti-human vWF antibody (Dakopatts, Glostrup, Denmark) for
detection. Recombinant pro-vWF calibrated against an uncleavable pro-vWF mutant (pro-vWFgly763)21 was used as a reference.
The specificity of this assay system for pro-vWF was shown by its lack
of cross-reactivity with a recombinant preparation containing only
mature vWF.24 The propeptide ELISA poorly recognized
pro-vWF, yielding values of approximately 0.05 nmol/L propeptide per
nmol/L pro-vWF. This 20-fold difference in reactivity allowed us to
monitor the quantitative increase of the propeptide in plasma samples.
Ristocetin cofactor activity (RCoF) was determined by measuring the
ristocetin-induced agglutination of formaldehyde-fixed human
platelets25 under conditions described in the
literature26 using a 570-VS whole blood aggregometer
(Chrono-Log, Havertown, PA) equipped with a chart recorder. FVIII
activity was measured with a 2-stage clotting method performed as
described by Austen and Rhymes27 using the reagents from
the 2-stage FVIII assay kit from Immuno. A reference plasma calibrated
against the 3rd International Standard 91/666 was used in the
determination of FVIII and RCoF, and the results were expressed in
human plasma-equivalent units as IU/mL.
Test Substance and Electrophoretic Analyses
The human recombinant preparation containing pro-vWF (rpvWF) was
produced by expression in a rodent cell line, Chinese hamster ovary
(CHO) cells.28 rpvWF was purified from cell culture
supernatant by ion-exchange chromatography followed by immuno-affinity
chromatography on an immobilized monoclonal antibody directed against
mature vWF as described by Mejan et al.29 Because of the
specificity of the final purification step, the preparation contained
only a minute quantity of free propeptide (<4% on a molar basis).
For the initial characterization of the test substance, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed under reducing conditions.30 Samples were incubated with
buffer containing dithiothreitol at 96°C for 5 minutes. Aliquots of
20 µL of these samples were subjected to electrophoresis on 8% gels in the presence of SDS. Protein bands were stained with Coomassie brilliant blue by a standard method.31
For analysis before and after the rpvWF infusion, plasma samples were
reduced at 70°C for 15 minutes, applied to gradient polyacrylamide
gels (4% to 12%; Novex, San Diego, CA) in the presence of SDS, and subjected to standard blotting procedures on PVDF membrane
(Pall Gelman Sciences, Ann Arbor, MI). A polyclonal rabbit antibody
against vWF (Dakopatts, Glostrup, Denmark), which cross-reacts with
porcine, canine, and baboon vWF, and reacts with both mature and
pro-vWF, was used as the primary antibody, and an alkaline phosphatase-labeled goat anti-rabbit IgG (Accurate, Westbury, NY), as
secondary antibody. The blots were developed with
5-bromo-4-chloro-3-indolyl-phosphate/nitroblue-tetrazolium substrate
(Bio-Rad, Richmond, CA).
For the mouse experiment, in which 125I-labeled rpvWF was
administered, the samples were separated by SDS-PAGE and the gels were dried and exposed to x-ray films (Hyperfilm MP; Amersham-Pharmacia, Uppsala, Sweden) to obtain autoradiograms. The "Full Range Rainbow Marker" (Amersham-Pharmacia) ranging from 10 to 250 kD
was used to determine the molecular mass.
To analyze the size distribution of vWF multimers in the rpvWF
preparation and in plasma before and after infusion of rpvWF, high-density horizontal SDS agarose gel electrophoresis was performed as previously described,32 except that 3% agarose was used
to obtain ultra-high resolution of the vWF multimers. This method allowed separation of the homo- and hetero-forms of the vWF polymers. The same anti-human vWF antibody as described above was used for immunodetection.
Radioiodination of rpvWF
Radioiodination of rpvWF was performed by labeling 100 µg of rpvWF
with 125I (Amersham-Pharmacia) according to the iodobeads
method of Markwell.33 The 125I-labeled rpvWF
had a specific activity of 6.3 × 105 Bq/vWF:Ag unit.
Animal Studies
We previously used the porcine model of severe von Willebrand disease
for infusion studies with a recombinant preparation of mature human vWF
(rvWF).34,35 In the present study, we administered the
pro-vWF-containing rpvWF intravenously to 2 previously untreated vWF-deficient pigs from this colony, 1 pig receiving 17 and the other
receiving 70 RCoF IU/kg body weight. With citrate for anticoagulation, blood samples were withdrawn from the venous catheters before infusion
and 30 minutes, 1, 2, 3, 6, 8, 24, 32, 48, 72, and 96 hours after
infusion of the rpvWF. The timing of the sampling was based on the
results of the previous studies. Plasma was separated by immediate
centrifugation at 1,000g for 20 minutes at 15°C and deep
frozen in aliquots for analysis of vWF parameters (total vWF antigen,
pro-vWF antigen, propeptide antigen, RCoF and FVIII activity, and
multimers) as described above.
We had also tested recombinant human mature vWF in an earlier study in
Dutch Kooiker dogs with severe vWF deficiency.32 We used
similar techniques in the present study, infusing 1 previously untreated dog from the same colony with rpvWF intravenously at a dose
of 35 RCoF IU/kg. Samples of citrated plasma obtained by venous
puncture before infusion and at timepoints 15, 30, and 45 minutes and
1, 2, 3, 6, 24, 48, 72, and 95 hours after the administration of the
rpvWF were then subjected to in vitro analyses as described for the
pigs, and in addition were analyzed by SDS-PAGE under reducing
conditions and Western blotting.
Mice with a targeted disruption of the vWF gene36 (breeding
pair for establishment of a colony kindly provided by Denisa Wagner,
Center for Blood Research, Boston, MA) were used for infusion studies
with 125I labeled rpvWF. Two homozygous vWF knockout mice
from this colony received 70 RCoF U/kg containing 1.3 × 105 Bq/vWF:Ag U rpvWF. Citrated samples were drawn by heart
puncture 60 minutes after intravenous administration and subjected to
SDS-PAGE under reducing and nonreducing conditions. Autoradiograms of
the dried gels were then evaluated.
We also treated 2 colony-bred 4-year-old healthy male baboons
(papio papio hamadryas), weighing 8.5 kg, with rpvWF
intravenously at a dose of 50 RCoF IU/kg each. The animals were
anesthetized by intramuscular injection of 10 mg/kg ketamine (Ketavet;
Parke Davis, Munich, Germany). Citrated blood was drawn by venous
puncture before infusion and 5, 10, 15, 30 minutes and 2, 4, 24, 48, and 72 hours postinfusion and subjected to analyses as described for the pigs.
The dog, baboon, and mouse studies were performed in accordance with
the Austrian Animal Experiments Act (BG 501/1989). The dog was housed
in the Animal Care Facility of the Medical Faculty of the University of
Vienna in Himberg, Austria. The baboon study was performed in the Hans
Popper Primate Center in Orth/Donau, Austria, which is fully certified
by the American Association of Accreditation of Laboratory Animal Care
(AAALAC). The study was in full compliance with guidelines for the care
and use of laboratory animals (Institute of Laboratory Animals
Resources, Commission on Life Sciences, National Research Council,
Washington, DC, 1996). The pig study was performed at the Institut de
la Recherche Agronomique (INRA) in Jouy en Josas, France, according to
the ethical guidelines established by the Institut National de la Recherche Medical (INSERM) and INRA institutional policies.
Biometrical Methods
Half-life calculations were performed using the method described by Lee
et al.37
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RESULTS |
rpvWF Preparation
SDS-PAGE analysis showed the rpvWF preparation to be composed of
approximately 50% pro-vWF and 50% mature vWF, with a specific activity of 50 RCoF IU/mg protein (Fig 1).
SDS agarose gel electrophoresis revealed that the homo- and
hetero-dimers of pro-vWF and mature vWF monomers were multimerized to
high-molecular-weight forms (see Figs 3, 6, and 9, rpvWF lanes). Trace
amounts of free propeptide (<2% of pro-vWF on a molar basis) could
be detected in the rpvWF preparation with the ELISA method. However, no
propeptide was visible on the autoradiogram (see control lane A of Fig
7) of the 125I-labeled rpvWF.
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| Fig 1.
SDS-PAGE (8% T) under reducing conditions of rpvWF (lane
B), consisting of 50% pro-vWF and 50% mature vWF. Molecular-weight
standard (lane A). The purified rpvWF in the gel was visualized by
Coomassie blue staining.
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Fate of rpvWF After Infusion
vWF-deficient pigs.
Before infusion of the rpvWF preparation, levels of vWF:Ag, RCoF
activity, free propeptide, and pro-vWF were below the limits of
detection in each pig (Fig 2A and B).
Plasma concentrations of vWF:Ag, RCoF, and free propeptide increased
promptly after infusion of the rpvWF preparation and reached maximum
values within 30 minutes, rising higher in the pig that received the
larger dose. Because no free propeptide was present in the rpvWF
preparation, this propeptide likely appeared due to proteolytic
processing of pro-vWF after infusion. The vWF:Ag remained in the
circulation for at least 96 hours in both pigs, with a half-life of
about 22 hours, which was comparable to our previous observations after infusion of mature rvWF.33,34 The free propeptide
disappeared within 24 hours, with a half-life of 3.3 hours in the pig
treated with 17 RCoF IU/kg, and 1.7 hours in the pig treated with 70 RCoF IU/kg. Pro-vWF was not detectable in any of the samples tested.
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| Fig 2.
Fate of rpvWF after injection of a single bolus into
vWF-deficient pigs: (A) 17 RCoF IU/kg body weight; (B) 70 RCoF IU/kg).
vWF:Ag ( ), pro-vWF ( ), free propeptide ( ), and vWF:RCoF
( ).
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As shown in Fig 3 (see page 1639), vWF
multimers were virtually absent before infusion of the rpvWF
preparation. Comparative analysis of the multimers of the rpvWF
preparation and of vWF multimers in the plasma samples taken after
infusion of rpvWF (70 RCoF IU/kg) indicated that the pro-vWF had
undergone proteolytic processing (Fig 3). The high-density agarose gel
used for this analysis allowed separation of the homo- and hetero-forms
of the vWF polymers. The resolution was sufficient to identify 3 and 5 bands on the dimer and the tetramer levels, respectively (D I-III and T
I-V in Fig 3; schematic illustration of the composition of the vWF
oligomers in Fig 4). Thirty minutes after
infusion of the rpvWF into the pigs, we could detect only 1 dominating band at each oligomer and multimer of the vWF. This band had the same
molecular weight as the fastest migrating band of the oligomer equivalent to the homopolymer of mature vWF and also correlated in
molecular size with the central band of the human vWF triplet or
quintuplet structure.38 All multimeric forms of vWF were then gradually removed from the circulation.
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| Fig 3.
Fate of rpvWF after injection of a single bolus (70 RCoF
IU/kg) into a vWF-deficient pig (see Fig 2B): vWF multimer
pattern over time. Plasma samples taken before and at specified
intervals after injection were analyzed electrophoretically on a 3%
SDS agarose gel. Multimers were visualized using an anti-human
polyclonal vWF antibody that cross-reacts with vWF from other species.
Control lanes: recombinant vWF preparation before injection (rpvWF),
normal pig plasma, normal human plasma. DI-III,
TI-V: variants of homo- and hetero-dimers and tetramers (D,
dimer; T, tetramer) as illustrated in Fig 4. Arrows indicate satellite
band formation. Mr, apparent relative molecular mass.
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| Fig 4.
Schematic illustration of homo- and hetero-dimers
(DI-III) and tetramers (TI-V) of partially processed recombinant vWF
(rpvWF) consisting of pro (p) and mature (m) subunits at an equal
ratio.
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In addition to the degradation of the pro-vWF multimers, we observed
satellite bands that were similar but not completely equivalent to
those of the human triplet structure (see satellite band formation of
dimers and tetramers in Fig 3). These satellites had a different
molecular size than the satellite bands of human plasma-derived vWF
multimers (Fig 3). Although the central band was equal in size to the
central band of each multimer of human vWF, the faster and slower
migrating satellite bands generated from the infused rpvWF had higher
and lower molecular masses, respectively, than those found in normal
human plasma, resulting in a more compact triplet structure than that
seen in normal human plasma.
vWF-deficient dog.
The results of the study in pigs prompted us to study the effects of
rpvWF in a Dutch Kooiker dog with type 3 von Willebrand disease.32,39 In this study, we increased the frequency of blood drawing to every 15 minutes. As in the pigs, vWF:Ag, RCoF, and
free propeptide levels increased rapidly in the dog from below the
limit of detection to peak levels within 15 minutes after infusion of
rpvWF (35 RCoF IU/kg) (Fig 5). The
calculated half-life was 9 hours for the vWF antigen and 5 hours for
RCoF activity. In contrast to the pigs, a small amount of pro-vWF could
be measured in the dog, but only in the blood sample taken 15 minutes
after injection of pro-rvWF. The free propeptide showed a peak 15 minutes after injection of the vWF preparation and decreased by 20% in the next 15 minutes, but stayed at this level for an additional 30 minutes. Thereafter it declined gradually, with a half-life of 3 hours.
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| Fig 5.
Fate of rpvWF after injection of a single bolus (35 RCoF
IU/kg) into a vWF-deficient dog: vWF:Ag (), pro-vWF (), free
propeptide (), and vWF:RCoF ().
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Consistent with these findings, electrophoretic analysis under reducing
conditions (Fig 6, top) showed that the
signal corresponding to that of the pro-vWF of the infused preparation
was still slightly visible after 15 minutes, but gradually disappeared
over time.
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| Fig 6.
Fate of rpvWF after injection of a single bolus (35 RCoF
IU/kg) into a vWF-deficient dog (see Fig 5): (Top)
SDS-PAGE under reducing conditions/Western blot. (Bottom) vWF multimer
pattern over time. Plasma samples taken before and at specified
intervals after injection were analyzed electrophoretically on a 3%
SDS agarose gel. Multimers were visualized using an anti-human
polyclonal vWF antibody that cross-reacts with vWF from other species.
Control lanes: recombinant vWF preparation before injection (rpvWF),
normal dog plasma, normal human plasma. DI-III,
TI-V: variants of homo- and hetero-dimers and tetramers (D,
dimer; T, tetramer) as illustrated in Fig 4. Arrows indicate satellite
band formation. Mr, apparent relative molecular mass.
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The analysis of the vWF multimers (Fig 6, bottom) for the timepoints up
to 72 hours postinfusion showed that the pro-vWF multimers had
disappeared completely after the first 15 minutes, resulting in a
single banded multimer picture. All vWF multimers were gradually removed from the circulation over time. The formation of triplet bands
was evident but not as clear here as it was in the pig studies.
vWF knockout mice.
Before the injection of rpvWF, the vWF knockout mice had zero levels of
vWF antigen, vWF propeptide, and pro-vWF. The plasma sample taken from
mice 60 minutes after injection of 70 RCoF U/kg rpvWF mixed with
125I-labeled rpvWF contained 0.86 U/mL total vWF:Ag, 3.14 nmol/L propeptide and 0.43 U/mL RCoF. No pro-vWF:Ag was detected by the ELISA. On the autoradiograms of both the nonreducing and reducing SDS-PAGE (Fig 7), a new band appeared that
was not present in the 125I-labeled rpvWF preparation and
had a molecular mass of 73 kD (nonreduced) or 96 kD (reduced),
corresponding to the molecular mass of the propeptide. No additional
bands were observed. The 125I-labeled rpvWF showed only 1 band with a molecular mass greater than 250 kD, even when the gel was
overloaded with 1,600 Bq per lane (data not shown).
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| Fig 7.
Autoradiogram of a plasma sample from a vWF-deficient
mouse treated with 125I-labeled rpvWF (B) after separation
on SDS-PAGE under reducing and nonreducing conditions;
control lane (A): 125I-labeled radiolabeled rpvWF
preparation.
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Healthy baboons.
We then administered rpvWF to 2 healthy baboons at a dose of 50 RCoF
IU/kg. Blood sampling was now performed every 5 minutes during the
first 15 minutes after the injection. As shown for 1 baboon in
Fig 8, rpvWF induced an immediate increase
in vWF:Ag and free propeptide levels. vWF:Ag decreased gradually to
baseline levels within 48 hours, with a half-life of 22 hours. Pro-vWF also showed a sharp increase and remained detectable during the first
30 minutes in both animals, but had disappeared by the 2-hour timepoint. Free propeptide levels began to decrease only after the
pro-vWF had disappeared from the circulation, with a half-life of 5 hours in each baboon. The pretreatment levels of vWF:Ag and RCoF were
around 5 U/mL, which is normal for baboons, but 5 times higher than the
level of our human reference plasma. After injection of the rpvWF, RCoF
increased by 50% in 1 baboon (Fig 8), but by only 10% in the other
animal (not shown). Other parameters were almost identical in the 2 animals.
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| Fig 8.
Fate of rpvWF upon injection of a single bolus (50 RCoF
IU/kg) into a normal baboon (K6): vWF:Ag (), pro-vWF (), free
propeptide (), and vWF:RCoF ().
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Multimer analysis of the plasma samples from the baboons
(Fig 9, see page
1642
) also showed the
typical triplet structure similar to that observed in humans. After
injection of rpvWF, a smear could be observed in the samples taken up
to
2
hours after injection at the positions where the slower-migrating,
higher-molecular-weight bands of each pro-vWF oligomer were seen in the
recombinant preparation (Fig
9
, DII-DIII, TII-TV). However, the
changes in the baboons were not as visible as those in the pigs and the
dog, and at the later timepoints no difference to the multimer pattern
prior to infusion could be seen.
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| Fig 9.
Fate of rpvWF upon injection of a single bolus (50 RCoF
IU/kg) into a normal baboon (K6): vWF multimer pattern over time.
Plasma samples were analyzed electrophoretically on a 3% SDS agarose
gel. Multimers were visualized using an anti-human polyclonal vWF
antibody that cross-reacts with vWF from other species, particularly
baboons. Braces (}) indicate rpvWF homo-and hetero-oligomer smearing
on the agarose gel. Control lanes: recombinant vWF preparation before
injection (rpvWF), normal human plasma. DI-III, TI-V: variants of homo-
and hetero-dimers and tetramers (D, dimer; T, tetramer) as illustrated
in Fig 4. Mr, apparent relative molecular mass.
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Effects of rpvWF on FVIII
A substantial increase in FVIII levels was observed after infusion of
rpvWF in all studies performed in vWF-deficient animals, reaching peaks
between 20 and 30 hours posttreatment
(Fig 10). The FVIII levels in the pigs
remained higher than the starting levels for 96 hours, in the dog for
72 hours. No effect on FVIII could be observed in the healthy baboons;
FVIII remained constant over the entire observation period despite the
2-fold increase in circulating levels of vWF.
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| Fig 10.
Effect of single-bolus injection of human rpvWF on FVIII
plasma levels in 2 vWF-deficient pigs: 17 RCoF IU/kg ( ), 70 RCoF
IU/kg ( ); 1 vWF-deficient dog: 35 RCoF IU/kg (); and 2 normal
baboons: 50 RCoF IU/kg each (K5 , K6 ).
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DISCUSSION |
The availability of a recombinant preparation containing unprocessed,
multimerized pro-vWF in addition to mature vWF allowed us to study the
fate of pro-vWF in plasma, both in porcine and canine models of von
Willebrand disease as well as in vWF knockout mice and normal nonhuman
primates. The preparation used in our previous canine and porcine
infusion studies32,34,35 consisted of fully processed and
polymerized rvWF obtained from CHO cells manipulated to overexpress
recombinant furin in addition to rvWF.24 The rpvWF used in
the experiments described here was expressed from CHO cells without
coexpression of recombinant furin.28 Because the amount of
endogenous furin is limited, these cells were only capable of partially
processing the expressed rvWF without additional recombinant furin,
which resulted in the formation of rvWF heteromultimers. Therefore, the
preparation contained a mixture of mature, completely processed,
propeptide-free subunits and unprocessed forms still containing the
covalently linked propeptide.
The infusion studies presented here indicate that propeptide cleavage
from unprocessed vWF can occur in the circulation. The infused pro-vWF
could not be detected at all in the experiments with pigs and mice, in
which the first postinfusion sample was not drawn until 30 and 60 minutes, respectively, and could be detected only for brief periods in
the dog and baboon experiments, in which the first postinfusion sample
was drawn after 15 and 5 minutes, respectively. In contrast, vWF:Ag and
free propeptide levels (which before infusion of rpvWF were below the
limit of detection in the pigs, dog, and mice) increased rapidly in all animals tested, indicating that the pro-vWF was quickly metabolized. The minute quantity of free propeptide contained in the rpvWF preparation was too small to explain the rapid increase in free propeptide seen after infusion. After the initial increase, the free
propeptide remained at relatively high levels for periods of about 1 to
3 hours in the pigs, the dog, and the mice, and 4 hours in the baboons,
then began to decrease, dropping below the limit of detection within 10 to 24 hours. This was consistent with continued propeptide release as
long as pro-vWF remained in the circulation. In the pigs, the dog, and
the mice, the levels of vWF:Ag paralleled those of the free propeptide,
and in the baboons vWF:Ag remained high for longer periods.
These observations were confirmed by analyses of the multimeric
composition of the infused vWF as shown on a 3% agarose gel that
allowed separation of homo- and hetero-forms of the vWF polymers. Within 30 minutes after infusion in the pigs and the dog, and within 2 hours in the healthy baboons, the multimer pattern had changed to that
typically seen in mature vWF. Electrophoretic analysis under reducing
conditions of the composition of vWF after infusion of rpvWF into the
vWF-deficient dog led to the same conclusions as those drawn from the
multimer analysis. Because the Western blotting did not allow us to
visualize the cleavage product of pro-vWF (ie, the propeptide), we used
experiments with 125I-labeled rpvWF injected into vWF
knockout mice to show the liberation of the propeptide in plasma. Under
reducing conditions, autoradiograms from plasma samples obtained 1 hour
after the injection showed bands of mature vWF as well as a band with a
molecular mass of 96 kD, which corresponds to free propeptide. Under
nonreducing conditions, the propeptide had a lower molecular mass of 73 kD, consistent with that previously described.21 These
observations argue against a preferential clearing of pro-vWF as an
explanation for the loss of pro-vWF from the circulation. Although the
possibility that the infused pro-vWF is taken up into the trans-Golgi
network and processed intracellularly cannot be completely ruled out, this also appears unlikely, considering the rapid time course of
propeptide generation in the present study.
In fact, this is not the first observation of extracellular processing
of vWF. Although previous studies13 had suggested that
furin can mediate the processing of vWF only intracellularly, in a
recent study performed in vitro,40 recombinant furin was shown to convert the heterogeneous multimeric pattern of incompletely processed rvWF into homogeneous and structurally intact multimers, even
in the absence of cells, showing that vWF precursor-processing can
occur extracellularly upon furin coexpression.
In addition to the rapid disappearance of the pro-vWF-containing
multimers, satellite bands with molecular masses lower and higher than
the central band appeared in both the pigs and the dog. This structure
resembled the triplet structure of multimers seen in human
plasma-derived vWF,38 although the satellite bands were
less widely separated than in plasma-derived vWF. The appearance of a
satellite band structure suggests that further proteolytic cleavage of
the mature vWF subunit occurred in addition to the propeptide removal.
We had not observed any satellite band formation after infusion of
recombinant mature vWF without the pro-moiety in our previous study in
dogs.32 However, we were not then using ultra-high-resolution 3% agarose gels, but standard electrophoretic techniques with 2% agarose gels, which were not sufficient to resolve
all homo- and hetero-multimers of the rpvWF. Therefore, we may have
missed any further proteolytic degradation that might have occurred in
the mature vWF. The present finding of satellite band formation is
consistent with the results of a recent study showing that a
plasma-derived protease known to be specific for vWF cleavage at
Tyr842Met843 in the vWF subunit41-43 can cleave recombinant
vWF in vitro.44 It remains to be investigated whether the
same protease is responsible for the in vivo degradation of mature vWF
observed in the present study.
As shown in the medium of cultured endothelial cells, vWF propeptide
and mature vWF are released in nearly equimolar amounts with both
constitutive and stimulated secretion.19,21 Because of its
shorter half-life, the concentration of the propeptide in normal human
plasma ( 5 nmol/L) is 1 order of magnitude lower than that of mature
vWF (50 nmol/L).21,45 After administration of DDAVP or
endotoxin in healthy volunteers or patients with von Willebrand disease
or acquired von Willebrand syndrome, plasma propeptide concentrations
increased 5- to 20-fold, while mature vWF increased 4- to
5-fold.20,21,46,47 Based on our findings that propeptide
covalently linked to mature vWF in the rpvWF preparation can be cleaved
off after infusion, the possibility exists that, rather than resulting
from a concerted release of free propeptide and mature vWF molecules,
increases in the plasma concentrations of propeptide and mature vWF
might be caused at least in part by increased secretion of pro-vWF,
which is processed in the circulation immediately after its release.
This would be consistent with the finding of increased pro-vWF levels
in normal subjects after stimulation with DDAVP or
endotoxin.21 The extent to which the extracellular processing of circulating pro-vWF actually occurs under physiological conditions remains to be clarified.
The significance of the propeptide in the intracellular multimerization
process of human vWF has been clearly shown.48,49 The
propeptide is essential for promoting the multimerization of vWF,
possibly by promoting interchain disulfide bond formation, because
specific sequences in the D1 and D2 domain of the propeptide resemble
functional sites of the thiol-disulfide oxidoreductases.50 In contrast, an extracellular function for the propeptide has not yet
been clearly established. The propeptide has been shown to bind to type
I collagen and to cross-link to laminin in the presence of FXIIIa,
suggesting that it could play a role as a matrix protein after release
from endothelial cells.51 Furthermore, the propeptide
inhibits collagen-induced platelet aggregation.52 Taking
into consideration the finding that mature vWF supports platelet
adhesion and platelet aggregation, free propeptide found in the
circulation could attenuate or regulate excess platelet aggregation by
collagen in the presence of vWF. Recently, a ligand function of the
propeptide for very late antigen-4 integrin present on lymphocytes and
leukocytes was described, suggesting a link between vWF and
inflammation.53 Propeptide deposition into the subendothelium could play a role in leukocyte recruitment at sites of
vascular injury. Further studies are necessary to investigate whether
the proteolytic cleavage of pro-vWF generates two polypeptide species
with distinct physiological functions and whether the circulating
propeptide plays a role in hemostasis.
Proteolytic removal of the propeptide is required for high-affinity
binding of FVIII to the D-domain of vWF, and the uncleaved propeptide
is believed to inhibit binding of FVIII sterically by preventing access
to the FVIII binding site.14,16 Alternatively, removal of
the propeptide may result in a conformational change in the binding
region of each vWF subunit, leading to appropriate exposure of the
FVIII binding site.15 We did not determine the precise site
of cleavage of the propeptide from pro-vWF after infusion of rpvWF.
Whether the new aminoterminus generated after the in vivo processing of
pro-vWF is identical to that obtained after intracellular processing
remains to be investigated.
In our earlier studies in vWF-deficient dogs and pigs, infusion of the
rvWF preparation containing only completely processed mature subunits
was associated with increased FVIII levels that were maintained even
after vWF antigen had returned to very low levels.32,34,35
In the present studies, comparable effects were achieved by infusion of
rpvWF containing both mature subunits and pro-vWF. The infused pro-vWF
was metabolized immediately, and the resulting mature vWF appears to
have acted in the same way as intracellularly processed mature vWF to
bind and stabilize FVIII. To test this hypothesis, however, an in vivo
study would have to be performed with a preparation containing only
pro-vWF without mature vWF subunits. In the present studies, the free propeptide at least did not appear to inhibit FVIII binding.
In conclusion, these studies show that processing of pro-vWF to a
molecular size similar to that generated intracellularly occurs in the
circulation of 4 different species by an as yet unknown enzyme,
suggesting that the vWF propeptide, besides being derived from the
Weibel-Palade bodies or other stores after stimulation, can also be
cleaved from pro-vWF in the circulation.
| |
ACKNOWLEDGMENT |
We are grateful to Dr Jürgen Siekmann, Herbert Gritsch,
Günter Richter, Manfred Billwein, Jutta Schreiner, and Ingrid
Neunteufel for their skilled contributions to the analyses, to
Christine Eder for expertise in performing the baboon study, and to
Sylvia C. Maurer for graphical assistance and Kathryn Nelson for
editorial assistance.
| |
FOOTNOTES |
Submitted November 30, 1998; accepted April 23, 1999.
Supported in part by the Dutch Thrombosis Foundation (Grant No.
96.001).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to H.P. Schwarz, MD, Baxter Hyland Immuno,
Industriestrasse 67, A-1221 Vienna, Austria; e-mail:
schwarh{at}baxter.com.
| |
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ABSTRACT
INTRODUCTION