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Prepublished online as a Blood First Edition Paper on September 19, 2002; DOI 10.1182/blood-2002-03-0814.
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Blood, 1 February 2003, Vol. 101, No. 3, pp. 946-948
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Brief report
Changes in von Willebrand factor-cleaving protease
(ADAMTS13) activity after infusion of desmopressin
Rosemarie A. Reiter,
Paul Knöbl,
Katalin Varadi, and
Peter L. Turecek
From the Department of Clinical Pharmacology and the
Department of Medicine 1, University of Vienna; Baxter Bio Science,
Vienna, Austria.
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Abstract |
von Willebrand factor-cleaving protease (ADAMTS13) cleaves von
Willebrand factor (VWF) and regulates its physiologic function. To
investigate the relation between ADAMTS13 activity and VWF, we compared
ADAMTS13 activity with the VWF-related parameters VWF antigen (VWF:Ag),
VWF collagen-binding activity (VWF:CBA), VWF-propeptide, proVWF, and
VWF multimeric composition in 10 healthy volunteers and 3 patients with
type 1 von Willebrand disease before and after infusing 0.3 µg/kg
desmopressin. The VWF-related parameters in the volunteers increased 60 minutes after start of infusion by 3.7-fold for VWF:Ag, 7.2-fold for
propeptide, and 2.2-fold for VWF:CBA. Unusually large VWF multimers and
traces of proVWF appeared. The ADAMTS13 activity decreased to about
half the initial value. After 24 hours values returned to baseline.
Patients with type 1 von Willebrand disease showed similar results. We
conclude that the inverse correlation between ADAMTS13 and VWF-related parameters suggests a consumption of ADAMTS13 after the
desmopressin-induced release of higher multimers of VWF.
(Blood. 2003;101:946-948)
© 2003 by The American Society of Hematology.
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Introduction |
The blood glycoprotein von Willebrand factor (VWF)
mediates the adhesion of platelets to the subendothelium of damaged
blood vessels. Circulating mature VWF is a multimeric protein with a molecular mass ranging from 450 kDa to more than 20 000
kDa.1,2 A deficiency or functional abnormality of VWF
multimers is associated with bleeding,3 whereas increased
concentrations of large and the presence of unusually large VWF
multimers, as in thrombotic thrombocytopenic purpura, cause excessive
platelet aggregation.4
The VWF-cleaving protease, a metalloprotease of the ADAMTS
family, (ADAMTS13)5 is important for maintaining the normal size distribution of VWF multimers. In the absence or functional inactivation of ADAMTS13, unusually large VWF multimers accumulate and
can induce platelet aggregation, thrombosis, and thrombocytopenia under
high fluid shear rates.6 Reduced ADAMTS13 activity has been reported in thrombotic thrombocytopenic purpura and metastasizing malignancies,7 liver disease, or after
surgery,8 but little is known about the physiologic
regulation of ADAMTS13.
Infusion of the vasopressin analog desmopressin (DDAVP) induces the
release of unusually large VWF multimers, increasing the plasma
concentrations of VWF and coagulation factor VIII. Thus, desmopressin
is used as a first-line therapy in patients with type 1 von Willebrand
disease and mild hemophilia A.9 Desmopressin improves
congenital or acquired platelet dysfunction as found in uremia, liver
cirrhosis, and drug-induced bleeding associated with heparin, hirudin,
dextrane, aspirin, or ticlopidine.9
To study the regulation of ADAMTS13 after a short-term increase in VWF
concentrations, we measured ADAMTS13 and VWF-related parameters in 10 healthy volunteers and 3 patients with type 1 von Willebrand disease
before and after infusion of desmopressin.
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Study design |
Participating in the study, which was approved by the
ethics committee of Vienna University, were 10 healthy volunteers (7 men and 3 women; median age, 29.5 years) and 3 female patients with
type 1 von Willebrand disease (mean age, 49.6 years). All participants
gave their written informed consent.
Each participant received an infusion of desmopressin (Octostim;
Aventis AG, Vienna, Austria) (0.3 µg/kg body weight in 50 mL saline
over 30 minutes). Blood was collected before and 1, 2, 6, and 24 hours
after the start of infusion. VWF antigen (VWF:Ag), VWF-propeptide,
proVWF, collagen-binding activity (VWF:CBA), and the multimeric
composition of VWF were determined as described elsewhere.10-12 ADAMTS13 activity was measured according
to Gerritsen et al,13 modified by using a purified
recombinant VWF (Baxter BioScience, Vienna, Austria)14 as
substrate, and a different enzyme-linked immunosorbent assay
plate (Exiqon, Vedbaek, Denmark) to measure residual
VWF:CBA.15 All samples were tested at dilutions from 1:10
to 1:40, and the mean values were calculated. Statistica statistical software package (StatSoft, Tulsa, OK) was used for statistical analysis. All groups of data were normally distributed as
tested with the Kolmogorov-Smirnov test. The results are presented as
means ± standard error of the mean (SEM). Paired Student
t test was used to compare values, and Pearson
correlation to calculate relations between variables. P
values < .05 were considered statistically significant.
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Results and discussion |
In the healthy volunteers, 1 hour after start of infusion of 0.3 µg/kg desmopressin, VWF:Ag increased 3.7-fold from 0.91 ± 0.10 (mean ± SEM) to 3.34 ± 0.22 U/mL (P < .0001)
(Figure 1). This increase was maintained
for more than 6 hours and returned to baseline within 24 hours. In
parallel, VWF propeptide increased from 4.47 ± 0.3 to 32.12 ± 1.92 nM (P < .0001) and returned to baseline values
within 24 hours. Furthermore, traces of proVWF were detected
(0.22 ± 0.04 nM), suggesting that unprocessed VWF also is liberated.
VWF:CBA increased from 0.82 ± 0.11 to 1.77 ± 0.13 U/mL
(P < .0001) and returned to baseline values within 24 hours of infusion. A transient appearance of unusually large VWF
multimers was observed 1 and 2 hours after the start of desmopressin infusion (Figure 1). These findings accord with the
literature9,16,17 and suggest that desmopressin induces
the release of unusually large VWF multimers from endothelial
cells.
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| Figure 1.
Effects of desmopressin (DDAVP) on von Willebrand factor
antigen levels (VWF:Ag), VWF-cleaving protease activity (ADAMTS13), VWF
propeptide (VWF:pp), VWF collagen-binding activity (VWF:CBA), and VWF
multimers pattern (VWF:MM).
Healthy volunteers (n = 10) received 0.3 µg/kg DDAVP over 30 minutes. VWF:Ag is indicated by  ; ADAMTS13,  ; VWF:CBA,  ; and
VWF:pp,  . Data are presented as means ± standard errors of
mean. Presence of unusually large (UL) VWF:MM is indicated by +;
absence of UL VWF:MM,  .
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The activity of ADAMTS13 decreased significantly from 1.38 ± 0.2 to 0.66 ± 0.08 U/mL after the infusion of desmopressin
(P = .004). It remained reduced for at least 6 hours,
returning to baseline values after 24 hours (Figure 1).
Correlation analysis found significant inverse correlations between
ADAMTS13 and VWF:CBA, VWF:Ag, and VWF-propeptide before (R = 0.63, P = .03;
R = 0.67, P = .02; R = 0.56,
P = .05) and 1 hour after start of desmopressin infusion
(R = 0.66, P = .02; R = 0.53, P = .05; R = 0.34,
P = .17).
In the 3 patients with type 1 von Willebrand disease, the desmopressin
infusion induced an increase of VWF:Ag from 0.47 to 1.59 U/mL, VWF:CBA
from 0.32 to 1.46 U/mL, and VWF propeptide from 3.44 to 35.38 nM (mean
values). With the exception of the lower baseline values, the time
course was similar to that in the healthy subjects. Unusually
large VWF multimers appeared 1 hour after the start of infusion, and
traces of proVWF (0.17 and 0.26 nM) were detected in 2 of the 3 patients. Mean activity of ADAMTS13 decreased from 1.68 U/mL to
1.08 U/mL after infusion of desmopressin (Figure
2).
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| Figure 2.
Effects of desmopressin on von Willebrand factor antigen
levels (VWF:Ag), VWF cleaving-protease activity (ADAMTS13), VWF
propeptide (VWF:pp), VWF collagen-binding (VWF:CBA), and VWF multimer
pattern (VWF:MM) in 3 patients with type 1 von Willebrand disease.
All patients received DDAVP (0.3 µg/kg) over 30 minutes
intravenously. Symbols show data of all 3 patients with von Willebrand
disease: patient 1, ; patient 2,  ; patient 3, . Presence of
unusually large (UL) VWF:MM is indicated by +; absence of UL
VWF:MM, .
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The inverse correlations between ADAMTS13 activity and the VWF-related
parameters in the healthy volunteers are in agreement with the
observations of Mannucci et al,8 who found similar correlations between ADAMTS13 and VWF:Ag or VWF:CBA. In contrast, our
results differ considerably from the findings of Mannucci et al of
unchanged protease values after infusion of desmopressin.8 The discrepant data can be explained by differences in the assays used
for the determination of ADAMTS13 activity. We used a purified recombinant VWF preparation14 with a defined multimer
number of 10-12 as substrate, whereas Mannucci et al8 used
normal plasma for the source of VWF, which possibly has a higher
multimer number (> 20), and the collagen-binding assay
applied15 seems to be more sensitive within that multimer
range. A comparison of our assay and the original assay of Gerritsen et
al13 showed a more accurate standard curve in the lower
range of ADAMTS13 activity when using our recombinant VWF as substrate
(data not shown). To exclude the possibility that the high endogenous
VWF level might interfere with the assay and that the observed
decreased ADAMTS13 activity might be an artifact, all plasma samples
were measured in different dilutions.
Our results also are supported by findings of lower activity of
ADAMTS13 during acute-phase reactions, such as metastasizing malignancies, liver disease, or after surgery known to be associated with high concentrations of VWF.7,8 One explanation for
the drop of ADAMTS13 activity after desmopressin infusion may be that unusually large VWF multimers, once released into the plasma, are
immediately cleaved by ADAMTS13 in order to dispose these more
platelet-adhesive and agglutinating forms of VWF. We cannot say from
our data whether ADAMTS13 activity is thereby exhausted by the excess
of substrate or it is consumed and eliminated from plasma. However,
this phenomenon would explain the inverse correlations between the
protease and its substrate and solve the questions raised in
Mannuccis's paper.8(p2734) The existence of a
strong inverse correlation possibly due to some interactions between
the 2 proteins is further supported by the observed high ADAMTS13
levels in 2 of the 3 patients with type 1 von Willebrand disease before treatment.
In conclusion, our data confirm that desmopressin induces the release
of VWF from endothelial cell storage pools, some of it as unusually
large multimers, together with some unprocessed proVWF. Simultaneously,
the activity of ADAMTS13 dropped significantly, suggesting a direct
interaction between the protease and its substrate at the time of a
short-term increase of VWF.
| |
Acknowledgments |
We are grateful for the technical assistance of Brigitte
Keil, Jutta Schreiner, Ingrid Neunteufl, and Sylvia
Peyrer-Heimstätt (Baxter BioScience, Vienna, Austria), and the
editorial assistance of Elise Langdon-Neuner.
| |
Footnotes |
Submitted March 15, 2002; accepted September 6, 2002.
Prepublished
online as Blood First Edition Paper, September 19, 2002; DOI
10.1182/blood-2002-03-0814.
Supported in part by a grant from the Medizinisch
Wissenschaftlicher Fonds des Bürgermeisters der Stadt Wien.
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: Paul Knöbl, Department of
Medicine 1, University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria; e-mail:
paul.knoebl{at}akh-wien.ac.at.
| |
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Abstract
Introduction