Severe dengue is associated with consumption of von Willebrand factor and its cleaving enzyme ADAMTS-13.
ABSTRACT Thrombocytopenia, bleeding and plasma leakage are cardinal features of severe dengue. Endothelial cell activation with exocytosis of Weibel-Palade bodies (WPBs) may play an etiological role in this condition.
In a cohort of 73 Indonesian children with dengue hemorrhagic fever (DHF), of which 30 with dengue shock syndrome (DSS), we measured plasma levels of the WPB constituents von Willebrand factor antigen (VWF:Ag), VWF propeptide and osteoprotegerin (OPG), together with activity levels of the VWF-cleaving enzyme ADAMTS-13 and the amount of VWF in a platelet binding conformation (VWF activation factor). Compared with healthy controls (n = 17), children with DHF/DSS had significantly higher levels of VWF:Ag, VWF propeptide and OPG and decreased ADAMTS-13 activity. The VWF activation factor was also significantly higher in DHF/DSS and highest in children who died. There were significant differences in the kinetics of the various WPB constituents: VWF propeptide and OPG levels decreased toward discharge, while VWF:Ag levels were lower than expected at enrollment with plasma levels increasing toward discharge. Moreover, VWF propeptide levels correlated better with markers of disease severity (platelet count, liver enzymes, serum albumin and pleural effusion index) than corresponding VWF levels. Together, these findings suggest that there is consumption of VWF in DHF/DSS. In 4 out of 15 selected children with low ADAMTS-13 levels on admission, we found a remarkable reduction in the large and intermediate VWF multimers in the discharge blood samples, consistent with an acquired von Willebrand disease.
These findings suggest that severe dengue is associated with exocytosis of WPBs with increased circulating levels of VWF:Ag, VWF propeptide and OPG. High circulating levels of VWF in its active conformation, together with low ADAMTS-13 activity levels, are likely to contribute to the thrombocytopenia and complications of dengue. During the convalescence phase, qualitative defects in VWF with loss of larger VWF multimers may develop.
- SourceAvailable from: Dasja Pajkrt[show abstract] [hide abstract]
ABSTRACT: The results of studies with cultured endothelial cells have shown that most von Willebrand factor (vWF) synthesized is directly secreted (constitutive pathway) and consists of both mature vWF, its precursor molecule pro-vWF, and the cleaved vWF prosequence. Only fully processed, functionally mature vWF is stored within the cell, together with the propeptide, and leaves the cell only on stimulation (regulated secretion). Both in resting and stimulated cultured endothelial cells, the stoichiometry of the released propeptide to the released mature vWF is essentially equimolar. In the present study, we have measured the molar ratio of propeptide to mature vWF in vivo, both under resting conditions and conditions that reflect activation of the endothelium. To this end, we devised a method that allows the measurement of the propeptide (vW antigen II) on a quantitative, is, molar basis, using purified recombinant propeptide as a standard. Our results show that the molar concentration of the propeptide in normal plasma is about one tenth of the concentration of mature vWF (expressed as half-dimer concentration). This ratio is approximately 1:1 in the medium of cultured endothelial cells. On administration in healthy subjects of either 1-deamino-8-D-arginine vasopressin or endotoxin, both agents being known to elicit an intravascular increase of vWF, the molar ratio of propeptide to mature vWF increased fourfold to fivefold. The propeptide concentration returned to baseline values after about 6 to 7 hours of injection of each stimulus, whereas the increase of mature vWF was much more sustained. Because the respective half-lives of mature vWF and its propeptide clearly differ, measurement of the concentration of these proteins could provide a means to assess the extent of activation of the endothelium under physiological and pathophysiological conditions.Blood 11/1996; 88(8):2951-8. · 9.06 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Thrombotic thrombocytopenic purpura (TTP) is a severe, occlusive, microvascular "thrombotic microangiopathy" characterized by systemic platelet aggregation, organ ischemia, profound thrombocytopenia, and erythrocyte fragmentation. Failure to degrade "unusually large" (UL) von Willebrand factor (VWF) multimers as they are secreted from endothelial cells probably causes most cases of familial TTP, acquired idiopathic TTP, thienopyridine-related TTP, and pregnancy-associated TTP. The emphasis in this review is the pathophysiology of familial and acquired idiopathic TTP. In each of these entities, there is a severe defect in the function of a plasma enzyme, VWF-cleaving metalloprotease (ADAMTS-13), that normally cleaves hyper-reactive ULVWF multimers into smaller and less adhesive VWF forms. In familial TTP, mutations in the ADAMTS13 gene cause absent or severely reduced plasma VWF-cleaving metalloprotease activity. Acquired idiopathic TTP, in contrast, is the result in many patients of the production of autoantibodies that inhibit the function of ADAMTS-13. Established, evolving, and some of the unresolved issues in TTP pathophysiology will be summarized.Seminars in Hematology 02/2004; 41(1):4-14. · 3.36 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: To highlight mechanisms that regulate the balance between latent and active von Willebrand factor (VWF), and describe pathological conditions leading to increased levels of active VWF. Levels of circulating active VWF are increased in von Willebrand disease type 2B, HELLP syndrome, malaria and antiphospholipid syndrome. Freshly secreted VWF consists of ultra-large multimers that interact spontaneously with platelets at the endothelial cell surface. Proteolysis of ultra-large VWF by a member of the disintegrin and metalloprotease with thrombospondin motif family (ADAMTS13) reduces both multimeric size and accessibility of platelet-adhesion sites. The resulting VWF molecules circulate as inactive multimers, which regain their platelet-adhesion capacity upon binding to the subendothelial matrix, in particular under conditions of high shear. Unfortunately, mechanisms responsible for suppression of circulating plasma levels of active VWF are hampered in a number of pathological conditions, leading to VWF-platelet aggregates associated with thrombotic complications or thrombocytopenia. A recently developed assay allowed us to monitor the presence of circulating active VWF and we found that several diseases are characterized by increased levels. Further analysis provided insight into mechanisms contributing to the presence of active VWF, which revealed that beta2-glycoprotein I may act as a natural regulator of VWF-platelet interactions.Current Opinion in Hematology 06/2007; 14(3):284-9. · 4.11 Impact Factor
Severe Dengue Is Associated with Consumption of von
Willebrand Factor and Its Cleaving Enzyme ADAMTS-13
Kis Djamiatun1, Andre J. A. M. van der Ven2, Philip G. de Groot3, Sultana M. H. Faradz4, D. Hapsari5,
Wil M. V. Dolmans2, Silvie Sebastian3, Rob Fijnheer3, Quirijn de Mast2*
1Department of Parasitology, Faculty of Medicine, Diponegoro University, Semarang, Indonesia, 2Department of General Internal Medicine, Radboud University
Nijmegen Medical Centre, Nijmegen, The Netherlands, 3Laboratory for Thrombosis and Haemostasis, Department of Clinical Chemistry and Haematology, University
Medical Centre, Utrecht, The Netherlands, 4Division of Human Genetics, Faculty of Medicine, Diponegoro University, Semarang, Indonesia, 5Department of Pediatrics,
Faculty of Medicine, Diponegoro University, Semarang, Indonesia
Background: Thrombocytopenia, bleeding and plasma leakage are cardinal features of severe dengue. Endothelial cell
activation with exocytosis of Weibel-Palade bodies (WPBs) may play an etiological role in this condition.
Methods and Principal Findings: In a cohort of 73 Indonesian children with dengue hemorrhagic fever (DHF), of which 30
with dengue shock syndrome (DSS), we measured plasma levels of the WPB constituents von Willebrand factor antigen
(VWF:Ag), VWF propeptide and osteoprotegerin (OPG), together with activity levels of the VWF-cleaving enzyme ADAMTS-
13 and the amount of VWF in a platelet binding conformation (VWF activation factor). Compared with healthy controls
(n=17), children with DHF/DSS had significantly higher levels of VWF:Ag, VWF propeptide and OPG and decreased
ADAMTS-13 activity. The VWF activation factor was also significantly higher in DHF/DSS and highest in children who died.
There were significant differences in the kinetics of the various WPB constituents: VWF propeptide and OPG levels
decreased toward discharge, while VWF:Ag levels were lower than expected at enrollment with plasma levels increasing
toward discharge. Moreover, VWF propeptide levels correlated better with markers of disease severity (platelet count, liver
enzymes, serum albumin and pleural effusion index) than corresponding VWF levels. Together, these findings suggest that
there is consumption of VWF in DHF/DSS. In 4 out of 15 selected children with low ADAMTS-13 levels on admission, we
found a remarkable reduction in the large and intermediate VWF multimers in the discharge blood samples, consistent with
an acquired von Willebrand disease.
Conclusion: These findings suggest that severe dengue is associated with exocytosis of WPBs with increased circulating
levels of VWF:Ag, VWF propeptide and OPG. High circulating levels of VWF in its active conformation, together with low
ADAMTS-13 activity levels, are likely to contribute to the thrombocytopenia and complications of dengue. During the
convalescence phase, qualitative defects in VWF with loss of larger VWF multimers may develop.
Citation: Djamiatun K, van der Ven AJAM, de Groot PG, Faradz SMH, Hapsari D, et al. (2012) Severe Dengue Is Associated with Consumption of von Willebrand
Factor and Its Cleaving Enzyme ADAMTS-13. PLoS Negl Trop Dis 6(5): e1628. doi:10.1371/journal.pntd.0001628
Editor: Maria G. Guzman, Tropical Medicine Institute Pedro Kourı ´ (IPK), Cuba
Received October 10, 2011; Accepted March 8, 2012; Published May 1, 2012
Copyright: ? 2012 Djamiatun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: QdM is supported by a junior researcher grant of the Radboud University Nijmegen Medical Centre. KD received an International Collaboration
Research grant 2009 from the Ministry of National Education, Republic of Indonesia, to support the present work. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Dengue has become a major international public health concern
a non-specific febrile illness, but its course may become complicated
by bleeding and a transient plasma leakage that may ultimately lead
to shock and death . Severe dengue with thrombocytopenia,
bleeding and plasma leakage is referred to as dengue hemorrhagic
fever (DHF). The most severe form of DHF, which is accompanied
by hemodynamic instability and shock is referred to as dengue shock
syndrome (DSS). DHF/DSS is most frequently seen in children and
tends to manifest at the time the fever subsides. The pathogenic
mechanisms responsible for the development of DHF/DSS are
still poorly understood. A central feature of DHF/DSS is the
development of a pronounced thrombocytopenia.
The large glycoprotein von Willebrand factor (VWF) plays a
central role in platelet-vessel wall interaction as it is responsible for
mediation of platelet adhesion at sites of endothelial injury. VWF
is predominantly synthesized in endothelial cells and, after
cleavage of a VWF propeptide, it is either released constitutively
or stored in specialized secretory granules, known as Weibel-
Palade bodies (WPBs). Injury or activation of the endothelium
leads to a rapid secretion of equimolar amounts of stored VWF
and VWF propeptide, and both proteins are regarded as markers
of endothelial cell activation . Freshly released VWF consists of
ultra-large prothrombogenic multimers (UL-VWF). The metallo-
protease ADAMTS-13 (a disintegrin and metalloproteinase with
thrombospondin-1-like domains) functions as a natural regulator
that de-activates the prothrombogenic UL-VWF by proteolysis
. The importance of ADAMTS-13 is illustrated by the notion
www.plosntds.org1May 2012 | Volume 6 | Issue 5 | e1628
that absence of ADAMTS-13 is associated with platelet-rich
microthrombi in the microvasculature, a disease known as
thrombotic thrombocytopenic purpura (TTP) . Under normal
circumstances, VWF circulates in the plasma in a knot-like
conformation in which the binding sites for platelet glycoprotein
(GP) receptor Ib is not exposed. A change in conformation into a
more elongated ‘activated’ form can be observed under different
conditions in which increased platelet-VWF interaction is thought
to play a role . Determination of the amount of this ‘active’
VWF in plasma is possible using a llama-derived nanobody
(designated AU/VWFa-11) that displays specific binding only to
the GPIba-binding conformation of the VWF.
Osteoprotegerin (OPG) is a member of the tumor necrosis
factor receptor superfamily, which is stored in WPBs and in
platelets [6,7], similar to VWF. OPG was traditionally known for
its role in bone remodeling, but a growing body of literature
suggests that OPG also has specific effects on the endothelium,
including stimulation of vasculogenesis , expression of adhesion
molecules  and adhesion of leukocytes . Moreover, OPG is
physically associated with the A1 domain of VWF, both in stored
and in circulating VWF, suggesting that OPG could interfere with
platelet-VWF binding .
Children with an acute dengue infection have elevated VWF
levels [12,13] and one study reported a moderate decrease in
ADAMTS13 activity using an indirect assay for ADAMTS13
activity . The aim of this study was to characterize WPB
exocytosis and changes in factors involved in VWF-platelet
interaction in patients with severe dengue. We determined
concentrations of VWF antigen (VWF:Ag), VWF propeptide,
OPG and ADAMTS-13 activity in serial obtained plasma samples
from a cohort of Indonesian children with DHF/DSS, together
with the multimeric pattern of VWF and the amount of VWF in a
platelet binding conformation (VWF activation factor).
The Research Ethics Committee of the Faculty of Medicine
Diponegoro University, Semarang, Indonesia, approved all legal,
ethical and laboratory aspects of the study. Written informed
consent was obtained from parents or legal guardians of the
Patients and study procedures
This observational study enrolled consecutive children aged 3–14
years who were admitted to the pediatric ward or intensive care unit
of the Dr. Kariadi University Hospital in Semarang, Indonesia
between July 2005 and July 2006 with a clinical diagnosis of
suspected DHF/DSS according to the 1997 WHO criteria . A
control group of 17 healthy Indonesian children, aged 6 to 14 years,
was also enrolled. Inclusion criteria and clinical characteristics for
this group have recently been described . All patients tested
positive for a dengue specific IgM in the discharge blood sample.
DSS was defined as DHF with evidence of circulatory failure.
Blood samples were collected in citrate anti-coagulated blood
tubes (Beckton-Dickinson) at hospital admission (enrollment; day 0),
on day 1 after admission and on dayof discharge. A full blood count
was performed daily in all patients as part of routine clinical care
until platelet counts had shown a substantialincrease. Therefore, no
platelet counts or other hematological values were usually available
on the day of discharge. A chest X-ray was performed with the
patient lying in right lateral decubitus position to detect pleural
effusion at enrollment and on day 2. The pleural effusion index
(PEI) was calculated by dividing 100 times the maximum width of
the pleural effusion by the maximum width of the hemi-thorax.
Citrate blood was centrifuged for 20 minutes at 1600 g and
plasma was stored at 280uC until further analysis. Plasma levels of
VWF:Ag and VWF propeptide were determined by enzyme-
linked immunosorbent assay (ELISA) as described previously .
Active VWF was determined by ELISA using a nanobody (AU/
VWFa-11) that specifically recognizes the GP-1ba binding
configuration of VWF, as described previously . The term
VWF activation factor was used to express the relative amount of
VWF that circulates in its active, platelet binding conformation.
VWF activation factor of normal pooled plasma was referred to as
1. The multimeric pattern of VWF was analyzed in a selection of
15 patients, among whom the patients with the lowest ADAMTS-
13 activity levels at enrollment or on day 1 using 2% agarose gel
electrophoresis, followed by in-gel immunostaining and infrared
imaging . ADAMTS-13 activity was determined using the
fluorescence resonance energy transfer (FRETS) assay for
ADAMTS-13 activity (Peptides International, Inc., USA) whereby
the ADAMTS-13 activity of normal pooled plasma (NPP) of
healthy Dutch donors was set at 100% . Values obtained in
the study participant samples were expressed as percentage of
NPP. Normal values in healthy volunteers in our laboratory were
in the range of 60–140%. OPG was measured by an in-house
ELISA using microtiter plates coated overnight at 4uC with a
mouse anti-human OPG antibody (MAB8051, R&D Systems,
Minneapolis, MN). Diluted plasma samples were incubated for 2 h
at room temperature. Detection was done by incubation with a
secondary goat polyclonal antibody (BAF805, R&D Systems,
Minneapolis, MN) and streptavidin-conjugated HRP (Sanquin,
Amsterdam, the Netherlands) and SuperSignal West Pico
Chemiluminescent Substrate (Thermo Scientific). For statistical
analyses, the upper limit of detection for OPG (1200 mg/L) was
used in samples with values above this detection limit. Serum
SGOT concentrations (normal value for 7 year old children #35
Unit/L) were measured using a colorimetric method (Hitachi
7050; Boehringer Ingelheim, Germany). Serum albumin concen-
trations (normal value $3.26 g/dl) were measured using Bromo-
cresol–Green method. Presence of dengue specific IgM and IgG
Severe dengue infections are characterized by thrombocy-
topenia, clinical bleeding and plasma leakage. Activation of
the endothelium, the inner lining of blood vessels, leads to
the secretion of storage granules called Weibel Palade
bodies (WPBs). We demonstrated that severe dengue in
Indonesian children is associated with a strong increase in
plasma levels of the WPB constituents von Willebrand factor
(VWF), VWF propeptide and osteoprotegerin (OPG). An
increased amount of the hemostatic protein VWF was in a
hyperreactive, platelet binding conformation, and this was
most pronounced in the children who died. VWF levels at
enrollment were lower than expected from concurrent VWF
propeptide and OPG levels and VWF levels did not correlate
wellwithmarkersof disease severity. Together, this suggests
that VWF is being consumed during severe dengue.
Circulating levels of the VWF-cleaving enzyme ADAMTS-13
were reduced. VWF is a multimeric protein and a subset of
children had a decrease in large and intermediate VWF
multimers at discharge. In conclusion, severe dengue is
associated with exocytosis of WPBs with consumption of
VWF and low ADAMTS-13 activitylevels. This may contribute
to the thrombocytopenia and complications of dengue.
VWF and ADAMTS-13 in Severe Dengue
www.plosntds.org2May 2012 | Volume 6 | Issue 5 | e1628
antibodies was determined by capture and indirect ELISA (Focus
Technologies, Cypress, Calif., USA), according to the manufac-
turer’s instructions . A full blood count was performed daily by
a standard hematology analyzer.
Data were expressed as medians with interquartile ranges
(IQR). Differences between groups were assessed by Mann-
Whitney tests; changes in laboratory parameters over time within
groups were evaluated by Wilcoxon matched-pairs signed rank
test. Relationships between continuous variables were examined
by Spearman’s rank correlation analysis. A p-value of ,0.05
indicated a significant difference. Statistical analyses were
performed with SPSS version 16.0.
Clinical characteristics and treatment
A total number of 73 children with severe dengue were enrolled,
of whom 43 were classified as having DHF grade I or II and 30 as
DSS (DHF grade III or IV). In addition, 17 healthy controls were
enrolled. Clinical characteristics of the patients are summarized in
table 1 (adapted from ). The median duration of fever at
enrollment was 4.0 days in both groups. All patients showed
plasma leakage on day two after enrollment as evidenced by
pleural effusion on a lateral chest X-ray. The nadir in platelet
counts was observed on day 1 with median (IQR) values of
456109/L (27–756109/L) and 506109/L (18–746109/L) for the
DHF and DSS group; by day 3, median platelet counts had raised
to 896109/L (43–1116109/L) and 806109/L (40–1536109/L),
respectively. Clinical bleeding occurred in 7 (16.3%) patients with
DHF (epistaxis, n=4; gum bleeding, n=1; hematemesis, n=2)
and in 2 (7.1%) patients with DSS (hematemesis, n=1; melena,
n=1). Intravenous fluids were administered to all but three
children. Free frozen plasma (FFP) was administered to 3 (7.0%)
children with DHF and to 8 (28.6%) children with DSS. Two
(4.7%) children from the DHF group and 4 (14.3%) from the DSS
group received a platelet transfusion. Six children, all from the
DSS group, died during the admission. No plasma sample was
available for analysis in two patients from the DHF group and one
Table 1. Patient characteristics and baseline data for children with dengue hemorrhagic fever/dengue shock syndrome.
Characteristic DHF (DHF I and II) DSS (DHF III and IV)
Male sex, n (%) 15 (35)10 (33)
Age, years8.0 (6.0–9.0) 7.0 (6.0–9.3)
Mortality, n (%)0 (0%) 6 (20%)
Body weight, kg20 (18–28) 22 (17–30)
Duration fever until admission, days4.0 (3.0–5.0)4.0 (4.0–5.0)
Tourniquet test positive, n (%)26/36 (72) 11/17 (65)
Petechiae per 2.5 cm2, n (%)26 (8)24 (5)
Enrollment13.1 (12.3–14.0)13.6 (12.2–14.4)
Day 113.0 (11.7–13.6)12.7 (10.9–13.4)
Platelet count, 6109/L
Enrollment 64 (41–85)38 (25–71)
Day 1 45 (27–75) 50 (18–74)
Albumin serum, g/dL
Enrollment 3.5 (2.9–3.8)2.8 (2.4–3.3)*
Day 23.5 (3.1–3.7) 3.3 (3.0–3.7)
Total protein serum, g/dL
Enrollment 5.8 (4.7–6.5) 4.8 (3.8–5.7)*
Day 26.0 (5.4–6.6)5.9 (5.0–6.3)
Enrollment103 (87–184)118 (77–199)
Day 293 (64–143) 126 (91–224)*
Pleural effusion, n (%)
Enrollment 25/33 (76)23/26 (88)
Day 239/39 (100) 24/24 (100)
Pleural effusion index
Enrollment10.0 (2.0–20.2)19.4 (12.7–29.4)*
Day 224.5 (15.4–32.7)30.9 (23.1–42.8)*
Data represent median with interquartile ranges or numbers with percentages or number with percentage. DHF, dengue hemorrhagic fever; DSS, dengue shock
syndrome; SGOT, serum glutamic oxaloacetic transaminase.
*p,0.05 by Mann-Whitney U-test.
VWF and ADAMTS-13 in Severe Dengue
www.plosntds.org3 May 2012 | Volume 6 | Issue 5 | e1628
patient from the DSS group at day 1 and in fifteen and six patients
of these respective groups at discharge.
VWF:Ag, VWF propeptide and OPG
Children from the DHF group had the highest VWF:Ag levels
at enrollment, followed by children with DSS and healthy controls
with median (IQR) values of 16.6 mg/mL (12.9–20.6 mg/mL),
12.5 (9.9–16.8 mg/mL) and 7.2 mg/mL (5.8–9.5 mg/mL), respec-
tively (Figure 1A). In contrast, the highest VWF propeptide levels
were found in the DSS group with a median level of 22.7 nM
(15.7–35.8 nM) compared to 21.9 nM (17.7–24.9 nM) in the
DHF group and 5.7 nM (4.7–6.4 nM) in healthy controls
(Figure 1B). There was also a clear difference in the kinetics of
VWF:Ag and VWF propeptide levels, despite the fact that both
proteins are released into the plasma in equimolar amounts. While
VWF propeptide levels decreased towards discharge, VWF:Ag
levels increased significantly. OPG levels followed the same
pattern as VWF propeptide: baseline levels were very high with
49% and 70% of the children in the DHF and DSS group,
respectively, having a value above the upper detection limit of the
assay (1200 mg/L), followed by a decrease towards discharge
VWF activation factor and ADAMTS-13
The median VWF activation factor was about twofold higher in
the DHF/DSS patients than in the healthy controls, indicating
that a higher amount of the circulating VWF was in a platelet-
binding conformation (Figure 2A). This increased activation status
of VWF persisted until discharge. A marked reduction in
ADAMTS-13 activity levels was also a common finding at the
time of enrollment (Figure 2B). At enrollment, 20/43 (46%) of
children with DHF and 20/30 (67%) of children with DSS had an
ADAMTS-13 activity level of #50%; a severe ADAMTS-13
deficiency (#10%) was found in 1/43 (2%) and in 3/30 (10%),
respectively. ADAMTS-13 activity levels recovered to near normal
values at the time of discharge.
VWF multimeric pattern
The multimeric pattern of VWF was determined in 15 patients.
These 15 patients included the 7 patients of the cohort with the
lowest ADAMTS-13 activity levels at enrollment in whom a blood
sample at discharge was available; the remaining 8 patients were
randomly selected. Despite the low ADAMTS13 levels, UL-VWF
was not observed in any of the blood samples. However, the
discharge blood sample of 4 patients with low ADAMTS-13 levels
at enrollment showed a reduction in large and intermediate VWF
multimers (Figure 3; patient 1 to 3, patient 4 not shown). None of
these 4 patients suffered from clinical bleeding.
Relation to severity of illness and correlation between
In the group of children with DSS, there was no significant
difference between the 6 children who died compared with those
who survived in median values at enrollment for VWF:Ag levels
(14.9 mg/mL vs. 12.3 mg/mL; p=0.53), VWF propeptide levels
(33.3 nM vs. 21.7 nM; p=0.10), ADAMTS-13 activity (30% vs.
49%; p=0.19) and platelet count (376109/L vs. 586109/L;
p=0.19). The most outspoken difference between these groups
was an almost twofold higher VWF activation factor (3.2 vs. 1.4;
p,0.01) at enrollment in the children who died. No significant
differences in these parameters were found between those with and
those without clinical bleeding (data not shown). Infusion of blood
products had only a minor influence on these median laboratory
values because only one of the six children who died received FFP
and platelets at the day of enrollment.
Correlations of VWF:Ag and VWF-related parameters with
clinical and laboratory markers are shown in table 2. There was no
significant correlation between VWF:Ag and markers of dengue
severity (platelets and SGOT levels) and plasma leakage (albumin
levels and PEI). In contrast, VWF propeptide levels had a much
stronger correlation with these parameters; VWF propeptide levels
were positively associated with severity of plasma leakage (negative
correlation with plasma albumin level and positive correlation with
PEI) and with liver enzyme disturbances and negatively associated
with platelet count.
Our study shows that DHF/DSS is associated with acute
endothelial cell activation with exocytosis of WPBs and release of
VWF:Ag, VWF propeptide and OPG in the circulation, combined
with a decrease in ADAMTS-13 activity. The circulating VWF
had a higher activation factor, indicating that an increased amount
of VWF was in an elongated, ‘active’ conformation enabling
Figure 1. Plasma levels of Weibel-Palade body constituents in Indonesian children with dengue. (A) VWF antigen, (B) VWF propeptide
and (C) osteoprotegerin levels (all determined by ELISA) in Indonesian children with dengue hemorrhagic fever (DHF) and dengue shock syndrome
(DSS) and in healthy controls. Horizontal lines represent median values. The upper limit of detection of the OPG assay was 1200 pg/ml; 49% and 70%
of children in the DHF and DSS group, respectively, had an OPG plasma level above this cut-off value at enrollment. No plasma available for analysis
in two patients from the DHF group and one from the DSS group at day 1 and in fifteen and six patients from the DHF and DSS group at discharge. P
values were determined by Wilcoxon matched-pairs signed rank test for data in time and Mann Whitney U- test for comparison with the control
group. *p value,0.05, **p value,0.01.
VWF and ADAMTS-13 in Severe Dengue
www.plosntds.org4May 2012 | Volume 6 | Issue 5 | e1628
spontaneous platelet-VWF binding. The patients who died had a
significantly higher amount of VWF in its most active conforma-
tion and in some of the patients with low ADAMTS-13 activity at
enrollment, qualitative defects in VWF with a pronounced loss of
larger VWF multimers was seen in the discharge blood samples.
There was a clear difference in the kinetics of VWF:Ag and the
other WPB constituents VWF propeptide and OPG: VWF:Ag
levels were relatively low at enrollment and increased towards
discharge, while VWF propeptide and OPG levels were very high
at baseline and decreased upon clinical recovery. In contrast to
VWF propeptide levels, VWF:Ag levels did not correlate well with
parameters for disease severity. Hence, VWF propeptide levels
seem to better reflect endothelial cell activation status and disease
severity in DHF/DSS than VWF:Ag levels. We hypothesize that
increased VWF consumption due to agglutinating platelets
underlies this phenomenon. It is unlikely that VWF would simply
leak out of the circulation during plasma leakage, just like albumin,
because of the very large size of VWF multimers (.10.000 kDA)
and because plasma levels of VWF propeptide would be expected
to leak out even more as it is a smaller molecule. The four- to
fivefold shorter half-life of VWF propeptide compared to mature
VWF could explain why VWF:Ag levels were still elevated in the
discharge samples, while VWF propeptide levels had returned to
normal . The VWF activation factor remained elevated across
the study period. VWF activation is a measure of the relative
amount of VWF that circulates in a platelet binding conformation.
This parameter does not take into account the circulating VWF
concentration, but total active VWF levels can be approximated
by multiplying the VWF activation factor by the VWF:Ag levels.
This approximation is hampered in this study by the consumption
of VWF. Nonetheless, the high VWF propeptide levels in the early
phase after enrollment and the fact that VWF:Ag and VWF
propeptide are released in equimolar amounts from the endothe-
lium suggest that total active VWF levels were higher early after
enrollment and decreased towards discharge.
Our study is the first to report OPG data in dengue. The
vascular effects of OPG have received increased attention in recent
years. High OPG levels have been related to atherosclerosis and
cardiovascular disease in epidemiological studies [22,23] and OPG
was shown to have specific effects on endothelial cells in vitro, such
as prevention of apoptosis and up-regulation of adhesion
molecules [8–10]. OPG is also closely linked to VWF: both
proteins are cohabitants of WPBs and remain associated after
release in the circulation. The notion that OPG binds selectively to
the VWF A1 domain suggests that it may interfere with the
binding of platelets to activated VWF, thereby preventing
excessive platelet adhesion and aggregation [11,24]. This process
may especially be relevant in inflammatory conditions, which are
usually accompanied by endothelial cell activation and release of
VWF, since inflammatory cytokines up-regulate the synthesis and
release of OPG . OPG may also influence dengue pathogenesis
in other ways. OPG is a decoy receptor that competes with both
tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)
and receptor activator of nuclear factor kappa-B (RANK) ligand
(RANKL). Patients with an acute dengue infection have elevated
TRAIL serum levels . This may be important in host defense
against dengue, because recent work by Warke et al. suggested
that TRAIL has dengue antiviral properties and suppresses the
production of pro-inflammatory mediators by dengue-virus
infected dendritic cells . Moreover, while the RANK/RANKL
system is predominantly known for its role in bone remodeling,
there is increasing evidence that RANKL, which is among others
expressed by activated T-cells, is also involved in regulation of
immunity . Hence, the high OPG levels may not only interfere
with VWF-platelet interaction, but also with the antiviral and
immune effects of TRAIL and the RANK/RANKL system.
While OPG levels increased during the acute phase of DHF/
DSS, a reduction in ADAMTS-13 activity was common.
ADAMTS13 regulates the multimeric size and function of VWF
by cleaving VWF within the A2 domain. To our knowledge, no
Figure 2. Plasma ADAMTS-13 activity level and VWF activation factor in Indonesian children with dengue. (A) ADAMTS-13 activity level
in children with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) and in healthy controls. The ADAMTS-13 activity levels were
determined by FRETS-VWF73 assay and are depicted in % of normal pool plasma. (B) VWF activation factors were determined by ELISA using the
llama-derived nanobody AU/VWFa-11. The VWF activation factor represents the relative amount of VWF that circulates in an active platelet-binding
conformation, whereby the VWF activation factor of normal pooled plasma was referred to as 1. Horizontal lines represent median values. No plasma
available for analysis in two patients from the DHF group and one from the DSS group at day 1 and in fifteen and six patients from the DHF and DSS
group at discharge. P values were determined by Wilcoxon matched-pairs signed rank test for data in time and Mann Whitney U-test for comparison
with the control group. *p value,0.05, **p value,0.01.
VWF and ADAMTS-13 in Severe Dengue
www.plosntds.org5May 2012 | Volume 6 | Issue 5 | e1628
previous study has so far reported data on both OPG and
ADAMTS13 in clinical samples. An acute increase in circulating
VWF levels in volunteers through administration of desmopressin
or endotoxin is followed by a decrease in ADAMTS-13 levels
[28,29] and lower ADAMTS-13 levels have been observed in
several physiologic and pathologic conditions with high VWF
levels, e.g. pregnancy and acute inflammatory states . Hence,
in our opinion, endothelial cell activation with release of VWF and
secondary ADAMTS-13 consumption is the most likely explana-
tion for the low ADAMTS-13 activity levels in these children.
Other factors may also be involved: in vitro studies have shown
that pro-inflammatory cytokines can reduce ADAMTS-13 syn-
Figure 3. VWF multimer pattern in Indonesian children with dengue. Plasma VWF multimer distribution was analysed by agarose gel
electrophoresis, followed by in-gel immunostaining. Electrophoresis was performed from the top to the bottom. Plasma at discharge (Dis) from 3
children with severe dengue (patient 1–3) demonstrated a reduction in large and intermediate VWF multimers compared to plasma at enrollment
(D0) or day 1 (D1), consistent with an acquired von Willebrand disease. No ultralarge VWF multimers were seen. NPP depicts normal pool plasma.
VWF:Ag, VWF antigen. * The last available platelet count before discharge is given.
Table 2. Spearman correlation (r) of VWF-related variables with laboratory and clinical parameters of dengue severity.
VWF:AgVWF propeptideVWF activation factorADAMTS-13
VWF propeptide0.280.150.22 0.06
VWF activation 0.260.03 0.22 0.06
Significant (P,0.05) correlations are shown in bold. VWF:Ag, von Willebrand factor antigen; PEI, pleural effusion index; SGOT, serum glutamic oxaloacetic transaminase.
VWF and ADAMTS-13 in Severe Dengue
www.plosntds.org6May 2012 | Volume 6 | Issue 5 | e1628
thesis, while thrombin and plasmin can inactivate ADAMTS-13
[31,32]. Finally, a case of dengue-associated microangiopathic
thrombocytopenia due to an inhibitor of ADAMTS-13 was
recently reported . The rapid normalization of ADAMTS-
13 levels in the children in our study, however, argues against a
role for ADAMTS-13 antibodies.
A remarkable observation in our study was the absence of large
and intermediate VWF multimers in the discharge blood sample
of some of the children. Loss of larger multimers is a prominent
feature of acquired type 2A von Willebrand disease (VWD). This
condition is associated with a number of different disease states,
including hematoproliferative and auto-immune diseases and
cardiac abnormalities such as aortic stenosis . One case report
described a transient acquired VWD in a child recovering from an
acute EBV infection . Different pathophysiologic mechanisms
may underly the loss of larger multimers . In essential and
reactive thrombocytosis, the increased number of circulating
platelets is associated with a reduction of large VWF multimers
. In dengue, platelet numbers start to rise abruptly in the
convalescent phase and we hypothesize that the combination of
rapidly rising platelet numbers, the restoration of ADAMTS-13
activity and ‘exhaustion’ of endothelial cells is responsible for the
transient disappearance of large and intermediate VWF multi-
mers. Loss of larger VWF multimers may result in an increased
bleeding tendency and, although clinical bleeding in DHF/DSS is
usually restricted to the critical phase around defervescence,
qualitative abnormalities of VWF should be considered in patients
with a persistent bleeding tendency.
Is the observed imbalance in the VWF-ADAMTS-13 system
clinically relevant? High circulating levels of VWF in an elongated,
active conformation together with reduced VWF proteolysis by
ADAMTS-13 may lead to increased platelet adhesion and
formation of platelet-rich thrombi. In patients with severe sepsis
and severe malaria, a similar imbalance in VWF and ADAMTS-
13 was found and this was considered to be related to
thrombocytopenia and organ dysfunction [17,37–40]. What
DHF/DSS distinguishes from these other severe infectious diseases
is that consumption of VWF;Ag and loss of larger VWF multimers
have not been observed in these other diseases [17,41,42]. The
notion that the AB blood group is associated with a higher risk for
severe dengue supports a role for VWF in dengue pathogenesis,
since blood group AB is associated with higher VWF levels .
Hence, even though typical features of thrombotic microangiop-
athy (e.g. schizocytes) are usually not found in dengue and the
etiology of dengue-associated thrombocytopenia is multifactorial,
we suggest that the observed changes in VWF-ADAMTS-13 play
a role in the pathogenesis of DHF/DSS and in the etiology of
thrombocytopenia in special. Disturbances in normal platelet-
endothelium interaction may especially be relevant for DHF/DSS,
since increasing evidence has shown platelet-endothelium interac-
tion to be important for vessel wall stability during inflammation
. There is currently no specific treatment for DHF/DSS
except careful fluid therapy. Although there is little evidence to
support the practice of transfusing fresh-frozen plasma (FFP), this
is sometimes done in practice for severe bleeding or for correction
of prolonged coagulation tests. An unintended advantage of FFP
infusion might be the replenishment of ADAMTS-13. One small
trial in patients with acute dengue from Sri Lanka indeed found a
small increase in platelet count in patients treated with 600 ml of
FFP compared with isotonic saline .
Several limitations to our study should be considered. First,
enrollment to our study was restricted to children with suspected
DHF/DSS and samples from children with a mild dengue infection
and uncomplicated dengue fever were not available for analysis.
Whether the reported abnormalities in the VWF-ADAMTS13
system are specific for DHF/DSS or can also be found in less severe
dengue infections therefore remains unknown. Second, platelet
counts at discharge and in the control group were unavailable.
In conclusion, our data show that WPB exocytosis of VWF in its
active conformation and consumption of VWF and ADAMTS-13
are prominent phenomena in severe dengue, which may
contribute to thrombocytopenia and organ dysfunction. Severe
dengue is also associated with very high plasma OPG levels, of
which the functional consequences need further study. Finally, a
transient reduction in larger VWF multimers may develop during
We thank all the children and their parents/caretakers for participating in
Conceived and designed the experiments: KD AJAMV PGG RF SMHF
DH WMVD QM. Performed the experiments: SS PGG RF. Analyzed the
data: KD QM. Contributed reagents/materials/analysis tools: PGG RF.
Wrote the paper: KD QM AJAMV.
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