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.
The authors have declared that no competing interests exist.
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.
Severe dengue infections are characterized by thrombocytopenia, 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 well with markers of 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 activity levels. This may contribute to the thrombocytopenia and complications of dengue.
Dengue has become a major international public health concern with up to 100 million annual cases worldwide. It usually manifests as 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
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
Osteoprotegerin (OPG) is a member of the tumor necrosis factor receptor superfamily, which is stored in WPBs and in platelets
Children with an acute dengue infection have elevated VWF levels
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.
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
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 day of discharge. A full blood count was performed daily in all patients as part of routine clinical care until platelet counts had shown a substantial increase. 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 −80°C until further analysis. Plasma levels of VWF:Ag and VWF propeptide were determined by enzyme-linked immunosorbent assay (ELISA) as described previously
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.
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
Characteristic | DHF (DHF I and II) | DSS (DHF III and IV) |
n = 43 | n = 30 | |
Male sex, n (%) | 15 (35) | 10 (33) |
Age, years | 8.0 (6.0–9.0) | 7.0 (6.0–9.3) |
Mortality, n (%) | 0 (0%) | 6 (20%) |
Body weight, kg | 20 (18–28) | 22 (17–30) |
Duration fever until admission, days | 4.0 (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) |
Hemoglobin, g/dL | ||
Enrollment | 13.1 (12.3–14.0) | 13.6 (12.2–14.4) |
Day 1 | 13.0 (11.7–13.6) | 12.7 (10.9–13.4) |
Platelet count, ×109/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 2 | 3.5 (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 2 | 6.0 (5.4–6.6) | 5.9 (5.0–6.3) |
SGOT, U/l | ||
Enrollment | 103 (87–184) | 118 (77–199) |
Day 2 | 93 (64–143) | 126 (91–224) |
Pleural effusion, n (%) | ||
Enrollment | 25/33 (76) | 23/26 (88) |
Day 2 | 39/39 (100) | 24/24 (100) |
Pleural effusion index | ||
Enrollment | 10.0 (2.0–20.2) | 19.4 (12.7–29.4) |
Day 2 | 24.5 (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.
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 µg/mL (12.9–20.6 µg/mL), 12.5 (9.9–16.8 µg/mL) and 7.2 µg/mL (5.8–9.5 µg/mL), respectively (
(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.
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 (
(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.
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 (
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.
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 µg/mL vs. 12.3 µg/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 (37×109/L vs. 58×109/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
VWF:Ag | VWF propeptide | VWF activation factor | ADAMTS-13 | |||||
r |
|
r |
|
r |
|
r |
|
|
VWF;Ag | 0.28 | 0.15 |
|
|
−0.03 | 0.83 | ||
VWF propeptide | 0.28 | 0.15 | 0.22 | 0.06 | −0.22 | 0.06 | ||
VWF activation |
|
|
0.22 | 0.06 | −0.13 | 0.29 | ||
ADAMTS-13 | −0.03 | 0.83 | −0.22 | 0.06 | −0.13 | 0.29 | ||
Platelets | −0.06 | 0.61 |
|
|
−0.20 | 0.09 | 0.21 | 0.08 |
Albumin | −0.05 | 0.71 |
|
|
−0.20 | 0.10 |
|
|
PEI | −0.25 | 0.06 |
|
|
−0.15 | 0.25 | −0.20 | 0.13 |
SGOT | 0.01 | 0.94 |
|
|
0.04 | 0.71 | −0.23 | 0.06 |
Significant (
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 spontaneous platelet-VWF binding. The patients who died had a significantly higher amount of VWF in its most active conformation 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
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
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 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
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
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
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 convalescence.
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We thank all the children and their parents/caretakers for participating in this study.