Journal of Medical Virology 63:247±251 (2001)

Activation of Coagulation and Fibrinolysis During

Dengue Virus Infection
Yng-Huey Huang,1 Ching-Chuan Liu,2 Shan-Tair Wang,3 Huan-Yao Lei,1 Hsiao-Sheng Liu,1
Yee-Shin Lin,1 Hua-Lin Wu,4 and Trai-Ming Yeh5*
1
Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan,
Taiwan, R.O.C.
2
Department of Pediatrics, College of Medicine, National Cheng Kung University, Tainan, Taiwan, R.O.C.
3
Department of Public Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan, R.O.C.
4
Department of Biochemistry, College of Medicine, National Cheng Kung University, Tainan, Taiwan, R.O.C.
5
Department of Medical Technology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, R.O.C.

Dengue virus infection can induce mild dengue
fever (DF) or severe dengue hemorrhagic fever
and dengue shock syndrome (DHF/DSS) in hu-
man. The pathogenesis of hemorrhage in dengue
virus infection is not fully understood. Since
hemostasis depends on the balance between
coagulation and ®brinolysis, alternation of some
coagulation parameters (platelet count and acti-
vated partial thromoboplastin time, APTT) as well
as ®brinolytic parameters (tissue plasminogen
activator, tPA and plasminogen activator inhibi-
tor-1, PAI-1) were compared in 8 DHF/DSS and 17
DF patients. Patients showed thrombocytopenia,
APTT prolongation, and tPA increase in the acute
stage of disease, indicating activation of coagula-
tion and ®brinolysis. The activation of coagula-
tion and ®brinolysis in DHF/DSS patients was
much more severe than DF patients. In the con-
valescent stage, a rise of PAI-1 level and platelet
count with concomitant decline of tPA level and
APTT returned to normal in both DHF/DSS and DF
patients. Therefore, the activation of coagulation
and ®brinolysis during the acute stage of dengue
virus infection is offset by the increase of platelet
and PAI-1 during convalescent stage. Taken toge-
ther, these results suggest that the degree of
coagulation and ®brinolysis activation induced
by dengue virus infection is associated with the
disease severity. J. Med. Virol. 63:247±251,
2001. ß 2001 Wiley-Liss, Inc.
KEY WORDS: dengue hemorrhagic fever/den-
gue shock syndrome (DHF/
DSS); tissue plasminogen acti-
vator (tPA); plas-minogen acti-
vator inhibitor-1 (PAI-1)
INTRODUCTION
Dengue viruses are the causative agents of dengue
fever (DF) characterized by fever, headache, muscle or
joint pain, and rash [Henchal and Putnak, 1990]. In
some cases, however, patients develop the life-threaten-
ing complication, dengue hemorrhagic fever and dengue
shock syndrome (DHF/DSS) [Halstead, 1982]. DHF is
characterized by a sudden onset of fever that lasts for
2±7 days and then subsides, at which time hemorrhage
manifestations, including a positive tourniquet test,
and petechial rash become evident. Sometimes, there is
an associated form of hypovolemic shock known as DSS.
Present evidence suggests that hemorrhage in DHF/
DSS is related to multiple factors that include throm-
bocytopenia, coagulopathy, and vasculopathy [Bhamar-
apravati, 1989]. However, the pathogenesis of
hemorrhage in dengue virus infection is still not fully
understood.
Hemostasis is maintained by the balance between the
activation of coagulation and ®brinolysis. The coagula-
tion system can be activated by intrinsic pathway and
extrinsic pathway to form thrombin that can convert
®brinogen to ®brin. The ®brinolytic system, on the other
hand, can break down ®brin into ®brin degradation
products. The human ®brinolytic system comprises a
proenzyme, plasminogen, which can be activated to the
active enzyme, plasmin, by several types of plasmino-
gen activators. The principal endogenous activator of
plasminogen is tissue-type plasminogen activator (tPA)
[Bachmann and Kruithof, 1984]. Circulating tPA is
derived mainly from the vascular endothelium and is
cleared rapidly by the liver [Emeis, 1995]. Plasminogen
activator inhibitor-1 (PAI-1), which was produced by
platelet, liver, and endothelium, on the other hand, is
the major inhibitor of tPA [Loskutoff et al., 1983].
Grant sponsor: National Science Council, Taipei, Taiwan; Grant
number: NSC88-2318-B006-004-M51.
*Correspondence to: Dr. Trai-Ming Yeh, Department of Medical
Technology, College of Medicine, National Cheng Kung Univer-
sity, Tainan 70101, Taiwan, R.O.C. E-mail address: today@-
mail.ncku.edu.tw.
Accepted 31 July 2000

ß 2001 WILEY-LISS, INC.

248
Generally, coagulation activation triggers a secondary
activation of ®brinolysis, which is rapidly shut off by
the release of large amounts of PAI-1 [van Gorp et al.,
1999]. Excess activation of ®brinolysis during infection
may cause the increased tendency of bleeding found in
patients. In this study, the dynamic responses of seve-
ral coagulation and ®brinolysis factors were monitored
in dengue patients to establish the sequential events of
coagulation and ®brinolysis responses that occur dur-
ing dengue virus infection. We measured several
coagulation factors such as platelet count, prothrombin
time (PT) and activated partial thromboplastin time
(APTT) as well as ®brinolytic factors such as tPA and
PAI-1 levels in 17 DF and 8 DHF/DSS patients during
the progress of the disease.
MATERIAL AND METHODS
Human samples
Serial blood samples were obtained from 25 dengue
patients during their hospitalization in Southern
Taiwan in 1998. All patients (6 female and 19 male;
age ranged from 4 to 75 years old with mean age of 29.5)
showed clinical symptoms such as fever, rash, and bone
pain. Seventeen patients were diagnosed as DF and
eight patients were diagnosed as DHF/DSS according
to WHO criteria [Anonymous, 1997; Surveillance
Report Monthly, 1999]. All patients with DHF have
thrombocytopenia ( < 100,000/mm3) and hemoconcen-
tration (hematocrit  20% of recovery value). Most
patients recovered and were discharged between 3±4
weeks after fever onset, except one of the DHF/DSS
patients who died from the hemorrhage and shock
syndromes at 24 days after fever onset. Dengue virus
infection of all patients was con®rmed by anti-dengue
ELISA-IgM or hemagglutination inhibition (HI) tests
by the National Institute of Preventing Medicine,
Center of Disease Control, Department of Health,
Taiwan. Patients were classi®ed as primary infection
if a convalescent serum had an HI titer  1:1280
against dengue virus type 1 antigen or secondary if HI
titer was  1:1280 after the onset of illness. Among the
16 cases tested, 5 cases were secondary infection, where
11 cases were primary infection. The serotype of den-
gue virus of these patients was determined as serotype
3 by serotype-speci®c RT-PCR and con®rmed by dengue
virus isolation with type-speci®c monoclonal antibodies
using C6/36 cell culture system. Samples that were
collected between 6±8 days after fever onset were
labeled as acute stage; those collected between 14±27
days after fever onset were labeled as convalescent
stage. Samples from 17 normal individuals without
antibody against dengue virus were used as controls.
Platelet count and measurement
of PT and APTT
Platelet counts were made on blood mixed with 3.8%
sodium citrate (9:1 vol/vol) using an electronic particle
counter STKS-2A (Coulter Electronics, Hialeah, FL).
Huang et al.
To assay the PT and APTT, citrated blood was centri-
fuged at 2,500 rpm for 10 minutes and platelet poor
plasma was collected. The PT was estimated using PT
reagent (thromboplastin-calcium) to evaluate the ex-
trinsic and common pathways. The APTT was mea-
sured using APTT reagent (freeze-dried cephalin and
buffered suspension of kaolin) and calcium to evaluate
the intrinsic and common pathways. The assays were
carried out with a coagulation instrument ACL-3000
(Instrumentation Laboratory, Milano, Italy).
tPA and PAI-1 antigen assay
The levels of tPA and PAI-1 antigen in plasma were
determined by commercial ELISA kits (American Diag-
nostica, Greenwich, CT) according to the manufac-
turer's instructions. The concentrations of tPA and
PAI-1 were calculated according to the standard curve.
Samples with levels above the maximum optical
density were diluted and retested.
Statistical analysis
Levels of hematological parameters in different gro-
ups were compared with one-way ANOVA, and differ-
ences were considered signi®cant if P values were
< 0.05.
RESULTS
Changes of platelet count, PT, and
APTT during dengue virus infection
Kinetic changes of several coagulational parameters,
including platelet count, PT, and APTT, in DF and
DHF/DSS patients were monitored. The normal range
of platelet count is 180,000±350,000/ml. Both DF and
DHF/DSS patients showed thrombocytopenia in the
acute stage; however, the degree of thrombocytopenia
in DHF/DSS patients was more severe than DF pat-
ients, and their platelet counts were not normal until
10 days after fever onset (Fig. 1). The extrinsic pathway
of coagulation was normal since PT was normal (10 to
11 seconds) in all patients except the patient who died
from DHF/DSS with a prolonged PT of 16.9 seconds at
17 days after fever onset (data not shown). Prolonged
APTT was much more severe in DHF/DSS than DF
patients during the acute stage of disease (Fig. 2). All
the DHF/DSS patients had prolonged APTT until 13
days after fever onset. APTT of the death case remained
very high in late stage (105 seconds at 17 days after
fever onset).
Dynamic responses of tPA and PAI-1 during
dengue virus infection
A hyper®brinolysis stage was noticed in the acute
stage of DHF/DSS patients. The average level of tPA in
the acute stage of DHF/DSS patients signi®cantly
increased (50.4  6.0 ng/ml) while the average level of
PAI-1 was unchanged (38.3  6.3 ng/ml) when com-
pared to normal controls (Table I). The level of tPA
Hemorrhage in DHF/DSS 249

Fig. 2. Kinetics of APTT in DHF/DSS and DF patients. Data

represent the mean  SEM of DHF/DSS and DF patients. Day 0
represents the ®rst day of fever onset. The horizontal line represents
the cut-off value of normal APTT (25 seconds).

APTT and tPA/PAI-1 ratio as markers

Fig. 1. Kinetics of platelet count in DHF/DSS and DF patients. Data for DHF/DSS early diagnosis
represent the mean  SEM of DHF/DSS and DF patients. Day 0
represents the ®rst day of fever onset. Normal range of platelet count When we compared data of APTT from different
is 180,000±350,000/ml.
dengue patients during their ®rst 48 hours of hospita-
lization, 50% of the patients who had prolonged APTT
between 36±45 seconds progressed to hemorrhagic
declined to 25.5  10.2 ng/ml and PAI-1 increased to
fever or shock syndromes later (Table II). All patients
79.3  10.6 ng/ml in convalescent stage of DHF/DSS
with APTT longer than 45 seconds progressed to
patients. The tPA level of the death case remained very
hemorrhagic fever or shock syndromes, suggesting that
high (62.2 ng/ml) even at 17 days after fever onset. A
APTT may serve as an indicator of disease prognosis. In
slightly increase of tPA (21.1  5.1 ng/ml) was also
addition, all patients with hemorrhage had tPA/PAI-1
found in the acute stage of DF patients, which returned
ratio three-fold higher than normal during the ®rst 48
to normal (6.4  1.3 ng/ml) at convalescent stage. On
hours of hospitalization (Table II). Eight out of nine
the other hand, level of PAI-1 in the acute stage of DF
patients who had tPA/PAI-1 ratio three-fold higher
patients increased to 63.2  8.3 ng/ml at the acute stage
than normal progressed to hemorrhagic fever or shock
and remained high in the convalescent stage (89.3  7.0
syndromes later.
ng/ml) (Table I). Furthermore, the kinetic change of the
tPA/PAI-1 ratio of DHF/DSS and DF patients was quite
DISCUSSION
different (Fig. 3). Normal tPA/PAI-1 ratio was about
0.27. In the acute stage of DHF/DSS patients, tPA/PAI- Hemostatic defects in DHF/DSS are a multifactorial
1 ratio was reversed and increased to as high as 2.0. In mechanism that include thrombopathy, coagulopathy,
addition, tPA/PAI-1 ratio in the acute stage of DHF/ and vasculopathy. Thrombocytopenia is a major ®nding
DSS patients was at least 2±3-fold higher than that in in a signi®cant number of cases of DHF/DSS and incre-
the acute stage of DF patients (Fig. 3). ases with severity of disease [WHO, 1973]. Our study

TABLE I. Comparison of Coagulation and Fibrinolysis Parameters in DF and DHF Patients in Acute and Convalescent Stages*

DHF/DSS (n ˆ 8) DF (n ˆ 17)

Acutea Convalescentb Acute Convalescent Normal

Platelet (104/ml) 5.8  1.3 30.4  8.8 13.2  5.1 55.6  22.9 18±35
APTT (second) 60.2  10.9 31.5  2.0 23±25
tPA (ng/ml) 50.4  6.0c 25.5  10.2 21.1  5.1d 6.4  1.3 10.4  1.2
PAI-1 (ng/ml) 38.6  6.3 79.3  10.6c 63.2  8.3c 89.3  7.0c 38.6  3.9
*Data are expressed as the mean  SEM.
a
Samples collected 6±8 days after fever onset were labeled as acute stage.
b
Samples collected 14±27 days after fever onset were labeled as convalescent stage.
c
Signi®cant difference compared with normal.
d
Signi®cant difference compared with DHF/DSS.
250
Fig. 3. Kinetics of tPA/PAI-1 ratio in DHF/DSS and DF patients.
Data represent the mean  SEM of DHF/DSS and DF patients. Day 0
represents the ®rst day of fever onset. The horizontal line represents
the normal ratio of tPA/PAI-1, which was about 0.27.
con®rmed that thrombocytopenia was observed early in
DHF/DSS patients but it also occurred slightly in acute
stage of some DF patients. The depression in the bone
marrow observed in DHF in the acute stage may
account for thrombocytopenia [Murgue et al., 1998]. In
addition, dengue virus infection of megakaryocytes
could also lead to an increase in the destruction of these
cells [Lin et al., 1989]. Therefore, both increase of
platelet consumption and decrease of platelet produc-
tion may cause thrombocytopenia during dengue virus
infection.
Coagulopathy is also found in most of DHF cases and
APTT and thrombin time (TT) are more frequently
abnormal than PT [Nimmannitya, 1987, Hathirat et al.,
1993]. In our study, PT was normal in all patients with
or without hemorrhage except a death case, while
prolonged APTT was observed in all DHF/DSS pat-
ients. Moreover, 50% of patients who had prolonged
APTT of 36±45 seconds during the ®rst 48 hours after
hospitalization and all the patients with APTT longer
than 45 seconds progressed to hemorrhagic fever or
shock syndromes. Therefore, there was a positive corre-
lation with the disease severity and APTT prolonga-
TABLE II. APTT Prolongation and tPA/PAI-1 Ratio Increase in Dengue Patients*
Case number
 25 26±35
DF 2 4
DHF/DSS 0 0
Fold of tPA/PAI-1 increase
< 1x 1±<2x
DF 9 2
DHF/DSS 0 0
*Patients' sera were collected during the ®rst 48 h of hospitalization. Normal range of APTT is 23±25
seconds. Normal value of t-PA/PAI-1 is 0.27.
Huang et al.
tion. Disseminated intravascular coagulation (DIC),
which occurs in DHF/DSS patients and consumes both
platelets and clotting factors, may contribute to the
prolongation of APTT [McKay and Margaretten, 1967].
The most striking ®nding in this study is the reverse
of tPA/PAI-1 ratio found in the acute stage of DHF/DSS.
In the acute stage of DHF/DSS, increased ®brin
degradation product (FDP) and decreased ®brinogen
levels have been found [Mitrakul, 1987]. Furthermore,
decrease in a2-antiplasmin with concomitant decrease
in plasminogen is also observed transiently in the acute
stage of DHF [Funahara et al., 1987]. Therefore, a
hyper®brinolysis state has been proposed to be involved
in the acute stage of DHF. Data of tPA and PAI found in
this study further support the hyper®brinolysis state
occurs in the acute stage of DHF/DSS. TPA increase in
dengue virus infection may result from dengue virus
infection of human monocytes and endothelial cells
[Avirutnan et al., 1998]. On the other hand, cytokine
production such as tumor necrosis factor (TNF) induced
during dengue virus infection can also in¯uence the
®brinolysis process. TNF has been shown to induce the
promotion and subsequent inhibition of plasminogen
activation in human [van der Poll et al., 1991] and high
serum level of TNF is found in the acute stage of dengue
patients [Hober et al., 1993]. In addition, plasminogen
cross-reactive anti-dengue antibodies that can interfere
with ®brinolysis have also been found in DHF/DSS
patients [Chungue et al., 1994, Huang et al., 1997].
Therefore, both virus infection and the immune res-
ponse induced by dengue virus may disturb the ®bri-
nolysis system and cause the hyper®brinolysis in the
acute stage of dengue virus infection.
In summary, there is now only supportive care avai-
lable for DHF/DSS patients. To improve therapy and
supportive care, a better understanding of the patho-
genesis of bleeding and early diagnostic markers of the
disease are very important. In our study, APTT prolon-
gation and tPA/PAI-1 ratio increase in the acute stage
of dengue virus infection showed good association with
disease severity, they can serve as early indicators. In
addition, therapeutic intervention to prevent plasmin
activation in the acute stage may be bene®cial to DHF/
DSS.
APPTT (seconds)
36±45 46±55 >55
2 0 0
2 2 2
2±<3x  3x
2 1
0 8
Hemorrhage in DHF/DSS
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