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Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection
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Microbes and Infection xx (2016) 1e7 www.elsevier.com/locate/micinf
Short communication
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Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection Elzinandes Leal de Azeredo a,*, Victor Edgar Fiestas Sol orzano a, Debora Batista de Oliveira a, Cintia Ferreira Marinho a, Luiz Jose de Souza b, Rivaldo Ven^ancio da Cunha c, Paulo Vieira Damasco d, Claire Fernandes Kubelka a, Luzia Maria de-Oliveira-Pinto a a
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Laboratory of Viral Immunology, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil b Referral Centre for Dengue, Campos de Goytacazes, Brazil c Department of Clinical Medicine, Universidade Federal do Mato Grosso do Sul, Campo Grande, Brazil d Rio-Laranjeiras Hospital, Gaffree Guinle University Hospital (The Federal University of Rio de Janeiro) and Pedro Ernesto University Hospital (The State University of Rio de Janeiro), Rio de Janeiro, Brazil Received 6 April 2016; accepted 22 August 2016 Available online ▪ ▪ ▪
Abstract Tissue Factor (TF) is the initiator of coagulation and Tissue Factor Inhibitor (TFPI) is the physiological inhibitor of the TF/FVIIa complex. Circulating levels of TF and TFPI were quantified in dengue patients and the relationships with disease severity and infecting serotype analysed. A significant decrease in TF and TPFI plasma levels was observed in mild DF patients compared with severe dengue. Furthermore, both factors were associated with haemorrhagic manifestations. Finally, TF levels were significantly increased in DENV-1/2 infected patients as compared with DENV-4. These findings suggest that activation of TF-pathway is an important component of DENV -related coagulation disorders. © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: TFPI; TF; Dengue
1. Introduction Blood extrinsic coagulation pathway is initiated by exposure to transmembrane glycoprotein Tissue Factor (TF), that functions as the cofactor for plasma FVIIa [1] leading thrombin generation, fibrin deposition and in conjunction with platelets a formation of a hemostatic plug. Tissue Factor Pathway Inhibitor (TFPI) regulates coagulation by inhibiting the catalytic activity of the TF/VIIa complex [2]. Dengue viruses (DENV) are members of the Flaviviridae family with four distinct serotypes designated DENV-1, DENV-2, DENV-3 and DENV-4. The clinical manifestation of dengue ranges from mild dengue fever to a severe life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). DHF/ * Corresponding author. E-mail address: [email protected] (E. Leal de Azeredo).
DSS is characterized by spontaneous bleeding and vascular leakage that can progress to hypovolemic shock and death [3]. Thrombocytopenia and coagulopathy are among the hematologic abnormalities characteristic of the severe forms of dengue [4]. Despite efforts and increasing data, the mechanisms involved in coagulopathy and hemorrhages are not well understood. Given the essential roles of TF and TFPI in the hemostasis, we measured circulating levels of these proteins in 148 DENV infected patients and the relationship with clinical manifestations and infecting serotype were analysed. 2. Materials and methods 2.1. Study population In 2010, 2011 and 2013, we evaluated 148 patients suspected of DENV infection at the Brazilian hospitals. Samples
http://dx.doi.org/10.1016/j.micinf.2016.08.005 1286-4579/© 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Leal de Azeredo E, et al., Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.08.005
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E. Leal de Azeredo et al. / Microbes and Infection xx (2016) 1e7
were collected during the acute phase of infection (1e15 days after disease onset). Cases of dengue were classified, according to the World Health Organization (WHO) criteria established in 2009, as DF (n ¼ 90); DF with warning signs (DF/ WS, n ¼ 41); or severe dengue (n ¼ 17). DF included cases in which the diagnosis were confirmed by laboratory tests and patients presented symptoms such as fever, headache, retroorbital pain, myalgia, arthralgia, rash, nausea, vomiting, positive tourniquet test, leukopenia and no warning signs. In addition to these symptoms, the patients classified into DF/WS presented abdominal pain or tenderness; persistent vomiting; clinically relevant fluid accumulation; mucosal bleeding; lethargy; restlessness; liver enlargement (>2 cm); and an increase in haematocrit concomitant with a rapid decrease in platelet count. Severe dengue included cases characterised by severe vascular leakage; fluid accumulation with respiratory distress; severe haemorrhage, such as massive vaginal bleeding (in women of childbearing age); gastrointestinal bleeding; and organ impairment, such as liver damage (aspartate aminotransferase or alanine aminotransferase > 1000 U/L); involvement of central nervous system (drop in the level of consciousness); as well as other organs, such as the heart [5]. DENV infection was confirmed by detection of antidengue IgM with capture enzyme-linked immunosorbent assay (ELISA; PanBio, Brisbane, Australia); detection of viral protein NS1 (Bio-Rad, Hercules, CA, USA); and detection of viral RNA by reverse-transcriptase polymerase chain reaction.
The pattern of primary or secondary response from dengue positive patients was determined based on the set of specific IgM tests, NS1 and or RT-PCR, specific IgG (EL1500G Focus Diagnostics, Cypress, CA, USA) as described previously [6]. Demographical, clinical and biochemical data related to the patients are shown. Fourteen healthy controls (HC) were included in the study. Table 1. The study was approved by the Ethics Committee of Plataforma Brasil, FIOCRUZ (CAAE 13318113.7.0000.5248). 2.2. Blood samples related to hemostasis determinations Blood samples were collected in blood collection tubes and kept on ice. Plasma was collected and stored at 70 C. Circulating levels of TF and TFPI from dengue patients and HC were tested for Human Coagulation Factor III/Tissue Factor (cat. DCF300) and TFPI (DTFP10) Quantikines by ELISAs (R&D Systems) according to the manufacturer's instructions. 2.3. Statistical analysis Statistical analyses were performed using GraphPad Prism software, version 6.0 (GraphPad Software Inc., San Diego, CA, USA). The non-parametric KruskaleWallis and ManneWhitney U tests were used to compare the differences between study groups (HC, DF, DF/WS and severe dengue). Correlation was estimated by Spearman regression analysis. Values of p < 0.05 were considered statistically significant.
Table 1 Demographic characteristics of the study population and clinical-laboratorial course of patients with dengue. Characteristic
Age; years Female sex; n (%) Post-infection daysa Clinical sign and symptoms Bleeding manifestations at enrolment,b n (%) Plasma leakage,c n (%) Laboratory test results DENV NS1 glycoprotein, n (%) anti-DENV IgM, n (%) anti-DENV IgG, n (%) serotype-specific by RT-PCR, n (%) Leukocytes count/mm3 Monocytes count/mm3 Lymphocytes count/mm3 AST IU/L ALT IU/L Platelets 103 count/mm3
Q3
Patients
Health controls (n ¼ 14)
DF
DF/WS
Severe DF
(n ¼ 90)
(n ¼ 41)
(n ¼ 17)
42 (26e54) 48 (53) 4 (2e6)
42 (25e55) 24 (59) 5 (3e7)
39 (23e54) 9 (53) 4 (3e6)
30 (26e31) 10 (71) e
9 (10) 0 (0)
20 (49) 8 (3)
12 (63) 8 (50)
e e
33 (75) 47 (57) 60 (76) 31 (57) 3400 (2700e4975)** 393 (294e644) 1128 (834e1614) 42 (29e82) 50 (32e80)¥ 149 (111e213)**, ¥¥¥
17 (94) 36 (90) 40 (98) 11 (39) 3200 (2200e3900)### 312 (174e421) 1229 (861e1819)$ 81 (41e232)$$ 83 (41e198) 77 (35e131)###
4 (57) 17 (100) 14 (100) 1 (33) 3690 (2820e4833)& 552 (260e741) 2193 (1226e3367)p p 97 (72e228) p 116 (66e215) 24 (14e53)&&&, ppp
e e e e 5800 (5292e6173) 480 (318e500) 1704 (1585e2146) e e 280 (242e332)
DF, dengue fever; DF/WS, DF with warning signs; AST, aspartate aminotransferase; ALT, alanine aminotransferase; nd, not determined. Data are presented as medians (interquartile range) or numbers (n) with percentage (%). ManneWhitney U tests were applied. A p-value <0.05 was considered significant and was indicated as *** (p < 0.001); **(p < 0.01); or *(p < 0.05). *DF vs Healthy Control; #DF/WS vs Healthy Control; &Severe DF vs Healthy Q4 Control; ¥ DF vs DF/WS; pDF vs Severe DF; $DF/WS vs Severe DF. a Days from disease onset until the interview. b Includes skin haemorrhages, epistaxis, gingival bleeding, gastrointestinal bleeding, urinary tract haemorrhage or metrorrhagia. c Signs of vascular leakage (pleural or pericardial effusion, ascites). Please cite this article in press as: Leal de Azeredo E, et al., Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.08.005
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3. Results 3.1. Patient characteristics Demographical, clinical and laboratorial data related to the patients enrolled in this study are shown in Table 1. Clinical manifestations of DF include fever, headache, retro-orbital pain, muscle pain, arthralgia, and nausea, vomiting and rash. Apart from petechiae, hemorrhagic manifestations were not common (10%). The following warning symptoms and signs were presented by DF/WS patients: severe abdominal pain, severe nausea and vomiting, some bleeding episodes as skin haemorrhages, epistaxis, gingival bleeding, gastrointestinal bleeding, urinary tract haemorrhage or metrorrhagia. Only 3% of DF/WS patients presented signs of vascular leakage as pleural or pericardial effusion, ascites. Among severe dengue cases, we documented serious manifestations, such as epistaxis, gingival bleeding, urinary tract haemorrhage or metrorrhagia (63%), vascular leakage and hypotension (50%). Among the serotypes identified, 14% were DENV-1, 28% were DENV-2 and 58% were DENV-4. As the majority of patients (85%) presented secondary infections, no correlation was observed between patients with primary or secondary infection with patients presenting neither clinical manifestations nor laboratory parameters. In relation to haematological parameters, the total leukocyte counts were lower in all DENV positive patients, regardless of disease severity when compared with HC. Lymphocyte counts were increased in severe dengue patients compared with DF, DF/WS patients and HC. No differences were observed in monocyte counts between groups of study. Severe dengue patients and DF/WS had low platelets counts as compared with both DF and HC. Levels of liver enzyme AST were significantly higher in severe cases compared with DF or DF/WS cases (Table 1). As a proposal for analysis, we compared patients from 2010/2011 (DENV-1 and DENV-2) outbreaks with those from 2013 (DENV-4) outbreak. In DENV-1/2 outbreak, laboratory findings indicated low lymphocyte count (1465 [1108e2420], median [25%e75% interquartile]) and low platelet count (85 [36e149]) when compared to HD. Moreover, high frequencies of hemorrhagic manifestations and/or plasma extravasation were noted in these patients (36 out 74, 48.6%). In comparison, patients of the 2013 DENV-4 outbreak showed an even lower frequency of lymphocyte count (1111 [802e1568], p ¼ 0.0023) but a higher platelet count (136.5 [95.8e192.3], p ¼ 0.0007) with less frequent bleeding and/or plasma leakage (8 out 72, 11.1%). 3.2. Circulating levels of TF, TFPI and TF/TFPI ratio in relation to dengue severity and DENV infecting serotype High circulating levels of TF were detected in severe dengue patients compared with DF fever patients (DF n ¼ 83 vs. severe n ¼ 14, p < 0.05), discriminating severe from mild patients (Fig. 1A). TFPI levels were higher in plasma from DF, DF/WS or Severe compared to HC and similar to TF levels, it
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also discriminate severe from mild patients [(DF n ¼ 73 vs. Severe n ¼ 13, p < 0.05) (DF n ¼ 73 vs. control n ¼ 20, p < 0.01) (DF/WS n ¼ 32 vs. control n ¼ 20, p < 0.001) (Severe n ¼ 13 vs. control n ¼ 20, p < 0.001)] (Fig. 1B). DENV-infected patients showed a decreased levels TF/ TFPI ratio as compared to controls, and decreased levels were statically significant in DF or DF/WS patients versus HC [(DF n ¼ 67 vs. control n ¼ 20, p < 005) (DF/WS n ¼ 26 vs. control n ¼ 20 p < 0.01**)] (Fig 1E). The analysis of these mediators by the prevalent serotype showed that DENV-1/2 infected patients presented high levels of TF compared with DENV-4 infected patients [(DENV-1/2 n ¼ 55 vs. control n ¼ 20, p < 0.01)(DENV-1/2 n ¼ 55 vs. DENV-4 n ¼ 59, p < 0001)(DENV-4 n ¼ 59 vs. control ¼ 20, p < 001)]. Interestingly, DENV-4 infected patients showed decreased TF levels than HC (DENV-4 n ¼ 61 vs. control n ¼ 20, p < 0001). In contrast to TF, despite significant difference observed in relation to controls TFPI levels did not discriminate DENV-1/2 from DENV-infected patients and no difference was detected between DENV-1/2 and DENV-4 infecting serotypes. [(DENV-1/2 n ¼ 55 vs. control n ¼ 20, p < 0001) (DENV-4 n ¼ 59 vs. control n ¼ 20, p < 0001)] (Fig. 1CeD). In addition, the analysis of TF/TFPI ratio demonstrated that DENV-4 infected patients presented a low ratio compared to DENV-1/2 and HC [(DENV-1/2 n ¼ 38 vs. DENV-4 n ¼ 56, p < 0.001) (DENV-4 n ¼ 56 vs. control n ¼ 20, P < 0001)] (Fig 1F). 3.3. Relationship of TF and TFPI with laboratorial parameters of gravity TFPI and TF levels were directly associated in DENV patients, regardless infecting serotype (r ¼ 0.255, p < 0.009, n ¼ 104). Additionally, an inverse correlation between postinfection days and TF levels were observed (r ¼ 0.226, p ¼ 0.017, n ¼ 124) suggesting an attempt to control overcoagulation at infection onset. We found an inverse relationship between TF levels and monocytes counts in DENV-1/2 infected patients. TF levels were inversely related to platelet count in all patients, particularly in those infected with DENV1/2. Similarly, TFPI levels were inversely correlated with platelet count, especially in DENV-1/2 infected patients. A positive correlation was observed between TFPI levels and liver transaminases in all groups of patients, including those infected with DENV-1/2 or DENV-4, suggesting a strong involvement of TFPI in clinical worsening (Table 2). 3.4. Circulating levels of TF and TFPI according to haemorrhagic manifestations and vascular leakage Following analyses of circulating TF and TFPI levels, DENV-infected patients with or without haemorrhagic manifestations or vascular leakage were compared. Briefly, no difference was observed among those with vascular leakage (n ¼ 9) compared those without (n ¼ 111, not shown). However the levels of TF and TFPI were significantly higher among patients with haemorrhagic manifestations compared
Please cite this article in press as: Leal de Azeredo E, et al., Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.08.005
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4
(B)
(A) *
400
80000
350
TFPI (pg/ml)
200 100 80 60 40 20 0
***
40000
20000
C on tr ol
D
Se ve re
D F
ol C on tr
re Se ve
F/ W S D
D
F/ W S
0
F
Dengue
(C)
(D)
400 350
*
***
300
**
80000
***
***
TFPI (pg/ml)
60000
250 200 100 80 60 40 20 0
40000
20000
D
ol on tr
EN
V1/ EN D
C
2
ro l on t C
D
D
EN
EN V-
V1/
4
2
0
V4
TF (pg/ml)
***
60000
250
Dengue
TF (pg/ml)
**
*
300
Dengue
Dengue
(E)
(F) *
0.025
*
**
0.08
***
***
0.07
0.020
Dengue
C
on
V4 EN D
V1/ D
EN
tr ol
0.05 0.010 0.008 0.006 0.004 0.002 0.000
ol C on tr
re Se ve
F/ W S D
D
F
0.015 0.005 0.004 0.003 0.002 0.001 0.000
2
TF/TFPI
0.06
TF/TFPI
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
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Dengue
Fig. 1. Levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in DENV-infected patients and in controls. (AeB, E) TF, TFPI and TF/TFPI ratio in plasma from DENV-patients according to severity and controls, respectively. (CeD, F) TF, TFPI and TF/TFPI ratio in plasma from DENV-patients according infecting serotype and controls, respectively. The KruskaleWallis non-parametric test was used and p < 0.05 were taken as being significant for all statistical analysis. *P < 0.05, **P < 0.01***P < 0.001.
Please cite this article in press as: Leal de Azeredo E, et al., Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.08.005
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Table 2 Tissue Factor (TF)/Tissue Factor Inhibitor Pathway (TFPI) association with: monocytes and platelet counts in blood and with circulating levels of liver transaminases. Monocytes
Platelets
AST
ALT
TF DENV Patientsb
nsn¼98
nsn¼87
nsn¼87
TF DENV1/2c
nsn¼40
nsn¼40
TF DENV 4d TFPI DENV Patients
r ¼ 0.349n¼42 p < 0.020 nsn¼56 nsn¼93
TFPI DENV1/2
nsn¼40
TFPI DENV4
nsn¼53
r ¼ 0.255n¼112 p < 0.007 r ¼ 0.319n¼52 p < 0.02 nsn¼62 r ¼ 0.270n¼107 p < 0.005 r ¼ 0.352n¼49 p < 0.01 nsn¼58
nsn¼47 r ¼ 0.528n¼79 p < 0.0001 r ¼ 0.549n¼37 p < 0.0004 r ¼ 0.548n¼40 p < 0.0004
nsn¼47 r ¼ 0.534n¼79 p < 0.0001 r ¼ 0.601n¼37 p < 0.0001 r ¼ 0.517n¼40 p < 0.0006
a b c d
a
Spearman correlation with r and P values (P < 0.05 in bold, significant). Dengue patients regardless infecting serotype. Patients from 2010/2011 DENV1/2 outbreak. Patients from 2013 DENV-4 outbreak.
with those without [TF 38.71 (30.44e54.28, n ¼ 33) vs. 16.31 (9.449e36.77, n ¼ 94), p < 0001] and [TFPI 29095 (19,081e34,365, n ¼ 33) vs. 22,289 (17,189e28,731, n ¼ 85), p < 005], respectively. 4. Discussion Dengue has emerged as most healthy problem in Brazil. After multiple epidemics of dengue, all 4 serotypes are circulating in the country. One of the major goals of the various groups studying dengue worldwide is to understand mechanisms that could promote the early identification of patients with severe disease and improve diagnostic and therapeutic strategies, thus limiting morbidity and mortality. Comparative analysis of two distinct periods, DENV-1/2 2010 outbreak and DENV-4 2013 showed that DENV-4 outbreak was associated with mild cases. These data indicate that, surprisingly, the reintroduction of DENV-4 did not result in a higher incidence of severe cases. Similarly, Huy and colls found a milder clinical presentation associated with DENV-4, confirming the existence of differences in virulence between the serotypes [7]. DENV-2 and DENV-1 were associated with disease severity in different studies [8,9]. In fact, in Brazil, DENV-2 caused DHF during a DENV-2 outbreak that was preceded by a DENV-1 [10] and the reintroduction of a new lineage of DENV-2 in Brazil in 2008 [11] was responsible for the most severe epidemic described in the country. During infection by DENV, there is an imbalance in the hemostatic system via activation of coagulation and fibrinolysis. TF is constitutively expressed by perivascular cells, brain, heart, lung, kidney, and placenta, which initially ensures rapid activation of coagulation at site of vascular damage providing additional tissue-specific hemostatic protection to these vital organs [12]. Furthermore, TF has been associated with disease states, including cancer, liver injury, sepsis, and HIV infection [13,14]. Viral hemorrhagic fevers are associated with increased coagulation and platelets consumption especially in Ebola [15] and Argentine hemorrhagic fever [16].
Importantly, the blockage of TF in Ebola virus infected rhesus macaque increased the survival of animals and clearly confirmed the relationship of coagulation activation with pathogenesis of Ebola Hemorrhagic Fever [17]. The regulation of TF and TFPI in human dengue infection is not well understood and few studies have analysed these factors in dengue-infected patients. So, we quantified TF and TFPI proteins in dengue patients observing initially its severity degree and then, with infecting serotype. We found that patients with a good outcome showed decreased circulating levels of TF than those with a poor outcome (Severe). Similarly to TF, TFPI levels were significantly lower in patients with a good outcome, but increased TFPI plasma levels were observed in severe patients. We also demonstrated that TF and TFPI levels were significantly higher among patients with haemorrhagic manifestations. In addition, a direct correlation between TF and TFPI levels in dengue patients suggested that TF in plasma increases before TFPI. The TF/TFPI plasma ratio became low in all DENV patients with high levels in healthy donors. It is likely that the hypercoagulability was improved in some patients due to the increase in TFPI plasma levels. Previous studies found increased levels of TF in DHF/ DSS patients [18,19] in agreement with our findings. Accordingly, DENV 1/2 patients were more likely to have increased levels of TF than DENV-4 patients. In fact, endothelial cells infected with DENV-2 originated from DHF induced TF and PAR1 expression [20]. We previously described an increase in surface expression of TF on monocytes in severe cases compared with mild ones. Inverse correlation between platelet counts and the TFexpressing monocytes was found [21]. Interestingly, we observed an inverse correlation between circulating TF levels with platelet and monocytes counts in DENV1/2 infected patients confirming our previous data. Activated monocytes are observed during dengue infection and produce inflammatory cytokines involved in dengue severity. Since that vast majority of TF came from activated monocytes [22], our findings are consistent with the notion that activation of TF-
Please cite this article in press as: Leal de Azeredo E, et al., Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.08.005
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pathways is an important component of DENV-related coagulation disorders. Furthermore, increased activated monocyte frequency in HIV patients was positively associated with D dimer levels and monocytes expressing TF [14]. Hepatic manifestations are frequently described during dengue infection and are either a result of direct viral toxicity or deregulated immunologic injury in response to the virus. The spectrum of involvement includes asymptomatic elevation of hepatic transaminases to occurrence of severe manifestation in the form of acute liver failure [23]. No difference was found in AST and ALT levels between DF and DF/WS groups from DENV-4 outbreak, while, in the 2010 DENV-1/2, severe dengue patients (95 [70e221]) showed a significant increase in AST, but not ALT, compared with DF patients (42 [27e95]). In severe dengue, the occurrence of fulminant hepatic failure has been the cause of death of both children and adults [23]. In addition, TFPI levels were positively associated with AST and ALT serum levels. DENV associated immune activation is known to play a role in the DENV pathogenesis and might contributes to the pathogenesis of plasma leakage and organ dysfunction as well [3]. Importantly, Increased TF activation is observed in response to tissue damage and cytokine release [24]. The intricate relationship between inflammation and coagulation may have major consequences in the pathogenesis of dengue as a result of systemic inflammatory response. TPFI is an anticoagulant that acts inhibiting TF-VIIa complex and factor X, but also possess antiinflammatory properties related to the inhibition of thrombin formation [25]. The impact of TF pathway on liver dysfunction during DENV infection has not been extensively explored and several important issues need to be clarified. There are some limitations in this study. Our study is observational and the association between TF and TFPI levels with platelet function abnormalities, bleeding and plasma leakage do not necessarily imply causation. Platelets count and presence or absence of bleeding was used in the analysis. Although, additional coagulation parameters released from vascular endothelial cells were not tested (i.e., thrombomodulin, tissue type plasminogen activator, plasminogen activator inhibitor-I, and von Willebrand Factor), we describe for the first time significant differences between TF and TFPI levels in relation to clinical presentation and infecting serotype that are important for prevention of severe cases. Further, an improvement of dengue may depend in part, on increases in plasma TFPI, reflecting the condition of vascular endothelial cells more prone to hemorrhagic manifestations. If TF levels exceed a certain threshold, it will overwhelm the inhibitory regulation by TFPI, resulting in the propensity to thrombosis. Conflicts of interest None of the authors has conflicts of interest to report. Acknowledgements We acknowledge the patients and healthy controls for their voluntary participation. We thank Claudia Kamel for the
manuscript review. This investigation was supported by Brazilian institutions: PROEP/IOC and PAPES, FIOCRUZ; Q2 CNPQ; CAPES and FAPERJ. References [1] Owens 3rd AP, Mackman N. Tissue factor and thrombosis: the clot starts here. Thromb Haemost 2010;104:432e9. [2] Camerer E, Kolsto AB, Prydz H. Cell biology of tissue factor, the principal initiator of blood coagulation. Thromb Res 1996;81:1e41. [3] Srikiatkhachorn A, Green S. Markers of dengue disease severity. Curr Top Microbiol Immunol 2010;338:67e82. [4] de Azeredo EL, Monteiro RQ, de-Oliveira Pinto LM. Thrombocytopenia in dengue: interrelationship between virus and the imbalance between coagulation and fibrinolysis and inflammatory mediators. Mediat Inflamm 2015;2015:313842. [5] WHO/TDR. Dengue: guidelines for diagnosis, treatment, prevention and control e new edition. World Health Organization (WHO) and the Special Programme for Research and Training in Tropical Diseases (TDR); 2009. http://whqlibdoc.who.int/publications/2009/ 9789241547871_eng.pdf. [6] Cordeiro MT, Braga-Neto U, Nogueira RM, Marques Jr ET. Reliable classifier to differentiate primary and secondary acute dengue infection based on IgG ELISA. PloS One 2009;4:e4945. [7] Huy NT, Van Giang T, Thuy DH, Kikuchi M, Hien TT, Zamora J, et al. Factors associated with dengue shock syndrome: a systematic review and meta-analysis. PLoS Negl Trop Dis 2013;7:e2412. [8] Macedo GA, de Araujo JM, Schatzmayr HG, Costa FA, de Filippis AM, Santos FB, et al. Virological surveillance for early warning of dengue epidemics in the State of Rio de Janeiro, Brazil. Trans R Soc Trop Med Hyg 2013;107:141e6. [9] Thomas L, Najioullah F, Besnier F, Valentino R, Cesaire R, Rosine J, et al. Clinical presentation of dengue by serotype and year of epidemic in Martinique. Am J Trop Med Hyg 2014;91:138e45. [10] Zagne SM, Alves VG, Nogueira RM, Miagostovich MP, Lampe E, Tavares W. Dengue haemorrhagic fever in the state of Rio de Janeiro, Brazil: a study of 56 confirmed cases. Trans R Soc Trop Med Hyg 1994; 88:677e9. [11] Faria NR, Nogueira RM, de Filippis AM, Simoes JB, Nogueira Fde B, da Rocha Queiroz Lima M, et al. Twenty years of DENV-2 activity in Brazil: molecular characterization and phylogeny of strains isolated from 1990 to 2010. PLoS Negl Trop Dis 2013;7:e2095. [12] Tilley R, Mackman N. Tissue factor in hemostasis and thrombosis. Semin Thromb Hemost 2006;32:5e10. [13] Langer F, Spath B, Haubold K, Holstein K, Marx G, Wierecky J, et al. Tissue factor procoagulant activity of plasma microparticles in patients with cancer-associated disseminated intravascular coagulation. Ann Hematol 2008;87:451e7. [14] Funderburg NT, Mayne E, Sieg SF, Asaad R, Jiang W, Kalinowska M, et al. Increased tissue factor expression on circulating monocytes in chronic HIV infection: relationship to in vivo coagulation and immune activation. Blood 2010;115:161e7. [15] Chen JP, Cosgriff TM. Hemorrhagic fever virus-induced changes in hemostasis and vascular biology. Blood Coagul Fibrinolysis 2000;11: 461e83. [16] Heller MV, Marta RF, Sturk A, Maiztegui JI, Hack CE, Cate JW, et al. Early markers of blood coagulation and fibrinolysis activation in Argentine hemorrhagic fever. Thromb Haemost 1995;73: 368e73. [17] Geisbert TW, Hensley LE, Jahrling PB, Larsen T, Geisbert JB, Paragas J, et al. Treatment of Ebola virus infection with a recombinant inhibitor of factor VIIa/tissue factor: a study in rhesus monkeys. Lancet 2003;362: 1953e8. [18] Wills BA, Oragui EE, Stephens AC, Daramola OA, Dung NM, Loan HT, et al. Coagulation abnormalities in dengue hemorrhagic fever: serial investigations in 167 Vietnamese children with dengue shock syndrome. Clin Infect Dis 2002;35:277e85.
Please cite this article in press as: Leal de Azeredo E, et al., Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.08.005
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[19] Sosothikul D, Seksarn P, Pongsewalak S, Thisyakorn U, Lusher J. Activation of endothelial cells, coagulation and fibrinolysis in children with dengue virus infection. Thromb Haemost 2007;97:627e34. [20] Huerta-Zepeda A, Cabello-Gutierrez C, Cime-Castillo J, MonroyMartinez V, Manjarrez-Zavala ME, Gutierrez-Rodriguez M, et al. Crosstalk between coagulation and inflammation during dengue virus infection. Thromb Haemost 2008;99:936e43. [21] de Azeredo EL, Kubelka CF, Alburquerque LM, Barbosa LS, Damasco PV, Avila CA, et al. Tissue factor expression on monocytes from patients with severe dengue fever. Blood Cells Mol Dis 2010;45: 334e5.
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[22] Osterud B, Bjorklid E. Sources of tissue factor. Semin Thromb Hemost 2006;32:11e23. [23] Samanta J, Sharma V. Dengue and its effects on liver. World J Clin Cases 2015;3:125e31. [24] Baker JV. Chronic HIV disease and activation of the coagulation system. Thromb Res 2013;132:495e9. [25] Kasthuri RS, Glover SL, Boles J, Mackman N. Tissue factor and tissue factor pathway inhibitor as key regulators of global hemostasis: measurement of their levels in coagulation assays. Semin Thromb Hemost 2010;36:764e71.
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Microbes and Infection xx (2016) 1e7 www.elsevier.com/locate/micinf
Short communication
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Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection Elzinandes Leal de Azeredo a,*, Victor Edgar Fiestas Sol orzano a, Debora Batista de Oliveira a, Cintia Ferreira Marinho a, Luiz Jose de Souza b, Rivaldo Ven^ancio da Cunha c, Paulo Vieira Damasco d, Claire Fernandes Kubelka a, Luzia Maria de-Oliveira-Pinto a a
Q1
Laboratory of Viral Immunology, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil b Referral Centre for Dengue, Campos de Goytacazes, Brazil c Department of Clinical Medicine, Universidade Federal do Mato Grosso do Sul, Campo Grande, Brazil d Rio-Laranjeiras Hospital, Gaffree Guinle University Hospital (The Federal University of Rio de Janeiro) and Pedro Ernesto University Hospital (The State University of Rio de Janeiro), Rio de Janeiro, Brazil Received 6 April 2016; accepted 22 August 2016 Available online ▪ ▪ ▪
Abstract Tissue Factor (TF) is the initiator of coagulation and Tissue Factor Inhibitor (TFPI) is the physiological inhibitor of the TF/FVIIa complex. Circulating levels of TF and TFPI were quantified in dengue patients and the relationships with disease severity and infecting serotype analysed. A significant decrease in TF and TPFI plasma levels was observed in mild DF patients compared with severe dengue. Furthermore, both factors were associated with haemorrhagic manifestations. Finally, TF levels were significantly increased in DENV-1/2 infected patients as compared with DENV-4. These findings suggest that activation of TF-pathway is an important component of DENV -related coagulation disorders. © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: TFPI; TF; Dengue
1. Introduction Blood extrinsic coagulation pathway is initiated by exposure to transmembrane glycoprotein Tissue Factor (TF), that functions as the cofactor for plasma FVIIa [1] leading thrombin generation, fibrin deposition and in conjunction with platelets a formation of a hemostatic plug. Tissue Factor Pathway Inhibitor (TFPI) regulates coagulation by inhibiting the catalytic activity of the TF/VIIa complex [2]. Dengue viruses (DENV) are members of the Flaviviridae family with four distinct serotypes designated DENV-1, DENV-2, DENV-3 and DENV-4. The clinical manifestation of dengue ranges from mild dengue fever to a severe life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). DHF/ * Corresponding author. E-mail address: [email protected] (E. Leal de Azeredo).
DSS is characterized by spontaneous bleeding and vascular leakage that can progress to hypovolemic shock and death [3]. Thrombocytopenia and coagulopathy are among the hematologic abnormalities characteristic of the severe forms of dengue [4]. Despite efforts and increasing data, the mechanisms involved in coagulopathy and hemorrhages are not well understood. Given the essential roles of TF and TFPI in the hemostasis, we measured circulating levels of these proteins in 148 DENV infected patients and the relationship with clinical manifestations and infecting serotype were analysed. 2. Materials and methods 2.1. Study population In 2010, 2011 and 2013, we evaluated 148 patients suspected of DENV infection at the Brazilian hospitals. Samples
http://dx.doi.org/10.1016/j.micinf.2016.08.005 1286-4579/© 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Leal de Azeredo E, et al., Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.08.005
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were collected during the acute phase of infection (1e15 days after disease onset). Cases of dengue were classified, according to the World Health Organization (WHO) criteria established in 2009, as DF (n ¼ 90); DF with warning signs (DF/ WS, n ¼ 41); or severe dengue (n ¼ 17). DF included cases in which the diagnosis were confirmed by laboratory tests and patients presented symptoms such as fever, headache, retroorbital pain, myalgia, arthralgia, rash, nausea, vomiting, positive tourniquet test, leukopenia and no warning signs. In addition to these symptoms, the patients classified into DF/WS presented abdominal pain or tenderness; persistent vomiting; clinically relevant fluid accumulation; mucosal bleeding; lethargy; restlessness; liver enlargement (>2 cm); and an increase in haematocrit concomitant with a rapid decrease in platelet count. Severe dengue included cases characterised by severe vascular leakage; fluid accumulation with respiratory distress; severe haemorrhage, such as massive vaginal bleeding (in women of childbearing age); gastrointestinal bleeding; and organ impairment, such as liver damage (aspartate aminotransferase or alanine aminotransferase > 1000 U/L); involvement of central nervous system (drop in the level of consciousness); as well as other organs, such as the heart [5]. DENV infection was confirmed by detection of antidengue IgM with capture enzyme-linked immunosorbent assay (ELISA; PanBio, Brisbane, Australia); detection of viral protein NS1 (Bio-Rad, Hercules, CA, USA); and detection of viral RNA by reverse-transcriptase polymerase chain reaction.
The pattern of primary or secondary response from dengue positive patients was determined based on the set of specific IgM tests, NS1 and or RT-PCR, specific IgG (EL1500G Focus Diagnostics, Cypress, CA, USA) as described previously [6]. Demographical, clinical and biochemical data related to the patients are shown. Fourteen healthy controls (HC) were included in the study. Table 1. The study was approved by the Ethics Committee of Plataforma Brasil, FIOCRUZ (CAAE 13318113.7.0000.5248). 2.2. Blood samples related to hemostasis determinations Blood samples were collected in blood collection tubes and kept on ice. Plasma was collected and stored at 70 C. Circulating levels of TF and TFPI from dengue patients and HC were tested for Human Coagulation Factor III/Tissue Factor (cat. DCF300) and TFPI (DTFP10) Quantikines by ELISAs (R&D Systems) according to the manufacturer's instructions. 2.3. Statistical analysis Statistical analyses were performed using GraphPad Prism software, version 6.0 (GraphPad Software Inc., San Diego, CA, USA). The non-parametric KruskaleWallis and ManneWhitney U tests were used to compare the differences between study groups (HC, DF, DF/WS and severe dengue). Correlation was estimated by Spearman regression analysis. Values of p < 0.05 were considered statistically significant.
Table 1 Demographic characteristics of the study population and clinical-laboratorial course of patients with dengue. Characteristic
Age; years Female sex; n (%) Post-infection daysa Clinical sign and symptoms Bleeding manifestations at enrolment,b n (%) Plasma leakage,c n (%) Laboratory test results DENV NS1 glycoprotein, n (%) anti-DENV IgM, n (%) anti-DENV IgG, n (%) serotype-specific by RT-PCR, n (%) Leukocytes count/mm3 Monocytes count/mm3 Lymphocytes count/mm3 AST IU/L ALT IU/L Platelets 103 count/mm3
Q3
Patients
Health controls (n ¼ 14)
DF
DF/WS
Severe DF
(n ¼ 90)
(n ¼ 41)
(n ¼ 17)
42 (26e54) 48 (53) 4 (2e6)
42 (25e55) 24 (59) 5 (3e7)
39 (23e54) 9 (53) 4 (3e6)
30 (26e31) 10 (71) e
9 (10) 0 (0)
20 (49) 8 (3)
12 (63) 8 (50)
e e
33 (75) 47 (57) 60 (76) 31 (57) 3400 (2700e4975)** 393 (294e644) 1128 (834e1614) 42 (29e82) 50 (32e80)¥ 149 (111e213)**, ¥¥¥
17 (94) 36 (90) 40 (98) 11 (39) 3200 (2200e3900)### 312 (174e421) 1229 (861e1819)$ 81 (41e232)$$ 83 (41e198) 77 (35e131)###
4 (57) 17 (100) 14 (100) 1 (33) 3690 (2820e4833)& 552 (260e741) 2193 (1226e3367)p p 97 (72e228) p 116 (66e215) 24 (14e53)&&&, ppp
e e e e 5800 (5292e6173) 480 (318e500) 1704 (1585e2146) e e 280 (242e332)
DF, dengue fever; DF/WS, DF with warning signs; AST, aspartate aminotransferase; ALT, alanine aminotransferase; nd, not determined. Data are presented as medians (interquartile range) or numbers (n) with percentage (%). ManneWhitney U tests were applied. A p-value <0.05 was considered significant and was indicated as *** (p < 0.001); **(p < 0.01); or *(p < 0.05). *DF vs Healthy Control; #DF/WS vs Healthy Control; &Severe DF vs Healthy Q4 Control; ¥ DF vs DF/WS; pDF vs Severe DF; $DF/WS vs Severe DF. a Days from disease onset until the interview. b Includes skin haemorrhages, epistaxis, gingival bleeding, gastrointestinal bleeding, urinary tract haemorrhage or metrorrhagia. c Signs of vascular leakage (pleural or pericardial effusion, ascites). Please cite this article in press as: Leal de Azeredo E, et al., Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.08.005
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3. Results 3.1. Patient characteristics Demographical, clinical and laboratorial data related to the patients enrolled in this study are shown in Table 1. Clinical manifestations of DF include fever, headache, retro-orbital pain, muscle pain, arthralgia, and nausea, vomiting and rash. Apart from petechiae, hemorrhagic manifestations were not common (10%). The following warning symptoms and signs were presented by DF/WS patients: severe abdominal pain, severe nausea and vomiting, some bleeding episodes as skin haemorrhages, epistaxis, gingival bleeding, gastrointestinal bleeding, urinary tract haemorrhage or metrorrhagia. Only 3% of DF/WS patients presented signs of vascular leakage as pleural or pericardial effusion, ascites. Among severe dengue cases, we documented serious manifestations, such as epistaxis, gingival bleeding, urinary tract haemorrhage or metrorrhagia (63%), vascular leakage and hypotension (50%). Among the serotypes identified, 14% were DENV-1, 28% were DENV-2 and 58% were DENV-4. As the majority of patients (85%) presented secondary infections, no correlation was observed between patients with primary or secondary infection with patients presenting neither clinical manifestations nor laboratory parameters. In relation to haematological parameters, the total leukocyte counts were lower in all DENV positive patients, regardless of disease severity when compared with HC. Lymphocyte counts were increased in severe dengue patients compared with DF, DF/WS patients and HC. No differences were observed in monocyte counts between groups of study. Severe dengue patients and DF/WS had low platelets counts as compared with both DF and HC. Levels of liver enzyme AST were significantly higher in severe cases compared with DF or DF/WS cases (Table 1). As a proposal for analysis, we compared patients from 2010/2011 (DENV-1 and DENV-2) outbreaks with those from 2013 (DENV-4) outbreak. In DENV-1/2 outbreak, laboratory findings indicated low lymphocyte count (1465 [1108e2420], median [25%e75% interquartile]) and low platelet count (85 [36e149]) when compared to HD. Moreover, high frequencies of hemorrhagic manifestations and/or plasma extravasation were noted in these patients (36 out 74, 48.6%). In comparison, patients of the 2013 DENV-4 outbreak showed an even lower frequency of lymphocyte count (1111 [802e1568], p ¼ 0.0023) but a higher platelet count (136.5 [95.8e192.3], p ¼ 0.0007) with less frequent bleeding and/or plasma leakage (8 out 72, 11.1%). 3.2. Circulating levels of TF, TFPI and TF/TFPI ratio in relation to dengue severity and DENV infecting serotype High circulating levels of TF were detected in severe dengue patients compared with DF fever patients (DF n ¼ 83 vs. severe n ¼ 14, p < 0.05), discriminating severe from mild patients (Fig. 1A). TFPI levels were higher in plasma from DF, DF/WS or Severe compared to HC and similar to TF levels, it
3
also discriminate severe from mild patients [(DF n ¼ 73 vs. Severe n ¼ 13, p < 0.05) (DF n ¼ 73 vs. control n ¼ 20, p < 0.01) (DF/WS n ¼ 32 vs. control n ¼ 20, p < 0.001) (Severe n ¼ 13 vs. control n ¼ 20, p < 0.001)] (Fig. 1B). DENV-infected patients showed a decreased levels TF/ TFPI ratio as compared to controls, and decreased levels were statically significant in DF or DF/WS patients versus HC [(DF n ¼ 67 vs. control n ¼ 20, p < 005) (DF/WS n ¼ 26 vs. control n ¼ 20 p < 0.01**)] (Fig 1E). The analysis of these mediators by the prevalent serotype showed that DENV-1/2 infected patients presented high levels of TF compared with DENV-4 infected patients [(DENV-1/2 n ¼ 55 vs. control n ¼ 20, p < 0.01)(DENV-1/2 n ¼ 55 vs. DENV-4 n ¼ 59, p < 0001)(DENV-4 n ¼ 59 vs. control ¼ 20, p < 001)]. Interestingly, DENV-4 infected patients showed decreased TF levels than HC (DENV-4 n ¼ 61 vs. control n ¼ 20, p < 0001). In contrast to TF, despite significant difference observed in relation to controls TFPI levels did not discriminate DENV-1/2 from DENV-infected patients and no difference was detected between DENV-1/2 and DENV-4 infecting serotypes. [(DENV-1/2 n ¼ 55 vs. control n ¼ 20, p < 0001) (DENV-4 n ¼ 59 vs. control n ¼ 20, p < 0001)] (Fig. 1CeD). In addition, the analysis of TF/TFPI ratio demonstrated that DENV-4 infected patients presented a low ratio compared to DENV-1/2 and HC [(DENV-1/2 n ¼ 38 vs. DENV-4 n ¼ 56, p < 0.001) (DENV-4 n ¼ 56 vs. control n ¼ 20, P < 0001)] (Fig 1F). 3.3. Relationship of TF and TFPI with laboratorial parameters of gravity TFPI and TF levels were directly associated in DENV patients, regardless infecting serotype (r ¼ 0.255, p < 0.009, n ¼ 104). Additionally, an inverse correlation between postinfection days and TF levels were observed (r ¼ 0.226, p ¼ 0.017, n ¼ 124) suggesting an attempt to control overcoagulation at infection onset. We found an inverse relationship between TF levels and monocytes counts in DENV-1/2 infected patients. TF levels were inversely related to platelet count in all patients, particularly in those infected with DENV1/2. Similarly, TFPI levels were inversely correlated with platelet count, especially in DENV-1/2 infected patients. A positive correlation was observed between TFPI levels and liver transaminases in all groups of patients, including those infected with DENV-1/2 or DENV-4, suggesting a strong involvement of TFPI in clinical worsening (Table 2). 3.4. Circulating levels of TF and TFPI according to haemorrhagic manifestations and vascular leakage Following analyses of circulating TF and TFPI levels, DENV-infected patients with or without haemorrhagic manifestations or vascular leakage were compared. Briefly, no difference was observed among those with vascular leakage (n ¼ 9) compared those without (n ¼ 111, not shown). However the levels of TF and TFPI were significantly higher among patients with haemorrhagic manifestations compared
Please cite this article in press as: Leal de Azeredo E, et al., Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.08.005
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4
(B)
(A) *
400
80000
350
TFPI (pg/ml)
200 100 80 60 40 20 0
***
40000
20000
C on tr ol
D
Se ve re
D F
ol C on tr
re Se ve
F/ W S D
D
F/ W S
0
F
Dengue
(C)
(D)
400 350
*
***
300
**
80000
***
***
TFPI (pg/ml)
60000
250 200 100 80 60 40 20 0
40000
20000
D
ol on tr
EN
V1/ EN D
C
2
ro l on t C
D
D
EN
EN V-
V1/
4
2
0
V4
TF (pg/ml)
***
60000
250
Dengue
TF (pg/ml)
**
*
300
Dengue
Dengue
(E)
(F) *
0.025
*
**
0.08
***
***
0.07
0.020
Dengue
C
on
V4 EN D
V1/ D
EN
tr ol
0.05 0.010 0.008 0.006 0.004 0.002 0.000
ol C on tr
re Se ve
F/ W S D
D
F
0.015 0.005 0.004 0.003 0.002 0.001 0.000
2
TF/TFPI
0.06
TF/TFPI
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
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Dengue
Fig. 1. Levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in DENV-infected patients and in controls. (AeB, E) TF, TFPI and TF/TFPI ratio in plasma from DENV-patients according to severity and controls, respectively. (CeD, F) TF, TFPI and TF/TFPI ratio in plasma from DENV-patients according infecting serotype and controls, respectively. The KruskaleWallis non-parametric test was used and p < 0.05 were taken as being significant for all statistical analysis. *P < 0.05, **P < 0.01***P < 0.001.
Please cite this article in press as: Leal de Azeredo E, et al., Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.08.005
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Table 2 Tissue Factor (TF)/Tissue Factor Inhibitor Pathway (TFPI) association with: monocytes and platelet counts in blood and with circulating levels of liver transaminases. Monocytes
Platelets
AST
ALT
TF DENV Patientsb
nsn¼98
nsn¼87
nsn¼87
TF DENV1/2c
nsn¼40
nsn¼40
TF DENV 4d TFPI DENV Patients
r ¼ 0.349n¼42 p < 0.020 nsn¼56 nsn¼93
TFPI DENV1/2
nsn¼40
TFPI DENV4
nsn¼53
r ¼ 0.255n¼112 p < 0.007 r ¼ 0.319n¼52 p < 0.02 nsn¼62 r ¼ 0.270n¼107 p < 0.005 r ¼ 0.352n¼49 p < 0.01 nsn¼58
nsn¼47 r ¼ 0.528n¼79 p < 0.0001 r ¼ 0.549n¼37 p < 0.0004 r ¼ 0.548n¼40 p < 0.0004
nsn¼47 r ¼ 0.534n¼79 p < 0.0001 r ¼ 0.601n¼37 p < 0.0001 r ¼ 0.517n¼40 p < 0.0006
a b c d
a
Spearman correlation with r and P values (P < 0.05 in bold, significant). Dengue patients regardless infecting serotype. Patients from 2010/2011 DENV1/2 outbreak. Patients from 2013 DENV-4 outbreak.
with those without [TF 38.71 (30.44e54.28, n ¼ 33) vs. 16.31 (9.449e36.77, n ¼ 94), p < 0001] and [TFPI 29095 (19,081e34,365, n ¼ 33) vs. 22,289 (17,189e28,731, n ¼ 85), p < 005], respectively. 4. Discussion Dengue has emerged as most healthy problem in Brazil. After multiple epidemics of dengue, all 4 serotypes are circulating in the country. One of the major goals of the various groups studying dengue worldwide is to understand mechanisms that could promote the early identification of patients with severe disease and improve diagnostic and therapeutic strategies, thus limiting morbidity and mortality. Comparative analysis of two distinct periods, DENV-1/2 2010 outbreak and DENV-4 2013 showed that DENV-4 outbreak was associated with mild cases. These data indicate that, surprisingly, the reintroduction of DENV-4 did not result in a higher incidence of severe cases. Similarly, Huy and colls found a milder clinical presentation associated with DENV-4, confirming the existence of differences in virulence between the serotypes [7]. DENV-2 and DENV-1 were associated with disease severity in different studies [8,9]. In fact, in Brazil, DENV-2 caused DHF during a DENV-2 outbreak that was preceded by a DENV-1 [10] and the reintroduction of a new lineage of DENV-2 in Brazil in 2008 [11] was responsible for the most severe epidemic described in the country. During infection by DENV, there is an imbalance in the hemostatic system via activation of coagulation and fibrinolysis. TF is constitutively expressed by perivascular cells, brain, heart, lung, kidney, and placenta, which initially ensures rapid activation of coagulation at site of vascular damage providing additional tissue-specific hemostatic protection to these vital organs [12]. Furthermore, TF has been associated with disease states, including cancer, liver injury, sepsis, and HIV infection [13,14]. Viral hemorrhagic fevers are associated with increased coagulation and platelets consumption especially in Ebola [15] and Argentine hemorrhagic fever [16].
Importantly, the blockage of TF in Ebola virus infected rhesus macaque increased the survival of animals and clearly confirmed the relationship of coagulation activation with pathogenesis of Ebola Hemorrhagic Fever [17]. The regulation of TF and TFPI in human dengue infection is not well understood and few studies have analysed these factors in dengue-infected patients. So, we quantified TF and TFPI proteins in dengue patients observing initially its severity degree and then, with infecting serotype. We found that patients with a good outcome showed decreased circulating levels of TF than those with a poor outcome (Severe). Similarly to TF, TFPI levels were significantly lower in patients with a good outcome, but increased TFPI plasma levels were observed in severe patients. We also demonstrated that TF and TFPI levels were significantly higher among patients with haemorrhagic manifestations. In addition, a direct correlation between TF and TFPI levels in dengue patients suggested that TF in plasma increases before TFPI. The TF/TFPI plasma ratio became low in all DENV patients with high levels in healthy donors. It is likely that the hypercoagulability was improved in some patients due to the increase in TFPI plasma levels. Previous studies found increased levels of TF in DHF/ DSS patients [18,19] in agreement with our findings. Accordingly, DENV 1/2 patients were more likely to have increased levels of TF than DENV-4 patients. In fact, endothelial cells infected with DENV-2 originated from DHF induced TF and PAR1 expression [20]. We previously described an increase in surface expression of TF on monocytes in severe cases compared with mild ones. Inverse correlation between platelet counts and the TFexpressing monocytes was found [21]. Interestingly, we observed an inverse correlation between circulating TF levels with platelet and monocytes counts in DENV1/2 infected patients confirming our previous data. Activated monocytes are observed during dengue infection and produce inflammatory cytokines involved in dengue severity. Since that vast majority of TF came from activated monocytes [22], our findings are consistent with the notion that activation of TF-
Please cite this article in press as: Leal de Azeredo E, et al., Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.08.005
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pathways is an important component of DENV-related coagulation disorders. Furthermore, increased activated monocyte frequency in HIV patients was positively associated with D dimer levels and monocytes expressing TF [14]. Hepatic manifestations are frequently described during dengue infection and are either a result of direct viral toxicity or deregulated immunologic injury in response to the virus. The spectrum of involvement includes asymptomatic elevation of hepatic transaminases to occurrence of severe manifestation in the form of acute liver failure [23]. No difference was found in AST and ALT levels between DF and DF/WS groups from DENV-4 outbreak, while, in the 2010 DENV-1/2, severe dengue patients (95 [70e221]) showed a significant increase in AST, but not ALT, compared with DF patients (42 [27e95]). In severe dengue, the occurrence of fulminant hepatic failure has been the cause of death of both children and adults [23]. In addition, TFPI levels were positively associated with AST and ALT serum levels. DENV associated immune activation is known to play a role in the DENV pathogenesis and might contributes to the pathogenesis of plasma leakage and organ dysfunction as well [3]. Importantly, Increased TF activation is observed in response to tissue damage and cytokine release [24]. The intricate relationship between inflammation and coagulation may have major consequences in the pathogenesis of dengue as a result of systemic inflammatory response. TPFI is an anticoagulant that acts inhibiting TF-VIIa complex and factor X, but also possess antiinflammatory properties related to the inhibition of thrombin formation [25]. The impact of TF pathway on liver dysfunction during DENV infection has not been extensively explored and several important issues need to be clarified. There are some limitations in this study. Our study is observational and the association between TF and TFPI levels with platelet function abnormalities, bleeding and plasma leakage do not necessarily imply causation. Platelets count and presence or absence of bleeding was used in the analysis. Although, additional coagulation parameters released from vascular endothelial cells were not tested (i.e., thrombomodulin, tissue type plasminogen activator, plasminogen activator inhibitor-I, and von Willebrand Factor), we describe for the first time significant differences between TF and TFPI levels in relation to clinical presentation and infecting serotype that are important for prevention of severe cases. Further, an improvement of dengue may depend in part, on increases in plasma TFPI, reflecting the condition of vascular endothelial cells more prone to hemorrhagic manifestations. If TF levels exceed a certain threshold, it will overwhelm the inhibitory regulation by TFPI, resulting in the propensity to thrombosis. Conflicts of interest None of the authors has conflicts of interest to report. Acknowledgements We acknowledge the patients and healthy controls for their voluntary participation. We thank Claudia Kamel for the
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