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. Author manuscript; available in PMC: 2014 Apr 1.
Published in final edited form as: J Thromb Haemost. 2013 May;11(5):951–962. doi: 10.1111/jth.12178

Dengue Induces Platelet Activation, Mitochondrial Dysfunction and Cell Death through Mechanisms that Involve DC-SIGN and Caspases

Eugenio D Hottz 1,2, Marcus F Oliveira 4, Priscila C G Nunes 3, Rita Maria R Nogueira 3, Rogério Valls-de-Souza 2, Andréa T Da Poian 4, Andrew S Weyrich 5,6, Guy A Zimmerman 6, Patricia T Bozza 1,*, Fernando A Bozza 2,*
PMCID: PMC3971842  NIHMSID: NIHMS566793  PMID: 23433144

Abstract

Background

Dengue is the most prevalent human arbovirus disease in the world. Dengue infection may cause a range of clinical manifestation from self-limiting febrile illness through life-threatening syndrome accompanied by bleeding and shock. Thrombocytopenia is frequently observed in mild and severe disease, however the mechanisms involved in DENV-induced platelet activation and thrombocytopenia are incompletely understood.

Patients/ Methods

Freshly-isolated platelets from patients with dengue were evaluated for markers of activation, mitochondrial alterations and activation of cell death pathways. In parallel, we determined whether DENV induced direct activation and apoptosis of platelets that were obtained from healthy subjects.

Results

We found that platelets from DENV-infected patients display increased activation when compared to control subjects. Moreover, platelets from DENV-infected patients exhibited classic signs of the intrinsic pathway of apoptosis that include increased surface phosphatidylserine exposure, mitochondrial depolarization and activation of caspase-9 and 3. Indeed, thrombocytopenia was shown to strongly associate with enhanced platelet activation and cell death in DENV-infected patients. Platelet activation, mitochondrial dysfunction and caspase-dependent phosphatidylserine exposure on platelets were also observed when platelets from healthy subjects were directly exposed to DENV in vitro. DENV-induced platelet activation was shown to occur through mechanisms largely dependent of DC-SIGN.

Conclusions

Together our results demonstrate that platelets from patients with dengue present signs of activation, mitochondrial dysfunction, and activation of apoptosis caspase cascade, which may contribute to the genesis of thrombocytopenia in patients with dengue. Our results also suggest the involvement of DC-SIGN as a critical receptor in DENV-dependent platelet activation.

Keywords: apoptosis, DC-SIGN, dengue, mitochondrial dysfunction, platelet activation, thrombocytopenia

Introduction

Dengue is an infectious disease caused by the dengue virus (DENV). Over 2.5 billion people live in high-risk transmission areas and dengue has emerged as a problem in the Southern United States [1]. It is estimated that over 50 million individuals are annually exposed to DENV [2]. The natural history of dengue is a self-limiting fever followed by a critical phase of defervescence, in which the patient may improve or progress to severe dengue that is associated with life-threatening increases in vascular permeability, hypovolemia, hypotension, and shock [2, 3]. Thrombocytopenia is commonly observed in both mild and severe dengue syndromes and correlates with clinical outcome [2–6].

The potential mechanisms inciting DENV-associated thrombocytopenia were recently reviewed [7]. These include impaired thrombopoiesis [8] and/or peripheral platelet destruction. The latter may involve antibody-induced platelet clearance [9], enhanced interactions of platelets with leukocytes or endothelium [10, 11], and activation of platelets as they contact the DENV [12, 13]. Recent studies suggest that DENV is able to interact with platelets inducing ultrastructural changes [12]. Platelets also become activated when they are exposed to DENV-infected endothelial cells [10] and increased numbers of platelet-monocyte aggregates have been observed in dengue patients [11]. These studies indicate that platelets are activated during dengue illness, but the mechanisms underlying this process and its clinical consequences remain unknown.

Mitochondria are known to regulate apoptotic pathways in activated platelets [14–16]. Therefore, we examined activities of platelets in patients with dengue, focusing on how dengue influences mitochondrial function and apoptosis. We found that platelet activation is significantly increased in dengue-infected patients, especially in individuals with thrombocytopenia. Platelets from patients with dengue also have impaired mitochondrial function and activation of apoptosis pathways. Consistent with these findings, in vitro DENV infection induces similar responses of activation, mitochondrial dysfunction and apoptosis through mechanisms involving DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin). Our results suggest that DENV-induced platelet activation and apoptosis occurs though DC-SIGN dependent mechanisms and may influence the development of thrombocytopenia in dengue.

Material and Methods

Human Subjects

We prospectively followed a cohort of 39 serologically/molecularly-confirmed DENV-infected patients from the Instituto Pesquisas Clínicas Evandro Chagas, FIOCRUZ, Rio de Janeiro, Brazil, whose characteristics are presented in Table 1. Peripheral vein blood samples were obtained at febrile (n=26), defervescence (n=26), and convalescence (n=13) phases of infection. The mean day of sample collection after onset of illness was 4.5±1.6 in febrile, 7.5±2.4 in defervescence and 25.3±12.9 in convalescence. Primary and secondary infections were distinguished using IgM/IgG antibody ratio as previously described [2, 17]. For virus typing and quantification, viral RNA was extracted (QIAamp Viral RNA, Quiagen) from plasma samples and processed as previously described [18, 19].

Table 1.

Characteristics of dengue-infected patients:

Control (22) Dengue (39)
Age, years 27 (26–35) 37 (26–46)
Gender, male 12 (40 %) 22 (56.4%)
Platelet count, × 1,000/mm3 – 107 (49–145)
Hematocrit, % – 43.5 (40.7–45.3)
Albumin, g/dL – 3.6 (3.1–3.9)
TGO/AST, IU/L – 66 (35–115)
TGP/ALT, IU/L – 69 (46–102)
Hemorrhagic manifestations1 – 11 (28.2%)
Venous hydration – 16 (41%)
Secondary infection – 24 (61%)
Mild Dengue – 20 (51.3%)
Mild Dengue with – 15 (38.5%)
Warning Signs2
Severe Dengue3 – 4 (10,2%)
PCR positive – 29
  DENV-1 – 27 (93%)
  DENV-2 – 02 (7%)
Viremia, × 105 copies/mL – 1.5±3.4
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Data are expressed as median (interquartile range) or number (%).

1

Gingival bleed, vaginal bleed, gastrointestinal bleed, petechiae and exanthema.

2

Abdominal pain or tenderness, persistent vomiting, clinical fluid accumulation, mucosal bleed, and/or increased hematocrit concurrent with rapid decrease in platelet count.

3

Severe plasma leakage, fluid accumulation, ascites, and/or massive bleeding.

2,3

According to WHO guidelines [2].

Peripheral vein blood was also collected from 30 aged-matched healthy subjects and 13 patients with non-dengue febrile illness (NDFI). The disease etiologies of the NDFI patients are listed in Table S1. Written informed consent was obtained from all participants prior to any study-related procedure. The study protocol was approved by the Institutional Review Board (IPEC#016/2010).

Platelet Isolation

Platelets were isolated as previously described [20] and re-suspended in medium 199 at 37°C. The purity of the platelet preparations (greater than 99% of CD41+ events) was confirmed by flow cytometry.

Flow Cytometric Analysis

Platelets were re-suspended in modified Tyrode’s (137 mM NaCl, 2.68 mM KCI, 5 mM HEPES, 1 mM MgCl2, 11.9 mM NaHCO3, 0.42 mM NaH2PO4, 4.7 mM glucose; pH 7.4). P-selectin (CD62-P) surface expression was determined using PE- or FITC-conjugated anti-human CD62P (BD Pharmingen) (0.25 μg/mL, 30 min); DC-SIGN surface expression was determined by incubating platelets with biotin-conjugated antibody against DC-SIGN (eBioscience eB-h209) (1.5 μg/mL, 20 min) and PECy5-conjugated streptavidin (0.2 μg/mL, 20 min); mitochondrial membrane potential (ΔΨm) was measured using the probe tetramethylrhodamine ethyl ester (TMRE, Fluka Analytical) (100 nM, 10 min), mitochondrial-derived reactive oxygen species (ROSm) were detected using MitoSoxRed (Invitrogen) (2.5 μM, 10 min); active caspase-9 was determined using green FAM-LEDH-FMK, FLICA (Immunochemistry Technologies); and phosphatidylserine exposure was stained with FITC-conjugated Annexin V (BD Pharminogen). A minimum of 10,000 gated events were acquired using a FACScalibur flow cytometer (BD Bioscience).

Assessment of mitochondrial function

TMRE and MitoSoxRed were used to labeling ΔΨm and ROSm, respectively. To control the mitochondrial specificity of the probes platelets were treated with the proton ionophore FCCP (0.5 µM) 15min before labeling. To assess the mitochondrial permeability, platelets were treated with the F1F0-ATP-synthase inhibitor Oligomycin (1 µg/mL) 15 min before ΔΨm labeling. Mitochondrial content was determined measuring citrate-synthase activity in platelet protein extracts (10 μg) by 412 nm spectrophotometric detection of CoA-TNB2, which formed with citrate as DTNB, acetyl-CoA and oxaloacetate interacted with one another.

Western blotting

Western blotting was performed as previously described [21]. The primary antibodies used for this work were: mouse anti-human caspase-9 and rabbit anti-human caspase-3 (Cell Signaling Technology, USA).

Virus Preparation

DENV serotype 2 strain 16881 was propagated in C6/36 Aedes albopictus mosquito cells and titrated by plaque assay on BHK cells [22]. The amount of infectious particles was expressed as plaque forming units (PFU)/mL. Supernatants from uninfected cell cultures (mock) were produced using the same conditions.

In vitro platelet stimulation

Platelets from healthy volunteers were incubated with thrombin (Sigma T1063) (0.1 U/mL) or with DENV-2 (1 PFU/platelet) at 37°C for the indicated times. In selected experiments platelets were pre-incubated for 30 min with neutralizing antibodies against DC-SIGN (R&D Systems 120507) (25 μg/mL), the integrin αV subunit, or an isotype-matched antibody; or with the pan-caspase inhibitor ZVAD-fmk (BioVision, USA) (20 μg/mL).

Statistical Analysis

Statistics were performed using GraphPad Prism 5.0 software (San Diego, CA). One way analysis of variance (ANOVA) was used to determine differences. Bonferroni’s post-hoc test was employed to identify the location of each difference between groups. Paired two-tail t-test was used to compare stimulated and unstimulated platelets from the same donor. Correlations were assessed using the Pearson’s test.

Results

Platelet activation in patients with dengue

The intensity of surface P-selectin expression (mean fluorescence intensity, MFI) on platelets was significantly (p<0.05) higher in samples from patients with dengue during the febrile (51.5±24.3 MFI) and defervescence (41.0±22.0 MFI) phases compared to the convalescence (22.9±6.3 MFI) phase or healthy volunteers (16.9±4.3 MFI). P-selectin expression was also higher on platelets isolated from febrile and defervescence DENV-infected patients compared to NDFI (33.8±8.7 MFI) even though surface P-selectin expression was greater in NDFI compared to healthy subjects (Figure 1A).

Figure 1. Platelet activation is increased during dengue illness.

Figure 1

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The mean fluorescence intensity (MFI) of P-selectin expression (A) and the percentage of annexin V-binding platelets (B) in platelets freshly-isolated from healthy subjects (control), patients with non-dengue febrile illness (NDFI), and dengue-infected patients in febrile (Feb), defervescence (Def), and convalescence (Conv) phases. Boxes indicate median and interquartile ranges and whiskers indicate 5-95 percentile. *p<0.05 versus control; #p<0.05 versus NDFI.

Phosphatidylserine exposure on platelets (percentage of positive platelets) was similarly increased (p<0.01) in patients in febrile (34.4±10.4%) and defervescence (31.6±10.7%) dengue phases versus patients in the convalescence phase (14.1±7.3%), healthy volunteers (9.1±4.2%), or NDFI (14.3±7.6%) (Figure 1B).

Platelet activation is associated with thrombocytopenia during dengue disease

Using platelet counts determined on the day of sample collection, patients were classified as thrombocytopenic (TCP) (<150.000/mm3) or non-thrombocytopenic (NTCP). Based on this grouping, 14 patients in the febrile phase were TCP while 12 were NTCP. The breakdown was similar in dengue patients in the defervescence (14 TCP and 10 NTCP patients). P-selectin surface expression was higher in TCP dengue patients (61.5±27.5 MFI for febrile and 49.3±25.2 MFI for defervescence) versus NTPC (39.8±13.0 for febrile and 29.8±6.6 MFI for defervescence) (Figure 2A).

Figure 2. Platelet counts in patients with dengue correlate with indices of platelet activation.

Figure 2

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(A) The mean fluorescence intensity (MFI) of P-selectin expression in thrombocytopenic (TCP) and non-thrombocytopenic (NTCP) dengue patients. Boxes indicate the median and interquartile ranges and whiskers indicate 5-95 percentile. (B-C) Platelet counts were plotted against the MFI of P-selectin expression (B) and the percent of annexin V-binding platelets (C) in febrile (Feb) defervescence (Def) and convalescence (Conv) phases. In Panels B and C the analysis was restricted to platelet counts obtained on the same day that P-selectin and phosphatidylserine were analyzed. (D) Platelet counts and the MFI of P-selectin expression were plotted against the day of illness in which each value was obtained. Non-linear regressions were traced according to the distribution of dots. *p<0.05 versus control; #p<0.01 TCP versus NTCP.

Moreover, we found that platelet counts in DENV-infected patients inversely correlated with surface P-selectin expression and phosphatidylserine exposure (Figure 2B and 2C). This relationship between thrombocytopenia and platelet activation was similarly observed during the course of the disease (Figure 2D).

Mitochondrial dysfunction in platelets from patients with dengue

Mitochondria are important regulators of the intrinsic pathways of apoptosis [23]. They also regulate activation responses and procoagulant activity in platelets [24], and loss of ΔΨm occurs in platelets that become apoptotic after activation [14, 15] or ageing during storage [16, 25]. As shown in Figure 3A, the proton ionophore FCCP significantly reduced ΔΨm in platelets isolated from healthy subjects or DENV-infected patients. However, basal ΔΨm was significantly (p<0.01) reduced in platelets isolated from dengue infected patients in febrile (14.6±3.5 MFI) and defervescence (14.6±4.1 MFI) compared to dengue patients in the convalescence (21.8±3.6 MFI), NDFI (20.5±4.6 MFI), or healthy volunteers (23.2±4.6 MFI) (Figure 3B).

Figure 3. Mitochondrial function is impaired in platelets from dengue-infected patients.

Figure 3

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(A-C) The mean fluorescence intensity (MFI) for TMRE, (D) the Citrate-Synthase activity, and (E-F) the MFI of MitoSoxRed in platelets from healthy subjects (control), patients with non-dengue febrile illness (NDFI), and dengue-infected patients in febrile (Feb), defervescence (Def), or convalescence (Conv) phases. (A, C and E) Mitochondrial responses in platelets treated with FCCP or oligomycin. Bars (A) depict the mean±SEM of 7-9 healthy participants and dengue patients; dots (C and E) represent TMRE or MitoSoxRed fluorescence in platelets from control or dengue patients before (basal) and after the treatments. Boxes (B, D and F) indicate median and interquartile ranges and whiskers indicate 5-95 percentile. +p<0.05 versus basal; *p<0.05 versus control; #p<0.05 versus NDFI.

As expected, ΔΨm was significantly increased when platelets from healthy subjects were incubated with oligomycin, which inhibits mitochondrial H+-ATP synthase. This increase, however, was not observed when platelets from dengue patients were exposed to oligomycin (Figure 3C). Next we assessed citrate-synthase activity in platelets and found no differences between dengue patients and controls (Figure 3D). These results indicate that perturbations in ΔΨm are due to increased mitochondrial permeability and not a reduction in mitochondrial content.

As shown in Figure 3E and 3F, platelets from DENV-infected patients constitutively generate ROSm. Treatment of the platelets with FCCP confirmed that ROS were derived from mitochondria (Figure 3E). Basal production of ROSm was significantly (p<0.05) higher in platelets from dengue patients at both febrile (14.2±3.8 MFI) and defervescence (11.8±2.7 MFI) phases compared to dengue patients at the convalescence (8.4±0.9 MFI), NFDI (9.7±2.2 MFI), and healthy subjects (8.4±1.3 MFI) (Figure 3F).

Platelet apoptosis in patients with dengue

Increased phosphatidylserine exposure and mitochondrial depolarization suggest that apoptotic pathways are more active in platelets from DENV-infected patients. Consistent with this possibility, we found that phosphatidylserine exposure negatively correlated with ΔΨm in platelets from dengue patients (Figure 4A). To assess activation of apoptosis pathways in more depth, we measured activation of caspase-9 and caspase-3 in platelets during dengue illness. Caspase-9 activation was significantly (p<0.01) higher in platelets isolated from febrile (15.3±3.2 MFI) and defervescence dengue patients compared to healthy subjects (8.5±1.7 MFI), dengue patients in convalescence (8.8±2.5 MFI), and NDFI (11.3±2.3 MFI) (Figure 4B). Higher amounts of cleaved caspase-9 and caspase-3 were also observed by western blot in DENV-infected patients compared to healthy volunteers (Figure 4C).

Figure 4. Platelets apoptosis in patients with dengue.

Figure 4

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(A) The mean fluorescence intensity (MFI) for TMRE was plotted against the percentage of annexin V-binding platelets in febrile (Feb), defervescence (Def) and convalescence (Conv) dengue phases. (B) The MFI of Caspase-9 activation in platelets freshly-isolated from healthy subjects (control), patients with non-dengue febrile illness (NDFI), and dengue-infected patients in Feb, Def, and Conv. Boxes indicate median and interquartile ranges and whiskers indicate 5-95 percentile. (C) Western analysis of pro- and cleaved caspase-9 (casp-9) and caspase-3 (casp-3), and β-actin in platelets isolated from control or dengue patients (representative of 5). *p<0.01 versus control; #p<0.05 versus NDFI.

DENV-2 induces activation, mitochondrial dysfunction and apoptosis in platelets

Next we determined whether DENV-2 directly activates platelets yielding similar responses to those of platelets from dengue-infected patients. Platelets from healthy donors were incubated with DENV-2 or mock for 1 min, 30 min, 1½ hour, 3 hours and 6 hours. Incubation of platelets with DENV-2 significantly increased surface P-selectin expression at 6 hours compared to mock (55.2±5.7 versus 30.7±12.4 MFI; n = 4, p = 0.019). The kinetic of P-selectin expression in platelets exposed to DENV-2 was distinct from that of thrombin stimulation, suggesting that DENV activates platelets through different mechanisms (Figure 5A).

Figure 5. DENV-2 induces platelet activation.

Figure 5

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The mean fluorescence intensity (MFI) of P-selectin expression (A, B and D) and the percentage (%) of annexin V-binding platelets (C and E) in platelets exposed to DENV in vitro. (A and C) Platelets were exposed to mock or DENV-2 for 1 min, 30 min, 1½ hour, 3 hours or 6 hours; or activated with thrombin for 1 min, 5 min, 15 min, 30 min or 1 hour. (B) Platelets were exposed (6 hours) to the filtrate or the retentate of DENV-2 purified through centrifugation in a Centricon Filter, or mock processed in parallel. (D and E) Platelets were exposed (6 hours) to mock, DENV or heat-inactivated DENV in the presence of the pan caspase inhibitor ZVAD-fmk. Dots and bars represent mean±SEM of 4 independent experiments from individual donors. *p<0.05 versus mock; #p<0.05 versus DENV filtrate or ZVAD-fmk treated platelets.

Six hours exposure to purified DENV-2 similarly activated platelets (Figure 5B). Virus purification was obtained using a Centricon YM-100 Centrifugal Filter (Merck Millipore) and platelets from healthy subjects were exposed separately to the filtrate or to the retentate (purified virus) ressuspended in medium. Exposure of platelets to purified DENV-2 significantly increased surface P-selectin expression compared to the virus filtrate or to mock sample processed in parallel (purified DENV-2, 49.6±12.5 MFI versus 23.6±14.5 MFI or 32.6±13.7 MFI for virus filtrate and mock retentate, respectively; n = 4, p<0.05).

Phosphatidylserine exposure on platelets was also increased after 6 hours of incubation with DENV-2 (68.7±11.5% versus 19.2±11.8% of positive platelets; n = 4, p = 0.052) (Figure 5C). To investigate the proportion of phosphatidylserine exposed due to platelet activation or apoptosis, platelets were incubated with DENV in presence of the pan caspase inhibitor ZVAD-fmk. Blocking caspases activity did not affect P-selectin expression (Figure 5D), but significantly (p<0.05) impaired phosphatidylserine exposure on platelets (Figure 5E). These data demonstrate that most of phosphatidylserine exposure depends of apoptosis pathways and only a small proportion depends of platelet activation.

In addition to infective DENV-2 we also exposed platelets to heat inactivated virus (56°C, 1 hour). Heat inactivation prevented DENV-2 from increasing P-selectin and phosphatidylserine on the surface of platelets (Figure 5D and 5E).

DENV-2 also significantly impaired mitochondrial function in platelets as evidenced by a reduction in TMRE fluorescence (infectious DENV-2, 18.2±7.4 MFI versus 31.6±18.3 MFI or 31.3±12.3 MFI for heat-inactivated and mock, respectively; n = 6, p<0.01)) and increased MitoSOX Red fluorescence (infectious DENV-2, 38.3±13.8 MFI versus 23.5±7.7 MFI or 24.6±6.4 MFI for heat inactivated-DENV-2 and mock, respectively; n = 6, p<0.01) (Figure 6A and 6B). Activated caspase-9 was also significantly higher in platelets exposed to DENV-2 (infectious DENV-2, 50.4±11.5 MFI versus 28.6±12.3 MFI or 29.0±5.6 MFI for heat-inactivated DENV-2 and mock, respectively; n = 4, p<0.01) (Figure 6C). Furthermore, a negative correlation was observed between ΔΨm and phosphatidylserine exposure (Figure 6D). We observed that platelets with high phosphatidylserine exposure concomitantly exhibited reduced TMRE fluorescence (Figure 6D insets).

Figure 6. DENV-2 induces mitochondrial dysfunction and apoptosis in platelets.

Figure 6

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The mean fluorescence intensity (MFI) for TMRE (A), MitoSoxRed (B) and Caspase-9 activation (C) in platelets exposed for 6 hours to mock, DENV-2 or heat-inactivated DENV-2. The bars represent mean±SEM of 4 to 6 independent experiments from individual donors. (D) The MFI for TMRE was plotted against the percentage of annexin V-binding platelets. Representative density plots are shown. *p<0.01 versus mock.

DENV-2 activates platelets through pathways that involve DC-SIGN

Previous studies in other cell types have shown that DENV particles interact with surface molecules that include DC-SIGN and integrin-αvβ3 [26, 27]. Although these receptors have been reported in platelets [28, 29], they have not been linked to DENV-induced platelet activation. To investigate their roles in platelet activation, platelets were incubated with neutralizing antibodies against DC-SIGN or αv integrin subunit prior to being exposed to DENV-2. Blocking of DC-SIGN, but not αv, prevented DENV-2 from increasing P-selectin expression on the surface of platelets (Figure 7A). Decreased ΔΨm elicited by DENV-2 was similarly rescued by anti-DC-SIGN antibodies (Figure 7B).

Figure 7. DENV-2 activates platelets through mechanisms that involve DC-SIGN.

Figure 7

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Platelets were exposed for 6 hours to mock, DENV-2 or heat-inactivated DENV-2 in the presence or absence of neutralizing antibodies against DC-SIGN or the αV integrin subunit. The percent increase in P-selectin expression (A) or percent decrease in TMRE fluorescence (B) related to mock values are shown. (C) Representative density plots showing the expression of DC-SIGN on platelets. (D) The percentage of DC-SIGN-expressing platelets exposed to mock or DENV. (E) Platelets exposed to mock or DENV-2 were stained for P-selectin and DC-SIGN. The mean fluorescence intensity (MFI) for P-selectin expression was assessed on platelets gated as DC-SIGN negative (DC-SIGN-) or positive (DC-SIGN+). Representative histograms and density plots are shown next to their corresponding graphs. Bars represent the mean±SEM of 4 independent experiments from individual donors. *p<0.05 versus mock; #p<0.05 between anti-DC-SIGN or IgG isotype (A and B); or between DC-SIGN- and DC-SIGN+ (E).

To clarify the role played by DC-SIGN-expressing platelets in DENV-induced platelet activation, we analyzed surface P-selectin expression in DC-SIGN stained platelets gated as DC-SIGN positive or negative. DC-SIGN positive platelets ranged from 13.4% to 37.5% (21.4±8.5%) (Figure 7C). DENV exposure did not affect DC-SIGN expression on platelets (Figure 7D). As shown in Figure 7E, DC-SIGN positive platelets exhibited increased P-selectin expression compared to DC-SIGN negative (51.8±14.6 MFI for DC-SIGN+ versus 31.4±3.2 MFI for DC-SIGN-; n = 4, p = 0.0398).

Discussion

Thrombocytopenia is a common manifestation of dengue illness. Increased platelet clearance may occur in dengue infection as consequence of platelet activation, however the mechanisms remain elusive. Here we show that platelets from dengue patients display classic signs of apoptosis that include increased phosphatidylserine exposure, mitochondrial depolarization and activation of caspase-9 and 3. Moreover, thrombocytopenia in patients with dengue strongly associates with enhanced platelet activation and apoptosis. Our data also implicate DC-SIGN as a critical receptor in DENV-dependent platelet activation.

Abnormal platelet function has been reported in dengue [30], but the underlying mechanisms are not fully understood. We consistently observed that platelets from DENV-infected patients were more activated compared to healthy subjects. Moreover, platelet activation was more severe in thrombocytopenic patients. This suggests that dengue may activate platelets and, as a result, facilitate platelet deposition in micro-vessels and/or clearance. Consistent with this hypothesis, it was recently reported increased phagocytosis of platelets by macrophages in patients with dengue [6]. Sequestration of platelets in the liver and spleen of dengue-infected patients have also been observed [31].

Many factors influence platelet survival, which is reduced in patients with dengue [31]. One of these involves the intrinsic pathway of apoptosis [16]. It is widely accepted that DENV induces apoptosis in host cells [32, 33]. When DENV infects hepatocytes it increases mitochondrial permeability and, as a consequence, induces apoptosis [33]. We similarly observed mitochondrial dysfunction and apoptosis in platelets from patients with dengue. Moreover, DENV-induced phosphatidylserine exposure in vitro was largely dependent on caspase activation as assessed by ZVAD blockade, further supporting apoptotic-dependent mechanisms of cell death. Conceivably, mitochondrial dysfunction followed by activation of apoptosis pathways in platelets may contribute to the genesis of thrombocytopenia in dengue. Consistent with this possibility, we found that platelet counts in dengue-infected patients inversely correlate with phosphatidylserine exposure (Figure 2C) and positively correlate with ΔΨm (r = 0.4612, p<0.001) (data not shown).

It was previously reported that platelet activation and apoptosis followed distinct kinetics during dengue illness [34]. We similarly observed that phosphatidylserine exposure remained higher while P-selectin expression was recovered to normal levels (data not shown). Platelet apoptosis has been shown to follow the platelet activation in vitro [35]. However, it was not the case in dengue illness, since P-selectin expression and phosphatidylserine exposure were concomitantly increased in initial phase of infection. Furthermore, the kinetics of P-selectin expression and phosphatidylserine exposure were similar in platelets incubated with DENV in vitro (Figure 5), suggesting that platelet apoptosis is directly induced by DENV. Other factors beyond DENV exposure may be responsible for platelet apoptosis in non-viremic phases. Although our results showed activation of the mitochondrial pathway of apoptosis, we cannot exclude participation of other cell death pathways as necrosis or apoptosis by extrinsic pathways.

In addition to mitochondrial dysfunction and activation of apoptosis pathways, we found that DENV-2 directly activated platelets isolated from healthy donors. Consistent with our observations, Ghosh et al. [12] previously demonstrated that DENV-2 induces morphological features of activation in platelets. Although these data indicate that the virus can directly activate platelets, we recognize that other factors can contribute to platelet activation including antibodies, cytokines, endothelial activation, and coagulation factors among others [7]. It was previously shown that DENV-specific antibodies may potentiate DENV binding to platelets [36]. Thus, increased serum IgG levels in patients with secondary dengue may also contribute to platelet activation, even though we did not find significant differences in platelet activation and apoptosis from primary or secondary dengue infections (Table S2). Also, although in our study patients with severe dengue exhibited a trend towards increased platelet activation and apoptosis (data not shown), it was not possible to determine whether it correlates or not with more severe clinical outcome because of the small size of our cohort.

The mechanisms by which dengue induces platelet activation are not known. Here we show that DC-SIGN supports DENV-induced P-selectin surface expression and mitochondrial dysfunction in platelets. Boukour and colleagues [28] previously demonstrated that platelets express DC-SIGN, a C-type lectin receptor that has demonstrated roles in DENV recognition in dendritic cells [26]. Blockade of DC-SIGN on the surface of platelets prevents DENV-2 from activating platelets and impairing ΔΨm. Whether this is due to outside-in activation of DC-SIGN signaling pathways and/or internalization of DENV-2 into platelets requires further investigation. Regarding the latter, DC-SIGN facilitates entry of HIV into platelets [28] and DENV has been detected in platelets [13, 37]. If DENV entry is necessary to induce platelet activation and apoptosis, other receptors as FCγRIIa may be also involved, since it can mediate DENV entry in macrophages [38] and has demonstrated roles in Staphylococcus aureus-induced platelet activation [39].

In summary, we show that platelets from dengue-infected patients have impaired mitochondrial function and evidence of apoptosis, which likely contributes to the development of thrombocytopenia in dengue disease. Because DENV can trigger mitochontrial dysfunction and platelet activation through mechanisms that involve DC-SIGN, therapeutic strategies that prevent DENV from interacting with DC-SIGN on platelets may have a role in the treatment of dengue-associated thrombocytopenia.

Acknowledgements

We thank Gisele B. Lima for virus production, and Alan B. Carneiro, Luiz Felipe G. Souza, Edson F. Assis and Monique R. Q. Lima for technical assistance.

Funding: This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), PAPES/FIOCRUZ, INBEB and PRONEX Dengue; and from the National Institutes of Health (HL066277, HL091754, and HL044525 awarded to A.S.W. and G.A.Z).

Footnotes

Addendum: E.D.Hottz – Performed the majority of experiments, data analyses, and manuscript drafting and preparation;

P.C.G.Nunes – Performed virological and immunological analyses from patient plasma; R.M.R. Nogueira – Experimental design and manuscript editing/review;

M.F. Oliveira – Experimental design and manuscript editing/review;

R. Valls-de-Souza- Patient enrolment and experimental analyses;

A.T. Da Poian – Experimental design and manuscript editing/review;

A.S. Weyrich - Experimental design and manuscript editing/review;

G.A. Zimmerman - Experimental design and manuscript editing/review;

P.T.Bozza and F.A.Bozza- Directed all aspects of the study, data analyses, and manuscript preparation and review.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

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