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Influenza Virus Infection Induces Platelet-Endothelial Adhesion

Which Contributes to Lung Injury
Michael G. Sugiyama,a,c Asela Gamage,a,b Roman Zyla,a Susan M. Armstrong,a,b Suzanne Advani,a Andrew Advani,a
Changsen Wang,a Warren L. Leea,b,c,d
Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute of St. Michael’s Hospital, Toronto, Canadaa; Institute of Medical Science, University of
Toronto, Toronto, Canadab; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canadac; Division of Respirology and
Interdepartmental Division of Critical Care Medicine, University of Toronto, Toronto, Canadad

ABSTRACT
Lung injury after influenza infection is characterized by increased permeability of the lung microvasculature, culminating in
acute respiratory failure. Platelets interact with activated endothelial cells and have been implicated in the pathogenesis of some
forms of acute lung injury. Autopsy studies have revealed pulmonary microthrombi after influenza infection, and epidemiologi-
cal studies suggest that influenza vaccination is protective against pulmonary thromboembolism; however, the effect of influ-
enza infection on platelet-endothelial interactions is unclear. We demonstrate that endothelial infection with both laboratory
and clinical strains of influenza virus increased the adhesion of human platelets to primary human lung microvascular endothe-
lial cells. Platelets adhered to infected cells as well as to neighboring cells, suggesting a paracrine effect. Influenza infection
caused the upregulation of von Willebrand factor and ICAM-1, but blocking these receptors did not prevent platelet-endothelial
adhesion. Instead, platelet adhesion was inhibited by both RGDS peptide and a blocking antibody to platelet integrin ␣5␤1, im-
plicating endothelial fibronectin. Concordantly, lung histology from infected mice revealed viral dose-dependent colocalization
of viral nucleoprotein and the endothelial marker PECAM-1, while platelet adhesion and fibronectin deposition also were ob-
served in the lungs of influenza-infected mice. Inhibition of platelets using acetylsalicylic acid significantly improved survival, a
finding confirmed using a second antiplatelet agent. Thus, influenza infection induces platelet-lung endothelial adhesion via
fibronectin, contributing to mortality from acute lung injury. The inhibition of platelets may constitute a practical adjunctive
strategy to the treatment of severe infections with influenza.

IMPORTANCE
There is growing appreciation of the involvement of the lung endothelium in the pathogenesis of severe infections with influenza
virus. We have recently shown that the virus can infect human lung endothelial cells, but the functional consequences of this
infection are unknown (S. M. Armstrong, C. Wang, J. Tigdi, X. Si, C. Dumpit, S. Charles, A. Gamage, T. J. Moraes, and W. L. Lee,
PLoS One 7:e47323, 2012, http://dx.doi.org/10.1371/journal.pone.0047323). Here, we show that this infection causes platelets to
adhere to the lung endothelium. Importantly, blocking platelets using two distinct antiplatelet drugs improved survival in a
mouse model of severe influenza infection. Thus, platelet inhibition may constitute a novel therapeutic strategy to improve the

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host response to severe infections with influenza.

D espite annual vaccination programs and widely available an-

tiviral drugs, seasonal influenza alone causes an estimated
tens of thousands of deaths in North America annually (1, 2).
chiolar epithelium, leading to alveolar damage and frank alveolar
denudement (10). The average thickness of the alveolar capillary
membrane is just over 1 ␮m, which includes a single layer of
Most deaths occur due to pulmonary complications, such as pri- alveolar epithelium, wisps of interstitial tissue, and a single layer of
mary viral pneumonia (3) or a superimposed bacterial pneumo- lung microvascular endothelium (11). In some areas, the mem-
nia (4). In both, respiratory deterioration is marked by acute lung brane is as thin as 200 nm (11). Thus, the death of infected epithe-
injury (3, 5), a potentially fatal syndrome of pulmonary edema lial cells will create gaps in the monolayer and give newly released
that occurs due to the increased permeability of the lung micro- virions access to the endothelium (10, 12), even in the absence of
vasculature (6, 7). Therapeutic options are limited. While antiviral
drugs exist, they only partially reduce mortality (8), they must be
administered early to be maximally effective, and their use is com- Received 9 October 2015 Accepted 25 November 2015
plicated by the rapid development of resistance. For instance, al- Accepted manuscript posted online 4 December 2015
most 100% of H3N2 strains of influenza A are already resistant to Citation Sugiyama MG, Gamage A, Zyla R, Armstrong SM, Advani S, Advani A,
Wang C, Lee WL. 2016. Influenza virus infection induces platelet-endothelial
amantadine (9). Thus, new therapies for these most severe cases of adhesion which contributes to lung injury. J Virol 90:1812–1823.
influenza are urgently needed. doi:10.1128/JVI.02599-15.
The influenza virus infects the bronchial epithelium, leading to Editor: R. M. Sandri-Goldin, University of California, Irvine
epithelial injury, apoptosis, and desquamation (10). In uncompli- Address correspondence to Warren L. Lee, [email protected].
cated infections, these changes to the airway epithelium are tran- M.G.S. and A.G. contributed equally.
sient. However, in primary viral pneumonia, the virus also infects Copyright © 2016, American Society for Microbiology. All Rights Reserved.
the distal lung, particularly type I pneumocytes and ciliated bron-

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 Influenza Virus Induces Platelet-Endothelial Adhesion

frank viremia. We have recently reported that human influenza is ticoagulated whole blood at 300 ⫻ g for 7 min. Platelets then were isolated
capable of infecting lung endothelial cells both in vitro (13) and in from the PRP with the use of a Sepharose 2B column in piperazine-N,N=-
vivo (7), although very little is known about the functional conse- bis(2-ethanesulfonic acid) (PIPES) buffer (5 mM PIPES, 1.37 mM NaCl,
quences of such infection. Certainly disruption or injury to either 4 mM KCl, 0.1% glucose, pH 7.0). Following isolation, platelets were
alveolar epithelium or the underlying endothelium or both may diluted to final concentrations required by each experimental condition.
contribute to the formation of lung edema (13). Platelets were fluorescently labeled with calcein acetoxymethyl ester (cal-
Importantly, whether viral infection affects the relationship of cein-AM) (1 ␮g ml⫺1; Molecular Probes, Eugene, OR). Platelet purity and
the lack of activation were verified by flow cytometry for CD41 (25);
the lung endothelium with other cells in the lung, such as platelets,
preparations were 99% single platelets, and ⬎95% were positive for CD41
is unknown. This is an important question, since platelets have
(not shown).
been identified to contribute to certain forms of lung injury (14, In the plate-based adhesion assay, endothelial cells were seeded on 96-well
15) through their recruitment of leukocytes (16, 17) and induc- plates and grown to confluence (3 to 4 days). Platelets were added to each well
tion of an inflammatory response (18). Importantly, the endothe- at the indicated concentrations and incubated for 1 h. After incubation, cells
lium normally is antithrombogenic and prevents platelet adhe- were washed with phosphate-buffered saline (PBS) and fixed with 4% para-
sion. An effect of the virus to induce platelet-endothelial adhesion formaldehyde (PFA) for 15 min. The adhesion of fluorescently labeled plate-
is plausible: numerous reports describe pulmonary thrombi as an lets was measured by using a fluorescent plate reader.
important complication of severe infections with influenza (10, In the flow cytometry adhesion assay, endothelial cells were trypsinized
19), while endothelial activation, which would be expected to in- and then labeled with LDS 751 (a cell-permeant nucleic acid stain; Life
duce platelet adhesion, was highly correlated with death from in- Technologies) before suspending them with calcein-labeled platelets for
fluenza in a murine model (20). Furthermore, a study of patients 15 min. Endothelium-platelet adhesion was detected by flow cytometry
with venous thromboembolism found that vaccination against using the MACSQuant analyzer (Miltenyi Biotec Inc., San Diego, CA).
influenza was associated with a protective effect against pulmo- Antibodies. Rabbit anti-von Willebrand factor (vWF) (ab6994),
nary clots (21). Determining how influenza infection affects plate- rabbit anti-fibronectin (ab2413), and goat anti-ICAM (EP1442Y) were
from Abcam (Cambridge, MA). Mouse anti-P-selectin (MAB2154)
let-endothelial interactions may have important clinical implica-
was from EMD Millipore, and mouse anti-VE-cadherin (sc-6458),
tions, since numerous platelet antagonists are available already rabbit anti-PECAM-1 (sc-8306), and mouse nuclear protein (NP; sc-
and might constitute useful therapies. 80481) were from Santa Cruz Biotechnology. To block integrin-fi-
In this study, we elucidated the effect of influenza virus infec- bronectin binding, platelets were treated with 170 ␮M Arg-Gly-Asp-
tion on the adhesion of platelets to the lung microvascular endo- Ser (RGDS) peptide (A9041; Sigma-Aldrich) for 30 min before
thelium. In addition, we determined whether inhibiting platelets incubation with endothelial cells. Anti-fibrinogen (SC-166968; Santa
using readily available platelet inhibitors exerted protective effects Cruz) and anti-fibronectin (SC-9068; Santa Cruz) were used in West-
on the degree of lung injury in a mouse model of severe influenza. ern blot analyses to detect expression levels of fibrinogen and fibronec-
tin. Antibodies to block cell surface ligands and integrins in adhesion
MATERIALS AND METHODS assays were obtained from the following sources: anti-vWF (3.1 g/liter;
Cell culture and influenza infection. Primary human microvascular en- Dako North America Inc., Carpinteria, CA), anti-ICAM (BBA3;
dothelial cells (HMVECs) derived from lung were obtained from R&D), anti-integrin ␣5␤1 (ab75472; Abcam), and anti-integrin ␣2␤3
Lonza and were cultured in EBM-2 media with the recommended (ab3919; Abcam). Cells were treated for 60 min prior to the adhesion
supplements and used in passages 6 to 8. Influenza A virus X31 (H3N2) assay.
was used, since the H3N2 subtype is most commonly associated with Immunofluorescence. For VE-cadherin and viral nucleoprotein (NP)
complications and death (1, 22); we also used an influenza A virus immunostains, cells were fixed in 4% PFA for 1 h at room temperature,

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clinical isolate (H3N2; from Susan Richardson). The effect of a labo- incubated in 0.15% glycine for 10 min, and then permeabilized in 0.1%
ratory strain of H1N1 (A/Puerto Rico/8/34, also known as PR8; a gift Triton X-100 for 20 min. After blocking, cells were treated with anti-VE-
from Conrad Liles) was tested as well. HMVECs were infected with cadherin and NP antibodies for 1 h, followed by incubation for 1 h with a
virus at different multiplicities of infection (MOI; defined as the ratio fluorescently labeled secondary antibody. Images were acquired by spin-
of PFU to endothelial cells). Virus was added to cells in serum-free
ning-disc confocal microscopy (Zeiss Axiovert 200M microscope). Mi-
media. After 1 h, 0.5% serum was added. All infections were for 24 h.
croscope settings were kept constant between conditions. All images were
To study any contributions from viral replication, we developed rep-
randomly chosen and were acquired as z-stack projections (z-stack inter-
lication-deficient virus by exposing it to UV light for 10 min as previ-
val, 0.5 ␮m).
ously reported (13). The amount of virus was quantified both by PFU
Apoptosis assays. Apoptotic cells were detected using an annexin V-
and hemagglutinin units (HAU) using published protocols (23).
Reagents. To inhibit virus-induced apoptosis, cells were treated with fluorescein isothiocyanate (FITC) apoptosis detection kit (BioVision,
100 ␮M ZVAD-FMK (Enzo Life Sciences) for 24 h simultaneously with Milpitas, CA) according to the manufacturer’s instructions. Annexin V-
infection. To evaluate the effect of influenza surface proteins on platelet FITC was detected using a flow cytometer (BD FACSCalibur cytometer;
adhesion, endothelial cells were exposed to recombinant hemagglutinin Becton Dickinson, Mississauga, ON, Canada), and the data were analyzed
(HA; 5 to 50 ␮g/ml; catalogue no. IT-003-00418DTMp; Immune Tech- using De Novo Software-FCS Express v 3.0.
nology Corp.) and neuraminidase (NA; 250 ␮g/␮l; 6875-NM; Cedarlane) Western blotting. Lysates were prepared with lysis buffer (62.5 mM
from H3N2 influenza virus for 30 min or 24 h. In other experiments, Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 10 mM dithiothreitol [DTT])
endothelial cells were exposed to the synthetic viral RNA mimetic and separated using 10% polyacrylamide gels. Proteins were transferred
poly(I·C) (2 ␮g/ml; tlr1-pic; Cedarlane) for 24 h prior to the addition of to nitrocellulose membranes, blocked for 1 h in 5% milk in Tris-buffered
platelets. In one experiment, thrombin (8 U/ml; T6884; Sigma) was used saline (TBS), and probed overnight with primary antibody at 4°C. After
as a positive control to induce platelet adhesion. washing, blots were incubated with horseradish peroxidase (HRP)-con-
Platelet-endothelial adhesion assays. Gel-filtered platelets were pre- jugated secondary antibodies for 1 h, washed, and then visualized by ad-
pared as previously described (24). Briefly, human blood was collected vanced chemiluminescence (Amersham). Band intensity was quantified
from healthy adult donors by venipuncture and anticoagulated with Na- using Image J (NIH) and normalized to the loading control after back-
citrate. Platelet-rich plasma (PRP) was obtained by centrifugation of an- ground correction.

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Sugiyama et al.

FIG 1 Influenza A virus induces a dose-dependent increase in platelet-endothelial adhesion. Endothelial monolayers infected with increasing MOIs of influenza
(Flu 1, MOI of 1; Flu 5, MOI of 5, etc.) have increased platelet adhesion, whether infected with a laboratory strain of H3N2 (A) (n ⫽ 5 to 7), a clinical H3N2 strain

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(B) (n ⫽ 3), or a laboratory strain of H1N1 (C) (n ⫽ 3). *, P ⬍ 0.05 versus control. (D to E) Influenza virus induces a dose-dependent increase in platelet-
endothelial interaction in studies using endothelial cells in suspension. (D) Endothelial cells bound to platelets appear in the upper right quadrant. (E)
Quantification of data from panel D. n ⫽ 3; *, P ⬍ 0.05 versus the control. (F) Influenza virus-induced platelet-endothelial interaction visualized by confocal
microscopy. Calcein-AM-labeled platelets are shown in green, while endothelial cell-cell junctions stained with VE-cadherin appear in red. The image is
representative of 2 experiments.

Immunohistochemistry. Mice were infected intranasally with 32 or viral NP and PECAM-1 was quantified using Manders’ coefficient (27)
64 HAU influenza virus and were sacrificed 5 days later. The lungs were as determined using Volocity software.
collected for histological analysis, fixed in formalin, and embedded in For experiments with acetylsalicylic acid (ASA; Sigma), mice re-
paraffin as previously described (26). For fibronectin, sections were ceived 3 mg ASA by intraperitoneal injection at the time of infection
stained with a mouse monoclonal antibody directed against fibronec- and again 24 h later. For adjuvant ASA, mice also received a dose of
tin (Santa Cruz Biotechnology). Slides were washed and subsequently amantadine by oral gavage twice daily at a concentration of 138 mg/kg
incubated with Alexa Fluor 488 donkey anti-mouse secondary anti- of body weight/day starting on the day of infection (28). For platelet
body from Life Technologies Inc. (Burlington, ON, Canada). For inhibition by ticlopidine, mice received a daily dose of 10 mg/kg ticlo-
platelet deposition, sections were stained in the same manner using pidine by intraperitoneal injection (29). Mice were monitored 3 times
anti-CD41 antibody (bs-2636R; Bioss, Woburn, MA) followed by an daily for the duration of the experiment (14 days after infection) and
anti-rabbit secondary antibody and then DAB staining (Dako). Ran- were scored for weight loss, hypothermia, hypoxemia, spontaneous
dom fields were acquired by spinning-disc confocal microscopy (Zeiss activity (scored from 1 [moribund] to 5 [normal], as described in
Axiovert 200M microscope) and quantified in a blinded fashion using reference 30), and other clinical features of influenza infection. Mice
ImageJ. For the staining of viral nucleoprotein and PECAM-1, antigen were euthanized if two or more of the following occurred: weight loss
retrieval was performed by boiling for 15 min in citric acid buffer, exceeded 30% of initial weight, temperature fell below 31°C, and/or
cooling for 45 min, and then blocking and permeabilization. Lung the animal appeared moribund. In one experiment, lung homogenates
sections were stained with rabbit anti-PECAM-1 (1:500) overnight from ASA-treated and untreated mice were measured for viral PFU 3
and with mouse anti-influenza NP (1:75) for 1 h. The colocalization of days postinfection using published protocols (23).

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 Influenza Virus Induces Platelet-Endothelial Adhesion

FIG 2 Exploring the components of the virus responsible for influenza virus-induced endothelium-platelet adhesion. Inhibition of caspases using ZVAD
does not affect influenza-induced platelet-endothelial interactions (n ⫽ 3; *, P ⬍ 0.05 versus the control [Ctrl]) (A), although ZVAD treatment

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successfully blocks influenza-induced endothelial cell apoptosis (B). The histogram is representative of 3 experiments. (C) Viral binding is not sufficient
to induce platelet-endothelial interaction, as the effect was not recapitulated by treatment with the protein hemagglutinin (HA; at 1 or 10 ␮g/ml). n ⫽ 3;
*, P ⬍ 0.05 versus control and HA groups. (D) Recombinant viral membrane enzyme neuraminidase (NA) cannot recapitulate influenza-induced
platelet-endothelial interaction. n ⫽ 3; *, P ⬍ 0.05 versus control and NA groups. (E) Viral RNA mimetic [poly(I·C)] did not recapitulate influenza-
induced platelet-endothelial interaction. n ⫽ 3; *, P ⬍ 0.05 versus control. (F) Viral replication is not required for influenza-induced platelet-endothelial
interaction. Replication-deficient (UV flu) and live virus induced similar degrees of platelet-endothelial adhesion. n ⫽ 3; *, P ⬍ 0.05 versus control. (G)
Influenza-induced platelet-endothelial interaction visualized by confocal microscopy reveals that influenza induces platelet adhesion to endothelial cells
in a paracrine fashion. Calcein-AM-labeled platelets are shown in green, while an influenza-infected endothelial cell is labeled using an antibody to viral
nucleoprotein and appears red. The image is representative of 2 experiments.

Pulse oximetry measurements. Arterial oxygen saturation was mea- were approved by the Animal Care Committee of St. Michael’s Hospital
sured using the MouseOx Plus device and software (Starr Life Sciences, (protocol 297). Care was taken to minimize animal discomfort per insti-
Oakmont, PA) on conscious (nonanesthetized) mice using a collar clip. tutional guidelines. Thus, mice were anesthetized with isoflurane for in-
Mice were allowed to acclimate to the collar clip for several minutes before tranasal instillation and were monitored regularly postinfection.
the maximal measurement was recorded.
Statistics. Experiments were repeated a minimum of three times, and RESULTS
data are presented as means and standard errors. Unless otherwise stated, Influenza induces platelet-endothelial adhesion. Confluent pri-
comparisons between greater than 2 groups were performed by one-way
mary human lung endothelial monolayers were infected with an
analysis of variance (ANOVA) with Bonferroni t tests for post hoc analyses;
unpaired student’s t tests were performed for comparisons between two H3N2 subtype of influenza A virus (X31) for 24 h; we and others
groups, and all tests were two-tailed. Statistics were performed using have previously reported that human influenza is capable of in-
GraphPad Prism software (La Jolla, CA). fecting and replicating in human endothelial cells both in vitro
Ethics statement. All mouse experiments were performed in accor- (13, 31, 32) and in vivo (7). This was followed by incubation with
dance with the regulations of the Canadian Council on Animal Care and purified calcein-labeled human platelets. After washing to remove

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Sugiyama et al.

FIG 3 Influenza virus upregulates endothelial vWF and ICAM-1 expression. (A to C) Endothelial expression of vWF (A), ICAM-1 (B), and P-selectin (C) after
influenza infection were measured by flow cytometry. Thrombin was used as a positive control in the P-selectin experiment, showing that influenza infection does
not affect endothelial P-selectin expression. Histograms and graphs of relative mean fluorescence are representative of 2 to 3 experiments. *, P ⬍ 0.05 versus the
control; isotype refers to isotype control antibody. (D) ICAM-1 and vWF are not required for flu-induced platelet-endothelial interaction, as the incubation of
HMVECs with anti-vWF or anti-ICAM-1 blocking antibodies did not prevent influenza-induced platelet-endothelial adhesion. n ⫽ 3; *, P ⬍ 0.05 versus the
control.

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unbound platelets, the degree of platelet adhesion was measured cells (data not shown). In contrast, influenza infection induced
by a fluorescent plate reader. Viral infection caused a dose-depen- the adhesion of platelets to areas of the endothelial monolayer that
dent increase in platelet-endothelial adhesion (Fig. 1A); impor- were both distant and adjacent to cell-cell junctions (Fig. 1F).
tantly, we observed a similar effect with a clinical isolate (H3N2) of Thus, influenza infection of endothelial cells induces true platelet-
the virus (Fig. 1B) as well as a laboratory H1N1 subtype of influ- endothelial adhesion.
enza A (PR8) (Fig. 1C). Influenza-induced platelet adhesion is not due to endothelial
We considered the possibility that infection could lead to the apoptosis and occurs with both infected and adjacent cells. We
retraction of the endothelial cells, exposing the extracellular ma- next considered the possibility that platelet-endothelial adhesion
trix and favoring the adhesion of platelets to the spaces between was a consequence of endothelial apoptosis, as viral infection is
cells rather than causing bona fide platelet-endothelial adhesion. known to trigger cell death (13), which itself increases adhesive-
To establish that true platelet-endothelial adhesion was occurring, ness to platelets (33). However, treatment with the pan-caspase
we studied platelet-endothelial interactions in suspension by flow inhibitor ZVAD had no effect on platelet adhesion (Fig. 2A), even
cytometry, thereby eliminating confounding from the extracellu- though it did prevent influenza-induced endothelial cell apoptosis
lar matrix. To do so, we briefly trypsinized control and infected (Fig. 2B).
endothelial monolayers before the addition of platelets. Under We then performed a series of experiments attempting to
these conditions, platelet-endothelial adhesion continued to be recapitulate platelet adhesion using different elements of the
strongly induced by infection with the virus in a dose-dependent influenza virus. The most abundant surface proteins of influ-
fashion (Fig. 1D and E). To confirm these findings visually, we enza are hemagglutinin (HA), which mediates viral binding,
performed immunofluorescence for the specific endothelial junc- and neuraminidase (NA), which cleaves sialic acid residues,
tional protein VE-cadherin on infected monolayers after the ad- allowing for the release of newly formed virions. We treated
dition of platelets and then imaged the monolayer along the entire endothelial monolayers with recombinant hemagglutinin or
z axis. As expected, platelets showed little adherence to uninfected recombinant neuraminidase to determine whether this would

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FIG 4 Integrin binding is required for flu-induced endothelium-platelet binding. (A) Platelets pretreated with RGDS peptide to block platelet integrin binding
prior to their addition to influenza-infected and control endothelium show impaired influenza-induced endothelium-platelet adhesion. n ⫽ 3; *, P ⬍ 0.05 versus
control and Flu⫹RGDS groups. (B) Integrin ␣IIb␤3 is not required for influenza-induced endothelium-platelet binding, as incubating platelets with an
anti-␣IIb␤3 antibody prior to their addition to endothelium does not block adhesion. n ⫽ 2; *, P ⬍ 0.05 versus control. (C) In contrast, blocking antibodies to
integrin ␣5␤1 inhibits influenza-induced endothelium-platelet binding. n ⫽ 3; *, P ⬍ 0.05 versus the control. (D, E, and F) Influenza A virus induces a
dose-dependent decrease in endothelial fibronectin monomer expression and increase in fibronectin multimer expression. HMW, high molecular weight. n ⫽
3; *, P ⬍ 0.05 versus the control. (G) Influenza induces endothelial fibrinogen expression. n ⫽ 3; *, P ⬍ 0.05.

be sufficient to induce platelet adhesion. However, at concen- ponents to induce platelet adhesion suggested that endothelium-
trations as high as or higher than those found for the intact platelet interactions occur via a paracrine effect rather than being
virus (34), exposure to recombinant HA and NA for 30 min to isolated to individual infected cells. Using immunofluorescence
24 h did not induce platelet adhesion (Fig. 2C and D and data for viral nucleoprotein (NP) to denote infected cells, we observed
not shown). Similarly, we treated monolayers with poly(I·C), a the adherence of platelets to both NP-positive and NP-negative
synthetic analogue of double-stranded RNA (35), to evaluate cells, suggesting that adhesion is mediated at least in part by para-
whether the recognition of viral RNA could be responsible for crine factors (Fig. 2G).
the effect. However, it did not recapitulate platelet adhesion Endothelial cells are activated after viral infection, and plate-
(Fig. 2E). We then generated replication-deficient virus using let adhesion is mediated by integrins. We next sought to deter-
UV irradiation, which we have shown is still capable of infect- mine the cellular mechanism of influenza-induced platelet-endo-
ing the endothelium and producing viral proteins (13). Under thelial adhesion. We reasoned that viral infection was likely to
these conditions, platelet-endothelial adhesion still occurred, activate the lung endothelium, leading to increased expression of
indicating that viral replication is not required for the effect endothelial ligands capable of engaging platelet receptors. Indeed,
(Fig. 2F). infected monolayers demonstrated a significant increase in sur-
The failure of ZVAD to inhibit and of the purified viral com- face expression of vWF and ICAM-1 but no change in P-selectin

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Sugiyama et al.

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FIG 5 Colocalization of viral nucleoprotein and PECAM-1 increases as a function of viral inoculum. Mice were infected with 32 or 64 HAU of influenza A virus
(X31), and immunofluorescence on lung sections was performed 5 days later. Sections were probed for viral nucleoprotein and PECAM-1, and colocalization
using Manders’ coefficient (M2) was quantified using Volocity software; the insets show enlarged areas. *, P ⬍ 0.01; n ⫽ 3 mice at 32 HAU and 4 mice in each
of 64-HAU and control groups; colocalization could not be calculated in control mice due to the absence of viral nucleoprotein. Images were acquired at ⫻600
magnification.

levels (Fig. 3A to C). However, treating endothelial cells with adhesion in this context is integrin mediated, as integrins have
blocking antibodies to vWF or ICAM-1 did not prevent platelet been reported to be critical in thrombus formation within injured
adhesion, suggesting that these receptors are not responsible for vessels (36). We pretreated platelets with synthetic RGDS peptide,
influenza-induced endothelium-platelet adhesion (Fig. 3D). which competitively inhibits integrin binding to fibronectin and
Instead, we considered the possibility that platelet-endothelial related proteins (37). When RGDS-treated platelets were added to

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 Influenza Virus Induces Platelet-Endothelial Adhesion

FIG 6 Endothelium-platelet interaction in a mouse model of influenza virus-induced lung injury. (A) Lung histology showing that influenza infection is
associated with increased platelet deposition in murine lungs. Platelets shown by DAB staining are labeled using an anti-CD41 antibody. Control group, n ⫽ 3
animals; flu group, n ⫽ 5 animals. (B) Immunostaining for fibronectin reveals increased fibronectin deposition on the lung endothelium of influenza-infected
mice compared to that of uninfected controls. Fibronectin is stained green. Control group, n ⫽ 3 mice; flu group, n ⫽ 5 mice. In all experiments, images are
representative of 10 (⫻200 magnification) fields per lung. *, P ⬍ 0.05.

infected endothelial monolayers, we observed almost complete this is in association with lung edema and alveolar neutrophil
abrogation of platelet-endothelial adhesion (Fig. 4A). The inhib- recruitment (7, 38), akin to severe influenza pneumonia that oc-

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itory ability of RGDS peptide suggested a role for endothelial fi- curs in humans. This strain of the virus is also capable of infecting
bronectin or possibly fibrinogen in mediating platelet adhesion. mouse lung endothelium in vitro (13); furthermore, we observed
We observed a dose-dependent reduction in monomeric fi- colocalization of viral NP and the endothelial membrane protein
bronectin in endothelial lysates from infected cells (Fig. 4D and E) PECAM-1 (CD31) in the lung sections of infected mice; the degree
coincident with a marked increase in multimeric fibronectin of colocalization increased significantly in a viral dose-dependent
(Fig. 4F) and a significant increase in endothelial fibrinogen (Fig. manner (Fig. 5). At a dose of the virus causing 100% mortality
4G); however, the latter was not dose dependent. To further es- within 8 days, over 25% of the viral NP colocalized with the en-
tablish the involvement of integrins, we pretreated platelets with dothelial marker. Examination by immunohistochemistry 5 days
antibodies blocking the platelet integrin glycoprotein ␣5␤1, which after infection revealed marked platelet deposition in the lungs of
recognizes fibronectin and fibrinogen. Anti-␣5␤1 antibodies sig- influenza-infected mice compared to that of uninfected controls
nificantly attenuated virus-induced platelet adhesion (Fig. 4C). (Fig. 6A). The lungs of influenza-infected mice also displayed a
This was not a nonspecific effect of the antibody, since an antibody marked increase in fibronectin (Fig. 6B), mirroring the in vitro
to another platelet integrin, ␣IIb␤3, had no effect (Fig. 4B). While findings.
knockdown of endothelial fibronectin using short interfering To determine the importance of platelet-endothelial inter-
RNA (siRNA) was achieved, unfortunately exposure to both con- actions to the pathophysiology of influenza-induced acute lung
trol (scrambled) and targeted siRNA prevented influenza-in- injury, we administered acetylsalicylic acid (ASA) to mice si-
duced platelet-endothelial adhesion (not shown), confounding multaneously with the onset of infection. ASA inhibits platelet
the experiment. cyclooxygenase, preventing the synthesis of thromboxane A2 and
Mice infected with influenza demonstrate dose-dependent thereby impairing platelet function (39). Treatment of flu-in-
colocalization of viral nucleoprotein and PECAM-1 as well as fected endothelial cells with ASA resulted in a significant reduc-
platelet and fibronectin deposition in the lung. To validate our in tion in platelet-endothelial interaction (Fig. 7A). Under these con-
vitro data, we infected mice intranasally with influenza A virus at a ditions, ASA significantly improved survival from influenza (Fig.
dose that results in 100% mortality by about 7 days after infection; 7B) and decreased arterial hypoxemia (Fig. 7C). Contrary to pre-

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Sugiyama et al.

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FIG 7 ASA treatment improves survival following severe influenza virus infection. (A) Influenza-induced platelet-endothelial interaction in vitro is attenuated
by treatment with ASA. n ⫽ 4 experiments. *, P ⬍ 0.01 for the control versus flu groups. (B) Kaplan-Meier survival curves showing that ASA treatment at the time
of influenza virus infection improves survival in mice infected with a severe dose of X31 H3N2 influenza virus. Flu group, n ⫽ 6; flu/ASA group, n ⫽ 7 mice; *,
P ⬍ 0.05 by Mantel-Cox test. (C) ASA treatment is associated with improved arterial oxygen saturation 5 days after influenza infection. This time point was
chosen as mice die shortly after. *, P ⬍ 0.05. (D) ASA treatment of influenza-infected mice has no effect on lung viral titer, as measured 3 days after influenza
infection. (E) Kaplan-Meier survival curves showing that ASA treatment in combination with the antiviral drug amantadine (Amant) significantly improves
survival following severe influenza infection in mice. Flu group, n ⫽ 5 mice; flu/Amant group, n ⫽ 11 mice; flu/Amant/ASA group, n ⫽ 12 mice; **, P ⬍ 0.01 by
Mantel-Cox test. (F) Platelet inhibition by ticlopidine (Ticlid) in combination with amantadine improves survival. Flu/Amant group, n ⫽ 6 mice; flu/Amant/
Ticlid group, n ⫽ 5 mice.

vious reports, the benefit was not due to a direct or indirect effect pidine administered to mice at the time of infection resulted in a
on viral replication (40, 41), as viral titers from lung homogenates trend toward increased survival (Fig. 7F).
were unchanged by treatment with ASA (Fig. 7D).
To more closely mimic the clinical situation, in which a drug DISCUSSION
like ASA would be given as an adjuvant, we repeated the experi- Acute lung injury, which manifests clinically as the acute respira-
ment but administered ASA in conjunction with the antiviral drug tory distress syndrome (ARDS), is a common cause of mortality
amantadine. While amantadine alone exhibited a trend toward from severe influenza. Even in combination with optimal sup-
modestly improved survival, adjuvant therapy with ASA increased portive care, therapy with antiviral drugs does not completely
survival from 0 to approximately 80% (Fig. 7E). Finally, to estab- prevent mortality (8), indicating that new therapeutic ap-
lish the beneficial effect of platelet inhibition by a second method, proaches are needed. Given reports that microthombi are ob-
we tested ticlopidine, a drug that inhibits platelet function via a served in the lungs of patients with severe influenza (10, 19)
different pathway (ADP receptor inhibition). As with ASA, ticlo- and that platelet inhibition ameliorates certain forms of lung

1820 jvi.asm.org Journal of Virology February 2016 Volume 90 Number 4

 Influenza Virus Induces Platelet-Endothelial Adhesion

injury (14, 15), we hypothesized that the influenza virus in- fection (53) and the proposed role of platelets in regulating innate
duces platelet adhesion to the lung endothelium and that this immunity (18, 51), it is at least theoretically possible that platelet
process contributes to the pathology of severe influenza. inhibition is harmful (54). Instead, we found that treatment with
In this study, we have shown using both laboratory and clinical ASA improved survival, increased arterial oxygenation, and did
isolates that influenza virus infection of the lung endothelium can not impair viral clearance in vivo. Furthermore, the synergistic
induce a dose-dependent adhesion of platelets; this effect was in- benefit of combined antiplatelet and antiviral therapy strongly
dependent of endothelial apoptosis and viral replication and was supports the notion that targeting the host rather than the patho-
not recapitulated by purified hemagglutinin or neuraminidase. gen is a useful strategy in the treatment of severe influenza (55).
Our data are most consistent with adhesion being induced by In conclusion, the infection of lung microvascular endothelial
paracrine mediators that are elaborated in response to infection, cells by human influenza is capable of inducing platelet adhesion
although the identity of this factor or factors currently is being via integrin binding. Using a murine model of severe influenza,
investigated. Virus-induced platelet adhesion is dependent on the inhibition of platelets significantly improves mortality, and
platelet integrins and is likely mediated by interaction with endo- this effect is even greater when combined with an antiviral drug.
thelial fibronectin and/or fibrinogen. Fibronectin exists in both The wide availability of antiplatelet agents suggests that this ap-
circulating and cellular forms, and it is the latter that exhibits a proach holds promise for the therapy of severe influenza infec-
tendency to multimerize (42); in turn, multimerized fibronectin is tions, although further study is required.
thought to be more thrombogenic (43). It is important to ac-
knowledge that while antibody blockade of vWF and ICAM-1 did ACKNOWLEDGMENTS
not attenuate in vitro platelet adhesion, this does not exclude their We thank Chris Spring and Caterina Di Ciano-Oliveria from the core
potential involvement in vivo; platelet adhesion in vivo is complex facilities of the Keenan Research Centre for Biomedical Science for tech-
and multifactorial, and our in vitro mechanistic approach is nec- nical help.
essarily reductionist. A particular issue that is the subject of ongo- This work was supported by an operating grant from the Canadian
ing work is the role of shear stress, since vWF is known to play a Institutes of Health Research (CIHR MOP 130564), bridge funds from the
critical role in platelet tethering under conditions of flow. Institute of Circulatory and Respiratory Health at the CIHR (OCN
We and others have previously reported the infection of endo- 126577), and an Early Researcher Award from the Government of On-
tario, all to W.L.L. M.G.S. was supported by a Queen Elizabeth II Gradu-
thelial cells in vitro by human influenza (13), a phenomenon that
ate Scholarship in Science and Technology and a graduate award from the
also occurs in vivo in the lungs of infected mice and which we now Ted Rogers Centre for Heart Research. R.Z. is supported by the CREMS
show increases with increasing viral inoculum. However, the Research Scholar program at the University of Toronto.
functional consequences of endothelial infection by influenza are
largely unexplored. Using multiple strains of the virus, our data FUNDING INFORMATION
clearly indicate that infection of endothelial cells is sufficient to Government of Ontario Early Researcher Award provided funding to
induce platelet adhesion. Our model does not preclude a role for Warren Lester Lee under grant number ERA-07. This work was funded by
other stimuli besides endothelial infection to induce platelet-en- Gouvernement du Canada | Canadian Institutes of Health Research
dothelial adhesion. For instance, influenza infection recently has (CIHR) under grant MOP130564. This work was funded by Gouverne-
been reported to induce lung epithelial cytokine production, in- ment du Canada | Canadian Institutes of Health Research (CIHR) under
cluding that of interleukin-6 (IL-6), IL-8, and granulocyte-mac- grant OCN 126577.
rophage colony-stimulating factor (GM-CSF) (44, 45, 46). Simi-
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