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SARS-CoV-2 binds platelet ACE2 to enhance thrombosis in COVID-19

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Background: Critically ill patients diagnosed with COVID-19 may develop a pro-thrombotic state that places them at a dramatically increased lethal risk. Although platelet activation is critical for thrombosis and is responsible for the thrombotic events and cardiovascular complications, the role of platelets in the pathogenesis of COVID-19 remains unclear. Methods: Using platelets from healthy volunteers, non-COVID-19 and COVID-19 patients, as well as wild-type and hACE2 transgenic mice, we evaluated the changes in platelet and coagulation parameters in COVID-19 patients. We investigated ACE2 expression and direct effect of SARS-CoV-2 virus on platelets by RT-PCR, flow cytometry, Western blot, immunofluorescence, and platelet functional studies in vitro, FeCl3-induced thrombus formation in vivo, and thrombus formation under flow conditions ex vivo. Results: We demonstrated that COVID-19 patients present with increased mean platelet volume (MPV) and platelet hyperactivity, which correlated with a decrease in overall platelet count. Detectable SARS-CoV-2 RNA in the blood stream was associated with platelet hyperactivity in critically ill patients. Platelets expressed ACE2, a host cell receptor for SARS-CoV-2, and TMPRSS2, a serine protease for Spike protein priming. SARS-CoV-2 and its Spike protein directly enhanced platelet activation such as platelet aggregation, PAC-1 binding, CD62P expression, α granule secretion, dense granule release, platelet spreading, and clot retraction in vitro, and thereby Spike protein enhanced thrombosis formation in wild-type mice transfused with hACE2 transgenic platelets, but this was not observed in animals transfused with wild-type platelets in vivo. Further, we provided evidence suggesting that the MAPK pathway, downstream of ACE2, mediates the potentiating role of SARS-CoV-2 on platelet activation, and that platelet ACE2 expression decreases following SARS-COV-2 stimulation. SARS-CoV-2 and its Spike protein directly stimulated platelets to facilitate the release of coagulation factors, the secretion of inflammatory factors, and the formation of leukocyte-platelet aggregates. Recombinant human ACE2 protein and anti-Spike monoclonal antibody could inhibit SARS-CoV-2 Spike protein-induced platelet activation. Conclusions: Our findings uncovered a novel function of SARS-CoV-2 on platelet activation via binding of Spike to ACE2. SARS-CoV-2-induced platelet activation may participate in thrombus formation and inflammatory responses in COVID-19 patients.
ncreased platelet activation in patients with SARS-CoV-2 infection. a-f Dot plot showing the correlation between platelet count and PT (a), platelet count and PTA (b), platelet count and INR (c), platelet count and APTT (d), platelet count and D-dimer (e), as well as platelet count and FDPs (F) in COVID-19 patients (n = 241). Each circle represents a different patient. g Dynamics of platelet count in COVID-19 patients with critically severe illness after hospital admission. The platelet counts values were obtained from 22 independent patients. Different colors were used for different patients. h Increased expression of platelet integrin αIIbβ3 activation (PAC-1 binding) and P-selectin (CD62P) expression in COVID-19 patients compared with healthy 11 donors and non-COVID-19 patients. Each circle represents a different individual from healthy donors (n = 166), non-COVID-19 cases (n = 60), the mild and moderate COVID-19 cases (n = 184) or the severe and critically severe COVID-19 cases (n = 57). I, PAC-1 binding and CD62P expression are correlated with platelet count in COVID-19 patients (n = 241). Each solid circle represents a different individual. j PAC-1binding and CD62P expression in severe and critically severe type COVID-19 patients with detectable blood virus RNA (detectable, n = 12) and with undetectable blood virus RNA (undetectable, n = 45). Statistical analyses were performed using Kruskal-Wallis test with Bonferroni correction in (h), Pearson's correlation analysis in (i) and nonparametric Mann-Whitney U test in (j). NS no significance; **P < 0.01. PT prothrombin time, PTA prothrombin time activity, INR international normalized ratio, APTT activated partial thromboplastin time, FDPs fibrinogen degradation products, undetectable: severe and critically severe type COVID-19 patients with undetectable blood virus RNA, detectable: severe and critically severe type COVID-19 patients with detectable blood virus RNA
… 
Both human 1 and mouse platelets express ACE2 and TMPRSS2. A, RT2 PCR detection of ACE2 (A1) and monocyte-specific CD14 (A2) in healthy human platelets. B, Western blot detection of ACE2 and monocyte-specific CD14 (B2) in healthy human platelets. For A and B, the human colon cell line Caco-2 and the human lung cell line Calu-3 were used as positive controls of ACE2, and the human Hela cell line was used as a negative control of ACE2. The peripheral blood mononuclear cells (PBMCs) from healthy human were used as a positive control of CD14. C, RT-PCR detection of ACE2 (C1) in lungs, hearts, and platelets from wild-type mice. D, Western blot detection of ACE2 in lungs, hearts, and platelets from wild-type mice. For C and D, PBMCs from mice were used as a positive control of CD14. E, RT-PCR detection of TMPRSS2 in platelets from healthy human and wild-type mice. F, Western blot detection of TMPRSS2 in platelets from healthy human and wild-type mice. For E and F, the colon cell line Caco-2 and the human lung cell line Calu-3 from human and the lungs from mice were used as positive controls of TMPRSS2, and the human prostate cell line PC-3 was used as a negative control of TMPRSS2. For A to F, platelet-rich plasma prepared as previously described was filtered through a Sepharose 2B column equilibrated in Tyrode’s solution to isolate platelets. Platelets1 in A, B, E left panel and F left panel were platelets from 1 healthy blood sample and platelets2 in A, B, E left panel and F left panel were platelets mixture from 20 healthy donors. Platelets1 in C, D, E right panel and F right panel were platelets from 1 wild-type mouse and platelets2 in C, D, E right panel and F right panel were platelets mixture from 5 wild-type mice. PBMCs were isolated by centrifugation on a Ficoll-Paque from two different blood samples of healthy donors (PBMCs1 and PBMCs2 in A and B) and from two different blood samples of wild-type mice (PBMCs1 and PBMCs2 in C and D). The two different lung (lung1 and lung2 in C, D, E and F) and heart (heart1 and heart2 in C and D) tissues were dissected from different wild-type mice. G, Western blot detection of ACE2 and TMPRSS2 in megakaryocyte cell line (Meg-01). H, Detecting ACE2 and TMPRSS2 expression on healthy human and wild-type mice platelets by flow cytometry. I, Imaging of ACE2 (I1) and TMPRSS2 (I2) expression in healthy human platelets using confocal microscopy. ACE2, the angiotensin converting enzyme 2; TMPRSS2, transmembrane protease serine 2; and RT-PCR, reverse transcription polymerase chain reaction. Images were representative of three independent RT-PCR, Western blot or flow cytometry experiments
… 
SARS-CoV-2 directly enhances 1 platelet activation in vitro. a SARS-CoV-2 dose-dependently potentiated platelets aggregation and ATP release in response to collagen, thrombin, and ADP in vitro. Washed platelets from healthy donors were preincubated with SARS-CoV-2 in the indicated concentration for 30 min, then stimulated with collagen (0.6 μg/mL), thrombin (0.025 U/mL), or ADP (5 μM). Aggregation and ATP release (with luciferase) were assessed under stirring at 1200 rpm. Representative results and summary data of 4 experiments are presented. b SARS8 CoV-2 induced PAC-1 binding and CD62P expression in the absence of agonist; and potentiated integrin PAC-1 binding and CD62P expression induced by thrombin in platelets. Platelets were preincubated with SARS-CoV-2 virus (1 × 10⁵ PFU, 60 min) or with SARS-CoV-2 virus (1×10⁵ PFU, 30 min), and treated with thrombin (0.025 U/mL, 10 min), and then analyzed using a flow cytometer. Representative flow cytometry histograms and summary data of 5 experiments are presented. c Representative confocal fluorescence images (phalloidin) showing that SARS-CoV-2 potentiated platelet spreading on immobilized fibrinogen (100 μg/mL). After preincubation with SARS-CoV-2 (1 × 10⁵ PFU) for 30 min, platelets were allowed to spread on the fibrinogen-coated surfaces at 37 °C for indicated times. Representative results and summary data of 4 experiments are presented. d SARS-CoV-2 potentiated clot retraction induced by thrombin. Platelets from healthy donors were normalized at a concentration of 4 × 10⁸/mL and preincubated with SARS-CoV-2 (1 × 10⁵ PFU) for 30 min, then stimulated with thrombin (1 U/mL). Representative results and summary data of 4 experiments are presented. e Immunofluorescent staining of Nucleocapsid protein (NP, red) and CD41 (green) in human platelets incubated with SARS-CoV-2 virus (1 × 10⁵ PFU) for 3 h. Representative images from 3 experiments using platelets from different healthy donors. f Scanning electron microscope (SEM) of SARS-CoV-2 particles on the surface of platelets. SEM of healthy human platelets (3 × 10⁸ platelets/mL) incubated with SARS-CoV-2 (1 × 10⁵ PFU) for 30 min. Platelets were washed for 3 times and fixed immediately after incubation and processed for SEM experiment. Representative images of single platelet from control group (platelet1) and SARS-CoV-2 treatment group (platelet2 and platelet3) are shown from three different experiments. Arrows point toward the SARS-CoV-2 virus. g. Transmission electron microscopy (TEM) of SARS-CoV-2 particles in platelets. TEM of healthy human platelets (3 × 10⁸ platelets/mL) incubated with SARS-CoV-2 (1 × 10⁵ PFU) for 3 h. Platelets were washed for 3 times and fixed immediately after incubation and processed for TEM experiment. Representative images from control group (platelet1) and SARS CoV-2 treatment group (platelet2 and platelet3) are shown from three different experiments. Arrows point toward the SARS-CoV-2 particles. Statistical analyses were performed using unpaired two-tailed Student’s t test in (a), (b) and (c). NS no significance; *P < 0.05; **P < 0.01. Two-way ANOVA and Tukey’s post hoc test was performed in (d); *P < 0.05 and **P < 0.01 compared with control group
… 
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R E S E A R C H Open Access
SARS-CoV-2 binds platelet ACE2 to enhance
thrombosis in COVID-19
Si Zhang
2*
, Yangyang Liu
1
, Xiaofang Wang
1
, Li Yang
3
, Haishan Li
4
, Yuyan Wang
5
, Mengduan Liu
1
,
Xiaoyan Zhao
1
, Youhua Xie
5
, Yan Yang
6
, Shenghui Zhang
7
, Zhichao Fan
8
, Jianzeng Dong
1
, Zhenghong Yuan
5
,
Zhongren Ding
1
, Yi Zhang
3*
and Liang Hu
1*
Abstract
Background: Critically ill patients diagnosed with COVID-19 may develop a pro-thrombotic state that places them
at a dramatically increased lethal risk. Although platelet activation is critical for thrombosis and is responsible for the
thrombotic events and cardiovascular complications, the role of platelets in the pathogenesis of COVID-19 remains
unclear.
Methods: Using platelets from healthy volunteers, non-COVID-19 and COVID-19 patients, as well as wild-type and
hACE2 transgenic mice, we evaluated the changes in platelet and coagulation parameters in COVID-19 patients. We
investigated ACE2 expression and direct effect of SARS-CoV-2 virus on platelets by RT-PCR, flow cytometry, Western
blot, immunofluorescence, and platelet functional studies in vitro, FeCl
3
-induced thrombus formation in vivo, and
thrombus formation under flow conditions ex vivo.
(Continued on next page)
© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data.
* Correspondence: fcchul@zzu.edu.cn;zhangsi@fudan.edu.cn;
yizhang@zzu.edu.cn
Si Zhang, Yangyang Liu, Xiaofang Wang, and Li Yang contributed equally to
this work.
2
Department of Biochemistry and Molecular Biology, NHC Key Laboratory of
Glycoconjugates Research, School of Basic Medical Sciences, Fudan
University, Shanghai 200032, China
3
Biotherapy Center, the First Affiliated Hospital of Zhengzhou University,
Zhengzhou 450052, China
1
Department of Cardiology, the First Affiliated Hospital of Zhengzhou
University, Cardiovascular Institute of Zhengzhou University, Zhengzhou
450052, China
Full list of author information is available at the end of the article
Zhang et al. Journal of Hematology & Oncology (2020) 13:120
https://doi.org/10.1186/s13045-020-00954-7
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
(Continued from previous page)
Results: We demonstrated that COVID-19 patients present with increased mean platelet volume (MPV) and platelet
hyperactivity, which correlated with a decrease in overall platelet count. Detectable SARS-CoV-2 RNA in the blood
stream was associated with platelet hyperactivity in critically ill patients. Platelets expressed ACE2, a host cell
receptor for SARS-CoV-2, and TMPRSS2, a serine protease for Spike protein priming. SARS-CoV-2 and its Spike
protein directly enhanced platelet activation such as platelet aggregation, PAC-1 binding, CD62P expression, α
granule secretion, dense granule release, platelet spreading, and clot retraction in vitro, and thereby Spike protein
enhanced thrombosis formation in wild-type mice transfused with hACE2 transgenic platelets, but this was not
observed in animals transfused with wild-type platelets in vivo. Further, we provided evidence suggesting that the
MAPK pathway, downstream of ACE2, mediates the potentiating role of SARS-CoV-2 on platelet activation, and that
platelet ACE2 expression decreases following SARS-COV-2 stimulation. SARS-CoV-2 and its Spike protein directly
stimulated platelets to facilitate the release of coagulation factors, the secretion of inflammatory factors, and the
formation of leukocyteplatelet aggregates. Recombinant human ACE2 protein and anti-Spike monoclonal antibody
could inhibit SARS-CoV-2 Spike protein-induced platelet activation.
Conclusions: Our findings uncovered a novel function of SARS-CoV-2 on platelet activation via binding of Spike to
ACE2. SARS-CoV-2-induced platelet activation may participate in thrombus formation and inflammatory responses
in COVID-19 patients.
Keywords: COVID-19, Thrombosis, Platelet activation, ACE2, TMPRSS2
Background
The COVID-19 pandemic has become a serious public
health crisis worldwide since December 2019 [1]. COVID-
19 has been linked to a number of critical cardiovascular
complications [2,3], and even individuals without a his-
tory of cardiovascular disease are at risk of cardiovascular
complications [4]. Patients with severe COVID-19 com-
monly experience thrombotic disorders, sepsis, and dis-
seminated intravascular coagulation (DIC), and these
conditions have been closely linked to higher mortality
rates [1,5,6]. Large-scale studies have revealed that 18.8%
to 36.2% of patients [7,8] present with thrombocytopenia
on admission. In addition, the cumulative incidence of
thrombotic complications for COVID-19 patients in the
ICU was 31%, while only 1.3% of non-COVID-19 ICU pa-
tients experience thrombotic complications [9]. Although
the evidence supports a link between COVID-19 and the
development of a hypercoagulable state, the underlying
mechanisms for this association remain elusive.
Platelets are known for their critical contributions to
thrombosis and hemostasis [1012]. During infection,
activated platelets adhere to the sub-endothelium, and
their hyperactivity results in thrombus formation, lead-
ing to arterial ischemia and even pulmonary embolisms.
Many viruses, including human immunodeficiency virus
(HIV), hepatitis C virus (HCV), influenza virus, Ebola,
and Dengue virus (DV), can directly lead to platelet
hyperactivity [1316]. Influenza virus directly activates
platelets and triggers uncontrolled coagulation cascades
and consequent lung injury [1719]. Although COVID-
19 is a respiratory disease, SARS-CoV-2 RNA can be de-
tected in the blood and used as an indicator of disease
severity [20,21]. Currently, whether the COVID-19 virus
can directly activate platelets, and therefore promote its
pro-thrombotic function remains unclear.
The pathogen causing COVID-19 is severe acute re-
spiratory syndrome coronavirus 2 (SARS-CoV-2), an
enveloped RNA virus, and the seventh member of the hu-
man coronavirus family [22]. SARS-CoV-2 uses its Spike
protein to enter host cells by binding to angiotensin-
converting enzyme 2 (ACE2) on the host cell membrane
[2326]. Meanwhile, transmembrane protease serine 2
(TMPRSS2), a serine protease, proteolytically cleaves and
activates the Spike protein to facilitate SARS-CoV-2 virus-
cell membrane fusions. Although the Spike protein from
SARS-CoV-2 has been reported to bind to ACE2 and ma-
nipulate various cell functions [2731], it has not been ad-
dressed if platelets express ACE2 and TMPRSS2.
Here, we report that platelets from COVID-19 patients
are hyperactive, and demonstrate, for the first time, that
platelets express ACE2 and TMPRSS2. SARS-CoV-2 and
its Spike protein directly bind platelet ACE2 and en-
hance platelet activation in vitro. The Spike protein also
potentiates thrombus formation in vivo. Moreover, we
were able to demonstrate that SARS-CoV-2 and its
Spike protein directly stimulate platelets resulting in co-
agulation factor release, inflammatory cytokine secretion,
and leukocyteplatelet aggregates (LPAs) formation. Fi-
nally, we provide evidence that treatment with recom-
binant human ACE2 protein and an anti-Spike
monoclonal antibody can reverse SARS-CoV-2 Spike
protein-induced platelet activation.
Methods
Detailed materials and methods are described in Add-
itional file 1: Expanded Materials and Methods.
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 2 of 22
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Study design
For this study, we recruited COVID-19 patients admit-
ted to the First Affiliated Hospital of Zhengzhou Univer-
sity, Henan Province, China and three centers of the
Affiliated Hospital of Anhui Medical University, Anhui
Province, China between January 10th and March 18th
2020. The detailed study design, including the inclusion
and exclusion criteria as well as laboratory data collec-
tion, is described in Additional file 1: Expanded Mate-
rials and Methods. This study was approved by the
Ethics Committee of the First Affiliated Hospital of
Zhengzhou University (2020-KY-121) and the Ethics
Committee of Anhui Medical University (2020-AH-114)
and complied with the Declaration of Helsinki and good
clinical practice guidelines. All participants provided
written informed consent.
Materials
Detailed descriptions of the reagents, commercial ELISA
kits, and viral RNA detection in the blood are provided
in Additional file 1: Expanded Materials and Methods.
The primary antibodies and dilutions used in this study
are listed in Additional file 1: Online Table 1.
Washed platelets and peripheral blood mononuclear cells
isolation
Platelets and peripheral blood mononuclear cells
(PBMCs) were prepared [32] as detailed in Additional
file 1: Expanded Materials and Methods. All experiments
using human subjects were performed in accordance
with the Declaration of Helsinki and approved by the
Ethics Committee of Zhengzhou University.
Platelet functional studies
Washed platelet aggregation and secretion in response
to thrombin (0.025 U/mL) or collagen (0.6 μg/mL) as
well as washed platelet aggregation in response to ADP
(5 μM with 10 μg/mL fibrinogen) were measured as pre-
viously described [33]. Spreading and clot retraction
were measured as previously described [34,35] and de-
tailed in Additional file 1: Expanded Materials and
Methods. To explore the effects of SARS-CoV-2 and its
Spike protein on platelet function, SARS-CoV-2 (1×10
5
PFU [36]) or Spike protein (2 μg/mL) was added to
platelets for the indicated times before agonist-induced
stimulation [3739].
Cell culture
The human colon cell line Caco-2, the human lung cell
line Calu-3, the human prostate cell line PC-3, and the
human cervical carcinoma cell line HeLa were all cul-
tured in the appropriate medium detailed in Additional
file 1: Expanded Materials and Methods. All cells were
collected and processed for RNA and protein extraction.
SARS-CoV-2 virus preparation and incubation
SARS-CoV-2 virus was isolated from a COVID-19 pa-
tient in Shanghai (GenBank accession No. MT121215)
and propagated in Vero E6 cells [23]. The supernatant
from the SARS-CoV-2 infected cells was aliquoted and
stored at 80 °C until use. The supernatant from mock-
infected cells was used as a control. For the platelet
functional studies, around 300 μL of platelets (2 × 10
8
/
mL) were incubated with 1 × 10
5
PFU SARS-CoV-2 in
Tyrodes buffer. The infectious titers have been used in
other cells [36]. All experiments involving live SARS-
CoV-2 virus were performed in a biosafety level-3 (BLS-
3) laboratory.
Reverse transcription polymerase chain reaction
Total RNA was isolated from different cells and 1 μgof
RNA was reverse transcribed to cDNA using an RNA
isolation kit and polymerase chain reaction (RT-PCR) kit
(TaKaRa, Japan), respectively [35]. PCR reactions were
performed using specific primers (Additional file 1: On-
line Table 2).
Flow cytometry analysis
Flow cytometry was used to evaluate platelet activity in
COVID-19 patients, non-COVID-19 patients and
healthy volunteers, and to evaluate platelet activity and
ACE2 and TMPRSS2 expression in SARS-CoV-2 virus
or Spike protein treated healthy platelets and leukocyte-
platelet aggregates. All of these assays were completed
using the previously described methods [23,40,41], and
detailed in Additional file 1: Expanded Materials and
Methods. The primary antibodies and dilutions used in
these assays are listed in Additional file 1: Online Table
1.
Electron microscopy
Sample preparation [42]
Platelet samples were incubated with SARS-CoV-2 and
submitted to scanning electron microscopy (SEM) and
transmission electron microscopy (TEM) as previously
described. Briefly, platelets were placed in 6-well plates
and incubated with 1 × 10
5
PFU SARS-CoV-2 in Tyr-
odes buffer under constant rotation for 30 min or 3 h at
37 °C. Platelets were then washed three times with PBS
to remove virus and subjected to SEM and TEM as de-
scribed previously [43], and detailed in the Additional
file 1: Expanded Materials and Methods.
Co-immunoprecipitation
Washed platelets subjected to different treatments were
lysed with equal volumes of chilled 2× NP-40 lysis buffer
(100 mM Tris-HCl pH 7.4, 300 mM NaCl, 2 mM NaF,
2% NP-40, 2 mM EDTA, and 2× protease and phosphat-
ase inhibitor solution) on ice for 30 min. The
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 3 of 22
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
supernatants were then precleared using protein A/G
agarose beads for 3 h at 4 °C and then centrifuged. Im-
munoprecipitation was carried out using an anti-
phospho-Ser/Thr antibody and then incubated with pro-
tein A/G-agarose beads overnight on a rocker at 4 °C.
The beads were then harvested and rinsed 3 times with
1× NP-40 lysis buffer. Bead-captured ACE2 was detected
by immunoblot and the primary antibodies and dilutions
used in these assays are listed in Additional file 1: Online
Table 1.
Confocal microscopy
Platelets were attached to poly-L-Lysine-coated cover-
slips and fixed with precooled methanol and then
blocked with BSA in PBS. After incubation with the pri-
mary antibodies and appropriate secondary antibodies,
confocal images were captured using a laser-scanning
confocal microscope as previously described [44] and de-
tailed in Additional file 1: Expanded Materials and
Methods. The primary antibodies and dilutions used in
these assays are listed in Additional file 1: Online Table 1.
ELISA assays
PF4, TNF-α, IL-8, IL-1βFactor V, and Factor XIII con-
centrations were determined using commercial ELISA
kits according to the manufacturers instructions. More
information is provided in Additional file 1: Expanded
Materials and Methods.
Animal studies
Wild-type C57BL/6 mice and hACE2 transgenic mice
(Cat No. T037657) were purchased from Jiangsu Gem-
pharmatech, China. All animal procedures were carried
out in accordance with the ethical approval granted by
the Ethics Committee of Zhengzhou University. Mouse
platelets were prepared as described previously [35].
Adoptive platelet transfusions were performed based on
previous reports [45]. More information is provided in
Additional file 1: Expanded Materials and Methods.
FeCl
3
-induced thrombosis formation in mouse mesenteric
arterioles
Intravital microscopy of FeCl
3
-injured thrombus forma-
tion in mouse mesenteric arterioles was performed as
previously described with minor modifications [34,46,
47]. SARS-CoV-2 Spike protein (200 μg/kg) was injected
intravenously into wild-type mice according to a previ-
ously reported method with minor modifications [30].
More information is provided in Additional file 1:Ex-
panded Materials and Methods.
Thrombus formation under flow conditions ex vivo
The flow chamber assay was prepared as described pre-
viously with minor modification [44,48], and detailed in
Additional file 1: Expanded Materials and Methods.
Statistical analysis
Given the inherent differences between patients, a series
of propensity score analyses were performed for the fol-
lowing variables: age, sex, history of smoking, hyperten-
sion, diabetes mellitus, hypercholesterolemia, stroke, and
COPD. When matched with the healthy group, variables
included age, sex, and history of smoking; when matched
with other groups, variables included age, sex, history of
smoking, hypertension, diabetes mellitus, hypercholes-
terolemia, stroke, and COPD. The propensity scores
were estimated using a logit model. Matching was con-
ducted using 1:1 or 1:2 nearest neighbor methods with a
caliper width of 0.25*SDs in the logit propensity score,
which yielded 28 severe and critically severe COVID-19
patients matched with 56 healthy subjects, 37 severe and
critically severe COVID-19 patients matched with 37
non-COVID-19 patients, 29 severe and critically severe
COVID-19 patients matched with 58 mild and moderate
COVID-19 patients, 64 mild and moderate COVID-19
patients matched with 64 healthy subjects, and 59 non-
COVID-19 subjects matched with 115 mild and moder-
ate subjects. All matching analyses were performed using
R software (version 3.6.0).
Continuous variables are expressed as the mean ±
standard deviation of the mean (SD) or median (inter-
quartile range was defined as the difference between
twenty-fifth and seventy-fifth centiles) depending on
data distribution, and compared using an unpaired Stu-
dentsttest or Mann-Whitney Utest, as appropriate.
Categorical variables were expressed as numbers and
percentages and compared using the Chi-square test or
Fishers exact test. All data were evaluated for normality
(Kolmogorov-Smirnov) and subjected to the Bartletts
test for homogeneity of group variances prior to statis-
tical analysis. Group comparisons were made using one-
way ANOVA, followed by Tukeys post hoc analysis or
Kruskal-Wallis test with Bonferroni correction. Pearsons
correlation analysis was used to investigate the relation-
ships between two variables. A Pvalue of less than 0.05
was considered statistically significant. Statistical ana-
lyses were performed using SPSS (version 21.0) and
GraphPad Prism (7.0).
Results
Increased platelet activation in COVID-19
Our study population comprised 201 healthy volunteers
and 589 patients suspected of having COVID-19. Of
these 589 patients, 422 were identified as SARS-CoV-2
infected, while the remaining 167 patients were
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 4 of 22
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
identified as non-SARS-CoV-2 infected. We excluded 35
healthy volunteers, 107 non-COVID-19 patients, and
181 COVID-19 patients leaving a total of 166 healthy
volunteers, 60 non-COVID-19 patients, and 241
COVID-19 patients including 184 mild and moderate
patients, and 57 severe and critically severe patients in
the study (see Flowchart in Additional file 1: Online Fig-
ure 1). The characteristics of the healthy group, non-
COVID-19 patients, mild and moderate COVID-19 pa-
tients, and severe and critically severe COVID-19 pa-
tients are provided in Additional file 1: Online Table 3.
Severe and critically severe COVID-19 patients pre-
sented with abnormal platelet parameters, including de-
creased platelet counts and plateletcrit (PCT), increased
mean platelet volume (MPV), and platelet distribution
width (PDW) as well as abnormal coagulation parame-
ters including increased prothrombin time (PT), inter-
national normalized ratio (INR), activated partial
thromboplastin time (APTT), D-dimer and fibrinogen
degradation products (FDPs), and decreased prothrom-
bin time activity (PTA) when compared with healthy do-
nors, non-COVID-19 patients, and mild and moderate
COVID-19 patients (Additional file 1: Online Table 3).
Similar results were observed after propensity score
matching of severe and critically severe COVID-19 pa-
tients to the other groups (Additional file 1: Online
Table 4). In addition, mild and moderate COVID-19 pa-
tients presented with decreased platelet counts and in-
creased MPV, compared with healthy donors and non-
COVID-19 patients. Similar results were observed after
propensity score matching (Additional file 1: Online
Table 3 and 5). MPV was shown to correlate with plate-
let activity and is considered a marker of platelet activity
[49,50]; therefore, increased MPV in COVID-19 sug-
gests that these platelets may present as hyperactive.
The relationship between the platelet count and
coagulation parameters in COVID-19 patients is de-
scribed in Fig. 1af. For those with a normal platelet
count (> 125 × 10
9
/L), the platelet count did not signifi-
cantly impact the outcomes of the PT, PTA, INR, APTT,
D-dimer, and FDPs tests. However, in patients with
thrombocytopenia (< 125 × 10
9
/L), when the platelet count
decreases, PT, INR, APTT, D-dimer, and FDPs increase ex-
ponentially, while PTA decreases exponentially. We further
examined platelet count over time in severe and crit-
ically severe COVID-19 patients (n= 22). Our results
suggest that platelet count decreases gradually after
hospital admission (Fig. 1g).
Consistent with the increased MPV, we found that in-
tegrin αIIbβ3 activation (PAC-1 binding) and P-selectin
(CD62P) expression were increased in platelets of
COVID-19 patients (Fig. 1h, Additional file 1: Online
Figure 2). The severe and critically severe COVID-19 pa-
tients presented the highest integrin αIIbβ3 activation
and P-selectin expression on platelets. Platelet activation
leads to platelet consumption which causes
thrombocytopenia [51]; notably, PAC-1 binding and
CD62P expression were both moderately correlated
with decreases in platelet count (Pearson rfor
PAC-1 = 0.50, P<0.01;Pearsonrfor CD62 =
0.64, P< 0.01; Fig. 1i) in COVID-19 patients.
Detectable COVID-19 viral RNA in blood is an indicator of
platelet hyperactivity in severe and critically severe
COVID-19 patients
Of the 241 COVID-19 patients, a total of 15 (6.22%) pa-
tients, including 12 severe and critically severe and 3
mild and moderate cases of COVID-19, were positive for
blood SARS-CoV-2 RNA. In severe and critically severe
patients, patients with detectable blood viral RNA pre-
sented with higher integrin αIIbβ3 activation and P-
selectin expression on platelets, compared with those
with undetectable blood viral RNA (Fig. 1j). To test the
effect of SARS-COV-2 RNA positive blood on platelet
aggregation, we incubated healthy platelets with SARS-
COV-2 RNA-positive platelet-poor plasma (PPP) and
SARS-COV-2 RNA-negative PPP from severe and critic-
ally severe patients, and used healthy PPP as control. We
found that SARS-COV-2 RNA-positive PPP enhanced
platelet aggregation, compared with SARS-COV-2 RNA-
negative PPP and healthy PPP (Additional file 1: Online
Figure 3). These results suggest that the presence of
SARS-CoV-2 viral RNA in blood is an indicator of plate-
let hyperactivity.
Human and mouse platelets express ACE2 and TMPRSS2
Since SARS-CoV-2 infects host cells via ACE2, we ex-
plored whether platelets express ACE2. We found that
human platelets exhibit robust expression of ACE2 at
both the RNA and protein levels as detected by RT-PCR
(Fig. 2(A1)) and Western blot (Fig. 2(B1)). These levels
were similar to the human colon cell line Caco-2 and
the human lung cell line Calu-3, which are used as
SARS-CoV-2-infected host cells [52,53]. The HeLa cell
line was used as a negative control for ACE2 expression
[54]. The detected ACE2 in platelets is not from the con-
taminated PBMCs, because the PBMCs-specific marker
CD14 was not detectable in our platelet samples when eval-
uated by RT-PCR (Fig. 2(A2)) or Western blot (Fig. 2(B2)).
ACE2 level in platelets is comparable with that in
PBMCs (Fig. 2(B2)). Mouse platelets exhibited abun-
dant expression of ACE2, similar to lung and heart tissues
at the RNA (Fig. 2(C)) and protein levels (Fig. 2(D)).
A recent study reported that cellular serine protease
TMPRSS2 (transmembrane protease serine 2) primes
SARS-CoV-2 for cell entry [53]. Interestingly, we de-
tected robust expression of TMPRSS2 in platelets com-
parable with that in human Caco-2 cells, human Calu-3
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 5 of 22
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Fig. 1 Increased platelet activation in patients with SARS-CoV-2 infection. afDot plot showing the correlation between platelet count and PT
(a), platelet count and PTA (b), platelet count and INR (c), platelet count and APTT (d), platelet count and D-dimer (e), as well as platelet count
and FDPs (F) in COVID-19 patients (n= 241). Each circle represents a different patient. gDynamics of platelet count in COVID-19 patients with
critically severe illness after hospital admission. The platelet counts values were obtained from 22 independent patients. Different colors were
used for different patients. hIncreased expression of platelet integrin αIIbβ3 activation (PAC-1 binding) and P-selectin (CD62P) expression in
COVID-19 patients compared with healthy 11 donors and non-COVID-19 patients. Each circle represents a different individual from healthy donors
(n= 166), non-COVID-19 cases (n= 60), the mild and moderate COVID-19 cases (n= 184) or the severe and critically severe COVID-19 cases (n=
57). I, PAC-1 binding and CD62P expression are correlated with platelet count in COVID- 19 patients (n= 241). Each solid circle represents a
different individual. jPAC-1binding and CD62P expression in severe and critically severe type COVID-19 patients with detectable blood virus RNA
(detectable, n= 12) and with undetectable blood virus RNA (undetectable, n= 45). Statistical analyses were performed using Kruskal-Wallis test
with Bonferroni correction in (h), Pearsons correlation analysis in (i) and nonparametric Mann-Whitney Utest in (j). NS no significance; **P< 0.01.
PT prothrombin time, PTA prothrombin time activity, INR international normalized ratio, APTT activated partial thromboplastin time, FDPs
fibrinogen degradation products, undetectable: severe and critically severe type COVID-19 patients with undetectable blood virus RNA,
detectable: severe and critically severe type COVID-19 patients with detectable blood virus RNA
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 6 of 22
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Fig. 2 (See legend on next page.)
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 7 of 22
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cells, and mouse lung tissues at the RNA (Fig. 2(E)) and
protein levels (Fig. 2(F)). Furthermore, abundant ACE2
and TMPRSS2 protein expression was confirmed in
platelet progenitor megakaryocyte cells (Meg-01 cell
line, Fig. 2(G)). Finally, ACE2 and TMPRSS2 protein ex-
pression was further confirmed in human and mouse
platelets using flow cytometry (Fig. 2(H)) and in human
platelets using confocal immunofluorescence (Fig. 2(I)).
SARS-CoV-2 virus directly potentiates platelet activation
After providing evidence that platelets express ACE2
and TMPRSS2, we went on to evaluate whether SARS-
CoV-2 virus could directly activate platelets. We found
that incubation with SARS-CoV-2 (1 × 10
5
PFU [36]) for
30 min did not induce platelet aggregation in human
washed platelets from healthy donors (Additional file 1:
Online Fig. 4), but in the range of 0.1 to 1 × 10
5
PFU,
SARS-CoV-2 dose-dependently potentiated platelet ag-
gregation in response to collagen (0.6 μg/mL), thrombin
(0.025 U/mL), and ADP (5 μmol/L). Consistently, SARS-
CoV-2 increased platelet dense granule secretion (ATP
release) in response to collagen and thrombin in a dose-
dependent manner (Fig. 3a). SARS-CoV-2 induced integ-
rin αIIbβ3 activation and P-selectin expression under
basal conditions and enhanced both integrin αIIbβ3 acti-
vation and P-selectin expression following agonist acti-
vation (Fig. 3b).
We then went on to assess platelet spreading on
immobilized fibrinogen and clot retraction and found
that pre-incubation with SARS-CoV-2 markedly en-
hanced platelet spreading (Fig. 3c) and clot retraction
(Fig. 3d). In addition, we observed SARS-CoV-2 particles
attached to platelet membrane using SEM (Fig. 3f), indi-
cating that SARS-CoV-2 can bind directly to platelets.
Moreover, fluorescent confocal microscopy and TEM
revealed that SARS-CoV-2 particles were present inside
the platelets (Fig. 3e, g), suggesting that SARS-CoV-2
can infect these cells.
SARS-CoV-2 virus directly induces decrease in platelet
ACE2
ACE2 degradation plays an important role in the patho-
genesis of COVID-19. SARS-CoV2 has been reported to
promote ACE2 internalization and subsequent degrad-
ation [55]. Similarly, we found that SARS-CoV-2 in-
duced a time-dependent decrease in ACE2 levels in
platelets (Additional file 1: Online Figure 5a), indicating
the degradation of ACE2 in platelets upon ACE2 activa-
tion. Consistently, we found that ACE2 levels were de-
creased in platelets from COVID-19 patients, compared
with healthy donors. Severe and critically severe
COVID-19 patients presented the lowest level of ACE2
expression (Additional file 1: Online Figure 5b).
SARS-CoV-2 Spike protein directly potentiates platelet
activation
Since SARS-CoV-2 binds to host cells via interactions
between the Spike protein and ACE2, we sought to ex-
plore whether Spike protein could regulate platelet func-
tion. Incubation with Spike protein for 9 min did not
induce platelet aggregation in human washed platelets
from healthy donors (Additional file 1:OnlineFigure
4). However, similar to the results from the SARS-
CoV-2 virus experiments, we were able to demon-
strate that the Spike protein dose-dependently en-
hanced platelet aggregation and ATP release
(Additional file 1: Online Figure 6). We further found
that SARS-CoV-2 Spike protein (2 μg/mL, 5 min),
Spike protein subunit 1 (S1, 2 μg/mL, 5 min), but not
Spike protein subunit 2 (S2, 2 μg/mL, 5 min),
(See figure on previous page.)
Fig. 2 Both human 1 and mouse platelets express ACE2 and TMPRSS2. A, RT2 PCR detection of ACE2 (A1) and monocyte-specific CD14 (A2) in
healthy human platelets. B, Western blot detection of ACE2 and monocyte-specific CD14 (B2) in healthy human platelets. For A and B, the human
colon cell line Caco-2 and the human lung cell line Calu-3 were used as positive controls of ACE2, and the human Hela cell line was used as a
negative control of ACE2. The peripheral blood mononuclear cells (PBMCs) from healthy human were used as a positive control of CD14. C, RT-
PCR detection of ACE2 (C1) in lungs, hearts, and platelets from wild-type mice. D, Western blot detection of ACE2 in lungs, hearts, and platelets
from wild-type mice. For C and D, PBMCs from mice were used as a positive control of CD14. E, RT-PCR detection of TMPRSS2 in platelets from
healthy human and wild-type mice. F, Western blot detection of TMPRSS2 in platelets from healthy human and wild-type mice. For E and F, the
colon cell line Caco-2 and the human lung cell line Calu-3 from human and the lungs from mice were used as positive controls of TMPRSS2, and
the human prostate cell line PC-3 was used as a negative control of TMPRSS2. For A to F, platelet-rich plasma prepared as previously described
was filtered through a Sepharose 2B column equilibrated in Tyrodes solution to isolate platelets. Platelets1 in A, B, E left panel and F left panel
were platelets from 1 healthy blood sample and platelets2 in A, B, E left panel and F left panel were platelets mixture from 20 healthy donors.
Platelets1 in C, D, E right panel and F right panel were platelets from 1 wild-type mouse and platelets2 in C, D, E right panel and F right panel
were platelets mixture from 5 wild-type mice. PBMCs were isolated by centrifugation on a Ficoll-Paque from two different blood samples of
healthy donors (PBMCs1 and PBMCs2 in A and B) and from two different blood samples of wild-type mice (PBMCs1 and PBMCs2 in C and D). The
two different lung (lung1 and lung2 in C, D, E and F) and heart (heart1 and heart2 in C and D) tissues were dissected from different wild-type
mice. G, Western blot detection of ACE2 and TMPRSS2 in megakaryocyte cell line (Meg-01). H, Detecting ACE2 and TMPRSS2 expression on
healthy human and wild-type mice platelets by flow cytometry. I, Imaging of ACE2 (I1) and TMPRSS2 (I2) expression in healthy human platelets
using confocal microscopy. ACE2, the angiotensin converting enzyme 2; TMPRSS2, transmembrane protease serine 2; and RT-PCR, reverse
transcription polymerase chain reaction. Images were representative of three independent RT-PCR, Western blot or flow cytometry experiments
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 8 of 22
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Fig. 3 (See legend on next page.)
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 9 of 22
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potentiated platelet aggregation and dense granule se-
cretion in response to different agonists (Fig. 4a).
Using flow cytometry, we found that Spike protein in-
duced integrin αIIbβ3 activation and P-selectin ex-
pression in the absence of agonist. Furthermore, Spike
protein and S1, but not S2, enhanced both integrin αIIbβ3
activation and P-selectin expression in the presence of
agonist (Fig. 4b). These data indicate that S1, but not S2,
binds ACE2 to regulate platelet function, which corrobo-
rates the finding that the receptor-binding domain (RBD)
of the Spike protein is found in the S1 subunit [56]. After
incubation with Spike or S1 protein, platelets also dis-
played markedly accelerated spreading (Fig. 4c) and clot
retraction (Fig. 4d).
SARS-CoV-2 directly activates the ACE2/mitogen-activated
protein kinase pathway to potentiate platelet activation
Mitogen-activated protein kinase (MAPK) has been well
documented in platelet activation and thrombosis.
ACE2/MAPK pathway activation has been reported to
mediate SARS-CoV-2-induced cytokine modulation in
lung cells [27,57]. Thus, we examined whether the
ACE2/MAPK pathway is involved in SARS-CoV-2-medi-
ated platelet activation. SARS-CoV-2 and Spike protein
stimulated platelet ACE2 phosphorylation at 15 and 3
min, respectively (Fig. 5(A) and Additional file 1: Online
Figure 7a). MAPK (Erk, p38, and JNK) kinase was phos-
phorylated apparently later than ACE2 phosphorylation
(Fig. 5(A) and Additional file 1: Online Figure 7b). These
results agree with a previous study conducted on lung
cells [27] and supports the hypothesis that the MAPK
pathway is activated downstream of ACE2 in platelets.
We investigated whether MAPK signaling mediates the
potentiating effects of SARS-CoV-2 on platelet activation.
As shown in Fig. 5(B and, C), the inhibition of Erk, p38,
and JNK with PD98059 (10 μM), SB203580 (10 μM), and
SP600125 (10 μM) abolished the potentiating effects of
SARS-CoV-2 (Fig. 5(B)) or Spike protein (Fig. 5(C)) on
agonist-induced platelet aggregation. In addition, we ob-
served that the accelerated clot retraction elicited by
SARS-CoV-2 or Spike protein was also prevented by Erk,
p38, or JNK inhibitors (Fig. 5(D)).
We further detected MAPK phosphorylation in plate-
lets from COVID-19 patients. Our results demonstrated
that COVID-19 patients presented increased phosphor-
ylation of Erk, p38, and JNK in platelets, compared with
healthy donors (Fig. 5).
Together, these data suggest that MAPK is phosphory-
lated downstream of ACE2 and may mediate the po-
tentiating effects of SARS-CoV-2 on platelet activation.
SARS-CoV-2 Spike protein enhances thrombosis potential
in vivo
We then tested whether SARS-COV-2 Spike protein ac-
tivates platelets and thereby enhances thrombus forma-
tion in vivo. To eliminate non-platelet hemostatic or
thrombotic factor variations, we injected 10
9
platelets
from transgenic mice bearing human ACE2 (hACE2) or
wild-type mice into thrombocytopenic wild-type mice.
We simultaneously intravenously injected 200 μg/kg
SARS-CoV-2 Spike protein into mice [30], and examined
(See figure on previous page.)
Fig. 3 SARS-CoV-2 directly enhances 1 platelet activation in vitro. aSARS-CoV-2 dose-dependently potentiated platelets aggregation and ATP
release in response to collagen, thrombin, and ADP in vitro. Washed platelets from healthy donors were preincubated with SARS-CoV-2 in the
indicated concentration for 30 min, then stimulated with collagen (0.6 μg/mL), thrombin (0.025 U/mL), or ADP (5 μM). Aggregation and ATP
release (with luciferase) were assessed under stirring at 1200 rpm. Representative results and summary data of 4 experiments are presented. b
SARS8 CoV-2 induced PAC-1 binding and CD62P expression in the absence of agonist; and potentiated integrin PAC-1 binding and CD62P
expression induced by thrombin in platelets. Platelets were preincubated with SARS-CoV-2 virus (1 × 10
5
PFU, 60 min) or with SARS-CoV-2 virus
(1×10
5
PFU, 30 min), and treated with thrombin (0.025 U/mL, 10 min), and then analyzed using a flow cytometer. Representative flow cytometry
histograms and summary data of 5 experiments are presented. cRepresentative confocal fluorescence images (phalloidin) showing that SARS-
CoV-2 potentiated platelet spreading on immobilized fibrinogen (100 μg/mL). After preincubation with SARS-CoV-2 (1 × 10
5
PFU) for 30 min,
platelets were allowed to spread on the fibrinogen-coated surfaces at 37 °C for indicated times. Representative results and summary data of 4
experiments are presented. dSARS-CoV-2 potentiated clot retraction induced by thrombin. Platelets from healthy donors were normalized at a
concentration of 4 × 10
8
/mL and preincubated with SARS-CoV-2 (1 × 10
5
PFU) for 30 min, then stimulated with thrombin (1 U/mL).
Representative results and summary data of 4 experiments are presented. eImmunofluorescent staining of Nucleocapsid protein (NP, red) and
CD41 (green) in human platelets incubated with SARS-CoV-2 virus (1 × 10
5
PFU) for 3 h. Representative images from 3 experiments using
platelets from different healthy donors. fScanning electron microscope (SEM) of SARS-CoV-2 particles on the surface of platelets. SEM of healthy
human platelets (3 × 10
8
platelets/mL) incubated with SARS-CoV-2 (1 × 10
5
PFU) for 30 min. Platelets were washed for 3 times and fixed
immediately after incubation and processed for SEM experiment. Representative images of single platelet from control group (platelet1) and
SARS-CoV-2 treatment group (platelet2 and platelet3) are shown from three different experiments. Arrows point toward the SARS-CoV-2 virus. g.
Transmission electron microscopy (TEM) of SARS-CoV-2 particles in platelets. TEM of healthy human platelets (3 × 10
8
platelets/mL) incubated
with SARS-CoV-2 (1 × 10
5
PFU) for 3 h. Platelets were washed for 3 times and fixed immediately after incubation and processed for TEM
experiment. Representative images from control group (platelet1) and SARS CoV-2 treatment group (platelet2 and platelet3) are shown from
three different experiments. Arrows point toward the SARS-CoV-2 particles. Statistical analyses were performed using unpaired two-tailed
Students t test in (a), (b) and (c). NS no significance; *P< 0.05; **P< 0.01. Two-way ANOVA and Tukeys post hoc test was performed in (d); *P<
0.05 and **P< 0.01 compared with control group
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 10 of 22
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Fig. 4 (See legend on next page.)
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 11 of 22
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its effects on FeCl
3
-induced thrombus formation in the
mesenteric arterioles. Representative images of the
thrombus development at various time points following
FeCl
3
injury are shown in Fig. 6a. Notably, Spike protein
had no effect on thrombus formation following mesen-
teric arteriole injury in mice transfused with platelets
from wild-type mice, which agreed with the results that
SAR-CoV-2 Spike protein cannot bind mouse ACE2
protein [53]. However, Spike protein did potentiate
thrombus formation in mice transfused with platelets
from hACE2 transgenic mice.
We then assessed the role of the Spike protein on mice
platelets for thrombus formation under arterial flow
conditions using a microfluidic whole-blood perfusion
assay. Throughout the perfusion period, thrombus for-
mation was significantly increased when pre-treated with
Spike protein (2 μg/mL, 5 min), in whole blood from
hACE2 transgenic mice, but not in whole blood from
wild-type mice (Fig. 6b). In addition, the Spike protein
potentiated platelet aggregation and ATP release in re-
sponse to agonists in vitro and enhanced thrombosis for-
mation in vivo on hACE2 transgenic mice, while it had
no effect on wild-type mice (Fig. 6c and Additional file
1: Online Figure 8).
SARS-CoV-2 directly induces the release of coagulation
factors, inflammatory cytokines, and the formation of
leukocyte-platelet aggregates
Platelets are an important source of coagulation factors
V and XIII in αgranules, and serve as adhesion sites for
coagulation factors activation via their surface exposure
of phosphatidylserine (PS) [58]. We demonstrated that
SARS-CoV-2 stimulated platelets to release both Factor
V (Additional file 1: Online Figure 9A) and Factor XIII
(Additional file 1: Online Figure 9B), at levels that were
comparable to thrombin stimulation. However, SARS-
CoV-2 had no effect on platelet PS exposure (Additional
file 1: Online Figure 9C). Similar results were also
observed in the Spike protein-treated platelets (Add-
itional file 1: Online Figure 9D, 9E, and 9F).
Platelets carry a large variety of inflammatory cytokines
in αgranules, which can be quickly secreted upon activa-
tion to participate in the immune response [59]. We found
that SARS-CoV-2 and its Spike protein stimulated PF4
(Additional file 1:OnlineFigure10A),TNF-α(Additional
file 1: Online Figure 10B), IL-8 (Additional file 1: Online
Figure 10C), and IL-1β(Additional file 1: Online Figure
10D) secretion from platelets. Consistently, we found that
plasma PF4 levels were increased in COVID-19 patients,
compared with healthy donors. The severe and critically
severe COVID-19 patients presented the highest plasma
PF4 levels (Additional file 1: Online Figure 10E).
P-selectin is a key adhesion molecule that mediates
the interaction between platelets and leukocytes via P-
selectin glycoprotein ligand 1 (PSGL-1). Consistent with
our finding that SARS-CoV-2 increased P-selectin ex-
pression, we found that SARS-CoV-2 and Spike protein
increased the proportion of leukocyteplatelet aggre-
gates (LPAs) (CD45
+
CD41
+
aggregates, Additional file 1:
Online Figure 11). Specifically, both monocyteplatelet
aggregates (MPAs, CD14
+
CD41
+
) and neutrophil-
platelet aggregates (NPAs, CD65
+
CD41
+
) were signifi-
cantly increased after SARS-CoV-2 or Spike protein
treatment.
Recombinant human ACE-2 protein and an anti-Spike
monoclonal antibody suppress SARS-CoV-2-induced
platelet activation
Recombinant human ACE2 protein has been reported to
inhibit SARS-CoV-2 infection in engineered human tis-
sues [60]. SARS-CoV-2 neutralizing antibodies produced
by the host immune response are a critical part of the
toolbox of therapies for COVID-19 [61]. We pretreated
platelets from healthy donors with ACE2 protein or an
anti-Spike antibody. Both the ACE2 protein and the
anti-Spike antibody suppressed SARS-CoV-2 (Fig. 7(A))
(See figure on previous page.)
Fig. 4 SARS-CoV-2 Spike 1 protein directly enhances human platelet activation. aSARS-CoV-2 Spike protein and Spike subunit 1 (S1) potentiated
platelet aggregation and ATP release in response to collagen, thrombin, and ADP in vitro, whereas Spike subunit 2 (S2) did not. Washed platelets
from healthy donors were preincubated with Spike protein, S1 or S2 at 2 μg/mL for 5 min, then stimulated with collagen (0.6 μg/mL), thrombin
(0.025 U/mL), or ADP (5 μM). Aggregation and ATP release (with luciferase) were assessed under stirring at 1200 rpm. Representative results and
summary data of 4 experiments are presented. bSpike protein stimulated platelets for PAC-1 binding and CD62P expression in the absence of
agonist. In addition, Spike protein and S1 increased PAC-1 binding and CD62P expression induced by thrombin in platelets. Platelets were
incubated with Spike protein (2 μg/mL, 60 min) in the absence of agonist, or preincubated with Spike protein, S1 or S2 at 2 μg/mL for 5 min and
stimulated with thrombin (0.025 U/mL) for 10 min, and then analyzed using a flow cytometer. Representative results and summary data of 4
experiments are presented. cSpike protein (2 μg/mL) and S1 (2 μg/mL) potentiated platelet spreading on immobilized fibrinogen. After
preincubation with Spike protein (2 μg/mL) for 5 min, platelets were allowed to spread on the fibrinogen-coated surfaces at 37 °C for the
indicated times. Representative results and summary data of 3 experiments are presented. dSpike protein and S1 potentiated clot retraction
induced by thrombin. Platelets from healthy donors were normalized at a concentration of 4 × 10
8
/mL and preincubated with Spike protein (2
μg/mL) or S1 (2 μg/mL) for 5 min, then stimulated with thrombin (1 U/mL). Representative images and summary data are presented from 3
experiments using platelets from different donors. Statistical analyses were performed using one-way ANOVA, followed by Tukeys post hoc
analysis in (a), (b) and (c). NS no significance; *P< 0.05; **P< 0.01. Two-way ANOVA and Tukeys post hoc test was performed in (d); ## significant
difference (P< 0.01) between Spike and control group; ** significant difference (P< 0.01) between S1 and control group
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 12 of 22
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Fig. 5 (See legend on next page.)
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 13 of 22
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or Spike protein (Fig. 7(B))-potentiated platelet aggrega-
tion and reversed SARS-CoV-2 or Spike protein-induced
PAC-1 binding and CD62P expression (Fig. 7(C and D)).
The accelerated platelet spreading (Fig. 7(E)) and clot re-
traction (Fig. 7(F)) elicited by exposure to SARS-CoV-2
or Spike protein were also prevented by pretreatment
with the ACE2 protein and anti-Spike antibody. Of note,
treatment with the ACE2 protein and anti-Spike anti-
body also suppressed the thrombus formation induced
by the Spike protein following mesenteric arteriole injury
(Fig. 7(G)). These data clearly show that SARS-CoV-2
stimulates platelet activation via the interaction of its
Spike protein and ACE2, which can be suppressed by ex-
ogenous addition of ACE2 or an anti-Spike antibody.
Discussion
Accumulating evidence indicates that COVID-19 predis-
poses patients to thromboembolic disorders, but a direct
association between SARS-CoV-2 and platelet dysregula-
tion has not been reported. In this study, we demon-
strated that (1) COVID-19 patients experience increased
in vivo platelet activation, as evidenced by increased
αIIbβ3 activation and P-selectin expression, and detect-
able virus RNA in the blood is associated with platelet
hyperactivity; (2) platelets robustly express ACE2 and
TMPRSS2; (3) SARS-CoV-2 and its Spike protein pro-
mote platelet function and thrombus formation via the
MAPK pathway downstream of ACE2; and (4) recom-
binant human ACE2 protein and anti-Spike monoclonal
antibody treatment may block SARS-CoV-2-induced
platelet activation and thrombus formation. Collectively,
these data suggest that SARS-CoV-2-activated platelets
may result in the pro-thrombotic state described in
COVID-19 patients. Our findings also suggest that
reducing platelet hyperactivity via the addition of ACE2
protein and anti-Spike neutralizing antibodies could be
an effective therapeutic strategy to prevent thrombotic
events in COVID-19 patients (Fig. 8, central illustration).
Emerging evidence has revealed a high composite inci-
dence of thrombotic events in critically ill COVID-19
patients, including venous and arterial thrombotic
events, and thrombocytopenia, all of which have been
associated with increased mortality [6264]. Anticoagu-
lation is associated with a reduced risk of mortality with-
out increased bleeding diathesis among patients
hospitalized with COVID-19 [65]. However, the under-
lying mechanism of thrombus formation in COVID-19
is still unclear. We provide evidence that the platelets,
key mediators of thrombosis, are hyperactivated in
COVID-19 patients. Other recent studies have reported
that various platelet activation events, including aggrega-
tion, adhesion, infiltration, and inflammatory response,
contribute to lung injury and microvascular thrombosis
in SARS-CoV-2-associated pneumonia [6668]. These
results, together with those from our study, draw atten-
tion to the role of platelet activation in the pathogenesis
of COVID-19.
It has long been accepted that viruses indirectly acti-
vate platelets during infection by creating an inflamma-
tory microenvironment and subsequent vascular
endothelial dysfunction. However, recent studies have
shown that there are some direct interactions between
certain viruses and platelets, and that these interactions
serve as an important supplement for the above-
mentioned activation [69]. Platelets bind to encephalo-
myocarditis virus via TLR7, to rotavirus via GPIa/IIa, to
hantavirus and adenovirus via GPIIb/IIIa, to HIV and
DV via lectin receptors such as CLEC-2 and DC-SIGN,
(See figure on previous page.)
Fig. 5 ACE2/MAPK mediates the 1 potentiating effects of SARS-CoV-2 on platelet activation. A SARS-CoV-2 virus and its Spike protein
phosphorylate ACE2, Erk, p38, and JNK in human platelets. Platelets from healthy donors were pretreated with SARS-CoV-2 (1 × 10
5
PFU) or its
Spike protein (2 μg/mL) for various times, as 5 indicated. For ACE2 phosphorylation detection, cell lysates were prepared and subjected to
immunoprecipitation (IP) with anti-phospho-Ser/Thr antibody, followed by immunoblotting analysis with anti-ACE2 antibody. Cell lysates without
the process of immunoprecipitation (10% of input) were analyzed in parallel as loading controls. For p-Erk, p-p38, and p-JNK detection, cell lysates
were prepared and directly subjected to immunoblotting. Representative results are presented from 4 experiments using platelets from different
healthy donors and summary data is presented in the Additional file 1: Online Figure 7. B Enhanced platelet aggregation by SARS-CoV- 2 is
abolished by MAPK inhibitors. The healthy human platelets were pretreated with 10 μM PD98059 (ERK1/2 inhibitor), 10 μM SB203580 (p38
inhibitor), or 10 μM SP600125 (JNK inhibitor) for 10 min, then treated with SARS-CoV-2 (1 × 10
5
PFU, 30 min) before stimulation by 0.025 U/mL
thrombin or 0.6 μg/mL collagen. Representative results are presented from 3 experiments using platelets from different donors. C Enhanced
platelet aggregation by Spike protein is abolished by MAPK inhibitors. Human platelets were pretreated with 10 μM PD98059, 10 μM SB203580,
or 10 μM SP600125 for 10 min, then treated with Spike protein (2 μg/mL, 5 min) or vehicle as control before stimulation by 0.025 U/mL thrombin
or 0.6 μg/mL collagen. Representative results are presented from 3 experiments using platelets from different healthy donors. D Accelerated clot
retraction by SARS-CoV-2 (D1) or its Spike protein (D2) is abolished by MAPK inhibitors. Platelets were pretreated with 10 μM PD98059, 10 μM
SB203580, or 10 μM SP600125 for 10 min, and then incubated with SARS26 CoV-2 (1 × 10
5
PFU, 30 min) or Spike protein (2 μg/mL, 5 min) as in B
and C. Clot retraction was initiated as in Fig. 3d. Representative images and summary data of 3 experiments are presented using platelets from
different healthy donors. Representative images and summary data are presented from 3 experiments using platelets from different donors. E
Increased phosphorylation of Erk, p38, and JNK in platelets from COVID-19 patients, compared with healthy donors. Representative results are
presented using platelets from 6 individuals from different COVID-19 patients (n= 3) and healthy donors (n= 3). Statistical analyses were
performed using unpaired two34 tailed Studentsttest in (D1) and (D2). *P< 0.05; **P< 0.01. MAPK indicates mitogen activated protein kinase;
PD, PD98059; SB, SB203580; SP, SP600125
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 14 of 22
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Fig. 6 (See legend on next page.)
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 15 of 22
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
and to influenza/IgG immune complexes by FcgRIIA
[70]. Here, we provide evidence that platelets express
abundant ACE2 and TMPRSS2, two major cellular com-
ponents responsible for SARS-CoV-2 cell entry.
The recent studies by Manne B K et al. did not detect
robust expression of ACE2 in platelets. However, plate-
lets used for the detection of ACE2 in their studies
mainly come from COVID-19 patients. Actually, the
COVID-19 virus can induce ACE2 internalization and
subsequent degradation, evidenced by our study of
SARS-CoV-2-induced ACE2 degradation in platelets
(Additional file 1: Online Figure 5) and the other studies
on SARS-CoV-2-induced ACE2 degradation [30]. More-
over, a recent study indicated that SARS-CoV-2 infec-
tion could downregulate ACE2 at the mRNA level and
this effect requires action directly on the ACE2 pro-
moter [71]. In consistent with the identification of plate-
let ACE2 in our results, Zaid et al. also found that ACE2
mRNA was presented in platelets from both healthy
people and COVID-19 patients [72].
ACE2 is the primary enzyme responsible for the con-
version of Ang II, a pivotal mediator of lung injury, to
Ang peptides. SARS-CoV-2 uses ACE2 as its cellular re-
ceptor, resulting in ACE2 degradation and ACE/ACE2
imbalance, which could drive Ang II-mediated lung in-
jury in COVID-19 [73,74]. In addition, the decline of
ACE2 in infected cells could confer to a host protective
mechanism from further viral attack.
Recent studies reported that platelets are hyperactivated
in COVID-19 patients [67,75,76]. The platelet activity
biomarkers are associated with the coagulation dysfunc-
tion [67] and the composite outcome of thrombosis or
death [75]. These studies emphasized the notion that cyto-
kine storm may trigger hyperinflammation and hyperco-
agulability. Previous studies have found that fibrinogen
level is higher in COVID-19 patients, which may bind and
activate platelets to exacerbate thrombotic disorder in ca-
pillaries [77]. Our studies are consistent with the recent
finding of platelet hyperactivity in COVID-19 patients.
We further extend those finding and suggested a possibil-
ity that SARS-CoV-2 virus could directly activate platelets
via the interaction of Spike protein and platelet ACE2. We
cannot rule out the other possibility that virus-containing
immune complex or virus-induced immune mediators
may also contribute to platelet hyperactivity in COVID-
19, which needs further investigation.
We found that MAPK was phosphorylated in SARS-
CoV-2-activated platelets, and ACE2 was phosphorylated
earlier than MAPK signaling, indicating that MAPK is ac-
tivated downstream of ACE2 activation. Supporting our
finding, MAPK activation was reported to be attributable
to ACE2 signaling in lung cells in an earlier study [27].
We also found that MAPK inhibitors can reverse SARS-
CoV-2-induced platelet activation, supporting our hypoth-
esis that MAPK mediates this activation. In line with our
findings, recent studies have shown that MAPK phosphor-
ylation promotes thromboxane generation by activating
cPLA2 in platelets [78]. MAPK activation also facilitates
platelet secretion and clot retraction by stimulating phos-
phorylation of myosin light chain [79]. We previously re-
ported that MAPK phosphorylation potentiated platelet
aggregation and clot retraction [35]. Meanwhile, MAPK
inhibition could abolish platelet aggregation induced by a
low concentration of agonist [80,81].
Platelets contain a set of coagulation factors and inflamma-
tory factors, stored in the α-granules that are released upon
activation to potentiate the coagulation cascade [82,83]. We
found that SARS-CoV-2 induced CD62P expression, indicat-
ing α-granule secretion. In addition, SARS-CoV-2 and its
Spike protein directly stimulated Factor V and XIII release as
well as LPAs formation. SARS-CoV-2 failed to induce PS ex-
posure, which is consistent with previous reports that suggest
that platelet activators are inefficient in inducing PS exposure
intheabsenceofashearforce[84].
Conclusions
This study showed that ACE2, a host cell receptor for
SARS-CoV-2, and TMPRSS2, a serine protease for
(See figure on previous page.)
Fig. 6 SARS-CoV-2 Spike 1 protein directly enhances thrombosis potential in vivo. aWashed platelets from wild-type or hACE2 transgenic mice
were infused into WT mice. After intravenous injection 200 μg/kg Spike protein or control (saline), FeCl3-induced arterial thrombus formation was
initiated, and the thrombus area was recorded. Representative image of thrombus formation and the relative fluorescence at different time points
are shown. Statistically analysis of FeCl3-induced thrombosis by assess thrombus area at 8 min (n= 10). bSpike protein-treated whole blood
from hACE2 mice showed accelerated thrombus formation over an immobilized collagen surface at a shear rate of 1000 s
1
, whereas Spike
protein-treated whole blood from wild type mice did not. The whole blood from mice was fluorescently labeled by mepacrine (100 μM, 30 min)
and incubated with Spike protein (2 μg/mL) for 5 min, and then perfused through fibrillar collagen-coated bioflux plates for 5 min. Representative
images and time courses of thrombus formation challenged with control or Spike protein at the indicated time points are presented. Dot plot
showing thrombus formation area (n= 6). cSpike protein-treated platelets from hACE2 mice presented increased platelet aggregation and ATP
release in response to collagen, thrombin, and ADP, whereas Spike protein-treated platelets from wild-type mice did not. Washed platelets from
mice were pretreated with control or Spike protein (2 μg/mL) for 5 min, and then stimulated with collagen (0.6 μg/mL), thrombin (0.025 U/mL),
or ADP (5 μM). Aggregation and ATP release (with luciferase) were assessed under stirring at 1200 rpm. Representative results are presented from
4 experiments using platelets from different mice and summary data are presented in Additional file 1: Online Figure 8. Statistical analyses were
performed using unpaired two-tailed Studentsttest in (a) and (b). NS no significance; **P< 0.01. WT indicates wild-type; hACE2 indicates
hACE2 transgenic
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 16 of 22
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Fig. 7 (See legend on next page.)
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 17 of 22
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
(See figure on previous page.)
Fig. 7 Recombinant human ACE2 protein and anti-Spike monoclonal antibody suppress SARS-CoV-2-induced platelet activation. A, Enhanced
platelet aggregation by SARS-CoV-2 is abolished by recombinant human ACE2 protein and anti-Spike monoclonal antibody (targeting the
receptor-binding domain [RBD] of SARS-CoV-2). Representative results are presented from 3 experiments using platelets from different donors. B,
Enhanced platelet aggregation by Spike protein is suppressed by ACE2 protein and anti-Spike antibody. Representative results are presented from
3 experiments using platelets from different healthy donors. For A and B, the healthy human platelets were pretreated with ACE2 protein (10 μg/
mL) or anti-Spike antibody (4 μg/mL) for 10 min, then treated with SARS-CoV-2 (1 × 10
5
PFU, 30 min) or Spike protein (2 μg/mL, 5 min) before
stimulation by 0.025 U/mL thrombin or 0.6 μg/mL collagen. C and D, The ACE2 protein and anti-Spike antibody reversed PAC-1 binding and
CD62P expression induced by SARS-CoV-2 (C) or Spike protein (D). Representative images and summary data are presented from 4 experiments
using platelets from different healthy donors. E, Enhanced platelet spreading induced by SARS-CoV-2 (E1) or its Spike protein (E2) are abolished
by ACE2 protein and anti-Spike monoclonal antibody. Representative images and summary data of 4 experiments are presented using platelets
from different healthy donors. F, Accelerated clot retraction induced by SARS-CoV-2 (F1) or its Spike protein (F2) are abolished by ACE2 protein
and anti-Spike monoclonal antibody. Representative images and summary data of 4 experiments are presented using platelets from different
healthy donors. For C, D, E and F, platelets were pretreated with ACE2 protein (10 μg/mL) or anti-Spike antibody (4 μg/mL) for 10 min, and
incubated with SARS-CoV-2 (1 × 10
5
PFU, 30 min) or Spike protein (2 μg/mL, 5 min), and then subjected to flow cytometry of thrombin-activated
platelets, platelet spreading assay, and clot retraction assay. G, Increased thrombus area induced by Spike protein in wild-type mice transfused
with platelets from hACE2 transgenic mice is suppressed by ACE2 protein and anti-Spike antibody. Representative photographs of FeCl3-induced
thrombus formation at the indicated time points within 30 min after intravenous administration of Spike protein (200 μg/kg) with ACE2 protein
(1 mg/kg) or anti-Spike monoclonal antibody (400 μg/kg). Dot plot showing thrombus area for control or Spike protein treated mice (n= 10).
Statistical analyses were performed using one-way ANOVA, followed by Tukeys post hoc analysis in (C), (D), (E), (F), and (G). *P< 0.05; **P< 0.01.
RhACE2 indicates recombinant human ACE2 protein; anti-S Ab, anti-Spike antibody
Fig. 8 Summary schemes illustrating SARS-CoV-2 activates platelets and enhances thrombosis in COVID-19. Global schema illustrating SARS-CoV-2
from alveolus binds and activates platelets, which enhances thrombosis formation and inflammatory reaction in capillaries, and subsequently
contributes to development of disseminated intravascular coagulation and acute respiratory distress syndrome. SARS-CoV-2 Spike protein binds to
ACE2 and phosphorylates ACE2, leading to MAPK signaling activation (phosphorylation of Erk, p-38, and JNK) and subsequent platelet activation,
coagulation factors release, and inflammatory cytokines secretion. Interaction between SARS-CoV-2 Spike protein and platelet ACE2 confers the
platelet activation, which is suppressed by the recombinant human ACE2 protein and anti-Spike monoclonal antibody (central illustration)
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 18 of 22
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
protein priming, are expressed in platelets, and that the
SARS-CoV-2 virus directly activates platelets and poten-
tiates their prothrombotic function and inflammatory re-
sponse via Spike/ACE2 interactions. Considering the
critical roles of platelets in thrombosis, coagulation, and
immune response, our study sheds new insight into pos-
sible anti-platelet treatment opinions for thrombosis in
COVID-19 patients.
Limitations of study
Our study has several limitations. Firstly, animal experiments
of virus invasion are lacked due to the limitations of our ex-
perimental conditions. Whether our in vivo thrombus for-
mationstudyusingSpikeproteincanbealsoreproduced
using living SARS-CoV-2 virus still needs to be further inves-
tigated. Secondly, platelets in infected lungs may meet differ-
ent concentrations of viruses in COVID-19; the in vivo effect
of SARS-CoV-2 on platelet activation needs to be further in-
vestigated in COVID-19 patients. Thirdly, whether our study
in washed platelets can be also reproduced using platelet-
rich plasma still needs to be further investigated. Fourthly,
the relationship between ACE2 degradation and disease se-
verity, platelet activity, or blood SARS-COV2 RNA existence
still needs further investigation.
Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s13045-020-00954-7.
Additional file 1: Expanded materials and methods.. Online Table 1.
Antibodies, staining chemicals and dilutions used in this study. Online
Table 2. The primers used for RT-PCR. Online Table 3. Characteristics of
Healthy, Non-COVID-19 patients, Mild and moderate type COVID-19 pa-
tients and Severe and critically severe type COVID-19 patients groups be-
fore propensity score matching. Online Table 4. Patient Characteristics
after propensity score matching Sever and critically severe type COVID-19
patients group to healthy group (1:2), Non-COVID-19 patients group (1:1)
or Mild and moderate type COVID-19 patients group (1:2). Online Table
5. Patient Characteristics after propensity score matching healthy group
to Mild and moderate type COVID-19 group (1:1) or Non-COVID-19 pa-
tients group to Mild and moderate type COVID-19 group (1:2). Online
Figure 1. The flowchart showing the strategy of groups enrollment. On-
line Figure 2. Analysis of platelet activation in COVID-19 patients after
propensity score matching. A and B, Dot plot showing increased platelet
t acti activation (A) and CD62P expression (B) in severe and critically se-
vere COVID-19 patients compared with healthy donors (1:2 matching),
non-COVID-19 patients (1:1 matching) or mild and moderate COVID-19
patients (1:2 matching). C and D, Dot plot showing increased PAC-1 bind-
ing (C) and CD62P expression (D) in mild and moderate COVID-19 pa-
tients compared with healthy donors (1:1 matching), non-COVID-19
patients (2:1 matching). **P< 0.01 vs control. Statistical analyses were
performed using Mann-Whitney U test. *P< 0.05; **P< 0.01. Online Fig-
ure 3. SARS-CoV-2 RNA positive platelet-poor plasma enhanced platelet
aggregation compared with SARS-CoV-2 RNA negative platelet-poor
plasma and healthy platelet-poor plasma. Healthy platelets were incu-
bated with healthy platelet-poor plasma (PPP), SARS-COV-2 RNA positive
PPP and SARS-COV-2 RNA negative PPP from severe and critically severe
COVID-19 patients at the concentration of 2oor plasma (PPP), SARS-COV-
2 RNA positive PPP and SARS-COV-2 RNA ne experiment. Statistical ana-
lyses were performed using one-way ANOVA followed by Tukeyiment.
Statistical analP< 0.05, n = 4. Online Figure 4. SARS-CoV-2 and its Spike
protein did not induce platelet aggregation in the absence of agonist.
Platelets were treated with SARS-CoV-2 ((1telets were treated with SARS-
CoV-2 not induce plateletway ANOVA followed bygat indicated time in
aggregometry. Online Figure 5. Platelet ACE2 is reduced upon SARS-
CoV-2 treatment. A, Platelets from healthy donors were pretreated with
SARS-CoV-2 (1×105 PFU) for various times, as indicated. The ACE2 protein
level was detected by Western blot. Representative images and summary
data of 3 experiments are presented using platelets from different healthy
donors. Statistical analyses were performed using two-way ANOVA and
Tukey's post hoc test. **P< 0.01 vs control. B, Decreased ACE2 expression
in platelets from COVID-19 patients. Representative images of 12 different
individuals from healthy group (n = 4), mild and moderate COVID-19
group (n = 4) and severe and critically severe COVID-19 group (n = 4) are
presented. Online Figure 6. SARS-CoV-2 Spike protein dose-
dependently enhance platelet activation. Washed platelets from healthy
donors were incubated with Spike protein in the indicated concentration
for 5 min, then stimulated with collagen (0.6 n = 4)e thrombin (0.025 U/
mL), or ADP (5 min, then stimulated with collagen (0.6 n = 4)ere were
assessed under stirring at 1200 rpm. Representative results and summary
data of 4 experiments are presented. Statistical analyses were performed
using One-way ANOVA, followed by Tukeyted. Statistical analyses were
performed using One-wayata o 0.01. Online Figure 7. SARS-CoV-2 and
its Spike protein phosphorylates ACE2, Erk, p38, and JNK in human plate-
lets. A, Platelets were challenged with SARS-CoV-2 (1 x 105 PFU) or Spike
protein (2 h SARS-CoV-2 (1tes ACE2, Erk,rmed using One-wayata ACE2
normalized to input ACE2, corresponding to Figure 5a, are provided from
4 experiments using platelets from different donors. B, Platelets were
challenged with SARS-CoV-2 (1 x 105 PFU) or Spike protein (2 nors. CE2,
Erk,rmed using One-wayata p-Erk normalized to Erk, p-p38 normalized to
p38, and p-JNK normalized to JNK, corresponding to Figure 5a, are pro-
vided from 4 experiments using platelets from different donors. Statistical
analyses were performed using One-way ANOVA, followed by Tukeyt do-
nors. Statistical anaA) and (B). NS, no significance; *P< 0.05; **P< 0.01.
Online Figure 8. Spike protein enhances in vitro platelet activation and
in vivo thrombosis potential in hACE2 transgenic mice. A, Quantitative
analysis of platelets aggregation and ATP release corresponding to Figure
6C. Data are provided from 4 experiments using platelets from different
mice. B, After intravenous injection 200 ter intravenous injectionelets from
different mice. ing One-way ANOVA, f formation was initiated, and the
thrombus area was recorded. Representative image of thrombus forma-
tion and the relative fluorescence at different time points are shown. Sta-
tistically analysis of FeCl3-induced thrombosis by assessing thrombus area
at 8 min (n = 5). Statistical analyses were performed using unpaired two-
tailed Studentbus test. NS, no significance; **P< 0.01. WT indicates wild-
type; hACE2 indicates hACE2 transgenic. Online Figure 9. SARS-CoV-2
directly stimulates platelets for coagulation factor release. A and B, SARS-
CoV-2 induced release of Factor V (A) and Factor XIII (B) from platelets.
Washed platelets from healthy donors were incubated with or without
SARS-CoV-2 (1×105 PFU) for 1 h at room temperature, and subject to
ELISA assays. Thrombin at 0.025 U/mL served as the positive control.
Summary data of 4 experiments using platelets from different healthy do-
nors are presented. C, SARS-CoV-2 had no effect on platelet phosphatidyl-
serine (PS) exposure. Washed platelets from healthy donors were
incubated with or without SARS-CoV-2 (1×105 PFU) for 1 h at room
temperature, and then stained with Annexin V-FITC for 30 min. PS expos-
ure was analyzed using flow cytometry. Thrombin at 0.05 U/mL served as
the positive control. Representative images and summary data are pre-
sented from 4 experiments using platelets from different healthy donors.
D and E, Spike protein (2 erent healthy donor release of Factor V (D) and
XIII (E) from platelets. Factor V and XIII in supernatant were assessed using
commercial ELISA kits. Thrombin at 0.025 U/mL served as the positive
control. Summary data of 4 experiments using platelets from different
healthy donors are presented. F, Spike protein (2 d. ary data of 4 experi-
ments using platelets after 1 h incubation. Thrombin at 0.05 U/mL served
as the positive control. Representative images and summary data are pre-
sented from 4 experiments using platelets from different healthy donors.
Statistical analyses were performed using One-way ANOVA, followed by
Tukeylthy donors. Statistical A), (B), (C), (D), (E) and (F). NS, no significance;
**P< 0.01. Online Figure 10. SARS-CoV-2 directly potentiates PF4 and
inflammatory cytokines secretion from platelets. A, SARS-CoV-2 and its
Spike protein stimulated PF4 release from platelets. Summary data of at
Zhang et al. Journal of Hematology & Oncology (2020) 13:120 Page 19 of 22
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
3-4 experiments using platelets from different healthy donors are pre-
sented. B, C and D, SARS-CoV-2 and its Spike protein stimulated TNF-α, IL-
8 and IL-1d its Spike proteinnted. of at 3-4 experim experiments using
platelets from different healthy donors are presented. Washed platelets
were separated from whole blood of healthy donors and adjusted to
3edfromO in platelet-poor plasma. Platelets were then challenged with
SARS-CoV-2 (1×105 PFU), Spike protein (2 . Platelets were then challenged
withh. E, Increased expression of PF4 in plasma from severe and critically
severe COVID-19 patients, compared with healthy donors or mild and
moderated COVID-19 patients. Concentrations of PF4 were measured in
plasma from healthy donors, mild and moderated COVID-19 patients and
severe and critically severe COVID-19 patients (n = 5). PF4 and TNF-α, IL-8
and IL-1-19 patients and severe and critically sever commercial ELISA kits.
Statistical analyses were performed using One-way ANOVA, followed by
Tukeykits. Sthoc analysis in (A), (B), (C), (D) and (E). *P< 0.05; **P< 0.01.
Online Figure 11. SARS-CoV-2 directly stimulates leukocyte-platelet ag-
gregates (LPAs) formation. A and B, SARS-CoV-2 (A) and Spike protein (B)
increased leukocyte-platelet aggregates (LPAs, CD45+CD41+), monocyte-
platelet aggregates (MPAs, CD14+CD41+) and neutrophil-platelet aggre-
gates (NPAs, CD65+CD41+). After stimulation with SARS-CoV-2 (1hil-plate-
let aggregates (NPAs, CD65+CD41+). After(no with different antibodies
for about 30 min, red blood cells were removed and the blood samples
were then subjected to flow cytometry. Summary data of 5 experiments
using blood samples from different healthy donors are presented. Statis-
tical analyses were performed using unpaired two-tailed Student are pres
in (A) and (B). **P< 0.01.
Abbreviations
ACE2: Angiotensin-converting enzyme 2; TMPRSS2: Transmembrane protease
serine 2; PBMCs: Peripheral blood mononuclear cells; SEM: Scanning electron
microscope; TEM: Transmission electron microscopy; MPV: Mean platelet
volume; PCT: Plateletcrit; PDW: Platelet distribution width;
PS: Phosphatidylserine; PT: Prothrombin time; PTA: Prothrombin time activity;
INR: International normalized ratio; APTT: Activated partial thromboplastin
time; FDPs: Fibrinogen degradation products; NP: Nucleocapsid protein;
S1: Spike protein subunit 1; S2: Spike protein subunit 2; RBD: receptor-
binding domain; MAPK: Mitogen-activated protein kinase; HIV: Human
immunodeficiency virus; DV: Dengue virus
Acknowledgements
We thank all members of our team for critical input and suggestions. We
also thank all patients participated in our study.
Authorscontributions
LH, SZ, and YZ designed and conceived this project. SZ, YL, XW, and LY
performed the research, analyzed the data, and wrote the manuscript. HL
ML, XZ, and SZ collected and analyzed the clinical data. YW, YX, YY, ZF, JD,
ZY, and ZD performed the research on SARS-CoV-2 and analyzed the data.
All authors contributed to and approved the manuscript.
Funding
This work was partially supported by the National Natural Science of
Foundation of China to Liang Hu (81803522, 81970305), Si Zhang (81770137,
81573423), Yangyang Liu (81903603, 81900378), Xiaofang Wang (81400323)
and China Postdoctoral Research Foundation to Yangyang Liu (Grant No.
2020M672292) and Emergency Prevention and Control of COVID-19 Project
of Henan Province to Yi Zhang (Grant no. 201100310900) and Novel Corona-
virus Prevention and Control Emergency Project of Zhongshan University to
Yan Yang.
Availability of data and materials
All data generated or analysed during this study are included in this
published article (and its supplementary information files).
Ethics approval and consent to participate
All patient samples and clinical data were obtained with informed consent
from the first affiliated hospital of Zhengzhou University, Henan Province,
China and three centers of affiliated hospital of Anhui Medical University,
Anhui Province, China. The study was approved by the Ethics Committee of
the First Affiliated Hospital of Zhengzhou University (2020-KY-121) and Ethics
Committee of Anhui Medical University (2020-AH-114), and complied with
the Declaration of Helsinki and good clinical practice guidelines. The mice
were maintained under specific pathogen-free conditions in the Laboratory
Animal Center of Zhengzhou University. Animal procedures were carried out
in accordance with the ethical approval of the Ethical Committee of Zheng-
zhou University.
Consent for publication
Not applicable for this article.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Cardiology, the First Affiliated Hospital of Zhengzhou
University, Cardiovascular Institute of Zhengzhou University, Zhengzhou
450052, China.
2
Department of Biochemistry and Molecular Biology, NHC Key
Laboratory of Glycoconjugates Research, School of Basic Medical Sciences,
Fudan University, Shanghai 200032, China.
3
Biotherapy Center, the First
Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
4
Department of Emergency, Affiliated Hospital of Anhui Medical University,
Hefei, China.
5
Key Laboratory of Medical Molecular Virology (MOE/NHC/
CAMS), and Department of Medical Microbiology and Parasitology, School of
Basic Medical Sciences, Shanghai Medical College, Fudan University,
Shanghai, China.
6
School of Public Health (Shenzhen), Sun Yat-sen University,
Guangzhou, China.
7
Department of Hematology, Wenzhou Key Laboratory of
Hematology, The First Affiliated Hospital of Wenzhou Medical University,
Wenzhou, China.
8
Department of Immunology, School of Medicine, UConn
Health, Farmington, CT 06030, USA.
Received: 17 July 2020 Accepted: 19 August 2020
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... Both the greater prevalence of these associated proteases in lung tissue and the primary method of COVID transmission via airborne particles likely account for the respiratory predominance of the disease process. , Zhang et al., 2020, Li et al., 2021 ...
... First, the pro-inflammatory state promoted by SARS-CoV-2 infection has been associated with platelet hyperactivity and aggregation. (Zhang et al., 2020, Manne et al., 2020, Hottz et al., 2020 High circulating plasma levels of CRP and fibrinogen have been correlated with platelet activation, while No consensus exists on the exact pathway by which COVID-19 promotes these effects, but several pathways have been suggested. Generally, COVID-19 patients have high circulating plasma levels of CRP and fibrinogen, which correlate with platelet activation. ...
... Second, a handful of studies have identified SARS-CoV-2 within platelets. (Zhang et al., 2020, Manne et al., 2020, Zaid et al., 2020 However, only Zhang et al (2020) definitively identified ACE-2 expression on platelets in both mice and humans. (Zhang et al., 2020) These discordant findings highlight the need for further analysis. ...
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This narrative review paper is aimed to critically evaluate recent studies of the associations between air pollution and the outcomes in the COVID-19 pandemic. The main air pollutants we have considered are carbon monoxide (CO), nitrogen dioxide (NO2), ground-level ozone (O3), particulate matter (PM2.5 and PM10), and sulfur dioxide (SO2). We, specifically, evaluated the influences of these pollutants, both individually and collaboratively, across various geographic areas and exposure windows. We further reviewed the proposed biological mechanisms underlying the association between air pollution and COVID-19. Ultimately, we aim to inform policy and public health practice regarding the implications of COVID-19 and air pollution.
... SARS-CoV-2 infects host cells by binding spike protein to angiotensin-converting enzyme 2 (ACE2), expressed on alveolar epithelial cells, enterocytes, monocytes/macrophages, platelets, and vascular endothelial cells. 18 The binding of endothelial cells, leukocytes, and platelets to spike protein-ACE2 shifts their functions to procoagulant and thrombogenic as part of a thromboinflammatory response. 19 SARS-CoV-2 uses ACE2 as a receptor to attach cells, and the endothelial cell reduces ACE2 expression after the binding that decreases angiotensin II conversion to angiotensin 1 to 7. Increased angiotensin II stimulates vascular constriction, and decreased angiotensin 1 to 7 reinforces the proinflammatory reaction and thrombogenicity via vasoconstriction and leucocyte-platelet adhesion. ...
... 20 SARS-CoV-2 also infects platelets and megakaryocytes by binding to ACE2 on their cellular membranes, 21 Further, spike protein-ACE2 binding stimulates platelet release of α and dense granules' contents that include von Willebrand factor (VWF), PF4, adenosine diphosphate, and serotonin that increase platelet aggregation and enhance thrombus formation. 18 When an anti-PF4 IgG antibody is formed as part of inflammatory responses, the PF4anti-PF4 IgG complex binds platelets, macrophages, and neutrophils via the binding to Fcγ receptor IIA, and upregulates cell-cell interaction and aggregation. 22 SARS-CoV-2 stimulated platelets are also known to facilitate the release of coagulation factors, inflammatory cytokines, and express the procoagulant phosphatidylserine to facilitate prothrombotic effects 23 (►Fig. ...
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Although thrombosis frequently occurs in infectious diseases, the coagulopathy associated with COVID-19 has unique characteristics. Compared with bacterial sepsis, COVID-19-associated coagulopathy presents with minimal changes in platelet counts, normal prothrombin times, and increased D-dimer and fibrinogen levels. These differences can be explained by the distinct pathophysiology of the thromboinflammatory responses. In sepsis-induced coagulopathy, leukocytes are primarily responsible for the coagulopathy by expressing tissue factor, releasing neutrophil extracellular traps, multiple procoagulant substances, and systemic endothelial injury that is often associated with vasoplegia and shock. In COVID-19-associated coagulopathy, platelet activation is a major driver of inflammation/thrombogenesis and von Willebrand factor and platelet factor 4 are deeply involved in the pathogenesis. Although the initial responses are localized to the lung, they can spread systemically if the disease is severe. Since the platelets play major roles, arterial thrombosis is not uncommon in COVID-19. Despite platelet activation, platelet count is usually normal at presentation, but sensitive biomarkers including von Willebrand factor activity, soluble P-selectin, and soluble C-type lectin-like receptor-2 are elevated, and they increase as the disease progresses. Although the role of antiplatelet therapy is still unproven, current studies are ongoing to determine its potential effects.
... Thrombosis is reported to contribute significantly to the severity of COVID-19 symptoms [27][28][29][30]. Our results provide evidence at the gene expression level for the role of thrombosis in defining the severity of the disease. ...
... Notably, the expression of the ACE-2 gene encoding the putative cell entry receptor of SARS-CoV-2 was found to be upregulated in whole blood of CI patients with respect to MI (Table 3) but ACE2 expression level did not differ from NCI. Indeed, it is proposed that SARS-CoV-2 may directly infect and activate megakaryocytes and platelets [30,31]. Yet, the mechanism of interaction and direct activation remains controversial with contrasting reports on the presence of ACE2 protein expression on platelets. ...
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OBJECTIVE: Coronavirus disease 2019 (COVID-19), leading to mild infection (MI), acute respiratory distress syndrome or death in different persons. Although the basis of these variabilities has not been fully elucidated, some possible findings have been encountered. In the present study, we aimed to reveal genes with different expression profiles by next-generation se-quencing of RNA isolated from blood taken from infected patients to reveal molecular causes of different response. METHODS: Two healthy, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-negative control individuals (NCI), two SARS-CoV-2-positive patients who have MI, and two patients who have critical infection (CI) were included in the study. Total RNA was extracted from blood samples and sequenced. Raw RNA-Seq data were analyzed on Galaxy platform for the identification of differentially expressed genes and their pathway involvements. RESULTS: We found that 199 and 521 genes were downregulated in whole blood of COVID-19-positive CI patients compared to NCI and MI patients, respectively. We identified 21 gene ontology pathways commonly downregulated in CI patients compared to both NCI and MI, mostly associated with innate and adaptive immune responses. Three hundred and fifty-four and 600 genes were found to be upregulated compared to NCI and MI, respectively. Upregulated six pathways included genes that function in inflammatory response and inflammatory cytokine release. CONCLUSION: The transcriptional profile of CI patients deviates more significantly from that of MI in terms of the number of differentially expressed genes, implying that genotypic differences may account for the severity of SARS-CoV-2 infection and inflammatory responses through differential regulation of gene expression. Therefore, further studies that involve whole genome analysis coupled with differential expression analysis are required in order to determine the dynamics of genotype-gene expression profile associations.
... Platelets have important roles to play in the innate immune system and can directly contribute to NET generation when stimulated by viral activation, the interaction of platelets and neutrophils likely to be a relevant factor for thrombogenesis (193). Multiple mechanisms involving a global inflammatory reaction in COVID-19 patients can serve to activate platelets, be it diseased endothelium, increased viscosity or viral infection, considered plausible, despite debate regarding whether the platelet ACE2 expression demonstrated in murine platelets in vitro (194) is consistent for humans in vivo (195). Extrinsic coagulation pathways ultimately lead to the formation of thrombin, a proteinase that initiates platelet activation, aggregation and secretion of proinflammatory and signalling molecules including thromboxane A 2 . ...
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A long-shared evolutionary history is congruent with the multiple roles played by purinergic signaling in viral infection, replication and host responses that can assist or hinder viral functions. An overview of the involvement of purinergic signaling among a range of viruses is compared and contrasted with what is currently understood for SARS-CoV-2. In particular, we focus on the inflammatory and antiviral responses of infected cells mediated by purinergic receptor activation. Although there is considerable variation in a patient’s response to SARS-CoV-2 infection, a principle immediate concern in Coronavirus disease (COVID-19) is the possibility of an aberrant inflammatory activation causing diffuse lung oedema and respiratory failure. We discuss the most promising potential interventions modulating purinergic signaling that may attenuate the more serious repercussions of SARS-CoV-2 infection and aspects of their implementation.
... As we found MKs, the platelet precursors, actively infected in the bone marrow and the lung in fatal forms of the disease, SARS-CoV-2 might likely associate with platelets during thrombopoiesis, in a process we and others demonstrated for HIV [22,63], Dengue and Influenza virus [21,23]. Alternatively, platelets could endocytose the virus in the circulation as they express SARS-CoV-2 receptors such as ACE2 [64] and DC-SIGN that participate in virus endocytosis, as we have shown in vitro [18,65,66]. It is, however, unlikely as free SARS-CoV-2 in platelet-poor plasma is not infectious and as platelets could not internalize SARS-CoV-2 in vitro. ...
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Full-text available
SARS-CoV-2, although not being a circulatory virus, spread from the respiratory tract resulting in multiorgan failures and thrombotic complications, the hallmarks of fatal COVID-19. A convergent contributor could be platelets that beyond hemostatic functions can carry infectious viruses. Here, we profiled 52 patients with severe COVID-19 and demonstrated that circulating platelets of 19 out 20 non-survivor patients contain SARS-CoV-2 in robust correlation with fatal outcome. Platelets containing SARS-CoV-2 might originate from bone marrow and lung megakaryocytes (MKs), the platelet precursors, which were found infected by SARS-CoV-2 in COVID-19 autopsies. Accordingly, MKs undergoing shortened differentiation and expressing anti-viral IFITM1 and IFITM3 RNA as a sign of viral sensing were enriched in the circulation of deadly COVID-19. Infected MKs reach the lung concomitant with a specific MK-related cytokine storm rich in VEGF, PDGF and inflammatory molecules, anticipating fatal outcome. Lung macrophages capture SARS-CoV-2-containing platelets in vivo. The virus contained by platelets is infectious as capture of platelets carrying SARS-CoV-2 propagates infection to macrophages in vitro, in a process blocked by an anti-GPIIbIIIa drug. Altogether, platelets containing infectious SARS-CoV-2 alter COVID-19 pathogenesis and provide a powerful fatality marker. Clinical targeting of platelets might prevent viral spread, thrombus formation and exacerbated inflammation at once and increase survival in COVID-19.
... Interestingly, in addition to the antithrombotic effect, the anti-inflammatory function of heparin may be relevant in this setting. Heparan sulfate proteoglycans bind to SARS-CoV2 spike proteins and may decrease binding to host protein, and reduce the proinflammatory activities of damage associated molecular patterns (DAMPs), chemokines, neutrophil chemotaxis and leukocyte migration (Thachil, 2020b;Liu et al., 2020;Lindahl and Li, 2020). Using surface plasmon resonance, it was shown that SARS-CoV-2 Spike S1 protein binds to heparin inducing a structural change (Mycroft-West et al., 2020). ...
Thesis
Le sepsis est défini depuis 2016 comme la présence d'une infection associée à la survenue de dommages tissulaires à distance du foyer infecté. Le choc septique en est une des complications les plus graves. Le sepsis est une pathologie inflammatoire médiée par l'activation du système immun inné avec la reconnaissance par l'hôte de produits microbiens et de signaux de danger par des récepteurs cellulaires spécifiques et l'activation de multiples voies de signalisation cellulaires. Le sepsis entraîne un état d'hypercoagulabilité caractérisé par l'apparition de thrombi dans la microcirculation, de dépôts de fibrine, la formation de NETs (Neutrophil Extracellular Traps) et de lésions endothéliales. La formation de thrombi particulièrement dans la microcirculation entraîne un défaut de perfusion des organes. L'inhibition de l'activation plaquettaire réduit les interactions plaquettes-cellules inflammatoires-cellules endothéliales, ce qui théoriquement pourrait bloquer la réaction en cascade entre l'inflammation et la coagulation. Le potentiel des médicaments antiplaquettaires pour réduire les défaillances d'organes et améliorer le pronostic du sepsis est discuté. Cependant, l'activation plaquettaire et le détail de la cinétique d'activation plaquettaire dans le sepsis restent peu décrits, particulièrement chez l'Homme. Parallèlement, la crise COVID-19 a soulevé de nombreuses interrogations quant à la pathogénicité du virus et son impact sur la coagulation des patients de réanimation, présentant plus d'évènements thrombo-emboliques que de façon habituelle. Le rôle des plaquettes dans la physiopathologie de l'infection grave à SARS-CoV-2 reste encore à préciser. Ainsi le but de ce travail de thèse était double, travailler sur les fonctions plaquettaires au cours du sepsis puis, dans un second temps, mieux comprendre le rôle des plaquettes dans la pathogenèse de l'infection à SARS-CoV-2. Dans un premier temps, nous avons tenté de 2 déterminer le modèle animal précis d'étude du sepsis nous permettant une analyse de la cinétique d'activation plaquettaire. Nous avons ensuite utilisé ce modèle pour caractériser la cinétique d'activation des plaquettes au cours du choc septique. Nous avons dans un troisième temps réalisé la comparaison de l'activation plaquettaire chez des patients admis en réanimation polyvalente pour choc septique avec des sujets sains. Nous avons parallèlement étudié si le volume moyen plaquettaire (VMP) pouvait constituer un biomarqueur d'intérêt pour prédire la mortalité du choc septique chez l'Homme. Enfin, nous avons caractérisé le rôle des plaquettes dans la physiopathologie du syndrome de détresse respiratoire aigu sévère COVID-19 en réanimation.
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The role of a dysregulated renin-angiotensin system (RAS) in the pathogenesis of COVID-19 is well recognized. The imbalance between angiotensin II (Ang II) and Angiotensin1-7 (Ang1,7) caused by the interaction between SARS-CoV-2 and the angiotensin converting enzyme 2 (ACE2) receptors exerts a pivotal role on the clinical picture and outcome of COVID-19. ACE2 receptors are not the exclusive angiotensinases in nature. Other angiotensinases (PRCP, and POP) have the potential to limit the detrimental effects of the interactions between ACE2 And the spike proteins. In the cardiovascular disease continuum, ACE2 activity tends to decrease, and POP/PRCP activity to increase, from the health status to advanced deterioration of the cardiovascular system. The failure of the counter-regulatory RAS axis during the acute phase of COVID-19 is characterized by a decrease of ACE2 expression coupled to unchanged activity of other angiotensinases, therefore failing to limit the accumulation of Ang II. COVID-19 vaccines increase the endogenous synthesis of SARS-CoV-2 spike proteins. Once synthetized, the free-floating spike proteins circulate in the blood, interact with ACE2 receptors and resemble the pathological features of SARS-CoV-2. It has been noted that an increased catalytic activity of POP/PRCP is typical in elderly individuals with comorbidities or previous cardiovascular events, but not in younger people. Thus, the adverse reactions to COVID-19 vaccination associated with Ang II accumulation are generally more common in younger and healthy subjects. Understanding the relationships between different mechanisms of Ang II cleavage and accumulation offers the opportunity to close the pathophysiological loop between the risk of progression to severe forms of COVID-19 and the potential adverse events of vaccination.
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Background Coronavirus disease 2019 (COVID-19) became pandemic in 2020 and recently, mutated coronaviruses have emerged in many countries. The aim of this study was to identify the clinical characteristics and risk factors for critical illness in hospitalized COVID-19 patients in Zhengzhou for clinical prevention and management. Materials and methods A total of 70 patients hospitalized with COVID-19 were enrolled between 21 January and 29 February 2020, in Zhengzhou, China. Clinical characteristics, hematological findings, neutrophil lymphocyte ratio (NLR), platelet lymphocyte ratio (PLR), and inflammatory index on admission were obtained from medical records, COVID-19 patients with different outcomes were compared. Results The median age was 55 years. Forty-three (61.0%) patients were classified as having severe or critical cases. Eighteen (25.7%) patients died in hospital and the remaining 52 were discharged. Patients who died tend to be old with expectoration and chronic obstructive pulmonary disease. Compared to survivor, non-survivor had significantly higher numbers of leucocytes and neutrophils, NLR, aspartate aminotransferase (AST), γ-glutamyl transpeptidase, total bilirubin, direct bilirubin, lactate dehydrogenase (LDH), prothrombin time, D-dimer, C-reactive protein, and decreased platelets, lymphocytes, uric acid, and albumin (ALB). Logistic regression analysis identified leucocytes, platelets, PLR, NLR, AST, and ALB as independent predictive factors for poor outcomes. The area under curve of the combination of leucocytes, PLR, NLR, and AST was 0.87, with a sensitivity of 0.83 and specificity of 0.81. Conclusion Our results identified risk factors among COVID-19 patients for in-hospital mortality. Leucocytes, PLR, NLR, and AST could have important reference value for predicting prognosis, especially in low-resource countries.
Chapter
Polyanions as polymers carrying multiple negative charges have been extensively studied with regard to their potential antiviral activity. Most studies to date focused on organic polyanionic polymers, both natural and synthetic. The inorganic polymer, polyphosphate (polyP), despite the ubiquitous presence of this molecule from bacteria to man, has attracted much less attention. More recently, and accelerated by the search for potential antiviral agents in the fight against the pandemic caused by the coronavirus SARS-CoV-2, it turned out that polyP disrupts the first step of the viral replication cycle, the interaction of the proteins in the virus envelope and in the cell membrane that are involved in the docking process of the virus with the target host cell. Experiments on a molecular level using the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the cellular angiotensin converting enzyme 2 (ACE2) receptor revealed that polyP strongly inhibits the binding reaction through an electrostatic interaction between the negatively charged centers of the polyP molecule and a cationic groove, which is formed by positively charged amino acids on the RBD surface. In addition, it was found that polyP, due to its morphogenetic and energy delivering activities, enhances the antiviral host innate immunity defense of the respiratory epithelium. The underlying mechanisms and envisaged application of polyP in the therapy and prevention of COVID-19 are discussed.
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In the current pandemic of coronavirus disease (COVID-19), the identification of the patients admitted with severe infection–who are disposed to a high risk of acute respiratory distress syndrome (ARDS) development, is of a major significance for the determination of the appropriate therapeutic strategy. Laboratory records in admission were retrospectively reviewed from 493 cases of severe COVID-19 divided into two groups: Group 1 with ARDS and Group 2 without ARDS. The platelet distribution width (PDW) difference between Group 1 and Group 2 is significant–15.10 ± 2.08 fl for those who developed ARDS versus 12.94 ± 2.12 fl for those without ARDS. The sensitivity and the specificity of this parameter is lower than that of D-dimer. After grouping of the PDW values into intervals and combining them with the rate of increase in D-dimer (D-PDWf index) to form a forecasting index, a significant increase in the specificity and sensitivity of the two parameters is identified–area under the ROC curve (AUC) is 0.874 for D-PDWf index, with respective AUC for PDW 0.768 and AUC for D-dimer 0.777. Conclusion: PDW is a significant predictive parameter at admission for subsequent development of ARDS in patients, with increased significance in combination with the degree of increase in D-dimer.
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Platelets, small anucleate cells circulating in the blood, are critical mediators in haemostasis and thrombosis. Interestingly, recent studies demonstrated that platelets contain both pro-inflammatory and anti-inflammatory molecules, equipping platelets with immunoregulatory function in both innate and adaptive immunity. In the context of infectious diseases, platelets are involved in early detection of invading microorganisms and are actively recruited to sites of infection. Platelets exert their effects on microbial pathogens either by direct binding to eliminate or restrict dissemination, or by shaping the subsequent host immune response. Reciprocally, many invading microbial pathogens can directly or indirectly target host platelets, altering platelet count or/and function. In addition, microbial pathogens can impact the host auto- and alloimmune responses to platelet antigens in several immune-mediated diseases, such as immune thrombocytopenia, and fetal and neonatal alloimmune thrombocytopenia. In this review, we discuss the mechanisms that contribute to the bidirectional interactions between platelets and various microbial pathogens, and how these interactions hold relevant implications in the pathogenesis of many infectious diseases. The knowledge obtained from “well-studied” microbes may also help us understand the pathogenesis of emerging microbes, such as SARS-CoV-2 coronavirus.
Preprint
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Background Coronavirus disease 2019 (COVID-19), caused by novel coronavirus SARS-CoV-2, has to date affected over 13.3 million globally. Although high rates of venous thromboembolism and evidence of COVID-19-induced endothelial dysfunction have been reported, the precise aetiology of the increased thrombotic risk associated with COVID-19 infection remains to be fully elucidated. Objectives Here, we assessed clinical platelet parameters and circulating platelet activity in patients with severe and non-severe COVID-19. Methods An assessment of clinical blood parameters in patients with severe COVID-19 disease (requiring intensive care), patients with non-severe disease (not requiring intensive care), general medical in-patients without COVID-19 and healthy donors was undertaken. Platelet function and activity were also assessed by secretion and specific marker analysis. Results We show that routine clinical blood parameters including increased MPV and decreased platelet:neutrophil ratio are associated with disease severity in COVID-19 upon hospitalisation and intensive care unit admission. Strikingly, agonist-induced ADP release was dramatically higher in COVID-19 patients compared with non-COVID-19 hospitalized patients and circulating levels of PF4, sP-selectin and TPO were also significantly elevated in COVID-19. Conclusion Distinct differences exist in routine full blood count and other clinical laboratory parameters between patients with severe and non-severe COVID-19. Moreover, we have determined that COVID-19 patients possess hyperactive circulating platelets. These data suggest that abnormal platelet reactivity may contribute to hypercoagulability in COVID-19. Further investigation of platelet function in COVID-19 may provide additional insights into the aetiology of thrombotic risk in this disease and may contribute to the optimisation of thrombosis prevention and treatment strategies.
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Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) is an emergent pathogen responsible for the coronavirus disease 2019 (COVID-19). Since its emergence, the novel coronavirus has rapidly achieved pandemic proportions causing remarkably increased morbidity and mortality around the world. A hypercoagulability state has been reported as a major pathologic event in COVID-19, and thromboembolic complications listed among life-threatening complications of the disease. Platelets are chief effector cells of hemostasis and pathological thrombosis. However, the participation of platelets in the pathogenesis of COVID-19 remains elusive. This report demonstrates that increased platelet activation and platelet-monocyte aggregates formation is observed in severe COVID-19 patients, but not in patients presenting mild COVID-19 syndrome. In addition, exposure to plasma from severe COVID-19 patients increased the activation of control platelets ex vivo. In our cohort of COVID-19 patients admitted to the ICU, platelet-monocyte interaction was strongly associated with TF expression by the monocytes. Platelet activation and monocyte TF expression were associated with markers of coagulation dysfunction as fibrinogen and D-dimers, and were increased in patients requiring invasive mechanical ventilation or patients that evolved with in-hospital mortality. Finally, platelets from severe COVID-19 patients were able to induce TF expression ex vivo in monocytes from healthy volunteers, a phenomenon that was inhibited by platelet P-selectin neutralization or integrin αIIb/β3 blocking with the aggregation inhibitor abciximab. Altogether, these data shed light on new pathological mechanisms involving platelet activation and platelet-dependent monocyte TF expression, which were associated with COVID-19 severity and mortality.
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(17/September/2020) This manuscript has now been peer-reviewed and published by Circulation Research: https://www.ahajournals.org/doi/10.1161/CIRCRESAHA.120.317703 ----------------------------------------------------------------------------------------------------------------- Rationale: In addition to the overwhelming lung inflammation that prevails in COVID-19, hypercoagulation and thrombosis contribute to the lethality of subjects infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Platelets are chiefly implicated in thrombosis. Moreover, they can interact with viruses and are an important source of inflammatory mediators. While a lower platelet count is associated with severity and mortality, little is known about platelet function during COVID-19. Objective: To evaluate the contribution of platelets to inflammation and thrombosis in COVID-19 patients. Methods and Results: We document the presence of SARS-CoV-2 RNA in platelets of COVID-19 patients. Exhaustive assessment of cytokines in plasma and in platelets revealed the modulation of platelet-associated cytokine levels in COVID-19, pointing to a direct contribution of platelets to the plasmatic cytokine load. Moreover, we demonstrate that platelets release their alpha- and dense-granule contents and phosphatidylserine-exposing extracellular vesicles. Functionally, platelets were hyperactivated in COVID-19 subjects, with aggregation occurring at suboptimal thrombin concentrations. Furthermore, platelets adhered more efficiently onto collagen-coated surfaces under flow conditions. Conclusions: These data suggest that platelets could participate in the dissemination of SARS-CoV-2 and in the overwhelming thrombo-inflammation observed in COVID-19. Thus, blockade of platelet activation pathways may improve outcomes in this disease.
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There is an urgent need to understand the pathogenesis of coronavirus disease 2019 (COVID-19). In particular, thrombotic complications in patients with COVID-19 are common and contribute to organ failure and mortality. Patients with severe COVID-19 present with hemostatic abnormalities that mimic disseminated intravascular coagulopathy associated with sepsis with the major difference being increased risk of thrombosis rather than bleeding. However, whether SARS-CoV-2 infection alters platelet function to contribute to the pathophysiology of COVID-19 remains unknown. In this study, we report altered platelet gene expression and functional responses in patients infected with SARS-CoV-2. RNA sequencing demonstrated distinct changes in the gene expression profile of circulating platelets of COVID-19 patients. Pathway analysis revealed differential gene expression changes in pathways associated with protein ubiquitination, antigen presentation and mitochondrial dysfunction. The receptor for SARS-CoV-2 binding, ACE2, was not detected by mRNA or protein in platelets. Surprisingly, mRNA from the SARS-CoV-2 N1 gene was detected in platelets from 2/25 COVID-19 patients, suggesting platelets may take-up SARS-COV-2 mRNA independent of ACE2. Resting platelets from COVID-19 patients had increased P-selectin expression basally and upon activation. Circulating platelet-neutrophil, -monocyte, and -T-cell aggregates were all significantly elevated in COVID-19 patients compared to healthy donors. Furthermore, platelets from COVID-19 patients aggregated faster and showed increased spreading on both fibrinogen and collagen. The increase in platelet activation and aggregation could partially be attributed to increased MAPK pathway activation and thromboxane generation. These findings demonstrate that SARS-CoV-2 infection is associated with platelet hyperreactivity which may contribute to COVID-19 pathophysiology.
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COVID-19 is an infectious disease caused by SARS-CoV-2, which enters host cells via the cell surface proteins ACE2 and TMPRSS2. Using normal and malignant models and tissues from the aerodigestive and respiratory tracts, we investigated the expression and regulation of ACE2 and TMPRSS2. We find that ACE2 expression is restricted to a select population of highly epithelial cells and is repressed by ZEB1, in concert with ZEB1's established role in promoting epithelial to mesenchymal transition (EMT). Notably, infection of lung cancer cells with SARS-CoV-2 induces metabolic and transcriptional changes consistent with EMT, including upregulation of ZEB1 and AXL, thereby downregulating ACE2 post-infection. This suggests a novel model of SARS-CoV-2 pathogenesis in which infected cells shift toward an increasingly mesenchymal state and lose ACE2 expression, along with its acute respiratory distress syndrome-protective effect, in a ZEB1-dependent manner. AXL-inhibition and ZEB1-reduction, as with bemcentinib, offers a potential strategy to reverse this effect.
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A novel coronavirus was recently discovered and termed SARS-CoV-2. Human infection can cause coronavirus disease 2019 (COVID-19), which has been rapidly spreading around the globe1,2. SARS-CoV-2 shows some similarities to other coronaviruses. However, treatment options and a cellular understanding of SARS-CoV-2 infection are lacking. Here we identify the host cell pathways modulated by SARS-CoV-2 infection and show that inhibition of these pathways prevent viral replication in human cells. We established a human cell culture model for infection with SARS-CoV-2 clinical isolate. Employing this system, we determined the SARS-CoV-2 infection profile by translatome3 and proteome proteomics at different times after infection. These analyses revealed that SARS-CoV-2 reshapes central cellular pathways, such as translation, splicing, carbon metabolism and nucleic acid metabolism. Small molecule inhibitors targeting these pathways prevented viral replication in cells. Our results reveal the cellular infection profile of SARS-CoV-2 and led to the identification of drugs inhibiting viral replication. We anticipate our results to guide efforts to understand the molecular mechanisms underlying host cell modulation upon SARS-CoV-2 infection. Furthermore, our findings provide insight for the development of therapy options for COVID-19.