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SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: Implications for microclot formation in COVID-19

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SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: Implications for microclot formation in COVID-19

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Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) -induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by unprecedented clinical pathologies. One of the most important pathologies, is hypercoagulation and microclots in the lungs of patients. Here we study the effect of isolated SARS-CoV-2 spike protein S1 subunit as potential inflammagen sui generis. Using scanning electron and fluorescence microscopy as well as mass spectrometry, we investigate the potential of this inflammagen to interact with platelets and fibrin(ogen) directly to cause blood hypercoagulation. Using platelet poor plasma (PPP), we show that spike protein may interfere with blood flow. Mass spectrometry also showed that when spike protein S1 is added to healthy PPP, it results in structural changes to β and γ fibrin(ogen), complement 3, and prothrombin. These proteins were substantially resistant to trypsinization, in the presence of spike protein S1. Here we suggest that, in part, the presence of spike protein in circulation may contribute to the hypercoagulation in COVID-19 positive patients and may cause substantial impairment of fibrinolysis. Such lytic impairment may result in the persistent large microclots we have noted here and previously in plasma samples of COVID-19 patients. This observation may have important clinical relevance in the treatment of hypercoagulability in COVID-19 patients.
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SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to
fibrinolysis: Implications for microclot formation in COVID-19
Lize M. Grobbelaar1, Chantelle Venter1, Mare Vlok2, Malebogo Ngoepe3, Gert Jacobus
Laubscher 4, Petrus Johannes Lourens4, Janami Steenkamp5 Douglas B. Kell1,6*7,
Etheresia Pretorius1*
1Department of Physiological Sciences, Faculty of Science, Stellenbosch University,
Stellenbosch, Private Bag X1 Matieland, 7602, South Africa;
2 Central Analytical Facility: Mass Spectrometry Stellenbosch University, Tygerberg
Campus, Room 6054, Clinical Building, Francie van Zijl Drive
Tygerberg, CAPE TOWN, 7505
3 Department of Mechanical Engineering, Faculty of Engineering and the Built Environment,
University of Cape Town, Cape Town, Rondebosch, 7701, South Africa
4Mediclinic Stellenbosch, Stellenbosch 7600, South Africa;
5 PathCare Laboratories, PathCare Business Centre, PathCare Park, Neels Bothma Street,
N1 City 7460, South Africa
6 Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and
Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Crown St,
Liverpool L69 7ZB, UK
7 The Novo Nordisk Foundation Centre for Biosustainability, Building 220, Chemitorvet 200,
Technical University of Denmark, 2800 Kongens Lyngby, Denmark
*Corresponding authors:
*Etheresia Pretorius
Department of Physiological Sciences, Stellenbosch University, Private Bag X1 Matieland,
7602, SOUTH AFRICA
resiap@sun.ac.za
http://www.resiapretorius.net/
ORCID: 0000-0002-9108-2384
*Douglas B. Kell
Department of Biochemistry, Institute of Integrative Biology, Faculty of Health and Life
Sciences, University of Liverpool, Crown St, Liverpool L69 7ZB, UK
dbk@liv.ac.uk
ORCID: 0000-0001-5838-7963
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NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
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ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) -induced infection, the
cause of coronavirus disease 2019 (COVID-19), is characterized by unprecedented clinical
pathologies. One of the most important pathologies, is hypercoagulation and microclots in
the lungs of patients. Here we study the effect of isolated SARS-CoV-2 spike protein S1
subunit as potential inflammagen sui generis. Using scanning electron and fluorescence
microscopy as well as mass spectrometry, we investigate the potential of this inflammagen
to interact with platelets and fibrin(ogen) directly to cause blood hypercoagulation. Using
platelet poor plasma (PPP), we show that spike protein may interfere with blood flow. Mass
spectrometry also showed that when spike protein S1 is added to healthy PPP, it results in
structural changes to and fibrin(ogen), complement 3, and prothrombin. These proteins
were substantially resistant to trypsinization, in the presence of spike protein S1. Here we
suggest that, in part, the presence of spike protein in circulation may contribute to the
hypercoagulation in COVID-19 positive patients and may cause substantial impairment of
fibrinolysis. Such lytic impairment may result in the persistent large microclots we have noted
here and previously in plasma samples of COVID-19 patients. This observation may have
important clinical relevance in the treatment of hypercoagulability in COVID-19 patients.
Keywords: COVID-19; Spike protein S1; Fibrin(ogen); Microclot; Microscopy
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INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the
cause of coronavirus disease 2019 (COVID-19), is characterized by unprecedented clinical
pathologies. Phenotypic vascular characteristics are strongly associated with various
coagulopathies that may result in either bleeding and thrombocytopenia or hypercoagulation
and thrombosis (Gupta et al., 2020, Perico et al., 2021). Various circulating and
dysregulated inflammatory coagulation biomarkers, including fibrin(ogen), D-dimer, P-
selectin and von Willebrand Factor (VWF), C-reactive protein (CRP), and various cytokines,
directly bind to endothelial receptors. Endotheliopathies are therefore a key clinical feature of
the condition (Goshua et al., 2020, Ackermann et al., 2020). During the progression of the
various stages of the COVID-19, markers of viral replication, as well as VWF and fibrinogen
depletion with increased D-dimer levels and dysregulated P-selectin levels, followed by a
cytokine storm, are likely to be indicative of a poor prognosis (Grobler et al., 2020, Pretorius
et al., 2020, Venter et al., 2020, Roberts et al., 2020). This poor prognosis is further
worsened as together with a substantial deposition of microclots in the lungs (Renzi et al.,
2020, Ciceri et al., 2020, Bobrova et al., 2020), plasma of COVID-19 patients also carries a
massive load of preformed amyloid clots (Grobler et al., 2020), and there are also numerous
reports of damage to erythrocytes (Lam et al., 2020, Berzuini et al., 2020, Akhter et al.,
2020), platelets and dysregulation of inflammatory biomarkers (Grobler et al., 2020,
Pretorius et al., 2020, Venter et al., 2020, Roberts et al., 2020).
The virulence of the pathogen is closely linked to its membrane proteins. One such protein,
found on the COVID-19 virus, is the spike protein, which is a membrane glycoprotein. The
spike proteins are the key factors for virus attachment to target cells, as they bind to the
angiotensin-converting 2 (ACE2) surface receptors (Bergmann and Silverman, 2020). Spike
proteins are class I viral fusion proteins (Kawase et al., 2019). They present as protruding
homotrimers on the viral surface and mediate virus entry into the target host cells (Walls et
al., 2020). A singular spike protein is between 180–200 kDa in size and contains an
extracellular N-terminal, a transmembrane domain fixed in the membrane of the virus, and a
short intracellular C-terminal segment (Kawase et al., 2019, Zhang et al., 2020). Spike
proteins are coated with polysaccharide molecules that serve as camouflage. This helps
evade surveillance by the host immune system during entry (Zhang et al., 2020). The S1
subunit is responsible for receptor binding (Watanabe et al., 2020), with subunit 2 (S2), a
carboxyl-terminal subunit, responsible for viral fusion and entry (Flores-Alanis et al., 2020)
(see Figure 1).
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Figure 1: Schematic representation of SARS-CoV-2 Spike glycoprotein. Adapted from (Duan et al.,
2020). Abbreviations: S1, subunit 1; S2, subunit 2; HR1, heptad repeat 1; HR2, heptad repeat 2.
This image was created with BioRender (https://biorender.com/).
Receptor binding is certainly responsible for cell-mediated pathologies, but does not of itself
explain the coagulopathies. Spike protein, can however be shed, and it has been detected
in various organs, including the urinary tract (George et al., 2021). S1 proteins can also
cross the blood-brain-barrier (Rhea et al., 2021). Free S1 particles may also play a role in
the pathogenesis of the disease (Letarov et al., 2020, Buzhdygan et al., 2020). Free spike
protein can potentially be released due to spontaneous “firing” of the S protein trimers on the
surface of virions, and infected cells liberates free receptor binding domain-containing S1
particles (Letarov et al., 2020).
Here we study the effect of isolated SARS-CoV-2 spike protein S1 subunit as potential pro-
inflammatory inflammagen sui generis. We investigate the potential of this inflammagen to
directly interact with platelets and fibrin(ogen) to cause fibrin(ogen) protein changes and
blood hypercoagulation. We also determine if the spike protein may interfere with blood
flow, by comparing naïve healthy PPP samples, with and without added spike protein, to
PPP samples from COVID-19 positive patients (before treatment). We conclude that the
spike protein may have pathological effects directly, without being taken up by cells. This
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provides further evidence that targeting it directly, whether via vaccines or antibodies, is
likely to be of therapeutic benefit.
MATERIALS AND METHODS
Ethical clearance
Ethical clearance for the study was obtained from the Health Research Ethics Committee
(HREC) of Stellenbosch University (South Africa) (reference: N19/03/043, project ID: 9521).
The experimental objectives, risks, and details were explained to volunteers both verbally
and in text and informed consent were obtained prior to blood collection. Strict compliance to
ethical guidelines and principles Declaration of Helsinki, South African Guidelines for Good
Clinical Practice, and Medical Research Council Ethical Guidelines for Research were kept
for the duration of the study and for all research protocols.
Sample demographics and considerations
Blood was collected from healthy volunteers (N=11; 3 males, 8 females; mean age 43.4 ±
11.7) to serve as controls. Individuals who smoke, who were diagnosed with cardiovascular
diseases, clotting disorders (coagulopathies), and/or any known inflammatory conditions
(e.g. T2DM, rheumatoid arthritis, tuberculosis, asthma, etc.) could not serve as control
volunteers. Furthermore, pregnancy, lactation, hormonal therapy, oral contraceptive usage,
and/or using anticoagulants, were also factors that would result in exclusion. Smoking was
excluded since it has been proven to impair coagulation, fibrinolysis, and the haemostatic
process (Pretorius et al., 2010). Microfluidic analysis included a preliminary analysis using
PPP samples from three COVID-19 positive patients, on day of first diagnosis and before
any treatment were resumed. All three patients were diagnosed with moderate to severe
COVID-19 symptoms (1 male; 2 females mean age 71 ± 14.1).
Blood sample collection
Either a qualified phlebotomist or medical practitioner drew the volunteers’ (control) blood via
venepuncture, adhering to standard sterile protocol. Blood samples were stored in two to
three 4.5mL sodium citrate (3.2%) tubes (BD Vacutainer®, 369714). After several gentle
inversions, the collected citrate tubes were allowed to rest at room temperature for a
minimum of 30 minutes to allow for adequate anticoagulant amalgamation before
commencing sample preparation. Whole blood (WB) was centrifuged at 3000xg for 15
minutes at room temperature to isolate erythrocytes. The supernatant, i.e. PPP, was
collected and stored in 1.5mL Eppendorf tubes at -80ºC, till needed for experiments.
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Spike protein preparation
SARS-CoV-2 (2019-nCoV) Spike protein S1 Subunit, mFcTag, was purchased from Sino
Biological (Beijing, China) (catalog number 40591-V05H1) and prepared using doubly
distilled water, following the instructions provided. 400µL diluent (endotoxin-free water) was
added to the 100µg spike protein to create a stock solution (A) of 0.25mg.mL-1. This stock
solution was diluted to working solutions. To determine the concentration of spike protein
needed to result in significant, but yet a high enough concentration to cause physiological
effects on the viscoelastic properties of blood, different concentrations of spike protein in
PPP were assessed with fluorescence microscopy. A healthy control blood sample were
separated into four 1.5mL Eppendorf tubes with different final exposure concentrations of
spike protein in the PPP of 100ng.mL-1, 50ng.mL-1, 10ng.mL-1 and 1ng.mL-1. PPP samples
were incubated with the various spike protein concentrations for 30 minutes at room
temperature.
Fluorescence microscopy of purified fibrinogen and platelet poor plasma (PPP) with
and without thrombin
Concentration verification
To verify which spike protein concentration will be effective, 5uL of the PPP exposed to the
varies spike protein concentrations was placed on a glass slide, after being exposure to the
fluorescent amyloid dye, Thioflavin T (ThT) (Sigma-Aldrich, St. Louis, MO, USA) for 30
minutes at room temperature. The final concentration of ThT in all prepared samples was
0,005mM. After the evaluation of the samples, with the varies spike protein concentrations, it
was found that the final exposure concentrations of 1ng.mL-1 was sufficient and used for the
rest of the study.
Amyloid protein and anomalous clotting in platelet poor plasma samples
To study spontaneous anomalous clotting of fibrin(ogen), in the naïve healthy PPP samples,
and in the presence of spike protein, 5 L PPP exposed to 1ng.mL-1 (final concentrations) of
spike protein, was smeared on a glass slide and covered with a coverslip. This was done
after it was exposure to the fluorescent amyloid dye, Thioflavin T (ThT) (final concentration:
0,005mM) (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes at room temperature. Fibrin
PPP clots, with and without spike protein and after exposure to ThT, were also prepared by
adding 2,5 L of thrombin (7U·mL-1, South African National Blood Service) to 5uL PPP and
was placed on a glass slide and covered with a coverslip. The excitation wavelength for ThT
was set at 450nm to 488nm and the emission at 499nm to 529nm and processed samples
were viewed using a Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat
63x/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany).
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Fluorescence microscopy images of healthy PPP with and without spike protein were
analysed using Fiji® (Java 1.6_0 24 [64-bit]) to numerically represent the images. The total
area of fluorescing particles or anomalous clotting (identified by the amyloid dye, ThT) (Kell
and Pretorius, 2017, Pretorius et al., 2016, Pretorius et al., 2013b) was determined using a
thresholding algorithm in Fiji®. Images were firstly set to scale in Fiji® according to the
magnification of the lens used on the fluorescent microscope, followed by the selection of
appropriate threshold value to regard as much of the foreground and disregard as much of
the background of the image as possible. In order to optimise the amount of images
thresholded, a program was written in Java to simultaneously analyse a group of images
(see supplementary material). The total percentage of anomalous clots in each image was
calculated and the average of all the images per sample was calculated. These average
values were used for statistical analysis.
Purified fibrin(ogen) clot model
To determine if spike protein causes changes in purified fibrinogen, our purified fibrin(ogen)
clot model of choice was fluorescent fibrinogen conjugated to Alexa Fluor™488
(ThermoFisher, F13191). A final fibrinogen concentration of 2mg.mL-1 was prepared in
endotoxin-free water and exposed to 1ng.mL-1 (final concentrations) spike protein for 30
minutes at room temperature. 5uL of the purified fibrinogen was placed on a glass slide, with
2,5uL of thrombin. The excitation wavelength for our fluorescent fibrinogen model was set at
450nm to 488nm and the emission at 499nm to 529nm and processed samples were viewed
using a Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63x/1.4 Oil
DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany).
Whole blood
Healthy WB was exposed to a final exposure concentration of 1ng.mL-1 spike protein. The
fluorescent marker, CD62P (platelet surface P-selectin) was added to WB to study platelet
activation. CD62P is found on the membrane of platelets and then translocate to the platelet
membrane surface. The translocation occurs after the platelet P-selectin is released from the
cellular granules during platelet activation (Venter et al., 2020, Grobler et al., 2020). 4 µL
CD62P (PE-conjugated) (IM1759U, Beckman Coulter, Brea, CA, USA) was added to 20 µL
WB (either naïve or incubated with spike protein). The WB exposed to the marker was
incubated for 30 minutes (protected from light) at room temperature. The excitation
wavelength for the CD62P was 540nm to 570nm and the emission 577nm to 607nm.
Processed samples were also viewed using a Zeiss Axio Observer 7 fluorescent microscope
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with a Plan-Apochromat 63x/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich,
Germany).
Scanning electron microscopy of whole blood samples
Scanning electron microscopy (SEM) was used to view healthy WB samples, with and
without the addition of spike protein. 10uL WB was placed on a glass cover slip and
prepared according to previously published SEM preparation methods (Pretorius, 2013,
Pretorius et al., 2013a), starting with washing steps in phosphate-buffered saline (PBS) (pH
= 7.4) (ThermoFisher Scientific, 11594516) for 20 minutes. Fixation was performed by
coating the slides in 4% formaldehyde (FA) for 30 minutes, followed by washing them in PBS
three times. For each wash, the PBS should be left on for 3 minutes before removing and
washing again. Osmium tetroxide (OsO4) (Sigma-Aldrich, 75632) was added for 15 minutes
and the slides were washed in PBS three times with 3 minutes in each once more. The next
step was to serially dehydrate the slides with ethanol followed by a drying step using
hexamethyldisilazane (HMDS) (Sigma-Aldrich, 379212). Samples were mounted on glass
slides and coated with carbon. The slides were viewed on a Zeiss MERLIN FE-SEM with the
InLens detector at 1kV (Carl Zeiss Microscopy, Munich, Germany).
Microfluidics
Microfluidic analysis was performed using healthy PPP and healthy pooled PPP samples (3
pooled PPP samples), with and without spike protein, and 3 COVID-19 PPP samples.
Pooled samples were used due to the volume required for this experiment.
Hardware
A microfluidic setup was used to simulate and investigate clot growth under conditions of
flow. A Cellix microfluidic syringe pump (Cellix Ltd, Dublin, Ireland) was used to drive flow
through Cellix Vena8 Fluoro+ biochips (Cellix Ltd, Dublin, Ireland), comprising eight straight
microfluidic flow channels each, at flow rates specified in the following paragraph. A single
microchannel has a width of 400 m and a height of 100 m (equivalent diameter of 207 m),
and a length of 2.8cm. The dimensions of the microchannel are in the same order of
magnitude as those of some vessels of the microvasculature, that is, under 300 m (Jacob et
al., 2016). In order to observe clot evolution in real-time, the biochips were placed under the
Zeiss Axio Observer 7 fluorescent microscope with a 10x/0.25 objective (Carl Zeiss
Microscopy, Munich, Germany).
Flow conditions
A new flow channel was used for every run. The channel was flushed with distilled water at a
flow rate of 1mL.min-1 for 1 minute. After 5 minutes, thrombin was infused through the
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microchannel at a flow rate of 50 L.min
-1
for 90 seconds (Figure 2). The channel was left to
stand for another five minutes and then the sample (control, control with spike, or COVID-19)
was infused at a flow rate of 10 L.min
-1
for 5 minutes, with a video recording of the channel
(Figure 2). The flow was then stopped after 5 minutes, and a set of micrographs was taken
across the channel. The sample was then left for another 5 minutes, to see if any additional
changes occur (Figure 2). This flow rate corresponds with a shear rate, 250s
-1
and a
Reynolds numbers, Re 1. One of the main challenges in attempting to achieve consistent
shear rates and Reynolds numbers was the variability in viscosity from sample to sample.
Furthermore, blood flowing through microvessels is known to behave in a non-Newtonian
manner, adding to the complexities of variable viscosity within a single sample. To achieve
standardisation between samples, a constant flow rate was used.
Figure 2: Experimental protocol for growing clots in a microfluidic system.
Proteomics
Four healthy PPP samples were analysed before and after addition of spike protein. The
samples were diluted in 10mM ammonium bicarbonate to 1mg.mL
-1
. A total of 1
g
trypsin
(New England Biosystems) was added to the plasma for 1:50 enzyme to substrate ratio. No
reduction or alkylation was performed.
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Liquid chromatography
Dionex nano-RSLC
Liquid chromatography was performed on a Thermo Scientific Ultimate 3000 RSLC
equipped with a 5 mm x 300 µm C18 trap column (Thermo Scientific) and a CSH 25cmx75
µm 1.7 m particle size C18 column (Waters) analytical column. The solvent system
employed was loading: 2% acetonitrile:water; 0.1% FA; Solvent A: 2% acetonitrile: water;
0.1% FA and Solvent B: 100% acetonitrile:water. The samples were loaded onto the trap
column using loading solvent at a flow rate of 2 µL/min from a temperature controlled
autosampler set at 7 C. Loading was performed for 5 minutes before the sample was eluted
onto the analytical column. Flow rate was set to 300nL/minute and the gradient generated as
follows: 5.0% -30% B over 60 minutes and 30-50%B from 60-80 minutes. Chromatography
was performed at 45°C and the outflow delivered to the mass spectrometer.
Mass spectrometry
Mass spectrometry was performed using a Thermo Scientific Fusion mass spectrometer
equipped with a Nanospray Flex ionization source. Plasma samples, before and after
addition of spike protein addition (1 ng.mL-1 final exposure concentration), from 4 of our
control samples were analysed with this method. The sample was introduced through a
stainless-steel nano-bore emitter Data was collected in positive mode with spray voltage set
to 1.8kV and ion transfer capillary set to 275°C. Spectra were internally calibrated using
polysiloxane ions at m/z = 445.12003. MS1 scans were performed using the orbitrap
detector set at 120 000 resolution over the scan range 375-1500 with AGC target at 4 E5
and maximum injection time of 50ms. Data was acquired in profile mode. MS2 acquisitions
were performed using monoisotopic precursor selection for ion with charges +2-+7 with error
tolerance set to +/- 10ppm. Precursor ions were excluded from fragmentation once for a
period of 60s. Precursor ions were selected for fragmentation in HCD mode using the
quadrupole mass analyser with HCD energy set to 30%. Fragment ions were detected in the
Orbitrap mass analyzer set to 30 000 resolution. The AGC target was set to 5E4 and the
maximum injection time to 100ms. The data was acquired in centroid mode.
Statistical analysis
Data analysis: plasma samples
Statistical analyses of data generated were performed using GraphPad Prism software
(version 9.0.0). The normality of the data was assessed using the Shapiro-Wilk normality
test. For analysis of data between two groups, paired t-tests (for pairwise statistical
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comparisons between data from untreated and treated control groups) and unpaired t-tests
(for non-pairwise statistical comparisons) were performed to assess statistical significance
for parametric data, whereas the Mann-Whitney test was utilized to test for statistical
significance in non-parametric data and the Wilcoxon test was used for significance in paired
parametric data. When comparing three or more experimental groups, the Kruskal-Wallis
test (nonparametric data) or one-way ANOVA (parametric data) test was applied to test for
statistical significance. A p-value of less than 0.05 was considered to be statistically
significant. Parametric data were presented as the mean and standard deviation (SD),
whereas non-parametric data were presented as the median and interquartile range (IQR).
Mass spectrometer data analysis
The raw files generated by the mass spectrometer were imported into Proteome Discoverer
v1.4 (Thermo Scientific) and processed using the Sequest and Amanda algorithms.
Database interrogation was performed against the 2019-nCOVpFASTA1 database. Semi-
tryptic cleavage with 2 missed cleavages was allowed for. Precursor mass tolerance was set
to 10ppm and fragment mass tolerance set to 0.02 Da. Demamidation (NQ), oxidation (M)
allowed as dynamic modifications. Peptide validation was performed using the Target-Decoy
PSM validator node. The search results were imported into Scaffold Q+ for further validation
(www.proteomesoftware.com) and statistical testing. A t-test was performed on the datasets
and the emPAI quantitative method used to compare the datasets.
Supplementary material and raw data
All supplementary material and raw data can be accessed here:
https://1drv.ms/u/s!AgoCOmY3bkKHisg5J0nb6wqsBzzWAQ?e=XAsc7w
RESULTS
Fluorescence microscopy of purified fibrinogen and platelet poor plasma
Fluorescence microscopy was utilized to visualize the fluorescent amyloid signals in
spontaneously formed anomalous clots, present in a fluorescent fibrinogen model, and also
in healthy PPP with and without spike protein. As a preliminary investigation PPP from a
single health control were incubated for 30 minutes with varying spike protein
concentrations, followed by 30 minutes incubation with ThT and lastly preparation for
viewing. It was found that the final exposure concentrations of 1 ng.mL-1 was sufficient and
used for the rest of the study (see supplementary raw data for other exposure concentrations
micrographs).
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Figure 3A and B show representative micrographs of purified fluorescent (Alexa Fluor™488)
fibrinogen with added thrombin and after exposure to 1 ng.mL-1 spike protein. A denser fibrin
clot formed in the presence of spike protein (Figure 3B). In PPP, with and without thrombin,
the green fluorescent ThT signal indicates areas of amyloid deposit formation. ThT is known
to bind to open hydrophobic areas in protein and these are amyloid in nature (Adams et al.,
2019, de Waal et al., 2018, Kell and Pretorius, 2017, Page et al., 2019). Figure 4A and D
shows representative micrographs of a healthy PPP smear, with and without thrombin, and
with added ThT where slight anomalous clotting is seen. In contrast, when spike protein is
added to PPP, with and without thrombin, a major increase in dense anomalous clotted
deposits, with an amyloid nature, were noted (referred to as amyloid deposits) (Figure 4B
and D). A thresholding algorithm was applied to the micrographs (with and without thrombin)
using Fiji® which was used to calculate the total area of amyloid deposits in each micrograph
(in total, 320 micrographs were analysed). Using this method, the average total percentage
of amyloid deposits per group was calculated (naïve healthy PPP, naïve healthy PPP +
thrombin, and PPP incubated with 1 ng.mL-1 spike protein, with and without added thrombin)
(See Table 1). As expected, there were no significant differences between % area amyloid
deposits of healthy PPP with and without thrombin However, there was a significant increase
in % area amyloid deposits in PPP before and after added spike protein, in both PPP
smears and fibrin clots (where thrombin was added).
Figure 3: Representative fluorescence micrographs of purified fluorescent (Alexa Fluor™488)
fibrinogen (note no ThT added) with added thrombin to form extensive fibrin clots. A) Fluorescent
fibrinogen with thrombin; B) fluorescent fibrinogen with added spike protein (final exposure
concentration 1 ng.mL-1) and thrombin.
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Figure 4: Representative fluorescence micrographs of platelet poor plasma (PPP) from healthy
individuals after addition of ThT (green fluorescent signal). A) PPP smear. B) PPP with spike protein.
C) PPP with thrombin to create extensive fibrin clot; D) PPP exposed to spike protein followed by
addition of thrombin. Final spike protein concentration was 1ng.mL-1.
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Table 1. Percentage average amyloid area in platelet poor plasma (PPP) with and without spike
protein and with and without thrombin.
Platelet activity
Fluorescence microscopy was used to visualize platelet activation in naive healthy WB and
WB incubated with spike protein (1 ng.mL-1 final concentration). Samples were incubated
with the platelet marker, CD62P-PE. Figure 5A shows representative platelets from naïve
control samples, while Figure 5B show micrographs after spike protein incubation. Spike
protein caused an increase in platelet hyperactivation (Figure 5B arrows).
Naïve healthy PPP vs naïve healthy PPP + added thrombin (n=10)
P value (Wilcoxon test, paired non
-
parametric data expressed as median
[Q1 – Q3] 0.2
Median of naïve healthy samples 0.3% [0.1% - 0.8%]
Median of naïve control samples (+ thrombin) 0.9% [0.3% - 1.5%]
Naïve healthy PPP + spike protein (1ng.mL
-
1
)
vs healthy PPP + spike protein (1ng.mL
-
1
) + added
thrombin) (n=10)
P value (Data normally distributed; paired t-test) 0.3
Mean percentage amyloid of healthy samples + spike 1.9% (± 1.2%)
Mean percentage amyloid of healthy samples + spike (+ Thrombin) 2.4% (±1.3%)
Naïve healthy PPP vs healthy PPP + spike protein (1ng.mL
-
1
) (n=10)
P value (Wilcoxon test, paired non
-
parametric data expressed as median [Q1
– Q3] 0.004 (**)
Median of healthy samples 0.3% [0.1% - 0.8%]
Median of healthy samples + spike 1.9% [1.2% - 2.4%]
Naïve healthy PPP + added thrombin vs healthy PPP + spike protein (1ng.mL
-
1
) + added
thrombin) (n=10)
P value (Data normally distributed; paired t-test) 0.0036 (**)
Mean percentage amyloid of control samples (+ Thrombin) 0.9% (± 0.6%)
Mean percentage amyloid of control samples + spike (+ Thrombin) 2.4 % (1.3%)
Statistical significance was established at p<0.05. (** = p<0,01).
Data is represented as either mean
±
standard deviation or median [Q1 – Q3].
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Figure 5A: Fluorescence microscopy micrographs of representative naïve whole blood (WB), where
platelets were incubated with fluorescent marker, CD62P-PE. B) WB after exposure to spike protein.
The white arrows point to hyperactivated activated platelets.
Scanning electron microscopy of whole blood
SEM was used to assess the erythrocyte and platelet ultrastructure after treatment with
spike protein (1ng.mL-1 final concentration). Figure 6A and B shows micrographs from
healthy WB samples, while Figure 6C to H shows micrographs of WB after incubation with
spike protein. The majority of erythrocytes from healthy untreated controls were normocytic
(regularly shaped) [Figure 6A (arrow)], and featured characteristic discoid shapes smooth,
regular membrane surfaces. Slight platelet activation is seen due to contact activation
(Figure 6B). The WB incubated with spike protein showed erythrocyte agglutination, despite
the very low concentration of the spike protein. An increase in platelet hyperactivation,
membrane spreading (Figure 6C and D), platelet-derived micro-particle formation were
noted due to spike protein exposure. The formation of spontaneous and anomalous
fibrin(ogen) deposits with an amyloid nature, were prominent in all the samples incubated
with spike protein, without the addition of thrombin [Figure 6E to H (arrows)].
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Figure 6A to H: Representative scanning electron micrographs of healthy control whole blood (WB),
with and without spike protein. A and B) Healthy WB smears, with arrow indicating normal erythrocyte
ultrastructure. C to H) Healthy WB exposed to spike protein (1 ng.mL-1 final concentration), with C
and D) indicating the activated platelets (arrow), E and F) showing the spontaneously formed fibrin
network and G and H) the anomalous deposits that is amyloid in nature (arrows) (Scale bars: E:
20µm; A: 10µm; F and G: 5µm; H: 2µm; C: 1µm; B and D: 500nm).
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Microfluidics
Figure 7 show the clots that formed in the flow chambers after five minutes of starting the
experiment. Healthy PPP formed a small clot along the bottom surface of the channel, as
seen in Figure 7A. In healthy plasma, clot formation was a relatively slow and gradual
process, resulting in the formation of a modest clot (see supplementary healthy PPP video
1). Clots formed in healthy PPP were relatively small and were limited to the walls of the flow
channel. The clot had orderly layers that did not disrupt flow through the centre of the
channel. As expected, clot formation was also less frequent than with the other samples
(Figure 7B). The PPP with added spike protein showed a combination of a fibrous laminar
clot and disorderly clotted mass (Figure 7E and F) (see supplementary healthy PPP with
added spike protein video 2). The COVID-19 PPP show disorderly clots that cover the bulk
of the channel and often protruding into the centre of the flow channel and disrupting flow
(Figure 7C and D). In COVID-19 PPP, the reaction between thrombin and PPP occurred
rapidly, resulting in large clots after approximately 90 seconds (see supplementary pooled
COVID-19 patient PPP video 3). Interestingly, these clots did not propagate much after the
initial burst, indicating that most of the thrombin was consumed in a short period of time.
Clots also formed with the PPP with the addition of the spike protein, but not as disruptive as
the COVID-19 PPP clots.
An interesting observation was that clots from healthy PPP could easily be dislodged by
flushing the flow channel with water at a rate of 1mL.min-1 (0.42m.s-1). Similarly, clots from
healthy PPP with added spike protein could be dislodged in a similar fashion. COVID-19
clots, on the contrary, could not be displaced or dislodged and remained intact, even with
the force of high-speed water flow in a small flow channel. This observation was consistent
for all the COVID-19 samples.
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Figure 7: Representative micrographs of PPP clots in the microfluidic chambers (black horizontal
lines are the outlines of the chambers) that were coated with thrombin. A) Healthy PPP clot, with
small clot formation (arrow), with B) no clot formed in the healthy PPP sample; C and D) examples of
clots from COVID-19 PPP samples and E and F) healthy PPP clot with spike protein.
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Mass spectrometry analysis
Figure 8 shows results from the mass spectrometry analysis. Mass spectrometry showed
that when spike protein is added to healthy PPP, it results in structural changes to and
fibrin(ogen), complement 3 and prothrombin. These proteins were substantially resistant to
trypsinization, in the presence of spike protein. (for sequence data see supplementary files).
Figure 8: Mass spectrometry showing platelet poor plasma of 4 samples with and without added
spike protein, Spike protein results in structural changes to and fibrin(ogen), complement 3 and
prothrombin.
DISCUSSION
In this laboratory analysis, we provide evidence that spike protein does indeed play a major
role in hypercoagulability seen in COVID-19 patients. It causes anomalous clotting in both
purified fluorescent fibrinogen and in PPP, where the nature of the clots were shown to be
amyloid (ThT as our amyloid dye of choice). An interesting observation was that these dense
deposits were noted both in smears exposed to spike protein, and when thrombin was
added. The addition of thrombin causes purified (Alexa Fluor™488) fibrinogen to polymerize
into fibrin networks. Typically, these networks are netlike (Figure 3A). In the presence of
spike protein, the structure changed to form dense clot deposits (Figure 3B). These deposits
were seen in our fluorescent fibrin(ogen) model and PPP from healthy individuals exposed
to spike protein. In healthy PPP exposed to spike protein, followed by incubation with ThT,
there was a significant increase in anomalous clots with an amyloid nature, (Figure 4D),
when compared to the health PPP. Spike protein also caused major ultrastructural changes
in WB (as viewed with the SEM), where platelet hyperactivation were noted (Figure 6C and
D). Increased in spontaneously formed fibrin network, as well as anomalous clot formation
were also observed in SEM micrographs (Figure 6E - H). Interestingly, extensive
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20
spontaneous fibrin network formation was noted, without the addition of thrombin. This is in
line with results that were recently published, where we showed similar ultrastructure in
blood smears form COVID-19 positive patients. In these patient’s platelet hyperactivation,
anomalous clotting with amyloid signal and spontaneous fibrin fibre formation were also
observed (Pretorius et al., 2020, Venter et al., 2020).
With the microfluidic flow system, clots were formed, by infusing the entire microchannel with
thrombin, thus simulating a hypercoagulable state, where endothelial damage was
extensive. Given that the flow channel was made entirely of plastic and was devoid of any
endothelial cells, the main component under investigation was the PPP (mostly fibrinogen
protein) itself, which, in the case of the COVID-19 samples, may have contained
downstream effects of some endothelial changes that would give rise to the hypercoagulable
state that is characteristic of the disease. The flow setup used in this study could not directly
account for endothelial changes but nonetheless demonstrated that COVID-19 also results
in changes in the clotting profile of the PPP. This was evident in the rapid rate of thrombin
consumption and fibrin formation in COVID-19 clots, and also in the nature of the PPP clots
that were formed.
The clots that were observed in the healthy PPP with added spike protein, were of particular
interest as they demonstrated a bridge between healthy PPP clots and COVID-19 clots. As
described in the results, the healthy PPP clots were relatively small and orderly, while
COVID-19 PPP clots were large, disorderly masses that formed rapidly and disrupted PPP
flow in the channel. The healthy PPP clots with added spike protein, were a combination of
the two, demonstrating disorderly clumped clot areas, co-existing with laminar fibrous PPP
clots (which were larger than the healthy PPP clots). This intermediate state may arise from
a number of factors, including the interaction of other biological actors which were absent
from the flow setup and the time of exposure to spike protein. Further investigations would
be beneficial for understanding the clotting mechanisms that are altered in the presence of
spike protein.
One of the obvious differences, which was inadvertently observed while trying to clean the
channels with high-speed water flow (i.e. by mechanical means), was the ease of healthy
PPP and healthy PPP with added spike protein clot dissolution. However, there was a
complete failure to dislodge or disturb COVID-19 PPP clots from the channels. Given the clot
lysis and dissolution is a complex interplay between biochemical and biophysical factors,
investigation of the effects of different therapeutic agents could elucidate this phenomenon
(Hudson, 2017). The flow protocol used in this study would be a useful platform for testing
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21
different treatments for clinical application. A further limitation of this exploration is the use
of PPP in investigating clot formation at a scale appropriate to the microvasculature. While
the protocol enables the study of fibrin microclots, which are of interest in COVID-19, it
excludes the influence of RBCs, which are known to heavily influence the non-Newtonian
flow behaviour of blood at that scale (McHedlishvili, 1998). The inaccuracy of the flow regime
arising from this exclusion and from the variability of viscosity introduces error into the
results. Nonetheless, the inclusion of flow an appropriate spatial scale has enabled us to
observe COVID-19 PPP clot formation over space and time, under dynamic conditions, and
has given insights which would otherwise prove difficult to glean.
Mass spectrometry confirmed that spike protein causes structural changes to and
fibrin(ogen), complement 3 and prothrombin. These proteins become less resistant to
trypsinization and changes the conformation, in such a way that there is a significant
difference in peptide structure before and after spike protein addition.
CONCLUSION
Scanning electron- and fluorescence microscopy revealed large dense anomalous and
amyloid masses in whole blood and PPP of healthy individuals where spike protein was
added to the samples. Mass spectrometry confirmed that when spike protein was added to
PPP, it interacts with plasma proteins, resulting in fibrin(ogen), prothrombin and other
proteins linked to coagulation, to become substantially resistant to trypsinization, resulting in
less fragments. Flow analysis confirmed that microclots may impair blood flow. Here we
suggest that, in part, the presence of spike protein in circulation may contribute to the
hypercoagulation in COVID-19 positive patients and may cause severe impairment of
fibrinolysis. Such lytic impairment may be the direct cause of the large microclots we have
noted here in SEM and fluorescence microscopy, and previously in plasma samples of
COVID-19 patients (Pretorius et al., 2020, Venter et al., 2020).
DECLARATIONS
Funding
DBK thanks the Novo Nordisk Foundation for funding (grant NNF10CC1016517).
Competing interests
The authors declare that they have no competing interests.
Consent for publication
All authors approved submission of the paper.
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22
TABLE AND FIGURE LEGENDS
Table 1. Percentage average amyloid area in platelet poor plasma (PPP) with and without spike
protein and with and without thrombin.
Figure 1: Schematic representation of SARS-CoV-2 Spike glycoprotein. Adapted from (Duan et al.,
2020). Abbreviations: S1, subunit 1; S2, subunit 2; HR1, heptad repeat 1; HR2, heptad repeat 2.
This image was created with BioRender (https://biorender.com/).
Figure 2: Experimental protocol for growing clots in a microfluidic system.
Figure 3: Representative fluorescence micrographs of purified fluorescent (Alexa Fluor™488)
fibrinogen (note no ThT added) with added thrombin to form extensive fibrin clots. A) Fluorescent
fibrinogen with thrombin; B) fluorescent fibrinogen with added spike protein (final exposure
concentration 1 ng.mL-1) and thrombin.
Figure 4: Representative fluorescence micrographs of platelet poor plasma (PPP) from healthy
individuals after addition of ThT (green fluorescent signal). A) PPP smear. B) PPP with spike protein.
C) PPP with thrombin to create extensive fibrin clot; D) PPP exposed to spike protein followed by
addition of thrombin. Final spike protein concentration was 1ng.mL-1.
Figure 5A: Fluorescence microscopy micrographs of representative naïve whole blood (WB), where
platelets were incubated with fluorescent marker, CD62P-PE. B) WB after exposure to spike protein.
The white arrows point to hyperactivated activated platelets.
Figure 6A to H: Representative scanning electron micrographs of healthy control whole blood (WB),
with and without spike protein. A and B) Healthy WB smears, with arrow indicating normal erythrocyte
ultrastructure. C to H) Healthy WB exposed to spike protein (1 ng.mL-1 final concentration), with C
and D) indicating the activated platelets (arrow), E and F) showing the spontaneously formed fibrin
network and G and H) the anomalous deposits that is amyloid in nature (arrows) (Scale bars: E:
20µm; A: 10µm; F and G: 5µm; H: 2µm; C: 1µm; B and D: 500nm).
Figure 7: Representative micrographs of PPP clots in the microfluidic chambers (black horizontal
lines are the outlines of the chambers) that were coated with thrombin. A) Healthy PPP clot, with
small clot formation (arrow), with B) no clot formed in the healthy PPP sample; C and D) examples of
clots from COVID-19 PPP samples and E and F) healthy PPP clot with spike protein.
Figure 8: Mass spectrometry showing platelet poor plasma of 4 samples with and without added
spike protein, Spike protein results in structural changes to and fibrin(ogen), complement 3 and
prothrombin.
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... Неврологические нарушения при этой патологии очевидно возникают как в результате прямого цитотоксического действия вируса, так и митохондриальной дисфункции и тромботических нарушений [4]. Тем не менее, у выживших больных основные нарушения центральной нервной системы очевидно будут связаны с последствиями тромбоза мелких сосудов коры головного и мозга и амилоидозом головного мозга и периферической нервной системы, поскольку быстрое образование амилоида, как оказалось, является первичной причиной тромбообразования при COVID-19 [9]. ...
... Амилоид как контактная поверхность в процессе образования тромба В пока единственной работе обнаружена на наш взгляд сенсационная связь взаимодействия спайк-белка капсида вируса COVID-19 на процесс одновременного образования тромбов и амилоида в цельной крови здоровых людей и перенесших COVID-19 [9]. Донорами цельной крови были выбраны предварительно обследованные относительно здоровые добровольцы без вредных привычек и заболеваний вен -трое мужчин и восемь женщин, средний возраст 43,4±11,7 года. ...
... Общая распространённость острых цереброваскулярных заболеваний при COVID-19 составила 2,3% (95% ДИ 1,0-3,6), из которых большинство составляли ишемический инсульт -2,1% (95% ДИ 0,9-3,3), геморрагический инсульт -0,4% (95% ДИ 0,2-0,6) и тромбоз церебральных вен -0,3% (95% ДИ 0,1-0,6). Среди других неврологических проявлений; нарушения обоняния -35,8% (95% ДИ 21,4-50,2), нарушения вкуса -38,5% (95% ДИ 24,0-53,0), миалгия -19,3% (95% ДИ 15,1-23,6), головная боль -14,7% (95% ДИ 10, [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]9), головокружение -6,1% (95% ДИ 3,1-9,2), -особо следует отметить синкопальные состояния, наблюдавшиеся у 1,8% (95% ДИ 0,9-4,6) пациентов, которые не описаны другими авторами и могут быть причиной внезапной смерти больных [64]. ...
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We have considered the mechanisms of activation of coagulation hemostasis in COVID-19. It follows from the literature data that after interacting with the cell membrane of target cells, the spike proteins of the COVID-19 virus that have passed into the amyloid form initiate the formation of amyloid nanotubes from amyloid monomers and together activate plasma factor XI. This causes the formation of blood clots. The variety of neurological disorders in COVID-19 is provided by multiple thrombosis of small and large vessels of the central nervous system.
... Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 , is characterized by acute clinical pathologies, including various coagulopathies that may result in either bleeding and thrombocytopenia, hypercoagulation, pulmonary intravascular coagulation, microangiopathy venous thromboembolism or arterial thrombosis [1][2][3][4][5][6][7][8][9]. Acute COVID-19 infection is also characterized by dysregulated, circulating inflammatory biomarkers, hyperactivated platelets, damaged erythrocytes and substantial deposition of microclots in the lungs [6,[8][9][10][11][12][13][14][15][16]. ...
... The excitation wavelength for ThT was set at 450 nm to 488 nm and the emission at 499 nm to 529 nm and processed samples were viewed using a Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63×/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany) [5,8,9]. ...
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Background Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by acute clinical pathologies, including various coagulopathies that may be accompanied by hypercoagulation and platelet hyperactivation. Recently, a new COVID-19 phenotype has been noted in patients after they have ostensibly recovered from acute COVID-19 symptoms. This new syndrome is commonly termed Long COVID/Post-Acute Sequelae of COVID-19 (PASC). Here we refer to it as Long COVID/PASC. Lingering symptoms persist for as much as 6 months (or longer) after acute infection, where COVID-19 survivors complain of recurring fatigue or muscle weakness, being out of breath, sleep difficulties, and anxiety or depression. Given that blood clots can block microcapillaries and thereby inhibit oxygen exchange, we here investigate if the lingering symptoms that individuals with Long COVID/PASC manifest might be due to the presence of persistent circulating plasma microclots that are resistant to fibrinolysis. Methods We use techniques including proteomics and fluorescence microscopy to study plasma samples from healthy individuals, individuals with Type 2 Diabetes Mellitus (T2DM), with acute COVID-19, and those with Long COVID/PASC symptoms. Results We show that plasma samples from Long COVID/PASC still contain large anomalous (amyloid) deposits (microclots). We also show that these microclots in both acute COVID-19 and Long COVID/PASC plasma samples are resistant to fibrinolysis (compared to plasma from controls and T2DM), even after trypsinisation. After a second trypsinization, the persistent pellet deposits (microclots) were solubilized. We detected various inflammatory molecules that are substantially increased in both the supernatant and trapped in the solubilized pellet deposits of acute COVID-19 and Long COVID/PASC, versus the equivalent volume of fully digested fluid of the control samples and T2DM. Of particular interest was a substantial increase in α(2)-antiplasmin (α2AP), various fibrinogen chains, as well as Serum Amyloid A (SAA) that were trapped in the solubilized fibrinolytic-resistant pellet deposits. Conclusions Clotting pathologies in both acute COVID-19 infection and in Long COVID/PASC might benefit from following a regime of continued anticlotting therapy to support the fibrinolytic system function.
... Частицы S-белка способны взаимодействовать напрямую с тромбоцитами и белком фибриногеном, вызывая в нем изменения и, как следствие, нарушения свертываемости крови: формирование более плотных фибриновых сгустков, склеивание эритроцитов, гиперактивацию тромбоцитов и разрастание их мембраны. В присутствии тромбина спайковый белок способствует отложению фибрина амилоидной природы, формируя волокнистые и бесформенные сгустки и препятствуя току жидкости в сосудах [14]. ...
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The ongoing COVID-19 pandemic has caused significant morbidity and mortality worldwide, as well as a profound impact on society. Among the nosologies that increase the risk of a severe course of COVID-19, coronary heart disease, chronic heart failure, cardiomyopathy. The main complications caused by coronavirus infection include thrombotic ones. Spike protein SARS-CoV-2 can interact directly with platelets and fibrin, causing blood hypercoagulation and obstructing blood flow. The presence of the spike protein in circulation leads to structural changes in fibrin, complement 3 and prothrombin, which can contribute to hypercoagulability in COVID-19 positive patients and cause a significant violation of fibrinolysis. Endothelial damage and systemic inflammation, being interrelated triggers of coagulopathy characteristic of COVID-19, trigger a cascade of reactions resulting in thrombotic complications against the background of endothelial dysfunction and hyperinflammation, which may be of clinical importance in the treatment of hypercoagulability in patients with COVID-19 (bibliography: 14 refs).
... In addition to trimerization, membrane anchoring seems to further improve immunogenicity, as transmembrane anchored prefusion-stabilized full-length S protein was reported to elicit higher VNA levels than corresponding secreted constructs [1,83]. Both in terms of immunogenicity and potential association of circulating SARS-Cov-2 S1 subunit with enhanced blood clotting [84], use of a small membrane-anchored antigen is rational. ...
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Vaccines of outstanding efficiency, safety, and public acceptance are needed to halt the current SARS-CoV-2 pandemic. Concerns include potential side effects caused by the antigen itself and safety of viral DNA and RNA delivery vectors. The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which might cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a trans-membrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype repli-con vaccine complemented with VSV G, VSVΔG-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients, and protected transgenic K18-hACE2 mice from COVID-19-like disease. Homolo-gous boost immunization further enhanced virus neutralizing activity. The results demonstrate that non-spreading rhabdovirus RNA replicons expressing minispike proteins represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen.
Article
State of the problem. Thrombotic complications are a common risk factor for a variety of diseases and are one of the leading causes of death. This leads to a strong interest in finding effective means of prevention and treatment. A characteristic feature of the last decade is the growing interest and numerous attempts to introduce into clinical practice fibrinolytic enzymes that are not functionally related to the hemostasis system. The aim of the work. Investigation of molecular mechanisms that cause the lack of efficiency of native fibrinolysis in relation to fibrin clots with impaired regularity of fibrin structure. Correlation of own achievements in the field of biochemistry and medicine with systematized literary material. The possibilities created by non-plasmin fibrinolytics and the advantages of their use are considered. Discussion and conclusions. The reasons of the complications caused by insufficient efficiency of both own fibrinolytic system, and the entered fibrinolytics are substantiated. It is shown that the leading role in such complications is played by violation of the regularity of the structure of fibrin clots. The mechanisms of action of leading fibrinolytic agents are considered and the expediency of using alternative non-plasmin fibrinolytics is substantiated. The properties and expediency of the use of fibrinolytics based on components of snake venoms and bacterial proteinases as effective means for the breakdown of fibrin with impaired regularity of structure are discussed. Key words: fibrosis, thrombotic therapy, fibrinolytic enzymes, hemostasis system.
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Течение COVID-19 у больных нередко осложняется гиперкоагуляцией и тромбозами магистральных сосудов. Как оказалось, вакцинирование против COVID-19 препаратом ChAdOx1 nCoV-19 (AstraZeneca) у ряда пациентов также вызвало тромбоцитопению и тромбообразование в нетипичных (церебральный венозный синус, воротная, чревная, печеночная вены) и типичных (глубокие вены бедра и голени, тромбоэмболия легочной артерии, острые артериальные тромбозы) местах. А использование мРНК вакцины (Moderna и Pfizer) иногда сопровождалось тромбоцитопенией и кровотечением, но без образования тромбов. Это обстоятельство послужило по-водом для поиска механизмов тромбообразования при использовании ранее никогда не применявшихся вакцин, разработанных против COVID-19. Цель публикации-информирование врачебной общественности о механизмах тромбообразования при COVID-19; обсуждение возможных патогенетических путей быстрого образования амилоида и амилоидогенной стимуляции системы коагуляционного гемостаза. В единственном завершенном на настоящий момент исследовании представлены сведения о запуске спайк-белком капсида вируса COVID-19 быстрого формирования амилоида с образованием плотных крупных фибриновых сгустков в цельной крови как здоровых людей, так и у находившихся в остром периоде заболевания COVID-19. Авторы, обнаружив факт прямого влияния спайк-белка на формирование тромбов, тем не менее не исследовали возможные патогенетические пути запуска тромбо-образования спайк-белком. Поскольку авторы прямо указали на роль быстрого образования амилоида в запуске коагуляции, механизм которого неизвестен практикующим специалистам, имеет смысл обсудить вопросы быстрого образования амилоида в сосудистом русле и роли амилоида как фактора запуска коагуляционного гемостаза. Обсуждаемая публикация подтверждается ранее проведенными исследованиями других авторов о влиянии -амилоида и АА-амилоида на процессы образования тромбов при болезни Альцгеймера и системных амилоидозах. На основании изученных литературных источников нами высказано предположение, что у какой-то части больных, перенесших COVID-19 в тяжелой форме, в последующем может развиться системный амилоидоз. Введение. COVID-19 часто сопровождается клинически значимыми тромбозами, в том числе тромбоэмболией легочной артерии, частота которой в 3,4 раза выше у больных COVID-19, чем у пациентов с заболеваниями, тя-жесть которых аналогична [25]. По этой причине тема активно обсуждается в ряде научных статей [4-6]. Из данных этих статей следует, что основной при-чиной тромботических осложнений при COVID-19 являются диссеминирован-ное внутрисосудистое свертывание, тромботическая микроангиопатия и гепа-рин-индуцированная тромбоцитопения. В большинстве случаев наблюдается удлинение активированного частичного тромбопластинового времени, что яв-ляется свидетельством снижения активности фактора Хагемана (FXII).
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Introduction The diagnosis of COVID-19 is normally based on the qualitative detection of viral nucleic acid sequences. Properties of the host response are not measured but are key in determining outcome. Although metabolic profiles are well suited to capture host state, most metabolomics studies are either underpowered, measure only a restricted subset of metabolites, compare infected individuals against uninfected control cohorts that are not suitably matched, or do not provide a compact predictive model. Objectives Here we provide a well-powered, untargeted metabolomics assessment of 120 COVID-19 patient samples acquired at hospital admission. The study aims to predict the patient’s infection severity (i.e., mild or severe) and potential outcome (i.e., discharged or deceased). Methods High resolution untargeted UHPLC-MS/MS analysis was performed on patient serum using both positive and negative ionization modes. A subset of 20 intermediary metabolites predictive of severity or outcome were selected based on univariate statistical significance and a multiple predictor Bayesian logistic regression model was created. Results The predictors were selected for their relevant biological function and include deoxycytidine and ureidopropionate (indirectly reflecting viral load), kynurenine (reflecting host inflammatory response), and multiple short chain acylcarnitines (energy metabolism) among others. Currently, this approach predicts outcome and severity with a Monte Carlo cross validated area under the ROC curve of 0.792 (SD 0.09) and 0.793 (SD 0.08), respectively. A blind validation study on an additional 90 patients predicted outcome and severity at ROC AUC of 0.83 (CI 0.74–0.91) and 0.76 (CI 0.67–0.86). Conclusion Prognostic tests based on the markers discussed in this paper could allow improvement in the planning of COVID-19 patient treatment.
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It is unclear whether severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019, can enter the brain. Severe acute respiratory syndrome coronavirus 2 binds to cells via the S1 subunit of its spike protein. We show that intravenously injected radioiodinated S1 (I-S1) readily crossed the blood–brain barrier in male mice, was taken up by brain regions and entered the parenchymal brain space. I-S1 was also taken up by the lung, spleen, kidney and liver. Intranasally administered I-S1 also entered the brain, although at levels roughly ten times lower than after intravenous administration. APOE genotype and sex did not affect whole-brain I-S1 uptake but had variable effects on uptake by the olfactory bulb, liver, spleen and kidney. I-S1 uptake in the hippocampus and olfactory bulb was reduced by lipopolysaccharide-induced inflammation. Mechanistic studies indicated that I-S1 crosses the blood–brain barrier by adsorptive transcytosis and that murine angiotensin-converting enzyme 2 is involved in brain and lung uptake, but not in kidney, liver or spleen uptake.
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Background Type 2 Diabetes Mellitus (T2DM) is a well-known comorbidity to COVID-19 and coagulopathies are a common accompaniment to both T2DM and COVID-19. In addition, patients with COVID-19 are known to develop micro-clots within the lungs. The rapid detection of COVID-19 uses genotypic testing for the presence of SARS-Cov-2 virus in nasopharyngeal swabs, but it can have a poor sensitivity. A rapid, host-based physiological test that indicated clotting severity and the extent of clotting pathologies in the individual who was infected or not would be highly desirable. Methods Platelet poor plasma (PPP) was collected and frozen. On the day of analysis, PPP samples were thawed and analysed. We show here that microclots can be detected in the native plasma of twenty COVID-19, as well as ten T2DM patients, without the addition of any clotting agent, and in particular that such clots are amyloid in nature as judged by a standard fluorogenic stain. Results were compared to ten healthy age-matched individuals. Results In COVID-19 plasma these microclots are significantly increased when compared to the levels in T2DM. Conclusions This fluorogenic test may provide a rapid and convenient test with 100% sensitivity (P < 0.0001) and is consistent with the recognition that the early detection and prevention of such clotting can have an important role in therapy.
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Progressive respiratory failure is seen as a major cause of death in severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection. Relatively little is known about the associated morphologic and molecular changes in the circulation of these patients. In particular, platelet and erythrocyte pathology might result in severe vascular issues, and the manifestations may include thrombotic complications. These thrombotic pathologies may be both extrapulmonary and intrapulmonary and may be central to respiratory failure. Previously, we reported the presence of amyloid microclots in the circulation of patients with coronavirus disease 2019 (COVID-19). Here, we investigate the presence of related circulating biomarkers, including C-reactive protein (CRP), serum ferritin, and P-selectin. These biomarkers are well-known to interact with, and cause pathology to, platelets and erythrocytes. We also study the structure of platelets and erythrocytes using fluorescence microscopy (using the markers PAC-1 and CD62PE) and scanning electron microscopy. Thromboelastography and viscometry were also used to study coagulation parameters and plasma viscosity. We conclude that structural pathologies found in platelets and erythrocytes, together with spontaneously formed amyloid microclots, may be central to vascular changes observed during COVID-19 progression, including thrombotic microangiopathy, diffuse intravascular coagulation, and large-vessel thrombosis, as well as ground-glass opacities in the lungs. Consequently, this clinical snapshot of COVID-19 strongly suggests that it is also a true vascular disease and considering it as such should form an essential part of a clinical treatment regime.
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The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a grave threat to global public health and imposes a severe burden on the entire human society. Like other coronaviruses, the SARS-CoV-2 genome encodes spike (S) glycoproteins, which protrude from the surface of mature virions. The S glycoprotein plays essential roles in virus attachment, fusion and entry into the host cell. Surface location of the S glycoprotein renders it a direct target for host immune responses, making it the main target of neutralizing antibodies. In the light of its crucial roles in viral infection and adaptive immunity, the S protein is the focus of most vaccine strategies as well as therapeutic interventions. In this review, we highlight and describe the recent progress that has been made in the biosynthesis, structure, function, and antigenicity of the SARS-CoV-2 S glycoprotein, aiming to provide valuable insights into the design and development of the S protein-based vaccines as well as therapeutics.
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Objective: In December 2019 a novel coronavirus (SARS-CoV-2) that is causing the current COVID-19 pandemic was identified in Wuhan, China. Many questions have been raised about its origin and adaptation to humans. In the present work we performed a genetic analysis of the Spike glycoprotein (S) of SARS-CoV-2 and other related coronaviruses (CoVs) isolated from different hosts in order to trace the evolutionary history of this protein and the adaptation of SARS-CoV-2 to humans. Results: Based on the sequence analysis of the S gene, we suggest that the origin of SARS-CoV-2 is the result of recombination events between bat and pangolin CoVs. The hybrid SARS-CoV-2 ancestor jumped to humans and has been maintained by natural selection. Although the S protein of RaTG13 bat CoV has a high nucleotide identity with the S protein of SARS-CoV-2, the phylogenetic tree and the haplotype network suggest a non-direct parental relationship between these CoVs. Moreover, it is likely that the basic function of the receptor-binding domain (RBD) of S protein was acquired by the SARS-CoV-2 from the MP789 pangolin CoV by recombination and it has been highly conserved.
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Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), also known as coronavirus disease 2019 (COVID-19)-induced infection, is strongly associated with various coagulopathies that may result in either bleeding and thrombocytopenia or hypercoagulation and thrombosis. Thrombotic and bleeding or thrombotic pathologies are significant accompaniments to acute respiratory syndrome and lung complications in COVID-19. Thrombotic events and bleeding often occur in subjects with weak constitutions, multiple risk factors and comorbidities. Of particular interest are the various circulating inflammatory coagulation biomarkers involved directly in clotting, with specific focus on fibrin(ogen), D-dimer, P-selectin and von Willebrand Factor (VWF). Central to the activity of these biomarkers are their receptors and signalling pathways on endothelial cells, platelets and erythrocytes. In this review, we discuss vascular implications of COVID-19 and relate this to circulating biomarker, endothelial, erythrocyte and platelet dysfunction. During the progression of the disease, these markers may either be within healthy levels, upregulated or eventually depleted. Most significant is that patients need to be treated early in the disease progression, when high levels of VWF, P-selectin and fibrinogen are present, with normal or slightly increased levels of D-dimer (however, D-dimer levels will rapidly increase as the disease progresses). Progression to VWF and fibrinogen depletion with high D-dimer levels and even higher P-selectin levels, followed by the cytokine storm, will be indicative of a poor prognosis. We conclude by looking at point-of-care devices and methodologies in COVID-19 management and suggest that a personalized medicine approach should be considered in the treatment of patients.
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Although COVID-19 is most well known for causing substantial respiratory pathology, it can also result in several extrapulmonary manifestations. These conditions include thrombotic complications, myocardial dysfunction and arrhythmia, acute coronary syndromes, acute kidney injury, gastrointestinal symptoms, hepatocellular injury, hyperglycemia and ketosis, neurologic illnesses, ocular symptoms, and dermatologic complications. Given that ACE2, the entry receptor for the causative coronavirus SARS-CoV-2, is expressed in multiple extrapulmonary tissues, direct viral tissue damage is a plausible mechanism of injury. In addition, endothelial damage and thromboinflammation, dysregulation of immune responses, and maladaptation of ACE2-related pathways might all contribute to these extrapulmonary manifestations of COVID-19. Here we review the extrapulmonary organ-specific pathophysiology, presentations and management considerations for patients with COVID-19 to aid clinicians and scientists in recognizing and monitoring the spectrum of manifestations, and in developing research priorities and therapeutic strategies for all organ systems involved.
Article
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) uses Angiotensin- converting enzyme 2 (ACE2) receptors to infect host cells which may lead to coronavirus disease (COVID-19). Given the presence of ACE2 receptors in the brain and the critical role of the renin-angiotensin system (RAS) in brain functions, special attention to brain microcirculation and neuronal inflammation is warranted during COVID-19 treatment. Neurological complications reported among COVID-19 patients range from mild dizziness, headache, hypogeusia, hyposmia to severe like encephalopathy, stroke, Guillain-Barre Syndrome (GBS), CNS demyelination, infarcts, microhemorrhages and nerve root enhancement. The pathophysiology of these complications is likely via direct viral infection of the CNS and PNS tissue or through indirect effects including post- viral autoimmune response, neurological consequences of sepsis, hyperpyrexia, hypoxia and hypercoagulability among critically ill COVID-19 patients. Further, decreased deformability of red blood cells (RBC) may be contributing to inflammatory conditions and hypoxia in COVID-19 patients. Haptoglobin, hemopexin, heme oxygenase-1 and acetaminophen may be used to maintain the integrity of the RBC membrane.
Article
Severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2) is the novel coronavirus responsible for the ongoing pandemic. It is known that SARS-CoV-2 infects the host through the cell surface receptor of angiotensin-converting enzyme 2 (ACE2), which is expressed in multiple organs, and in the arterial and venous endothelial cells. We have recently proposed the use of the term MicroCLOTS ( Microvascular COVID-19 lung vessels obstructive thromboinflammatory syndrome) to describe the unique type of ARDS seen in patients affected by SARS-COV-2. After a multidisciplinary assessment of more than 850 COVID-19 patients admitted to our Hospital with several bilateral pneumonia, we have collected evidences supporting a key role of vascular inflammation and microthrombosis in the pathophysiology of the multisystemic clinical manifestations that have been associated with COVID-19. There is now a general consensus on the recommendation of anticoagulation in patient with severe SARS-Cov2 infections, although the dose of the prophylaxis and even the choice between a prophylactic and a treatment regimen remains controversial. Randomized controlled trials are urgently needed to help clarifying the many therapeutic challenges associated with the management of SARS-Cov-2 patients.