https://doi.org/10.1042/BSR20210611
Received: 12 March 2021
Revised: 13 July 2021
Accepted: 29 July 2021
Accepted Manuscript Online:
30 July 2021
Version of Record published:
20 August 2021
Research Article
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 Ngoepe3,4, Gert Jacobus Laubscher5,
Petrus Johannes Lourens5, Janami Steenkamp1,6, Douglas B. Kell1,7,8 and Etheresia Pretorius1
1Department of Physiological Sciences, Faculty of Science, Stellenbosch University, Stellenbosch, Private Bag X1, Matieland 7602, South Africa; 2Central Analytical Facility: Mass
Spectrometry Stellenbosch University, Tygerberg Campus, Room 6054, Clinical Building, Francie van Zijl Drive, Tygerberg, Cape Town 7505, South Africa; 3Department of
Mechanical Engineering, Faculty of Engineering and the Built Environment, University of Cape Town, Cape Town, Rondebosch 7701, South Africa; 4Stellenbosch Institute for
Advanced Study, Wallenberg Research Centre, Stellenbosch University, Stellenbosch, South Africa; 5Private practice clinician, Mediclinic Stellenbosch, Stellenbosch 7600, South
Africa; 6PathCare Laboratories, PathCare Business Centre, PathCare Park, Neels Bothma Street, N1 City 7460, South Africa; 7Department of Biochemistry and Systems Biology,
Institute of Systems, Molecular and Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZB, U.K.; 8The Novo Nordisk Foundation Centre
for Biosustainability, Technical University of Denmark, Kemitorvet 200, Kgs Lyngby 2800, Denmark
Correspondence: Etheresia Pretorius (resiap@sun.ac.za) or Douglas B. Kell (dbk@liv.ac.uk)
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 largemicroclotswe 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.
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 [1,2]. 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 [3,4]. 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 [5–8]. This poor prognosis is further worsened as together with a
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Figure 1. Schematic representation of SARS-CoV-2 Spike glycoprotein
Adapted from[21]. Abbreviations: HR1, heptad repeat 1; HR2, heptad repeat 2; S1, subunit 1; S2, subunit 2. This image was created
with BioRender (https://biorender.com/).
substantial deposition of microclots in the lungs [9–11]. Plasma of COVID-19 patients also carries a massive load of
preformed amyloid clots [5], and there are also numerous reports of damage to erythrocytes [12–14], platelets, and
dysregulation of inflammatory biomarkers [5–8].
The virulenceof thepathogen is closelylinkedtoitsmembraneproteins.One suchprotein, foundontheCOVID-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 [15]. Spike proteins are
class I viral fusion proteins [16]. They are present as protruding homotrimers on the viral surface and mediate virus
entry into the target host cells [17]. A singular spike protein is between 180 and 200 kDa in size and contains an extracellularN-
terminal, a transmembrane domain fixed in themembrane of the virus, and a short intracellularC-terminal
segment [16,18]. Spike proteins are coated with polysaccharide molecules that serve as camouflage. This helps evade
surveillance by the host immune system during entry [18]. The S1 subunit is responsible for receptor binding [19],
with subunit 2 (S2), a carboxyl-terminal subunit, responsible for viral fusion and entry [20] (see Figure 1).
Receptor binding is certainly responsible for cell-mediated pathologies, but does not itself explain the coagulopathies.
Spike protein, can however be shed, and it has been detected in various organs, including the urinary tract
[22]. S1 proteins can also cross the blood–brain barrier [23]. Free S1 particles may also play a role in the pathogenesis
of the disease [24,25]. 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 [24].
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 interactwith 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 na¨ıve healthy platelet-poor plasma (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 provides further evidence that targeting
it directly, whether via vaccines or antibodies, is likely to be of therapeutic benefit.
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Materials and methods
Sample demographics and considerations
Blood was collected from healthy volunteers (n=11; 3 males and 8 females; mean age: 43.4 +−
11.7 years) to serve
as controls. Individuals who smoke, who were diagnosed with cardiovascular diseases, clotting disorders (coagulopathies),
and/or any known inflammatory conditions (e.g. Type 2 DiabetesMellitus (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 resulted in exclusion. Smoking was
excluded since it has been proven to impair coagulation, fibrinolysis, and the hemostatic process [26]. Microfluidics
analysis included a preliminary analysis using PPP samples from two COVID-19 positive patients, on the day of first
diagnosis and before any treatment was given. Both patients were diagnosed with moderate to severe COVID-19
symptoms (1 male and 1 female, mean age: 78.5+−
7.7 years).
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 4.5-ml sodium citrate (3.2%) tubes
(BD Vacutainer®, 369714). After several gentle inversions, the collected citrate tubes were allowed to rest at room
temperature foraminimumof 30min to allowfor adequate anticoagulant amalgamation before commencing sample
preparation.Whole blood (WB) was centrifuged at 3000×g for 15 min at room temperature to isolate erythrocytes.
The supernatant, i.e. PPP, was collected and stored in 1.5-ml Eppendorf tubes at −80◦C, till needed for experiments.
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 double distilled water, following the instructions provided. A
total of 400 μl diluent (endotoxin-free water) was added to the 100 μg spike protein to create a stock solution (A) of
0.25 mg.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 1.5-ml Eppendorf tubes with different final exposure concentrations
of spike protein in the PPP of 100, 50, 10, and 1 ng.ml−1. PPP samples were incubated with the various spike
protein concentrations for 30 min at room temperature.
Fluorescence microscopy of purified fibrinogen and PPP with and without
thrombin
Concentration verification
To verify which spike protein concentration will be effective, 5 μl of the PPP exposed to the varies spike protein
concentrations was placed on a glass slide, after being exposed to the fluorescent amyloid dye, Thioflavin T (ThT)
(Sigma–Aldrich, St. Louis,MO,U.S.A.) for 30min at roomtemperature. The final concentration of ThT in all prepared
sampleswas 0.005mM.After the evaluation of the samples, with the varying spike protein concentrations, itwas found
that the final exposure concentration of 1 ng.ml−1 was sufficient and used for the rest of the study.
Amyloid protein and anomalous clotting in PPP samples
To study spontaneous anomalous clotting of fibrin(ogen), in the na¨ıve healthy PPP samples, and in the presence of
spike protein, 5 μl PPP exposed to 1 ng.ml−1 (final concentrations) of spike protein, was smeared on a glass slide and
covered with a coverslip. This was done after it was exposed to the fluorescent amyloid dye, ThT (final concentration:
0.005mM)(Sigma–Aldrich, St. Louis,MO,U.S.A.) for 30 min at roomtemperature. Fibrin PPP clots, with andwithout
spike protein and after exposure to ThT, were also prepared by adding 2.5 μl of thrombin (7 U.ml−1, South African
National Blood Service) to 5 μl PPP and was placed on a glass slide and covered with a coverslip. The excitation
wavelength for ThT was set at 450–488 nm and the emission at 499–529 nm and processed samples were viewed
using a Zeiss Axio Observer 7 fluorescentmicroscope with a Plan-Apochromat 63×/1.4 Oil DIC M27 objective (Carl
Zeiss Microscopy,Munich, Germany).
Fluorescence microscopy images of healthy PPP with and without spike protein were analyzed 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) [27–29] 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
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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 optimize the amount of images thresholded, a program was written
in Java to simultaneously analyze a group of images (see SupplementaryMaterial). 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) clotmodel of choice was
fluorescent fibrinogen conjugated to Alexa Fluor™ 488 (Thermo Fisher, F13191). A final fibrinogen concentration of
2 mg.ml−1 was prepared in endotoxin-free water and exposed to 1 ng.ml−1 (final concentrations) spike protein for
30 min at room temperature. A total of 5 μl of the purified fibrinogen was placed on a glass slide, with 2.5 μl of
thrombin. The excitation wavelength for our fluorescent fibrinogen model was set at 450–488 nm and the emission
at 499–529 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).
WB (hematocrit)
Hematocrit was exposed to a final exposure concentration of 1 ng.ml−1 spike protein. The fluorescentmarker, CD62P
(platelet surface P-selectin) was added to the hematocrit to study platelet activation. CD62P is found on the membrane
of platelets and then translocated to the platelet membrane surface. The translocation occurs after the platelet
P-selectin is released fromthe cellular granules during platelet activation [5,7]. A total of 4 μl CD62P (PE-conjugated)
(IM1759U, Beckman Coulter, Brea, CA, U.S.A.) was added to 20 μl WB (either na¨ıve or incubated with spike protein).
The hematocrit exposed to the marker was incubated for 30 min (protected from light) at room temperature.
The excitation wavelength for the CD62P was 540–570 nm and the emission 577–607 nm. Processed samples were
also 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).
Scanning electron microscopy of WB samples
Scanning electron microscopy (SEM) was used to view healthyWB samples, with and without the addition of spike
protein. A total of 10 μl WB was placed on a glass coverslip and prepared according to previously published SEM
preparation methods [30,31], starting with washing steps in phosphate-buffered saline (PBS) (pH = 7.4) (Thermo
Fisher Scientific, 11594516) for 20 min. Fixation was performed by coating the slides in 4% formaldehyde (FA) for
30 min, followed by washing them in PBS three times. For each wash, the PBS should be left on for 3 min before
removing and washing again. Osmiumtetroxide (OsO4) (Sigma–Aldrich, 75632) was added for 15 min and the slides
were washed in PBS three-times with 3 min 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 coatedwith carbon.The slides were viewed on a ZeissMERLINFE-SEMwith the InLens
detector at 1 kV (Carl Zeiss Microscopy, Munich, Germany).
Microfluidics
Microfluidic analysis was performed using healthy PPP and healthy pooled PPP samples (three pooled PPP samples),
with and without spike protein, and two COVID-19 PPP samples. Pooled samples were used due to the volume
required for this experiment.
Hardware
Amicrofluidic 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.Asinglemicrochannel had awidth of 400 μmandaheight of 100 μm(equivalent diameter of 207 μm), and
a length of 2.8 cm. The dimensions of themicrochannel were in the same order of magnitude as those of some vessels
of themicrovasculature, that is, under 300 μm [32]. In order to observe clot evolution in real-time, the biochips were
placed under the Zeiss Axio Observer 7 fluorescent microscope with a 10×/0.25 objective (Carl Zeiss Microscopy,
Munich, Germany).
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Figure 2. Experimental protocol for growing clots in a microfluidics system
Flow conditions
A new flow channel was used for every run. The channel was flushed with distilled water at a flow rate of 1 ml.min−1
for 1 min. After 5 min, thrombin was infused through the microchannel at a flow rate of 50 μl.min−1 for 90 s (Figure
2). The channel was left to stand for another 5 min and then the sample (control, control with spike, or COVID-19)
was infused at a flow rate of 10 μl.min−1 for 5 min, with a video recording of the channel (Figure 2). The flow was
then stopped after 5min, and a set of micrographs was taken across the channel. The sample was then left for another
5min, to see if any additional changes occur (Figure 2). This flow rate corresponds with a shear rate, γ ≈ 250 s−1 and
a Reynolds number, Re ≤ 1. One of the main challenges in attempting to achieve consistent shear rates and Reynolds
number was the variability in viscosity from sample to sample. Furthermore, blood flowing through microvessels is
known to behave in a non-Newtonianmanner, adding to the complexities of variable viscosity within a single sample.
To achieve standardization between samples, a constant flow rate was used.
Proteomics
Four healthy PPP samples were analyzed before and after addition of spike protein. The samples were diluted in 10
mMammoniumbicarbonate to 1mg.ml−1. A total of 1 μg trypsin (NewEngland Biosystems) was added to the plasma
for 1:50 enzyme to substrate ratio. No reduction or alkylation was performed.
Liquid chromatography
Dionex nano-RSLC
Liquid chromatography was performed on a Thermo ScientificUltimate 3000 RSLC equipped with a 5mm×300 μm
C18 trap column (Thermo Scientific) and a CSH 25 cm × 75 μm, 1.7 μmparticle size C18 column (Waters) analytical
column. The solvent systememployed 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 on to 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 min
before the sample was eluted onto the analytical column. Flow rate was set to 300 nl/min and the gradient generated
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as follows: 5.0–30% B over 60 min and 30–50% B from 60–80 min. 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), four of our control samples were analyzed with this method. The sample was introduced through a
stainless-steel nano-bore emitter.Datawere collected inpositivemodewith spray voltage set to1.8kVandion 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 120000 resolution over the scan range 375–1500 with AGC target at 4 E5
and maximum injection time of 50 ms. Data were acquired in profile mode. MS2 acquisitions were performed using
monoisotopic precursor selection for ion with charges +2 to +7 with error tolerance set to+ −10 ppm. Precursor ions
were excluded from fragmentation once for a period of 60 s. Precursor ions were selected for fragmentation in HCD
mode using the quadrupole mass analyzer with HCD energy set to 30%. Fragment ions were detected in the Orbitrap
mass analyzer set to 30000 resolution. The AGC target was set to 5E4 and the maximum injection time to 100 ms.
The data were 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 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’s
test was used for significance in paired parametric data. When comparing three or more experimental groups, the
Kruskal–Wallis test (non-parametric data) or one-wayANOVA(parametric data) test was applied to test for statistical
significance.AP-value of less than 0.05 was considered to be statistically significant. Parametric datawere 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 two missed cleavages was allowed for. Precursor mass
tolerance was set to 10 ppm and fragment mass tolerance set to 0.02 Da. Demamidation (NQ), oxidation (M) were
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) andstatistical
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=lmObMy
Results
Fluorescence microscopy of purified fibrinogen and PPP
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 healthy control were incubated for 30 min with varying spike protein
concentrations, followed by 30-min 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).
Figure 3A,B shows 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
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Figure 3. Representative fluorescence micrographs of purified fluorescent (Alexa Fluor™ 88) 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.
Table 1 Percentage average amyloid area in PPP with and without spike protein and with and without thrombin
Na¨ıve healthy PPP vs na¨ıve healthy PPP + added thrombin (n=10)
P-value (Wilcoxon’s test, paired non-parametric data expressed as median [Q1 − Q3]0.2
Median of na¨ıve healthy samples 0.3% [0.1–0.8%]
Median of na¨ıve control samples (+ thrombin) 0.9% [0.3–1.5%]
Na¨ıve healthy PPP + spike protein (1 ng.ml−1) vs healthy PPP + spike protein (1 ng.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%)
Na¨ıve healthy PPP vs healthy PPP + spike protein (1 ng.ml−1) (n=10)
P-value (Wilcoxon’s 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%]
Na¨ıve healthy PPP + added thrombin vs healthy PPP + spike protein (1 ng.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 are represented as either mean +−
SD or median [Q1 − Q3].
formation. ThT is known to bind to open hydrophobic areas in protein and these are amyloid in nature [27,33–35].
Figure 4A,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,
amajor increase in dense anomalous clotted deposits, with an amyloid nature, were noted (referred to as amyloid
deposits) (Figure 4B,D). Athresholding algorithmwas applied to themicrographs (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 analyzed). Using this method, the average total percentage of amyloid deposits per group was calculated (na¨ıve
healthy PPP, na¨ıve healthy PPP + thrombin, and PPP incubated with 1 ng.ml−1 spike protein, with and without added
thrombin) (see Table 1).As expected, therewere 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).
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Figure 4. Representative fluorescence micrographs of 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 1 ng.ml−1.
Platelet activity
Fluorescence microscopy was used to visualize platelet activation in naive healthy hematocrit and hematocrit incubatedwith
spike protein (1 ng.ml−1 final concentration). Sampleswere incubated with the plateletmarker, CD62P-PE.
Figure 5A shows representative platelets from na¨ıve control samples, while Figure 5B shows micrographs after spike
protein incubation. Spike protein caused an increase in platelet hyperactivation (Figure 5B arrows).
SEM of WB
SEMwas used to assess the erythrocyte and platelet ultrastructure after treatment with spike protein (1 ng.ml−1 final
concentration). Figure 6A,B shows micrographs fromhealthyWBsamples, while Figure 6C–H shows micrographs of
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Figure 5. Fluorescence microscopy micrographs of platelets, before and after exposure to spike protein
(A) Representative platelets from hematocrit incubated with fluorescent marker, CD62P-PE. (B) Representative micrographs showing
activated platelets after exposure to spike protein. The white arrows point to hyperactivated activated platelets. White arrows
show hyperactivated platelets clumping together.
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). TheWB 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,D), platelet-derived microparticle 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–H (arrows)].
Microfluidics
Figure 7 show the clots that formed in the flow chambers after 5 min 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
S1). 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 clottedmass (Figure 7E,F) (see Supplementary healthy PPP with added spike protein video
S2). The COVID-19 PPP show disorderly clots that cover the bulk of the channel and often protruding into the center
of the flow channel and disrupting flow (Figure 7C,D). In COVID-19 PPP, the reaction between thrombin and PPP
occurred rapidly, resulting in large clots after approximately 90 s (see Supplementary pooled COVID-19 patient PPP
video S3). Interestingly, these clots did not propagatemuch after the initial burst, indicating thatmost 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 1 ml.min−1 (0.42 m.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.
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 β and γ fibrin(ogen), complement 3, and prothrombin.
©2021 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
9
Bioscience Reports (2021) 41 BSR20210611
https://doi.org/10.1042/BSR20210611
Figure 6. Whole blood sample of healthy volunteers, before and after exposure to spike protein
(A–H) Representative scanning electron micrographs of healthy control WB, with and without spike protein. (A,B) Healthy WB
smears, with arrow indicating normal erythrocyte ultrastructure. (C–H) Healthy WB exposed to spike protein (1 ng.ml−1 final concentration),
with (C,D) indicating the activated platelets (arrow), (E,F) showing the spontaneously formed fibrin network and (G,H)
the anomalous deposits that is amyloid in nature (arrows) (scale bars: (E) 20 μm; (A) 10 μm; (F,G) 5 μm; (H) 2 μm; (C) 1 μm; (B,D)
500 nm).
10 ©2021 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).

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