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It is well known that a variety of inflammatory diseases are accompanied by hypercoagulability, and a number of more-or-less longer-term signalling pathways have been shown to be involved. In recent work, we have suggested a direct and primary role for bacterial lipopolysaccharide (LPS) in this hypercoagulability, but it seems never to have been tested directly. Here, we show that the addition of tiny concentrations (0.2 ng l⁻¹) of bacterial LPS to both whole blood and platelet-poor plasma of normal, healthy donors leads to marked changes in the nature of the fibrin fibres so formed, as observed by ultrastructural and fluorescence microscopy (the latter implying that the fibrin is actually in an amyloid b-sheet-rich form that on stoichiometric grounds must occur autocatalytically). They resemble those seen in a number of inflammatory (and also amyloid) diseases, consistent with an involvement of LPS in their aetiology. These changes are mirrored by changes in their viscoelastic properties as measured by thromboelastography. As the terminal stages of coagulation involve the polymerization of fibrinogen into fibrin fibres, we tested whether LPS would bind to fibrinogen directly. We demonstrated this using isothermal calorimetry. Finally, we show that these changes in fibre structure are mirrored when the experiment is done simply with purified fibrinogen and thrombin (±0.2 ng l⁻¹ LPS). This ratio of concentrations of LPS: fibrinogen in vivo represents a molecular amplification by the LPS of more than 10⁸-fold, a number that is probably unparalleled in biology. The observation of a direct effect of such highly substoichiometric amounts of LPS on both fibrinogen and coagulation can account for the role of very small numbers of dormant bacteria in disease progression in a great many inflammatory conditions, and opens up this process to further mechanistic analysis and possible treatment. © 2016 The Author(s) Published by the Royal Society. All rights reserved.
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Research
Cite this article: Pretorius E, Mbotwe S,
Bester J, Robinson CJ, Kell DB. 2016 Acute
induction of anomalous and amyloidogenic
blood clotting by molecular amplification of
highly substoichiometric levels of bacterial
lipopolysaccharide. J. R. Soc. Interface 13:
20160539.
http://dx.doi.org/10.1098/rsif.2016.0539
Received: 6 July 2016
Accepted: 16 August 2016
Subject Category:
Life SciencesPhysics interface
Subject Areas:
biophysics, systems biology, biochemistry
Keywords:
bacterial lipopolysaccharide, plasma,
thromboelastography, electron microscopy,
amyloid, fibrin
Authors for correspondence:
Etheresia Pretorius
e-mail: resia.pretorius@up.ac.za
Douglas B. Kell
e-mail: dbk@manchester.ac.uk
Acute induction of anomalous and
amyloidogenic blood clotting by
molecular amplification of highly
substoichiometric levels of bacterial
lipopolysaccharide
Etheresia Pretorius1, Sthembile Mbotwe1, Janette Bester1,
Christopher J. Robinson2,4,5 and Douglas B. Kell3,4,5
1
Department of Physiology, Faculty of Health Sciences, University of Pretoria, Arcadia 0007, South Africa
2
Faculty of Life Sciences,
3
School of Chemistry,
4
The Manchester Institute of Biotechnology, and
5
Centre for
Synthetic Biology of Fine and Speciality Chemicals, The University of Manchester, 131, Princess Street,
Manchester M1 7DN, Lancs, UK
EP, 0000-0002-9108-2384
It is well known that a variety of inflammatory diseases are accompanied by
hypercoagulability, and a number of more-or-less longer-term signalling path-
ways have been shown to be involved. In recent work, we have suggested a
direct and primary role for bacterial lipopolysaccharide (LPS) in this hyper-
coagulability, but it seems never to have been tested directly. Here, we show
that the addition of tiny concentrations (0.2 ng l
21
) of bacterial LPS to both
whole blood and platelet-poor plasma of normal, healthy donors leads to
marked changes in the nature of the fibrin fibres so formed, as observed by
ultrastructural and fluorescence microscopy (the latter implying that the
fibrin is actually in an amyloid b-sheet-rich form that on stoichiometric
grounds must occur autocatalytically). They resemble those seen in a
number of inflammatory (and also amyloid) diseases, consistent with an invol-
vement of LPS in their aetiology. These changes are mirrored by changes in
their viscoelastic properties as measured by thromboelastography. As the
terminal stages of coagulation involve the polymerization of fibrinogen into
fibrin fibres, we tested whether LPS would bind to fibrinogen directly. We
demonstrated this using isothermal calorimetry. Finally, we show that these
changes in fibre structure are mirrored when the experiment is done simply
with purified fibrinogen and thrombin (+0.2 ng l
21
LPS). This ratio of concen-
trations of LPS : fibrinogen in vivo represents a molecular amplification by the
LPS of more than 10
8
-fold, a number that is probably unparalleled in biology.
The observation of a direct effect of such highly substoichiometric amounts of
LPS on both fibrinogen and coagulation can account for the role of very small
numbers of dormant bacteria in disease progression in a great many inflamma-
tory conditions, and opens up this process to further mechanistic analysis and
possible treatment.
1. Introduction
‘LPS’ describes a variety of cell wall lipopolysaccharides shed by Gram-negative
bacteria; also known as ‘endotoxin’, they have been found in various fluids, includ-
ing whole blood (WB). The ‘concentrations’ are typically assayed using the Limulus
amoebocyte lysate assay (e.g. [1–3]). However, although satisfactory in simple
matrices, this test is not considered very reliable in blood [4,5]. Indeed, because it
is so hydrophobic, little or no LPS is actually free (unbound), and so it is not even
obvious what its ‘concentration’ in blood might mean (see [5]). Although the
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quantitative assessment of LPS concentrations in WB can be pro-
blematic, its presence in this fluid may have important and
clinically relevant effects on the blood microenvironment, and
maybe centralin the treatment of inflammatory conditions[5 –8].
LPS molecules are potent inflammagens (e.g. [911])
and may be both cytotoxic and/or neurotoxic [5,12–15].
They are known to induce the production of a variety of pro-
inflammatory cytokines [1619] that are involved in various
apoptotic, programmed necrosis and pyroptotic pathways
[5,20,21]. Indeed, cytokine production [16] is central to the
development of inflammation [22]. A furtherch aracteristic of sys-
temic inflammation is a hypercoagulatory state [23–27]. Such
hypercoagulability is a common pathology underlying all throm-
botic conditions, including ischaemic heart disease, ischaemic
stroke and venousthromboembolism [28]. Furthermore, a hyper-
coagulable state is typicallyassociated with pathological changes
in the concentrations of fibrin(ogen) [29,30], and in particular an
increase in the level of the fibrin degradation product D-dimer
is seen as a reliable biomarker for cardiovascular risk [31,32].
Considering its cytokine-dependent effects, the question
then arises as to whether LPS can cause hypercoagulation
by acting on the coagulation pathway more directly. One
route is via tissue factor (TF) upregulation; TF is related to
the cytokine receptor class II family, and is active early in
the (extrinsic) coagulation cascade, where it is necessary to
initiate thrombin formation from prothrombin [33]. Recently,
it was shown that LPS may upregulate TF; 100 ng ml
21
LPS
added to healthy cord WB of newborns or the WB of healthy
adults induced TF-mediated activation of haemostasis [34].
LPS from Escherichia coli (100 ng ml
21
) also activated the
coagulation system when added to WB, via a complement-
and CD14-dependent upregulation of TF, leading to pro-
thrombin activation and hypercoagulation [35]; however,
this was noted after 2 h, and therefore it was not an acute pro-
cess [35]. Note that in these studies, the anticoagulant was
lepirudin, which prevents thrombin activation such that the
effects of thrombin could not be evaluated. In this work,
we used citrate as an anticoagulant.
It occurred to us that, in addition to changes in TF
expression by LPS, the process might also involve the direct
binding of the lipophilic LPS to circulating plasma proteins,
particularly fibrinogen, and that this (potentially rapid) bind-
ing might also cause pathological changes in the coagulation
process. This would be independent of the slower TF acti-
vation, and thus an acute and relatively immediate process
(figure 1). This indeed turned out to be the case. A preprint
has been lodged at bioRxiv [36].
2. Results
2.1. Scanning electron microscopy of whole blood,
plasma and purified fibrinogen
To investigate our hypothesis that LPS may cause hypercoagu-
lation via an acute and direct binding reaction (by interaction
with plasma proteins directly involved in the clotting cas-
cade), we investigated the effect of two LPS preparations
from E. coli (viz. O111:B4 and O26:B6). These were added to
WB of healthy individuals, to platelet (and cell)-poor plasma
(PPP), and to purified fibrinogen.
Although the physiological levels of LPS are said to be
10–15 ng l
21
, and little or none of it is free [5], in our
hands the addition of LPS at these concentrations caused
immediate coagulation when they were added to WB.
Figure 2 shows the effect of 0.2 ng l
21
O111:B4 LPS when
added to WB and incubated for 10 min. Dense matted depos-
its are spontaneously formed; these are not seen in healthy
WB. Fibrinogen with added O111:B4 or O26:B6 LPS with
just 30 s exposure (no thrombin added) also spontaneously
formed matted deposits (results not shown).
2.1.1. Platelet poor plasma and lipopolysaccharide
Figure 3 shows the effects of thrombin on clot formation for
a healthy control (figure 3a) and when the PPP was pre-
incubated for 10 min with 0.2 ng l
21
O111:B4 LPS (two
increased presence
of LPS in blood
systemic inflammatory profile
from genetics, environment,
lifestyle, etc.
activated inflammatory
pathways via cytokine production
changed coagulation cascade
hypercoagulable state of the
coagulation system
indirect activation of
hypercoagulation via
LPS
activation of
hypercoagulation
via TF expression by LPS
acute activation of
hypercoagulation
via LPS
1A
1B
2
*LPS is lipophilic
LPS binds to plasma
proteins e.g. fibrinogen
LPS-bound fibrinogen
clots abnormally
Figure 1. High-level effects of systemic inflammation on the coagulation system and the pathologic effects of LPS when present in blood and how it influences
coagulation via a direct or indirect activation. Processes 1A and B are currently known for LPS activity in blood, while process 2 is a newly proposed acute reaction
effect of LPS on blood and plasma. (Online version in colour.)
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examples in figure 3b,c). Figure 3dshows the distribution of
fibre thicknesses for the 30 individuals, with and without
added LPS. The fibre thickness is much more heterogeneous
after LPS is added. Clearly, these tiny amounts of LPS
are having enormous effects on the clotting process. These
kinds of netted structures, which we have also termed ‘dense
matted deposits’, were previously seen in inflammatory con-
ditions such as diabetes [37– 39], iron overload and stroke
[37,40– 42]. Typically, healthy fibrin fibre networks form a
net where individual fibrin fibres are seen, but with added
LPS a matted net develops. There is a significant difference
(p,0.0001) between fibre thickness before and after LPS
treatment in the presence of thrombin. Note, though, that the
distribution of the fibre thickness in the LPS-treated group
varies from very thick to very thin. In some cases, continuous
fibre plates are formed, where no individual fibres could
be seen or measured. This explains the difference in nbet-
ween the ‘before’ and ‘after’ treatment (1450 versus 1330
measured fibres).
The experiment with the O111:B4 LPS was also repeated
with a shorter 30 s exposure time. PPP with LPS and added
thrombin showed fibre agglutination starting to happen in
only 30 s of LPS exposure. These shorter experiments are to
be contrasted with previously reported experiments that
showed the much longer-term effect of LPS, involving cytokine
production, including increased TF production via monocytes.
By adding LPS to PPP (with thrombin), we bypass the possi-
bility that LPS can stimulate TF production via the monocyte
route suggested in [35,43]. To determine if another type of
LPS would also cause the changes noted above, we also
added E. coli O26:B6 LPS to PPP of five individuals (30 s and
10 min exposure time), followed by addition of thrombin. Scan-
ning electron microscopy (SEM) results showed the same
trends as noted with the O111:B4 LPS (results not shown).
2.1.2. Confocal microscopy
The reaction, and presumed binding, of the hydrophobic LPS
within fibrinogen fibres implies that they contain, or expose,
significant hydrophobic elements. Such elements, also
common in amyloid-like fibrils [44], can be stained fluores-
cently using dyes such as thioflavin T (ThT) [45]. We
therefore studied the effect of 0.2 ng l
21
LPS on the ability of
the fibrin fibres formed following thrombin treatment of PPP
to bind ThT (figure 4). In contrast with the LPS-free controls
(figure 4a), there is a very substantial binding of ThT to the
fibrin fibres formed in the presence of the LPS (figure 4b).
The lipopolysaccharide binding protein (LBP) is a potent
binder of LPS, and would therefore be expected to inhibit the
amyloidogenic effects on blood clotting observed. Thus, we
also studied the effect of 2 ng l
21
LBP and 2 ng l
21
LBP þ
0.2 ng l
21
LPS mixture on ThT binding (figure 4c,d) [7].
2.1.3. Purified fibrinogen
It is worth rehearsing just how big an effect this is in molar
terms: fibrinogen (MW 340 kDa) is present at in plasma at con-
centrations of approximately 2–4 g l
21
(Weisel’s authoritative
review [46] gives 2.5 g l
21
), and its levels are increased
during inflammation (see above), while the LPS (MW 10–
20 kDa) was added at a concentration of 0.2 ng l
21
. We will
assume 15 kDa for the MW of LPS and 30 fg LPS per cell.
Thus, 0.2 ng l
21
¼13 fM and 2.5 g l
21
fibrinogen approxi-
mately 7.35 mM which is a molar ratio of LPS; fibrinogen
monomer in the WB of less than 10
28
: 1. As we are here only
looking at the terminal stages of clotting, we considered that
fibrinogen might be an important mediator of the LPS-induced
hypercoagulation. Thus, we also added both LPS types to pur-
ified fibrinogen (30 s and 10 min exposure time) with added
thrombin. Even the 30 s exposure time changed the fibrin
fibres to form fibrils or dense matted deposits without any
individual fibres visible (figure 5).
It is also worth rehearsing what 0.2 ng l
21
of LPS means in
terms of the bacterial equivalents. Watson and co-workers [47]
showed in laboratory cultures that LPS amounted to some 50 fg
per cell in a logarithmic growth phase, falling to 29 fg per cell in
stationary phase. As remarked previously [5], this shows at
once that LPS contents per cell can be quite variable and that
bacteria can shed a considerable amount of LPS at no major
harm to themselves. On the basis that 1 mg dry weight of
bacteria is about 10
9
cells, each cell is about 1 pg, so 50 fg
LPS per cell equates to about 5% of its dry weight, a reasonable
and self-consistent figure for approximate calculations. We
shall take the ‘starved’ value of 30 fg per cell. Thus, 0.2 ng l
21
(200 pg l
21
) LPS is equivalent to the LPS content of approxi-
mately 7 10
3
cells l
21
. Most estimates of the dormant blood
microbiome (that is derived mainly by dysbiosis from the gut
and from the oral cavity as summarized in [5–7]) (some are
much greater [48]) imply values of 10
3
–10
4
ml, i.e. approxi-
mately 1000 times greater. In other words, a bacterial cell
need lose only a small amount of its LPS to affect blood clotting
in the way we describe here.
2.2. Isothermal titration calorimetry
ITC is a sensitive and convenient method for detecting biomo-
lecular interactions by measuring the heat that is released or
absorbed upon binding [49]. Measurements are conducted
directly in solution, without modification of immobilization
of the interacting species. We used ITC to study potential inter-
actions between human plasma fibrinogen and LPS from E. coli
O111:B4. Titration of fibrinogen into LPS resulted in strong
endothermic injection heats with a clear sigmoidal saturation
curve indicating a direct binding interaction (figure 6a).
Assuming molecular weights of 340 kDa for fibrinogen and
20 kDa for monomeric LPS, we determined a binding stoichi-
ometry (n) of approximately 0.135. This is consistent with
1mm
Figure 2. Effect of O111:B4 LPS (0.2 ng l
21
) on whole blood (without
thrombin), where dense matted deposits were spontaneously formed, not
seen in control whole blood smears.
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each fibrinogen monomer binding to a micelle formed from
approximately 75 LPS monomers. Reverse titrations were con-
ducted, injecting LPS into plasma concentrations of fibrinogen
(3 g l
21
¼8.8 mM). Titration of 2.5 mM LPS into fibrinogen
yielded endothermic injection heats greater than those
observed for titration of 2.5 mM LPS into buffer alone
(figure 6b), again clearly indicating a direct binding of LPS
to fibrinogen. Each injection added 125 ng of LPS into the
instrument cell, increasing the LPS concentration by approxi-
mately 30 nM per injection. Although we expect that LPS
binds fibrinogen at sub-nanomolar concentrations, interactions
at these concentrations are below the detection limits of the
ITC instrument.
2.3. Thromboelastography of whole blood and platelet
poor plasma
Thromboelastography (TEG
w
) is a viscoelastic technique for
measuring the clotting properties of WB [50,51]. TEG of
WB and PPP was performed. TEG was not performed with
purified fibrinogen because the coagulation activator in the
TEG is CaCl
2
and fibrinogen is only activated by thrombin,
not calcium. Figure 7 shows a typical TEG trace from a con-
trol WB with and without added LPS, overlaid with lines
that explain the parameters extracted by the instrument and
the values for those traces. The statistics are given in table 1.
2.3.1. Whole blood
TEG analysis of WB (10 min incubation timewith O111:B4 LPS)
showed that the R, TMRTG and TTG are all significantly
decreased (table 1). Changes to Rindicate that the clot forms
quicker and a decreased TMRTG indicates that the time to
maximum thrombus generation is also faster, suggesting a
hypercoagulable state. The TTG is also significantly decreased
suggesting total thrombus generation, thus implying clot
strength is decreased, although the clot forms faster. The SEM
fibrin fibre thickness results show areas where no individual
fibres are formed; instead, a matted homogeneous layer
forms, and there are also areas of fine and short fibres. Here,
we suggest that this morphology is probably related to the
decreased TTG, where the fibrin structure results in a clot
with decreased strength. We have not measured lyses, but a
decreased TTG is likely to indicate a hypofibrinolytic nature of
the clot. We have previously shown that the same concentration
of LPS as used in this paper, when added to naive uncitrated
healthy blood but without added CaCl
2
, also had an effect on
coagulation after only 30 s incubation time [52]. Both TMRTG
and Rof naive WB were also significantly shorter, also showing
hypercoagulation, and the TTG was increased, but not signifi-
cantly increased. However, in this study, the blood was not
drawn in citrated tubes and therefore not re-calcified.
2.3.2. Platelet-poor plasma
We also performed similar TEG experiments with PPP (table 1).
After 10 min exposure, just the initial clotting time Rwas chan-
ged. The results were not specific for O111:B4 LPS as O26:B6
LPS added to PPP behaved similarly. The decreased Rtime is
indicative of reaction time, and therefore the time to first mea-
surable clot formation is also significantly decreased (as the
initiation of the clot starts faster with than without LPS), con-
firming a hypercoagulable state with added LPS. After 30 s
exposure time, both the Rand the TMRTG were shorter (in
the five patients tested). This confirms our hypothesis that
LPS causes (near) instant hypercoagulability. Here also, the
results were not specific for O111:B4 LPS as O26:B6 LPS
added to PPP also showed a decreased R-time. TTG of PPP
was increased (but not significantly increased) after 30 s, as
1 mm
fibre diameter
600
500
400
211
347
165
86
0
40
103
8
control control + LPS
79
133
300
200
100
0
(b)(a)
(c)
(d)
Figure 3. The effect of 0.2 ng l
21
O111:B4 LPS on the morphology of fibrin fibres in the platelet-poor plasma (PPP) of healthy individuals (with added thrombin).
(a) Healthy fibres; (b,c) PPP with added LPS. (d) Fibre distribution of the control fibres and of controls with added LPS of 30 individuals. Note: in samples with
added LPS, there were areas of matted layers with no visible fibres to measure. Fibres were measured using ImageJ as described in Material and methods.
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well as 10 min, which compared with results of PPP from
patients with Alzheimer’s type dementia [52].
Table 1 shows a summary of the various viscoelastic
properties (TEG experiments) after LPS has been added to
WB and PPP. There are very substantial changes in a
number of the clotting parameters. Following these 10 min
exposure experiments, we shortened our experimental time
to 30 s and we repeated the experiments with five samples,
using PPP, where significant changes were still observed.
The results were not specific for O111:B4 LPS as O26:B6
LPS. We note that WB with added LPS showed a more
pronounced change in relevant viscoelastic parameters than
when LPS was added to PPP (table 1).
3. Discussion
In the introduction, we suggested that LPS might contribute to
excessive blood clotting (or an activated coagulation state) via
two possible routes: (i) via a direct and acute binding to plasma
proteins (e.g. fibrinogen) or (ii) by an indirect or chronic
(longer-term) process where it participates in an inflammatory
5 mm
5 mm
(b)
(a)
(c)(d)
Figure 4. (a) Control PPP with ThT and thrombin and (b) as panel (a) but pre-incubated with 0.2 ng l
21
LPS; (c) as panel (a) but pre-incubated with 2 ng l
21
LBP;
(d) as panel (a) but pre-incubated with 0.2 ng l
21
LPS and 2 ng l
21
LBP. (Online version in colour.)
(b)
(a)(c)
1 mm
Figure 5. (a) Purified fibrinogen with added thrombin but no LPS; (b) purified fibrinogen with added O111:B4 LPS (30 s exposure) and 0.2 ng l
21
LPS; (c) as panel
(b) but 10 min exposure.
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activation via cytokine production. Here, we showed that the
first process is indeed possible, using tiny amounts of LPS
that amounted in molar terms to less than 10
28
relative to
fibrinogen, and demonstrated it by both viscoelastic and ultra-
structural methods. We also confirmed that LPS can change the
viscoelastic properties of PPP within 30 s of its addition.
Furthermore, WB with added LPS, but without thrombin acti-
vation, showed spontaneously formed, amyloid-like matted
deposits. Purified fibrinogen experiments with O111:B4 LPS
and O26:B6 LPS, with and without added thrombin showed
0
0.40
0.30
0.20
0.10
0
250
200
150
Kcal mol–1 of injectant mcal s–1
100
50
0
0 0.01
molar ratio
LPS (ng ml–1); fibrinogen (mgml–1)
0.02 0.03 0.04
20 40 60 80 100
time (min) 0.30
50 3
03
50 0
00
0.20
0.10
0
0.30
0.20
0.10
0
0.30
0.20
0.10
0
0.30
0.20
0.10
0
2468
injection no.
10 12 14 16
120 140 160
(b)(a)
Figure 6. ITC analysis of the LPS–fibrinogen interaction. (a) Titration of 8.8 mM human plasma fibrinogen (black circles) or buffer (green open circles) into 100 mM
of E. coli O111:B4 LPS. (b) Titration of 50 ng ml
21
LPS (2.5 mM) or buffer into 3 mgml
21
fibrinogen (8.8 mM) or buffer as indicated. Experiments were conducted
at 378C in phosphate buffered saline. (Online version in colour.)
WB (green trace)
WB with LPS
(white trace)
MA
angle
20 mm
10 min
K
9.5control
whole
blood
with
added
LPS
5.8 46 55.5 2.27 16.33 627.06
6.3 4.7 54.6 47.4 2.58 8.08 452.08
R
(min)
K
(min)
angle
(°)
MA
(mm)
MRTG
(dyn. cm–2.s–1)
TMRTG
(s)
TGG
(dyn. cm–2)
R
Figure 7. TEG overlay from a control whole blood sample with and without added LPS. R, reaction time, first measurable clot formation; K, achievement of clot
firmness; angle, kinetics of clot development; MA, maximum clot strength; MRTG, maximum rate of thrombus generation; TMRTG, time to maximum rate of throm-
bus generation; TTG, final clot strength. (Online version in colour.)
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Table 1. Demographics of blood from healthy individuals with and without added LPS. Medians, standard deviation and p-values (values lower than 0.05 are
indicated in italic) obtained using the MannWhitney U-test are shown for iron profiles, and TEG of whole blood and plasma. R, reaction time, first measurable
clot formation; K, achievement of clot firmness; angle, kinetics of clot development; MA, maximum clot strength; MRTG, maximum rate of thrombus generation;
TMRTG, time to maximum rate of thrombus generation; TTG, final clot strength.
variables healthy individuals (n530) healthy individuals with added LPS (n530) p-value
age (years) 29.5 (+13.81)
gender
male 15 (50%)
female 15 (50%)
iron profiles
iron mM 16.9 (+6.18)
transferrin g l
21
2.75 (+0.46)
% saturation 25.0 (+10.94)
serum ferritin ng ml
21
42.5 (+92.47)
fibrin fibre thickness n¼1450 n¼1330
fibre thickness (nm) 103 (+40) 86 (+82) ,0.0001
TEG
w
TEG of whole blood—recalcified with CaCl
2
with added O111:B4 LPS (10 min)
MRTG (dyn cm
22
s
21
) 2.61 (+1.13) 2.89 (+0.90) 0.33
TMRTG (min) 13.9 (+3.53) 9.6 (+3.01) ,0.0001
TTG (dyn cm
22
) 615.0 (+179.55) 527.9 (+146.65) 0.049
R(min) 8.0 (+1.64) 6.2 (+1.77) ,0.0001
K(min) 4.9 (+2.63) 4.2 (+1.23) 0.07
angle (8) 49.8 (+5.27) 56.2 (+7.02) 0.0066
MA (mm) 55.0 (+8.07) 51.3 (+6.90) 0.092
TEG of platelet-poor plasma—recalcified with CaCl
2
with added O111:B4 LPS (10 min)
MRTG (dyn cm
22
s
21
) 3.6 (+4.35) 4.2 (+2.13) 0.36
TMRTG (min) 10.6 (+3.22) 9.3 (+3.68) 0.50
TTG (dyn cm
22
) 203.9 (+137.51) 211.6 (+103.67) 0.70
R(min) 8.2 (+2.64) 7.1 (+2.70) 0.026
K(min) 4.4 (+3.51) 3.8 (+2.42) 0.18
angle (8) 63.2 (+2.70) 54.4 (+10.67) 0.23
MA (mm) 28.4 (+8.34) 30.1 (+9.27) 0.196
TEG of platelet-poor plasma—recalcified with CaCl
2
with added O111:B4 LPS (30 s)
n¼5n¼5
MRTG (dyn cm
22
s
21
) 5.94 (+1.8) 8.2 (+2) 0.166
TMRTG (min) 11.58 (+1.2) 9 (+1.3) 0.0159
TTG (dyn cm
22
) 244.4 (+69.9) 290.2 (+66.5) .0.99
R(min) 9.8 (+1.2) 7 (+1.5) 0.031
K(min) 2.8 (+1.6) 2 (+0.9) 0.119
angle (8) 63.6 (+6.5) 68.8 (+8.2) 0.095
MA (mm) 32.8 (+6.5) 36.7 (+5.5) .0.99
TEG of platelet-poor plasma—recalcified with CaCl
2
with added O26:B6 LPS (30 s)
n¼5n¼5
MRTG (dyn cm
22
s
21
) 6.3 (+2.6) 6.2 (+3.6) 0.70
TMRTG (min) 11.6 (+2.1) 8.9 (+1.5) 0.15
TTG (dyn cm
22
) 276.7 (+43.1) 230.8 (+202.2) 0.69
R(min) 9.8 (+1.5) 6.4 (+1.4) 0.05
K(min) 2.1 (+0.5) 2.1 (+1.1) 0.88
angle (8) 64.4 (+4.3) 70.8 (+10.4) .0.99
MA (mm) 35.5 (+3.3) 31.5 (+13) 0.60
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a changed ultrastructure, suggesting that LPS indeed binds to
the 340 kDa fibrinogen molecule and that the effects of this
are visible ultrastructurally.
LPS, and especially its lipid A component, is highly lipophi-
lic, and it therefore may be able to bind directly to plasma
proteins, in an acute way. This might be one reason underlying
the hypercoagulability [5], as well as a denser clot structure
[53,54], as seen in various inflammatory diseases. Although
we show here that exposure to even tiny amounts of LPS
leads to an immediate (acute) change in the coagulability par-
ameters, we recognize that this may happen simultaneously
with chronic (longer-term) reactions (figure 1). Fibrinogen mol-
ecules are roughly 545 nm, and their self-assembly is a
remarkable process (some 5800 are involved in generating a
fibre of 80 90 nm diameter and 1 mmlength).Thiswould
explain why the highly substoichiometric binding of LPS can
have such considerable effects, especially as observed in WB.
Following Anfinsen [55], it is assumed that most proteins
adopt their conformation of lowest free energy. However, this
is not true for amyloid fibre formation [56] nor in the case of
the autocatalytic conversion of prion protein conformations
[57,58]. At present, the exact mechanisms of action of these
small amounts of LPS are not known, although it is indeed sim-
plest to recognize fibrinogen polymerization as a cascade effect,
much as occurs for amyloid and prion proteins whose initial
conformation is not in fact that of their lowest free energy [59].
Specifically [60], the ‘normal’ conformational macrostate of
such proteins is not in fact that of the lowest free energy, and
its transition to the energetically more favourable ‘rogue’ state
is thermodynamically favourable but under kinetic control,nor-
mally (in terms of transition state theory) with a very high
energy barrier DG
of maybe 36–38 kcal mol
21
[60]. Indeed, it
is now known that quite a number of proteins of a given
sequence can exist in at least two highly distinct conformations
[61]. Typically the normal (‘benign’) form, as produced initially
within the cell, will have a significant a-helical content, but the
abnormal (‘rogue’) form, often in the form of aninsoluble amy-
loid, will have a massively increased amount of b-sheet [62],
whether parallel or antiparallel. In the case of blood clotting,
we at least know that this is initiated by the thrombin-catalysed
loss of fibrinopeptides from fibrinogen monomers (e.g. [41,63]).
The massive adoption of a b-sheet conformation, as revealed
here for the first time by the thioflavin T staining, demonstrates
directly that virtually every fibrinogen molecule in the fibrin
fibril must have changed its conformation hugely; it is not just
a question of static ‘knobs and holes’ as usually depicted. We
also showed that LBP, and a mixture of LPS and LBP, shows
decreased ThT binding, compared with LPS alone.
Previously, we coined the term ‘atopobiotic’ microbes to
describe microbes that appear in places other than where
they should be, e.g. in the blood, forming a blood microbiome
[6]. Here, we suggest that the metabolic and cell membrane
products of these atopobiotic microbes correlate with, and
may contribute to, the dynamics of a variety of inflammatory
diseases [6467], and that LPS, in addition to (possibly low-
grade) long-term inflammation via cytokine production, may
lead an acute and direct hypercoagulatory effect by binding
to plasma proteins, especially fibrinogen. Specifically, we
showed here that, even with very low levels and highly sub-
stoichiometric amounts of LPS, a greatly changed fibrin fibre
structure is observed. An urgent task now is to uncover the
mechanism(s) of this acute and immediate effect, with its
remarkable molecular amplification.
4. Material and methods
4.1. Sample population
In total, 30 healthy individuals were included in the study. Exclu-
sion criteria were known inflammatory conditions such as
asthma, human immunodeficiency virus (HIV) or tuberculosis,
and risk factors associated with metabolic syndrome, smoking,
and, if female, being on contraceptive or hormone replacement
treatment. Full iron tests were performed, as high serum ferritin
and low transferrin levels are acute phase inflammatory protein
markers [68] and indicative of inflammation. We included con-
trols only if their iron levels were within normal ranges. WB of
the participants was obtained in citrate tubes and either WB or
platelet-poor plasma was used in this study for TEG, confocal
and SEM experiments.
4.2. Lipopolysaccharide types, purified fibrinogen and
thrombin concentration used
The LPS used was from E. coli O111:B4 (Sigma, L2630) and also
E. coli O26:B6 (Sigma L2762). A final LPS exposure concentration
of 0.2 ng l
21
(well below its critical micelle concentration [69])
was used in all experiments bar as noted for some of the ITC
measurements. A final LBP exposure concentration of 2 ng l
21
LBP and a mixture with final exposure concentration of LPS
(0.2 ng l
21
) and LBP (2 ng l
21
), incubated for 10 min with PPP,
were also used (only confocal studies).
For ITC experiments, a micellar suspension of 10 mg l
21
was
vortexed, followed by multiple serial dilutions. The South African
National Blood Service (SANBS) supplied human thrombin,
which was at a stock concentration of 20 U ml
21
and was made
up in a PBS containing 0.2% human serum albumin. In exper-
iments with added thrombin, 5 ml of thrombin was added to
10 ml of PPP or fibrinogen (with and without LPS exposure).
Human fibrinogen was purchased from Sigma (F3879–250MG).
A working solution of 0.166 mg ml
21
purified fibrinogen was
prepared. This concentration was found to be the optimal concen-
tration to form fibrin fibres in the presence of thrombin, similar to
that of platelet-rich plasma fibres from healthy individuals [70].
As noted by a referee, LPS is a common laboratory contaminant,
and care is needed; however, this was not an issue here as the
‘no-added-LPS’ controls showed.
4.3. Addition of lipopolysaccharide +thrombin to
whole blood, plasma and purified fibrinogen
LPS-incubated WB and purified fibrinogen were prepared for SEM
without added thrombin (LPS exposure concentration: 0.2 ng l
21
).
LPS-incubated PPP and purified fibrinogen samples were pre-
pared as above, but with added thrombin to create an extensive
fibrin fibre network (also with LPS exposure concentration:
0.2 ng l
21
before addition of thrombin).
4.4. Isothermal titration calorimetry
E. coli O111:B4 LPS and human plasma fibrinogen were purchased
from Sigma-Aldrich. Samples were reconstituted in warm phos-
phate buffered saline and incubated for 1 h at 378Cwith
shaking. LPS was then sonicated for 1 h at 608C. Fibrinogen sol-
utions were passed through a 0.2 mm polyethersulfone syringe
filter and concentrations were determined by UV absorbance
(E
1%
¼15.1 at 280 nm). Samples were then diluted with buffer to
the required concentration and degassed. ITC experiments were
performed at 378C on a MicroCal Auto-iTC200 system (GE Health-
care) in high-gain mode at a reference power of 10 mcal s
21
,with
an initial 0.5 ml (1 s) injection followed by 15 2.5 ml (5 s) injections
with 300 s spacing. For longer titrations, the syringe was refilled
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and injections continued into the same cell sample. Control runs
were performed in which cell samples were titrated with buffer
and syringe samples were titrated into buffer, and data from
these runs were subtracted from the experimental data as appro-
priate. Data analysis was performed in Origin, using the
supplied software (MicroCal).
4.5. Thromboelastography
TEG was used to study the viscoelastic properties of the partici-
pants’ blood, before and after addition of LPS. WB TEG was
performed on day of collection (after 10 min incubation time
with LPS—final exposure concentration 0.2 ng l
21
and PPP was
stored in 500 ml aliquots in a 2708C freezer. The thawed citrated
PPP (with and without LPS—where LPS was added to PPP at a
final exposure concentration of 0.2 ng l
21
. The incubation time of
LPS and PPP was 10 min, as with WB. Standard TEG procedures
were followed with addition of CaCl
2
to activate the coagulation
process as previously described [50,51,71,72]. TEG was also
performed on 5 PPP samples, 30 s after adding O111:B4 LPS or
O26:B6 LPS.
4.6. Confocal microscopy
Thioflavin T (ThT) was added at an exposure concentration of
5mM to 200 ml of PPP (incubated for 1 min, and protected from
light). A second sample was also prepared by adding an exposure
concentration of 0.2 ng l
21
LPS (incubate for 10 min, at room temp-
erature) before the addition of ThT. After an incubation time of
1 min and incubation protected from light, 10 ml of the PPP
(with and without LPS) was mixed with 5 ml of thrombin (see
SEM preparation of extensive fibrin fibres—as described above).
To determine if ThT binding will happen in the presence of LBP,
2ngl
21
LBP was pre-incubated for 10 min with PPP, followed
by 1 min ThT exposure, and fibrin fibre preparation by adding
thrombin. A mixture of LPS and LBP was also made (final PPP
exposure concentration: 0.2 ng l
21
LPS and 2 ng l
21
LBP). PPP
was exposed for 10 min to this mixture, followed by a 1 min
exposure of ThT and fibrin fibre formation by adding thrombin
to mixture-exposed PPP. Samples were viewed under a Zeiss
LSM 510 META confocal microscope with a Plan-Apochromat
63/1.4 Oil DIC objective, excitation was at 488 nm and emission
measured at 505–550.
4.7. Statistical analysis
The non-parametric Mann–Whitney U-test was performed using
STATSDIRECT software.
Ethics. Ethical clearance was obtained from the Human Ethics
Committee of the University of Pretoria.
Competing interests. We declare we have no competing interests.
Funding. We thank the Biotechnology and Biological Sciences Research
Council (grant no. BB/L025752/1) as well as the National Research
Foundation (NRF) of South Africa for supporting this collaboration.
This is also a contribution from the Manchester Centre for Synthetic
Biology of Fine and Speciality Chemicals (SYNBIOCHEM) (BBSRC
grant BB/M017702/1).
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... We have also found that during the presence of systemic inflammation, reflected in an increased presence of inflammagens, the biochemistry of the fibrin(ogen) molecule changes its folding characteristics considerably (Figure 3), to produce amyloid forms. We could visualize these changes using fluorescence markers [32,57,[79][80][81]. The fluorescence markers we have used to show these structural changes in the fibrin(ogen) biochemistry included thioflavin T (ThT) and various Amytracker dyes. ...
... The fluorescence markers we have used to show these structural changes in the fibrin(ogen) biochemistry included thioflavin T (ThT) and various Amytracker dyes. These fluorescence markers are typically used to show amyloid changes to proteins [32,57,[79][80][81], suggesting the misfolding seen in fibrin(ogen) during the presence of inflammagens in the blood, could also be described as amyloid. ThT binds to open hydrophobic regions on damaged protein [57,81]. ...
... These fluorescence markers are typically used to show amyloid changes to proteins [32,57,[79][80][81], suggesting the misfolding seen in fibrin(ogen) during the presence of inflammagens in the blood, could also be described as amyloid. ThT binds to open hydrophobic regions on damaged protein [57,81]. We showed, that when healthy fibrinogen is exposed to increased levels of inflammatory biomarkers and bacterial (viral) inflammagens, PPP TEG ® traces was significantly hypercoagulable [79][80][81][82]. ...
Article
Full-text available
An important component of severe COVID-19 disease is virus-induced endothelilitis. This leads to disruption of normal endothelial function, initiating a state of failing normal clotting physiology. Massively increased levels of von Willebrand Factor (VWF) lead to overwhelming platelet activation, as well as activation of the enzymatic (intrinsic) clotting pathway. In addition, there is an impaired fibrinolysis, caused by, amongst others, increased levels of alpha-(2) antiplasmin. The end result is hypercoagulation (proven by thromboelastography® (TEG®)) and reduced fibrinolysis, inevitably leading to a difficult-to-overcome hypercoagulated physiological state. Platelets in circulation also plays a significant role in clot formation, but they themselves may also drive hypercoagulation when they are overactivated due to the interactions of their receptors with the endothelium, immune cells or circulating inflammatory molecules. From the literature it is clear that the role of platelets in severely ill COVID-19 patients has been markedly underestimated or even ignored. We here highlight the value of early management of severe COVID-19 coagulopathy as guided by TEG®, microclot and platelet mapping. We also argue that the failure of clinical trials, where the efficacy of prophylactic versus therapeutic clexane (low molecular weight heparin (LMWH)) were not always successful, which may be because the significant role of platelet activation was not taken into account during the planning of the trial. We conclude that, because of the overwhelming alteration of clotting, the outcome of any trial evaluating an any single anticoagulant, including thrombolytic, would be negative. Here we suggest the use of the degree of platelet dysfunction and presence of microclots in circulation, together with TEG®, might be used as a guideline for disease severity. A multi-pronged approach, guided by TEG® and platelet mapping, would be required to maintain normal clotting physiology in severe COVID-19 disease.
... This transition between the two forms can itself be catalysed by the PrP Sc . The key point of importance in microclot formation in Long COVID/PASC is that fibrin(ogen) too can, in the presence of various trigger substances, fold into an amyloid form that has a very different macrostructure characterised by different fibre diameters and pore sizes 49 . We noted that e.g. in type 2 Diabetes Mellitus (T2DM) clots have a netlike appearance 50,51,52,53 while in Alzheimer's disease 16,54,55,56 and Parkinson's disease the fibres may be larger in size 15,22 ; they are also much more resistant to proteolysis 17 , and so are much more prevalent in the steady state. ...
... In this earlier literature we often referred to these anomalous clots as 'dense matted deposits'. In 2016, we showed that this anomalous resistance to fibrinolysis was because the anomalous structures were in fact amyloid in nature 49 . Such structures are easily observed under the optical microscope, and in particular may be stained with the fluorogenic dye thioflavin T and by the more recently developed oligothiophene dyes marketed by Ebba Biotech as Amytrackers 15,19,21 . ...
Preprint
Full-text available
This is study of microclots in long-COVID patients, including treatment thereof, and a data-driven correlation of comorbidities with long-COVID symptoms. This is a corrected version. The preprint original is also at: https://doi.org/10.21203/rs.3.rs-1205453/v1
... This transition between the two forms can itself be catalysed by the PrP Sc . The key point of importance in microclot formation in Long COVID/PASC is that fibrin(ogen) too can, in the presence of various trigger substances, fold into an amyloid form that has a very different macrostructure characterised by different fibre diameters and pore sizes 49 . We noted that e.g. in type 2 Diabetes Mellitus (T2DM) clots have a netlike appearance 50,51,52,53 while in Alzheimer's disease 16,54,55,56 and Parkinson's disease the fibres may be larger in size 15,22 ; they are also much more resistant to proteolysis 17 , and so are much more prevalent in the steady state. ...
... In this earlier literature we often referred to these anomalous clots as 'dense matted deposits'. In 2016, we showed that this anomalous resistance to fibrinolysis was because the anomalous structures were in fact amyloid in nature 49 . Such structures are easily observed under the optical microscope, and in particular may be stained with the fluorogenic dye thioflavin T and by the more recently developed oligothiophene dyes marketed by Ebba Biotech as Amytrackers 15,19,21 . ...
Preprint
Full-text available
We recognise that fibrin(ogen) amyloid microclots and platelet hyperactivation, that we have previously observed in COVID-19 and Long COVID/Post-Acute Sequelae of COVID-19 (PASC) patients, might form a suitable set of foci for the clinical treatment of the symptoms of long COVID/PASC. We first report on the comorbidities and symptoms found in a cohort of 845 South African Long COVID/PASC patients who filled in the South African Long COVID/PASC registry, of which hypertension and high cholesterol levels (dyslipidaemia) were the most important comorbidities. The gender balance (70% female) and the most commonly reported Long COVID/PASC symptoms (fatigue, brain fog, loss of concentration and forgetfulness, shortness of breath, as well as joint and muscle pains) were comparable to those reported elsewhere. This suggests that our sample was not at all atypical. Using a previously published scoring system for fibrin amyloid microclots and platelet pathology, we analysed blood samples from 70 patients, and report the presence of significant fibrin amyloid microclots and platelet pathology in all cases; these were associated with Long COVID/PASC symptoms that persisted after the recovery from acute COVID-19. A subset of 24 patients was treated with one month of dual antiplatelet therapy (DAPT) (Clopidogrel 75mg/Aspirin 75mg) once a day, as well as a direct oral anticoagulant (DOAC) (Apixiban) 5 mg twice a day. A proton pump inhibitor (PPI) pantoprazole 40 mg/day was also prescribed for gastric protection. Such a regime must only be followed under strict and qualified medical guidance to obviate any dangers, especially haemorrhagic bleeding, and of the therapy as a whole. Thromboelastography (TEG®) was used to assist in determining their clotting status. Each of the 24 treated cases reported that their main symptoms were resolved and fatigue as the main symptom was relieved, and this was also reflected in a decrease of both the fibrin amyloid microclots and platelet pathology scores. Nine patients were genotyped for genetic variation in homocysteine metabolism implicated in hypertension, a common COVID-19 co-morbidity reported in both patients found to be homozygous for the risk-associated MTHFR 677 T-allele. Fibrin amyloid microclots that block capillaries and inhibit the transport of O2 to tissues, accompanied by platelet hyperactivation, provide a ready explanation for the symptoms of Long COVID/PASC. The removal and reversal of these underlying epitheliopathies underlying this provide an important treatment option that seems to be highly efficacious, and warrants controlled clinical studies.
... Another is the continued release of sequestered microbially derived substances than can act as stimuli for continuing microclot formation. Here, the finding [202] that S1 spike protein can itself persist in CD16 + Monocytes in PASC for up to 15 months post-infection is highly relevant, as the amplification of trigger proteins to make microclots as part of the clotting mechanism means that miniscule (and highly substoichiometric) amounts of suitable triggers can suffice [26,47]. This alone is sufficient to account for the chronic nature of such diseases. ...
Article
Full-text available
Post-acute sequelae of COVID (PASC), usually referred to as 'Long COVID' (a phenotype of COVID-19), is a relatively frequent consequence of SARS-CoV-2 infection, in which symptoms such as breathlessness, fatigue, 'brain fog', tissue damage, inflammation, and coagulopathies (dysfunctions of the blood coagulation system) persist long after the initial infection. It bears similarities to other post-viral syndromes, and to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Many regulatory health bodies still do not recognize this syndrome as a separate disease entity, and refer to it under the broad terminology of 'COVID', although its demographics are quite different from those of acute COVID-19. A few years ago, we discovered that fibrinogen in blood can clot into an anomalous 'amyloid' form of fibrin that (like other β-rich amyloids and prions) is relatively resistant to proteolysis (fibrinolysis). The result, as is strongly manifested in platelet-poor plasma (PPP) of individuals with Long COVID, is extensive fibrin amyloid microclots that can persist, can entrap other proteins, and that may lead to the production of various autoantibodies. These microclots are more-or-less easily measured in PPP with the stain thioflavin T and a simple fluorescence microscope. Although the symptoms of Long COVID are multifarious, we here argue that the ability of these fibrin amyloid microclots (fibrinaloids) to block up capillaries, and thus to limit the passage of red blood cells and hence O2 exchange, can actually underpin the majority of these symptoms. Consistent with this, in a preliminary report, it has been shown that suitable and closely monitored 'triple' anticoagulant therapy that leads to the removal of the microclots also removes the other symptoms. Fibrin amyloid microclots represent a novel and potentially important target for both the understanding and treatment of Long COVID and related disorders.
... LPS can also bind to fibrinogen and induce misfolding. 118,119 In addition to a role in coagulation, thrombin can cleave pro-IL-1α and initiates the IL-1α inflammatory cascade. 120 Indeed, LPS-induced inflammatory processes promote coagulation (see 4,86 ). ...
Article
Full-text available
Lipopolysaccharide (LPS) is the main structural component of the outer membrane of most Gram-negative bacteria and has diverse immunostimulatory and procoagulant effects. Even though LPS is well described for its role in the pathology of sepsis, considerable evidence demonstrates that LPS-induced signalling and immune dysregulation are also relevant in the pathophysiology of many diseases, characteristically where endotoxaemia is less severe. These diseases are typically chronic and progressive in nature and span broad classifications, including neurodegenerative, metabolic, and cardiovascular diseases. This Review reappraises the mechanisms of LPS-induced signalling and emphasises the crucial contribution of LPS to the pathology of multiple chronic diseases, beyond conventional sepsis. This perspective asserts that new ways of approaching chronic diseases by targeting LPS-driven pathways may be of therapeutic benefit in a wide range of chronic inflammatory conditions.
... Dabigatran suppresses thrombin accumulation in the substantia nigra, decreasing the expression of pro-inflammatory cytokines and reducing oxidative stress [142,143]. Lipopolysaccharidebinding protein has also been shown to reverse the amyloid accumulation of fibrin induced by bacterial lipopolysaccharide and occurring in the blood of patients with PD [144,145]. Additional studies are necessary to characterize the utility of these treatments. ...
Article
Full-text available
Thrombin is a Na+-activated allosteric serine protease of the chymotrypsin family involved in coagulation, inflammation, cell protection, and apoptosis. Increasingly, the role of thrombin in the brain has been explored. Low concentrations of thrombin are neuroprotective, while high concentrations exert pathological effects. However, greater attention regarding the involvement of thrombin in normal and pathological processes in the central nervous system is warranted. In this review, we explore the mechanisms of thrombin action, localization, and functions in the central nervous system and describe the involvement of thrombin in stroke and intracerebral hemorrhage, neurodegenerative diseases, epilepsy, traumatic brain injury, and primary central nervous system tumors. We aim to comprehensively characterize the role of thrombin in neurological disease and injury.
Article
Fibrinolysis is the enzymatic digestion of fibrin, the primary structural component in blood clots. Mechanisms of fibrin fiber digestion during lysis have long been debated and obtaining detailed structural knowledge of these processes is important for developing effective clinical approaches to treat ischemic stroke and pulmonary embolism. Using dynamic fluorescence microscopy, we studied the time-resolved digestion of individual fibrin fibers by the fibrinolytic enzyme plasmin. We found that plasmin molecules digest fibers along their entire lengths, but that the rates of digestion are non-uniform, resulting in cleavage at a single location along the fiber. Using mathematical modeling we estimated the rate of plasmin arrival at the fiber surface and the number of digestion sites on a fiber. We also investigated correlations between local fiber digestion rates, cleavage sites, and fiber properties such as initial thickness. Finally, we uncovered a previously unknown tension-dependent mechanism that pulls fibers apart during digestion. Taken together these results promote a paradigm shift in understanding mechanisms of fibrinolysis and underscore the need to consider fibrin tension when assessing fibrinolytic approaches. Statement of Significance : We developed a method for interrogating lysis of individual fibrin fibers, enabling the time-resolved observation of individual fiber digestion for the first time. Our results resolve longstanding disagreements about fibrinolytic processes and reveal previously unknown mechanisms that also play a role. Also, we developed the first microscale mathematical model of plasmin-fibrin interaction, which predicts the number of plasmin molecules on each fiber and can serve as a framework for investigating novel therapeutics.
Article
Full-text available
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.
Preprint
Full-text available
The coronavirus disease 2019 (COVID-19) (SARS-Cov-2) has caused a worldwide, sudden and substantial increase in hospitalizations for pneumonia with multiorgan problems. An important issue is also that there is still no unified standard for the diagnosis and treatment of COVID-19. Substantial vascular events are significant accompaniments to lung complications in COVID-19 patients. Various papers have now also shown the significance of thromboelastrography (TEG) as point-of-care technology to determine the levels of coagulopathy (both clotting and bleeding) in COVID-19, in managing COVID-19 patients. Here we present two treatment protocols that may used to treat thrombotic and bleeding or thrombocytopenia pathologies. We also present a case study, where the thrombotic pathology was successfully treated with the thrombotic protocol. Both the protocols use clinical parameters like D-dimer and CRP, as well as the TEG, to closely follow the daily clotting propensity of COVID-19 patients. We conclude by suggesting that the treatment of COVID-19 patients, should be based on a combination of blood biomarkers, and results from point-of-care analyses like the TEG. Such a combination approach closely follow the physiological responses of the immune system, the haematological, as well as the coagulation system, in real-time.
Chapter
Textbooks of biochemistry will explain that the otherwise endergonic reactions of ATP synthesis can be driven by the exergonic reactions of respiratory electron transport, and that these two half-reactions are catalyzed by protein complexes embedded in the same, closed membrane. These views are correct. The textbooks also state that, according to the chemiosmotic coupling hypothesis, a (or the) kinetically and thermodynamically competent intermediate linking the two half-reactions is the electrochemical difference of protons that is in equilibrium with that between the two bulk phases that the coupling membrane serves to separate. This gradient consists of a membrane potential term Δψ and a pH gradient term ΔpH, and is known colloquially as the protonmotive force or pmf. Artificial imposition of a pmf can drive phosphorylation, but only if the pmf exceeds some 150–170 mV; to achieve in vivo rates the imposed pmf must reach 200 mV. The key question then is ‘does the pmf generated by electron transport exceed 200 mV, or even 170 mV?’ The possibly surprising answer, from a great many kinds of experiment and sources of evidence, including direct measurements with microelectrodes, indicates it that it does not. Observable pH changes driven by electron transport are real, and they control various processes; however, compensating ion movements restrict the Δψ component to low values. A protet-based model, that I outline here, can account for all the necessary observations, including all of those inconsistent with chemiosmotic coupling, and provides for a variety of testable hypotheses by which it might be refined.
Preprint
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The chief and largely terminal element of normal blood clotting is considered to involve the polymerisation of the mainly α-helical fibrinogen to fibrin, with a binding mechanism involving ‘knobs and holes’ but with otherwise littl change in protein secondary structure. We recognise, however, that extremely unusual mutations, or mechanical stressing, can cause fibrinogen to adopt a conformation containing extensive β-sheets. Similarly, prions can change morphology from a largely alpha-helical to a largely β-sheet conformation, and the latter catalyses both the transition and the self-organising polymerisation of the β-sheet structures. Many other proteins can do this, where it is known as amyloidogenesis. When fibrin is formed in samples from patients harbouring different diseases it can have widely varying diameters and morphologies. We here develop the idea, and summarise the evidence, that in many cases the anomalous fibrin fibre formation seen in such diseases actually amounts to amyloidogenesis. In particular, fibrin can interact withthe amyloid-β (Aβ) protein that is misfolded in Alzheimer's disease. Seeing these unusual fibrin morphologies as true amyloids explains a great deal about fibrin(ogen) biology that was previously opaque, and provides novel strategies for treating such coagulopathies. The literature on blood clotting can usefully both inform and be informed by that on prions and on the many other widely recognised (β)-amyloid proteins. “Novel but physiologically important factors that affect fibrinolysis have seldom been discovered and characterized in recent years” [1]
Preprint
Full-text available
It is well known that a variety of inflammatory diseases are accompanied by hypercoagulability, and a number of more-or-less longer-term signalling pathways have been shown to be involved. In recent work, we have suggested a direct and primary role for bacterial lipopolysaccharide in this hypercoagulability, but it seems never to have been tested directly. Here we show that the addition of tiny concentrations (0.2 ng.L ⁻¹ ) of bacterial lipopolysaccharide (LPS) to both whole blood and platelet-poor plasma of normal, healthy donors leads to marked changes in the nature of the fibrin fibres so formed, as observed by ultrastructural and fluorescence microscopy (the latter implying that the fibrin is actually in an amyloid β-sheet-rich form. They resemble those seen in a number of inflammatory (and also amyloid) diseases, consistent with an involvement of LPS in their aetiology. These changes are mirrored by changes in their viscoelastic properties as measured by thromboelastography. Since the terminal stages of coagulation involve the polymerisation of fibrinogen into fibrin fibres, we tested whether LPS would bind to fibrinogen directly. We demonstrated this using isothermal calorimetry. Finally, we show that these changes in fibre structure are mirrored when the experiment is done simply with purified fibrinogen and thrombin (± 0.2 ng.L ⁻¹ LPS). This ratio of concentrations of LPS:fibrinogen in vivo represents a molecular amplification by the LPS of more than 10 ⁸ -fold, a number that is probably unparalleled in biology. The observation of a direct effect of such highly substoichiometric amounts of LPS on both fibrinogen and coagulation can account for the role of very small numbers of dormant bacteria in disease progression, and opens up this process to further mechanistic analysis and possible treatment. Significance statement Most chronic diseases (including those classified as cardiovascular, neurodegenerative, or autoimmune) are accompanied by long-term inflammation. Although typically mediated by ‘inflammatory’ cytokines, the origin of this inflammation is unclear. We have suggested that one explanation is a dormant microbiome that can shed the highly inflammatory lipopolysaccharide LPS. Such inflammatory diseases are also accompanied by a hypercoagulable phenotype. We here show directly (using 6 different methods) that very low concentrations of LPS can affect the terminal stages of the coagulation properties of blood and plasma significantly, and that this may be mediated via a direct binding of LPS to a small fraction of fibrinogen monomers as assessed biophysically. Such amplification methods may be of more general significance.
Article
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Background Gestational diabetes mellitus (GDM) is increasing partly due to the obesity epidemic. Adipocytokines have thus been suggested as first trimester screening markers for GDM. In this study we explore the associations between body mass index (BMI) and serum concentrations of adiponectin, leptin, and the adiponectin/leptin ratio. Furthermore, we investigate whether these markers can improve the ability to screen for GDM in the first trimester. Methods A cohort study in which serum adiponectin and leptin were measured between gestational weeks 6+0 and 14+0 in 2590 pregnant women, categorized into normal weight, moderately obese, or severely obese. Results Lower concentrations of adiponectin were associated with GDM in all BMI groups; the association was more pronounced in BMI<35 kg/m Conclusions Low adiponectin measured in the first trimester is associated with the development of GDM; higher BMI was associated with lower performance of adiponectin, though this was insignificant. Leptin had an inverse relationship with GDM in severely obese women and did not improve the ability to predict GDM.
Article
Full-text available
The chief and largely terminal element of normal blood clotting is considered to involve the polymerisation of the mainly α-helical fibrinogen to fibrin, with a binding mechanism involving ‘knobs and holes’ but with otherwise little change in protein secondary structure. We recognise, however, that extremely unusual mutations or mechanical stressing can cause fibrinogen to adopt a conformation containing extensive β-sheets. Similarly, prions can change morphology from a largely α-helical to largely β-sheet conformation, and the latter catalyses both the transition and the self-organising polymerisation of the β-sheet structures. Many other proteins can also do this, where it is known as amyloidogenesis. When fibrin is formed in samples from patients harbouring different diseases it can have widely varying diameters and morphologies. We here develop the idea, and summarise the evidence, that in many cases the anomalous fibrin fibre formation seen in such diseases actually amounts to amyloidogenesis. In particular, fibrin can interact with the amyloid-β (Aβ) protein that is misfolded in Alzheimer's disease. Seeing these unusual fibrin morphologies as true amyloids explains a great deal about fibrin(ogen) biology that was previously opaque, and provides novel strategies for treating such coagulopathies. The literature on blood clotting can usefully both inform and be informed by that on prions and on the many other widely recognised (β-)amyloid proteins. A preprint has been lodged in bioRXiv (Kell and Pretorius, 2016).
Article
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For bacteria, replication mainly involves growth by binary fission. However, in a very great many natural environments there are examples of phenotypically dormant, non-growing cells that do not replicate immediately and that are phenotypically 'nonculturable' on media that normally admit their growth. They thereby evade detection by conventional culture-based methods. Such dormant cells may also be observed in laboratory cultures and in clinical microbiology. They are usually more tolerant to stresses such as antibiotics, and in clinical microbiology they are typically referred to as 'persisters'. Bacterial cultures necessarily share a great deal of relatedness, and inclusive fitness theory implies that there are conceptual evolutionary advantages in trading a variation in growth rate against its mean, equivalent to hedging one's bets. There is much evidence that bacteria exploit this strategy widely. We here bring together data that show the commonality of these phenomena across environmental, laboratory and clinical microbiology. Considerable evidence, using methods similar to those common in environmental microbiology, now suggests that many supposedly non-communicable, chronic and inflammatory diseases are exacerbated (if not indeed largely caused) by the presence of dormant or persistent bacteria (the ability of whose components to cause inflammation is well known). This dormancy (and resuscitation therefrom) often reflects the extent of the availability of free iron. Together, these phenomena can provide a ready explanation for the continuing inflammation common to such chronic diseases and its correlation with iron dysregulation. This implies that measures designed to assess and to inhibit or remove such organisms (or their access to iron) might be of much therapeutic benefit.
Article
Full-text available
Alzheimer-type dementia (AD) is a neurodegenerative disorder and the most common form of dementia. Patients typically present with neuro- and systemic inflammation and iron dysregulation, associated with oxidative damage that reflects in hypercoagulability. Hypercoagulability is closely associated with increased fibrin(ogen) and in AD patients fibrin(ogen) has been implicated in the development of neuroinflammation and memory deficits. There is still no clear reason precisely why (a) this hypercoagulable state, (b) iron dysregulation and (c) increased fibrin(ogen) could together lead to the loss of neuronal structure and cognitive function. Here we suggest an alternative hypothesis based on previous ultrastructural evidence of the presence of a (dormant) blood microbiome in AD. Furthermore, we argue that bacterial cell wall components, such as the endotoxin lipopolysaccharide (LPS) of Gram-negative strains, might be the cause of the continuing and low-grade inflammation, characteristic of AD. Here, we follow an integrated approach, by studying the viscoelastic and ultrastructural properties of AD plasma and whole blood by using scanning electron microscopy, Thromboelastography (TEG®) and the Global Thrombosis Test (GTT®). Ultrastructural analysis confirmed the presence and close proximity of microbes to erythrocytes. TEG® analysis showed a hypercoagulable state in AD. TEG® results where LPS was added to naive blood showed the same trends as were found with the AD patients, while the GTT® results (where only platelet activity is measured), were not affected by the added LPS, suggesting that LPS does not directly impact platelet function. Our findings reinforce the importance of further investigating the role of LPS in AD.
Article
Background Prostate-specific antigen (PSA) test is of paramount importance as a diagnostic tool for the detection and monitoring of patients with prostate cancer. In the presence of interfering factors such as heterophilic antibodies or anti-PSA antibodies the PSA test can yield significantly falsified results. The prevalence of these factors is unknown. Methods We determined the recovery of PSA concentrations diluting patient samples with a standard serum of known PSA concentration. Based on the frequency distribution of recoveries in a pre-study on 268 samples, samples with recoveries <80% or >120% were defined as suspect, re-tested and further characterized to identify the cause of interference. Results A total of 1158 consecutive serum samples were analyzed. Four samples (0.3%) showed reproducibly disturbed recoveries of 10%, 68%, 166% and 4441%. In three samples heterophilic antibodies were identified as the probable cause, in the fourth anti-PSA-autoantibodies. The very low recovery caused by the latter interference was confirmed in serum, as well as heparin- and EDTA plasma of blood samples obtained 6 months later. Analysis by eight different immunoassays showed recoveries ranging between <10% and 80%. In a follow-up study of 212 random plasma samples we found seven samples with autoantibodies against PSA which however did not show any disturbed PSA recovery. Conclusions About 0.3% of PSA determinations by the electrochemiluminescence assay (ECLIA) of Roche diagnostics are disturbed by heterophilic or anti-PSA autoantibodies. Although they are rare, these interferences can cause relevant misinterpretations of a PSA test result.
Article
Alzheimer's disease (AD) is associated with dementia, brain atrophy and the aggregation and accumulation of a cortical amyloid-β peptide (Aβ). Chronic bacterial infections are frequently associated with amyloid deposition. It had been known from a century that the spirochete Treponema pallidum can cause dementia in the atrophic form of general paresis where. It is noteworthy that the pathological hallmarks of this atrophic form are similar to those of AD. Recent observations showed that bacteria, including spirochetes contain amyloidogenic proteins and also that Aβ deposition and tau phosphorylation can be induced in vitro or in vivo following exposure to bacteria or LPS. Bacteria or their poorly degradable debris are powerful inflammatory cytokine inducers, activate complement, affect vascular permeability, generate nitric oxide and free radicals, induce apoptosis and are amyloidogenic. All these processes are involved in the pathogenesis of AD. Old and new observations, reviewed here, indicate that to consider the possibility that bacteria, including several types of spirochetes highly prevalent in the population at large or their persisting debris may initiate cascade of events leading to chronic inflammation and amyloid deposition in AD is important, as appropriate antibacterial and antiinflammatory therapy would be available to prevent dementia.
Article
Background: Recent studies have revealed that the blood of healthy humans is not as sterile as previously supposed. The objective of this study was to provide a comprehensive description of the microbiome present in different fractions of the blood of healthy individuals. Study design and methods: The study was conducted in 30 healthy blood donors to the French national blood collection center (Établissement Français du Sang). We have set up a 16S rDNA quantitative polymerase chain reaction assay as well as a 16S targeted metagenomics sequencing pipeline specifically designed to analyze the blood microbiome, which we have used on whole blood as well as on different blood fractions (buffy coat [BC], red blood cells [RBCs], and plasma). Results: Most of the blood bacterial DNA is located in the BC (93.74%), and RBCs contain more bacterial DNA (6.23%) than the plasma (0.03%). The distribution of 16S DNA is different for each fraction and spreads over a relatively broad range among donors. At the phylum level, blood fractions contain bacterial DNA mostly from the Proteobacteria phylum (more than 80%) but also from Actinobacteria, Firmicutes, and Bacteroidetes. At deeper taxonomic levels, there are striking differences between the bacterial profiles of the different blood fractions. Conclusion: We demonstrate that a diversified microbiome exists in healthy blood. This microbiome has most likely an important physiologic role and could be implicated in certain transfusion-transmitted bacterial infections. In this regard, the amount of 16S bacterial DNA or the microbiome profile could be monitored to improve the safety of the blood supply.
Article
Background Microglial activation due to a variety of stimuli induces secretion of neurotoxic substances including inflammatory cytokines and nitric oxide (NO). Clinical studies indicate a cross-link between inflammatory and hypoxia-regulated pathways suggesting that bacterial infections markedly sensitize the immature brain to hypoxic injury. Methods The impact of inflammation and hypoxia on interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), and NO secretion and microglia-induced cytotoxicity was investigated exposing BV2 cells to lipopolysaccharides (LPS) and hypoxia (1% O2). Cytotoxicity, NO, and cytokine release was quantified by MTS and Griess assays and by enzyme-linked immunosorbent assays, respectively. Results LPS exposure of BV2 cells induced a significant, persistent production of NO, IL-1β, IL-6, and TNF-α. Even after LPS removal, ongoing NO and cytokine secretion was observed. Hypoxia mediated exclusively a significant, short-term IL-1β increase, but enhanced LPS-induced cytokine and NO secretion significantly. In addition, LPS-induced supernatants exhibited a stronger cytotoxic effect in glial and neuronal cells than LPS exposition (p < 0.001). Hypoxia potentiated LPS-induced cytotoxicity. Conclusion Present data prove that LPS-induced soluble factors rather than LPS exposure mediate microglial toxicity under conditions of hypoxia in vitro. Apart from potential protective effects of the hypoxia-inducible transcription factor (HIF)-1α system, activation of proinflammatory pathways may markedly sensitize microglial cells to promote hypoxia-induced injuries of the developing brain.