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Title: Plasma non-esterified fatty acids contribute to increased coagulability in type-2
diabetes through altered plasma zinc speciation
Authors: Amélie I. S. Sobczak1, Kondwani G. H. Katundu1,2, Fladia A. Phoenix3, Siavash
Khazaipoul1, Ruitao Yu1,4, Fanuel Lampiao2, Fiona Stefanowicz5, Claudia A. Blindauer6,
Samantha J. Pitt1, Terry K. Smith7, Ramzi A. Ajjan3, and Alan J. Stewart1*
Affiliations: 1School of Medicine, University of St Andrews, St Andrews, United Kingdom.
2College of Medicine, University of Malawi, Blantyre, Malawi. 3Leeds Institute of
Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, United Kingdom. 4Key
Laboratory of Tibetan Medicine Research, Northwest Plateau Institute of Biology, Chinese
Academy of Sciences, 23 Xinning Road, Xining, Qinghai 810001, China. 5Scottish Trace
Element and Micronutrient Reference Laboratory, Department of Biochemistry, NHS Greater
Glasgow and Clyde, Glasgow, United Kingdom. 6Department of Chemistry, University of
Warwick, Coventry, United Kingdom. 7School of Biology, Biomedical Sciences Research
Complex, University of St Andrews, St Andrews, United Kingdom.
Running head: NEFAs and coagulability in type-2 diabetes
*Corresponding author: Alan. J. Stewart. School of Medicine, University of St Andrews,
Medical and Biological Sciences Building, St Andrews, Fife, KY16 9TF, United Kingdom.
E-mail: ajs21@st-andrews.ac.uk; Tel: +44 (0) 1334 463546; Fax: +44 (0) 1334 463482.
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Abstract
Zn2+ is an essential regulator of coagulation and its availability in plasma is fine-tuned
through buffering by human serum albumin (HSA). Non-esterified fatty acids (NEFAs)
transported by HSA reduce its ability to bind/buffer Zn2+. This is important as plasma NEFA
levels are elevated in type-2 diabetes mellitus (T2DM) and other diseases with an increased
risk of developing thrombotic complications. The presence of 5 mol. eq. of myristate,
palmitate, stearate, palmitoleate and palmitelaidate reduced Zn2+ binding to HSA. Addition of
myristate and Zn2+ increased thrombin-induced platelet aggregation in platelet-rich plasma
and increased fibrin clot density and clot time in a purified protein system. The
concentrations of key saturated (myristate, palmitate, stearate) and monounsaturated (oleate,
vaccinate) NEFAs positively correlated with clot density in subjects with T2DM (and
controls). Collectively, these data strongly support the concept that elevated NEFA levels
contribute to an increased thrombotic risk in T2DM through dysregulation of plasma zinc
speciation.
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Introduction
Zinc is an essential modulator of coagulation controlling multiple aspects (1). Molecular
regulation of coagulation by zinc is complex, as it binds numerous plasma proteins to
influence their activities (1). More specifically, zinc has been shown to enhance platelet
aggregation (2), accelerate clotting (3), and delay clot lysis (4). The fraction of zinc
responsible for these effects is the “free” aquo ion of Zn2+. Low nanomolar free Zn2+ is
cytotoxic (5, 6); therefore, extracellular zinc is well-buffered. In blood, this buffering role is
overwhelmingly performed by human serum albumin (HSA). Approximately 75% of the total
15-20 µM Zn2+ is bound to HSA (7), constituting >99% of the labile Zn2+ pool (8). Most of
the remaining labile Zn2+ is bound to small molecules, leaving ~1-3 nM free Zn2+ under
resting conditions (9). However, labile Zn2+ concentrations are subject to highly dynamic
spatial and temporal variations, increasing sharply during coagulation around activated
platelets that release Zn2+ from α-granule stores (4, 10). Zn2+ is also released by damaged
epithelial cells, neutrophils, lymphocytes and erythrocytes and from ruptured atherosclerosis
plaques (11).
In addition to binding/buffering Zn2+, HSA also transports non-esterified fatty acids (NEFAs)
at 5 medium- to high-affinity binding sites (FA1-5), one of which (FA2) is located close to
the main Zn2+ binding site (site A) (12-14). A secondary Zn2+ site with weaker affinity is also
present but is unlikely to contribute greatly to Zn2+ binding under normal conditions (15).
Figure 1A shows the structure of HSA with myristate bound. When a NEFA molecule binds
to FA2, the conformation changes, causing site A to be disrupted (the Zn2+-coordinating
nitrogen of His67 moves ~8 Å relative to His247 and Asp249; Figure 1B), dramatically
reducing the Zn2+ affinity of HSA (12-14). Thus, when plasma NEFA levels are elevated, the
Zn2+ buffering ability of HSA is compromised.
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Several disease states are associated with elevated plasma NEFA levels including cancer
(16), obesity (17), non-alcoholic fatty liver disease (18) and type 2 diabetes mellitus (T2DM)
(19). Interestingly, these disorders all associate with an increased risk of thrombotic
complications. We hypothesise that high plasma NEFA concentrations disrupt Zn2+ binding
by HSA, causing more Zn2+ to interact with coagulation proteins and consequently enhancing
thrombotic risk (20, 21). Here, the ability of plasma NEFA levels to impact on Zn2+ handling
and blood coagulability is examined. To assess this, Zn2+ binding to HSA in the presence of
various NEFAs was measured by isothermal titration calorimetry (ITC). The impact of these
interactions on platelet aggregation, fibrin clot formation and clot lysis was explored.
Moreover, the relationship between plasma NEFA concentration and coagulability in plasma
samples taken from individuals with T2DM and controls (without diabetes) was determined.
The results support the concept that elevated NEFA levels contribute to an increased
thrombotic risk in T2DM through mishandling of plasma Zn2+.
Results
Influence of various NEFAs on Zn2+ binding to HSA
The ability of different NEFAs (octaonate (C8:0), laurate (C12:0), myristate (C14:0),
palmitate (C16:0), palmitoleate (C16:1-cis), palmitelaidate (C16:1-trans) and stearate
(C18:0)) to influence Zn2+ binding to HSA was assessed by ITC. Building on previous work
(13, 15), a “two sets-of-sites” model was chosen to monitor changes to site A stoichiometry
in the presence of NEFAs. Fitted isotherms are shown in Figure 2, raw data in Figures S1-25,
fitting parameters in Table S1 and fitting results in Table S2. Addition of up to 5 mol. eq. of
octanoate had little effect on Zn2+ binding to HSA, but a change was seen with laurate and
longer chain saturated NEFAs, where the fitting results suggested a reduction in the
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stoichiometry of site A with increasing NEFA concentration. Indeed, 4 and 5 mol. eq. of
laurate reduced site A availability to 0.24 and 0.00, respectively whereas 3 mol. eq. had little
effect. In the presence of myristate, palmitate and stearate, 3-5 mol. eq. of these NEFAs
reduced binding of Zn2+ to HSA in a concentration-dependent manner; 3 mol. eq. myristate or
palmitate reduced site A availability to about 50%, and 4 mol. eq. palmitate or stearate
sufficient to abolish binding at site A. Almost no Zn2+ binding was observed at site A in the
presence of 5 mol. eq. of myristate, palmitate or stearate. The unsaturated NEFA,
palmitelaidate led to reduced Zn2+ binding in a similar manner to palmitate while
palmitoleate had less effect than palmitate at the concentrations examined, with the exception
of 5 mol. eq. of NEFA where almost no binding was detected.
Effect of Zn2+ and NEFAs on platelet aggregation
The effect of Zn2+ and NEFAs on platelet aggregation in washed-platelets and platelets-in-
plasma was assessed (Figure 3 and S26). In washed-platelets, addition Zn2+ increased
maximum aggregation (p=0.0080, as measured with one-way ANOVA), with a trend
observed at 50 µM and a significant increase at 100 µM Zn2+ (p=0.0566 and p=0.0047
respectively, measured by Dunnet’s multiple comparison tests). In platelets re-suspended in
plasma, no difference was observed upon addition of Zn2+, presumably due to Zn2+ buffering
by HSA. The effect of both octanoate and myristate on platelet aggregation was examined to
compare effects of NEFAs with differing abilities to perturbs Zn2+ binding to HSA (while
aware that Zn2+ is already present in plasma). Addition of octanoate had no effect on
maximum aggregation. In contrast, addition of 4 mol. eq. of myristate increased maximum
aggregation (p=0.0006). The zinc-selective chelator, TPEN, abolished the effects of
myristate. Finally, we assessed the effect of 100 µM Zn2+ in combination with 4 mol. eq.
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myristate. This further increased maximum aggregation (compared to 4 mol. eq. myristate
alone, p=0.0208).
Effect of Zn2+ and NEFAs on clot formation and lysis
To examine whether NEFAs alter the effect of Zn2+ on fibrin clot formation and lysis, we
utilised a validated turbidimetric assay employing both purified protein (fibrinogen and HSA)
and plasma samples. In the purified system, addition of 20-100 µM Zn2+ significantly
increased maximum absorbance and lysis time concentration-dependently, while 20 and 40
µM Zn2+ increased clot time and 100 µM Zn2+ decreased it (Figure 4 A-C). Zn2+ significantly
affected all three parameters (p<0.0001 for all). Addition of 4 mol. eq. of myristate also
increased maximum absorbance and clot time (p=0.0046 and p=0.0060 respectively;
calculated by Sidak’s multiple comparison tests) suggesting a direct effect on these
parameters but did not affect lysis time. To highlight the effect of added Zn2+, these
parameters values were calculated relative to “no Zn2+ added” for the samples with/without
NEFA (Figure 4 D-F). Addition of 20 µM Zn2+ led to a higher maximum absorbance in the
presence of myristate and the decrease in clot time at higher Zn2+ concentrations (40 and 100
µM) was more pronounced with myristate present. Zn2+ did not affect lysis time with or
without myristate differently. Myristate significantly affected maximum absorbance and clot
time (p=0.0455 and p=0.0194 respectively), but not lysis time. The effect of Zn2+ on clot
ultrastructure was examined by measuring fibrin fibre thickness using SEM; addition of 20
µM Zn2+ significantly increased fibrin fibre diameter (p<0.0001; Figure 4 G, I, S31 and S32).
For the experiments with citrated pooled plasma, 4-(2-pyridylazo)resorcinol was used to
calculate the amount of Zn2+ required to obtain the equivalent available Zn2+ concentration as
in the purified system; this is to account for the Zn2+-buffering capacity of citrate. The
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turbidimetric clot assay was performed on pooled-plasma (Figure S28), where addition of
Zn2+ increased maximum absorbance and clot time, while 20 µM available Zn2+ increased
lysis time and 40 and 100 µM available Zn2+ decreased it. Zn2+ significantly affected all three
parameters (p<0.0001 for all). Addition of myristate decreased clot time, increased lysis time
(p<0.0001 and p=0.0002 respectively; calculated by Sidak’s multiple comparison tests) and
did not affect maximum absorbance. The effects of the addition of Zn2+ on maximum
absorbance, clot time and lysis time were more pronounced in the presence of myristate.
Myristate significantly affected maximum absorbance, clot time and lysis time (p=0.0013,
p<0.0001 and p<0.0001 respectively).
Differences in clot formation in plasma from subjects with T2DM and controls
Elevated plasma NEFA levels are associated with T2DM (19, 22), To explore whether NEFA
levels in individuals with T2DM may impact on Zn2+ handling and coagulability, we
analysed plasma from individuals with T2DM and controls. In each sample, NEFA, total zinc
and HSA concentrations were measured (Figure 5 A-C). Demographic information and
plasma concentrations of lipids and glucose for the two groups are presented (Table S3). The
groups were matched for age but not sex (although no sex difference in NEFA concentration
was found, Figure S29); the T2DM group had significantly higher BMI, total plasma NEFA
and HSA concentrations than controls (p<0.0001, p=0.0011 and p<0.0001 respectively). The
T2DM group had higher concentrations of HbA1c, plasma glucose and triglycerides and a
higher cholesterol/LDL ratio (p<0.0001, p<0.0001, p=0.0313 and p=0.0198 respectively), but
had lower concentrations of cholesterol, HDL and LDL (p<0.0001 for all). Total zinc and
fibrinogen concentrations were comparable between groups.
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Turbidimetric assays were performed on all samples without added Zn2+ and with 20 µM
available Zn2+, as shown in Figure 5 D-F. Maximum absorbance was higher in the T2DM
group compared to controls regardless of the presence or absence of additional Zn2+
(p<0.0001; two-way ANOVA). Lysis time was prolonged in T2DM subjects (p=0.0448; two-
way ANOVA), but the differences between control samples and those with T2DM at 0 and at
20 µM available Zn2+ were not significant (Sidak’s multiple comparison tests). No
differences in clot time were observed. Positive correlations between NEFA concentration
and both BMI and maximum absorbance were observed (Figure 5 G, H; p=0.0420 and
p=0.03010 respectively). There was no correlation between NEFA concentration and either
clot time or lysis time. Comparisons of maximum absorbance, clot time and lysis time
between sexes revealed no significant differences (Figure S30). SEM studies were performed
to examine the fibrin fibre thickness in each group (Figure 6 A-C and S33-36), six plasma
samples from each group were randomly selected and pooled. Fibres were found to be
significantly thicker in the presence of 20 µM available Zn2+ in both groups and were thicker
in the T2DM group when compared to controls (p<0.0001 for both).
Differences in plasma concentrations of specific NEFA species and associations with fibrin
clot parameters
The plasma concentration of major NEFA species in the clinical samples was measured using
GC-MS (Figure 7). The majority of NEFAs measured were elevated in subjects with T2DM
compared to controls (p=0.0010 for myristate, p=0.0023 for palmitate, p=0.0003 for
linolenate (18:3), p=0.0054 for oleate (18:1c9), p=0.0029 for vaccinate (18:1c11), p=0.0002
for stearate, p=0.0092 for eicosapentaenoate (20:5) and p=0.0099 for arachidonate (20:4)),
with the exception of palmitoleate (16:1), linoleate (18:2), dihomo-γ-linoleate (20:3) and
docosahexanoate (22:6) species. The association between plasma concentrations of those
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NEFAs and fibrin clot maximum absorbance was then assessed. The concentrations of
myristate, palmitate, oleate, vaccinate and stearate positively correlated with maximum
absorbance (p=0.0313, p=0.0202, p=0.0307, p= 0.0067, p=0.0184 respectively).
Discussion
We previously demonstrated that myristate (14:0) impacts upon Zn2+ binding to HSA at the
highest affinity Zn2+ site using ITC (15), whilst mutagenesis and X-ray crystallography
studies confirm this to be site A (23). Longer-chain saturated NEFAs, which are more
abundant in plasma than myristate (e.g. palmitate and stearate) (24) bind to HSA with higher
affinity (25), while unsaturated NEFAs bind HSA at the FA2 site with high affinity as long as
the degree of unsaturation is low (26). Here, we assessed the ability of various NEFAs to
influence binding of Zn2+ to HSA. A concentration of 4 mol. eq. of palmitate or stearate
almost completely abrogated Zn2+ binding to site A, with 3 mol. eq. also reducing Zn2+
binding capacity. Palmitoleate had less of an effect on Zn2+ binding compared to palmitate
and palmitelaidate (no X-ray crystallographic structure of palmitoleate or palmitelaidate
binding to HSA is currently available to explain this phenomenon). The total NEFA
concentrations in plasma from subjects with T2DM measured in this work were as high as 2.7
mM, which is equivalent to >4 mol. eq. of total NEFA relative to HSA concentration.
Although our binding experiments examined the effects of NEFAs on Zn2+-HSA interactions
in isolation (whilst in plasma there is a mixture of different NEFAs), it would appear that this
allosteric interaction nevertheless has the potential to strongly impact on plasma Zn2+
handling in vivo: when platelets release Zn2+ during coagulation, HSA is likely to
buffer/control its action in the vicinity of injury sites.
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The importance of Zn2+ for platelet aggregation is well established (2), with its effects on
platelet behaviour exercised through both extracellular and intracellular interactions (27).
Here we found that Zn2+ concentration-dependently enhanced maximum aggregation in
washed platelets. However, in platelets-in-plasma, where HSA and other zinc-binding
molecules can buffer Zn2+, addition of up to 100 µM Zn2+ (0.17 mol. eq. compared to HSA)
had no effect on these parameters. Addition of 4 mol. eq. myristate to platelets-in-plasma
resulted in an increase in maximum aggregation, which further increased with added Zn2+.
The observation that the myristate-mediated effects were abolished by zinc-chelating TPEN
(Figure 3B), gives strong support to the conclusion that the myristate-mediated effects are
due to loss of Zn2+-buffering capacity by HSA. The observation that 4 mol. eq. of the
octanoate exerted no observable effect on aggregation of platelets-in-plasma is also consistent
with the Zn2+-NEFA crosstalk hypothesis, as octanoate is too short to elicit the allosteric
switch (28), and does not affect Zn2+ binding to HSA (Figure 2A).
The effect of Zn2+ on fibrin clot characteristics was previously examined by Henderson et al
(3, 4), who found that Zn2+ accelerated clot formation and increased fibrin fibre thickness and
clot porosity. This was suggested to promote clot lysis by allowing increased flow of plasma
components inside the clot. However, the same studies showed that Zn2+ reduced
plasminogen activation, resulting in delayed clot lysis. These studies were performed using a
purified system similar to ours or in dialysed plasma. The range of Zn2+ concentrations was
small, up to 6 or 15 µM. Here, we expand on those results by investigating a wider range of
Zn2+ concentrations (up to 100 µM), as the maximum total Zn2+ concentration that can be
reached in proximity of activated platelets, although still unknown, will be much higher than
basal physiological Zn2+ concentrations. In our pooled-plasma experiments, to avoid losing
any smaller molecules, we chose not to dialyse the plasma, but instead to estimate the amount
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of Zn2+ to be added in order to obtain the available Zn2+ concentrations desired. Our results
indicate that in plasma, maximum absorbance and clot time increase at physiological and
higher Zn2+ concentrations. The lysis time increased in the presence of 20 µM Zn2+ as shown
previously (4), however it decreased at higher concentrations (40-100 µM Zn2+). Also, we
confirmed that fibrin fibre thickness increases in the presence of Zn2+ (3). The potential
effects of NEFA-binding to HSA on coagulation had not been investigated before, but
addition of 4 mol. eq. myristate in a purified system that did not include HSA, increased
maximum absorbance and clot time (29). Addition of 4 mol. eq. myristate to plasma (which
includes physiological zinc) increased lysis time, decreased clot time and did not affect
maximum absorbance. To differentiate the standalone-effect of NEFAs from their effect on
Zn2+-binding by HSA, we calculated parameter values relative to the values in the absence of
added Zn2+. In plasma, the addition of 4 mol. eq. myristate results in more pronounced
changes in maximum absorbance and clot time when Zn2+ is added. This shows that,
independently to their own effect, NEFAs influence buffering of Zn2+ by HSA to
consequently alter Zn2+ speciation in plasma.
To confirm that pathophysiological concentrations of NEFAs are present in individuals with
T2DM and determine the potential impact of elevated NEFA levels on clot formation, we
examined plasma samples taken from individuals with T2DM and controls. In accordance
with other studies (19, 22), we found that those with T2DM had significantly elevated plasma
NEFAs. The T2DM group also had a higher concentration of plasma HSA. No difference in
total zinc concentration was observed between the groups, contrary to some previous reports
documenting a small decrease in total plasma zinc in individuals with T2DM (30). In our
cohort we observed an increase in fibrin fibre diameter in clots formed from pooled-plasma
from subjects with T2DM compared to controls. However, the higher fibrinogen
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concentrations in plasma from T2DM subjects likely contributed to this. A previous
examination of clots formed from fibrinogen purified from individuals with T2DM and
controls found the T2DM-derived samples exhibited denser and less porous clots (31). This
would likely contribute to the higher maximum absorbance (higher density) and longer lysis
time (lower porosity) that we observed. We also demonstrate that NEFA concentration
associates with clot maximum absorbance, similar to our observation in the purified and the
pooled-plasma systems upon addition of myristate. In addition, an examination of the plasma
concentrations of major NEFA species confirmed that most were elevated in individuals with
T2DM and showed a positive correlation between maximum absorbance and some NEFA
species, in particular saturated NEFAs (myristate, palmitate and stearate) and mono-
unsaturated NEFAs (oleate and vaccinate), which although we did not look at those cis-
unsaturated NEFAs specifically, is generally in accord with our ITC results. We did not find
any significant difference in clot parameters between males and females with T2DM.
However, other studies found maximum absorbance to be higher in females with T2DM (32).
It thus appears from the correlations between (particularly saturated) NEFA levels and clot
maximum absorbance that plasma NEFAs induce changes in the speciation of plasma Zn2+ to
influence Zn2+-dependent clotting. The effects of NEFAs and Zn2+ on coagulation
demonstrated here are summarised in Figure 8. In other studies individuals with T2DM have
been reported to be mildly deficient in zinc (30). This could be due to impaired transport of
zinc by HSA, resulting in altered zinc distribution and/or partial clearance of the “excess”
zinc. Plasma zinc levels have been negatively correlated with the risk of developing
cardiovascular diseases in individuals with T2DM (33). Zinc is important for insulin storage
and zinc supplementation in T2DM has beneficial effects (improved insulin and glucose
levels and decreased risk of developing T2DM) (34-36). However, while supplementation
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may increase zinc availability, it may also increase zinc binding to other plasma proteins,
including coagulation proteins, with further consequences for thrombotic risk in individuals
with T2DM. Heparin, an important anticoagulant, has been shown to be increasingly
neutralised in the presence of elevated Zn2+ levels (11, 37). Thus, the effect of high plasma
levels of NEFA and altered Zn2+ speciation may need to be carefully considered when
choosing an antithrombotic treatment for individuals with T2DM.
T2DM is not the only condition associated with high plasma NEFAs (22), and although
correlations await to be established, it seems prudent to suggest that plasma NEFA levels
need to be carefully controlled in all cardiometabolic disorders. This could be achieved
through several means, including: 1. A diet low in saturated fat - the heterogeneity in plasma
NEFA levels in our T2DM subjects is likely in part due to diet. In addition, dietary intake of
saturated fatty acids and polyunsaturated omega-3 fatty acids have been associated with
respectively an increase and a decrease in T2DM occurrence (38-40), however this is now
being increasingly challenged (40-45). 2. The use of statins or fenofibrate as, in addition to
affecting cholesterol or triglyceride levels, such drugs have been shown to reduce plasma
NEFA levels (46, 47). 3. Targeting fatty acid synthase in order to reduce de-novo synthesis of
NEFAs - this could be particularly important in cancer-associated thrombosis, as expression
of this enzyme is frequently increased in tumour cells (48). 4. Design and administration of a
small molecule able to inhibit binding of NEFAs to the FA2 site on HSA.
In conclusion, this study shows that plasma NEFA levels correlate with increased
coagulability in T2DM. It is also revealed that the NEFA species which exhibit the largest
plasma concentration increase in T2DM can perturb zinc binding to HSA and that addition of
zinc to plasma induces similar changes in coagulatory functioning as is observed in T2DM.
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Thus it appears that NEFAs are likely to influence available plasma Zn2+ concentrations by a
mechanism that involves the FA2 site on HSA, such as to exert a pro-coagulatory effect.
Given that plasma NEFA levels can be controlled pharmacologically or via dietary
intervention, we believe these findings not only increase our understanding of T2DM but will
be useful for the treatment and management of thrombotic complications associated with the
disease.
Materials and Methods
Full details of the methods can be found in the Online Supplementary Appendix.
Ethical statement
Recruitment of healthy volunteers and blood sample collection for the platelet aggregation
study was approved by the School of Medicine Ethics Committee, University of St Andrews.
Plasma samples from subjects with T2DM and controls were collected following approval by
the National Research Ethics Service Committee Yorkshire & The Humber – Leeds East. All
blood samples were taken after obtaining written informed consent.
Isothermal titration calorimetry
Experiments were performed using a MicroCal iTC200 (Malvern Pananalytical, Malvern,
UK) and 50 mM Tris (tris(hydroxymethyl)aminomethane), 140 mM NaCl, pH 7.4 buffer.
Two sets of experiments were carried out: (1) titrating 1.5 mM ZnCl2 into 60 µM HSA in
presence of 0-5 molar equivalents (mol. eq.) of octanoate, laurate, myristate or palmitate; (2)
for less soluble FFAs, titrating 0.75 mM ZnCl2 into 25 µM HSA in presence of 0-5 mol. eq.
of palmitate, palmitoleate, palmitelaidate or stearate. The FFAs were diluted in either
methanol or ethanol before being incubated with HSA in the reaction buffer for 2 h at 37°C
(1% final alcohol concentration). Heats of dilution were accounted for with blank titrations
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15
performed by injecting ligand solution into reaction buffer and subtracting the averaged heat
of dilution from the main experiments. Data fitting was performed using AFFINImeter
(Santiago de Compostela, Spain). Initial fitting was performed using the Zn2+/FFA-free HSA
titration and the values obtained were used to fix K1, ΔH1 and N2 for the other titrations.
Platelet aggregation assays
Platelets were isolated from whole blood collected in acid citrate dextrose collecting tubes
from healthy donors recruited from the student body. The blood was spun twice at 23°C, once
at 700 × g for 8 min to isolate platelet-rich-plasma and once at 400 × g for 20 min to pellet
the platelets. The platelets were washed and re-suspended in buffer solution (145 mM NaCl,
5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 1 mM CaCl2, 10 mM D-Glucose, pH 7.4) for
washed-platelet experiments, or re-suspended in platelet-poor-plasma prepared from hirudin-
coated tubes for platelet experiments requiring whole plasma. Hirudin-coated collection tubes
were used to avoid chelation of Zn2+ by other agents (e.g. citrate or EDTA).
Platelet aggregation experiments were performed to assess the effect of Zn2+ and FFAs
(octanoate or myristate) on platelet aggregation. Solutions of ZnCl2 (10, 50 or 100 µM),
sodium octanoate or sodium myristate (2 or 4 mol. eqs.) and N,N,N′,N′-tetrakis(2-
pyridinylmethyl)-1,2-ethanediamine (TPEN, 50 µM diluted in ethanol) were added to
washed-platelets or platelets-in-plasma. Vehicle control experiments were also performed.
Platelet aggregation was elicited with 2 µM of γ-thrombin (Merck, Watford, UK, final
volume 200 µL). Absorbance was monitored at 430 nm every 55 s for 35 min using an
Optima plate-reader (BMG Labtech, Ortenberg, Germany) while incubating the plate at 37oC
and shaking it in orbital mode. Data were recorded as a negative change in absorbance from
baseline (0%) and expressed as a percentage of the maximum response (100%). Calibration
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of 100 % aggregation was achieved using platelet-poor-plasma. From the recorded responses,
a maximum aggregation response was obtained.
Clinical sample collection
A total of 54 patients with T2DM and 18 age-matched controls were recruited from Leeds
Teaching Hospital Trust. Individuals with known diagnosis of T2DM and aged between 18-
75 years were recruited into the study. Given that aspirin may affect clot structure
characteristics (49), all individuals recruited into the study were on aspirin treatment for
primary or secondary cardiovascular protection. Exclusion criteria included: any type of
diabetes other than T2DM, any coagulation disorder, current or previous history of neoplastic
disease, history of acute coronary syndrome or stroke within 3 months of enrolment, active
history of transient ischaemic attacks, history of deep venous thrombosis or pulmonary
embolism, treatment with oral anticoagulant or non-steroidal anti-inflammatory drugs,
abnormal liver function tests defined as alanine transferase >3 fold upper limit of normal, or
previous or current history of gastrointestinal pathology. Baseline fasting blood samples were
collected in trisodium citrate- or in lithium heparin-coated tubes. Plasma was separated
within 2 h of collection by centrifugation at 2,400 × g for 20 min at 4°C, snap frozen in liquid
nitrogen and stored at -40°C until analysis.
Turbidimetric fibrin clotting and lysis assays
Clot assays were performed as previously described (50, 51), using purified proteins, pooled-
plasma from controls, as well as plasma samples from subjects with T2DM and age-matched
controls. To compensate for the complexation of Zn2+ by the citrate present in plasma, 4-(2-
pyridylazo)resorcinol was used to generate a calibration curve at 490 nm for different Zn2+
concentrations in buffer (50 mM Tris, 100 mM NaCl, pH 7.4) and citrated plasma. The
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17
amount of Zn2+ that should be added in plasma to yield particular concentrations of available
Zn2+ was calculated (116 µM in citrated plasma was equivalent to 20 µM in buffer, 232 µM
to 40 µM and 580 µM to 100 µM).
Sodium myristate (4 mol. eq.) was incubated for 15 min at 37 °C with HSA (in buffer) or
plasma. ZnCl2 was added to a final concentration of up to 100 µM Zn2+ in the purified system
or to available Zn2+ concentrations in plasma up to 100 µM. Final concentrations were: (1) In
the purified system, 0.5 mg/mL fibrinogen (plasminogen-depleted, Merck), 100 µM HSA, 2.5
mM CaCl2, 0.05 U/mL thrombin, 39 ng/mL tissue plasminogen activator (tPA, Technoclone,
Vienna, Austria) and 3.12 µg/mL plasminogen (Stratech, Ely, UK). (2) In pooled-plasma
(First Link (UK) Ltd, Wolverhampton, UK), plasma diluted 6-fold in buffer, 7.5 mM CaCl2,
0.03 U/mL thrombin and 20.8 ng/mL tPA. (3) In plasma from subjects with T2DM for the
clot formation assays, plasma diluted 3-fold in buffer, 7.5 mM CaCl2 and 0.03 U/mL
thrombin. For the clot formation and clot lysis assays, plasma diluted 6-fold in buffer, 3.75
mM CaCl2, 0.03 U/mL thrombin and 20.8 ng/mL tPA. The absorbance at 340 nm was read
every 12 s at 37°C using a Multiskan FC plate-reader (Thermo Scientific, Paisley, UK).
Maximum absorbance, clot time and lysis time were calculated from the raw data.
Scanning electron microscopy (SEM)
Clots were formed in duplicate from either: 1) 20 µM purified fibrinogen and 600 µM HSA
in the presence and absence of 40 µM Zn2+. 2) Pooled-plasma from 6 randomly chosen
patients with T2DM and age-matched controls in the presence and absence of 20 µM
available Zn2+. Clots were prepared and processed by stepwise dehydration as previously
described (50). All clots were viewed and photographed at ×10,000 magnification using a
SU8230 scanning electron microscope (Hitachi, Maidenhead, UK) in 5 different areas. The
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18
diameter of 50 fibres per image was measured using Adobe Photoshop (Adobe Systems, San
Jose, CA). The mean diameters of each image were used to compare each sample types.
Measurement of total FFA, HSA and total zinc concentrations
FFAs were extracted from citrated plasma from subjects with T2DM and controls with
Dole’s protocol (52). FFA concentrations were measured using the FFA Assay Kit -
Quantification (Abcam, Cambridge, UK). Zn2+ concentrations were measured in lithium-
heparin plasma from subjects with T2DM and controls by inductively coupled plasma-mass
spectrometry as described previously (53). HSA levels were measured in heparinised plasma
with the bromocresol purple method using an automated analyser (Architect; Abbot
Diagnosis, Maidenhead, UK). Plasma concentrations of fibrinogen, high density lipoprotein
(HDL), low density lipoprotein (LDL), cholesterol, triglyceride, HbA1c, fasting glucose and
platelets were measured with routine methods.
Measurement of the plasma concentration of specific FFA species by GC-MS
The FFA were characterised and quantified by their conversation to fatty acid methyl esters
(FAME) followed by gas chromatography-mass spectrometry analysis. Citrated plasma from
subjects with T2DM and controls was thawed and spiked with an internal standard fatty acid
C17:0 (100 pM) to allow for normalisation. The FFA were extracted with Dole’s protocol
(52). The fatty acids were converted to FAME using 1,500 µl of methanol, 200 µl of toluene
and 300 µl of 8% HCl, followed by incubation for 5 hr at 45°C. After cooling, samples were
evaporated to dryness with nitrogen. The FAME products were extracted by partitioning
between 500 µl of water and 500 µl of hexane and the samples were left to evaporate to
dryness in a fume hood. The FAME products were dissolved in 30 µl dichloromethane and
1–2 µl analysed by GC-MS (gas chromatography-mass spectrometry) on a Agilent
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19
Technologies (GC-6890N, MS detector-5973) with a ZB-5 column (30 M x 25 mm x 25 mm,
Phenomenex), with a temperature program of at 70 oC for 10 min followed by a gradient to
220oC at 5oC /min and held at 220oC for a further 15 min. Mass spectra were acquired from
50-500 amu and the identity of FAMEs was carried out by comparison of the retention time
and fragmentation pattern with a various FAME standard mixtures (Supelco) as previously
described (54).
Data analysis and representation
Data are shown as mean ± standard deviation (SD). Graphs were generated and statistical
analysis was performed using Prism 7.0 (GraphPad Software, La Jolla, CA). Differences
between groups were analysed using multiple Student’s t-tests or analysis of variance
(ANOVA) with Dunnet’s or Sidak’s multiple comparisons test, while correlations between
linear variants were analysed with Pearson’s correlation. Significance threshold: p≤0.05.
Acknowledgements
This work was supported by the British Heart Foundation (grant numbers PG/15/9/31270,
FS/15/42/31556) and travel grants from the Commonwealth Scholarship Commission (grant
number MWCN-2017-294) and the International Co-operation project of Qinghai Province
(grant number 2017-HZ-811).
Conflict-of-interest: The authors have no conflicts of interest to declare
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Figures
Figure 1.
Figure 1. Effects of NEFA binding at the FA2 site on the primary Zn2+-binding site of
HSA. (A) Structure of HSA with 5 molecules of myristate bound at its high affinity fatty acid
binding sites (FA1-5; PDB 1BJ5).(28) The surface of the protein is shown in grey. (B)
Overlay of Zn2+-binding site in myristate-bound HSA structure (in green, PDB 1BJ5) with
the Zn2+-bound HSA structure (in blue, PDB 5IJF).(23) The zinc atom is shown in purple and
the oxygen from the water participating in Zn2+-coordination is in red. A structural change in
domain II is induced by the binding of myristate to the FA2 binding site. This triggers the
Zn2+-coordinating residue His67 to move ~8 Å away from the site.
BA
Myr
(FA2)
Myr
(FA1)
Myr (FA5)
Myr (FA4)
Myr (FA3)
Myr (FA2)
His247 Asp249
His67
His67
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Figure 2.
Figure 2. Binding of Zn2+ to HSA in presence of different NEFAs. ITC experiments were
conducted in a buffer containing 50 mM Tris, 140 mM NaCl, pH 7.4. A first set of
experiments were performed with 1.5 mM ZnCl2 titrated into 60 µM HSA, in the presence of
either 0 (black), 2.5 (purple), 3 (blue), 4 (green) or 5 (red) mol. eq. of different NEFAs: (A)
octanoate, (B) laurate, (C) myristate and (D) palmitate. The settings used were 25°C, 39
injections (first injection was 0.4 µL, the remaining injections were 1 µL), initial delay 60 s
and spacing 120 s. A second set of experiments were performed with 750 µM Zn2+ titrated
into 25 µM HSA, in the presence of (E) palmitate, (F) palmitoleate, (G) palmitelaidate and
(H) stearate. For those ITC experiments, the settings were adjusted to 25°C, 19 injections
(first injection was 0.4 µL, the remaining injections were 2 µL), initial delay 60 s and spacing
120 s. Each fit corresponds to a two-sets-of-sites model. All NEFAs except octanoate
perturbed Zn2+ binding to the protein, with the effect increasing with the concentration of
NEFAs.
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Figure 3.
Figure 3. Effects of Zn2+ and NEFAs on maximum platelet aggregation and aggregation
rate in washed-platelets and platelets re-suspended in plasma. (A) Effect of Zn2+ on
maximum platelet aggregation (n=11). Maximum aggregation was higher in washed-platelets
than in platelets re-suspended in plasma. Addition of Zn2+ also increased maximum
aggregation in washed-platelets (p=0.0080) but not in platelets re-suspended in plasma. (B)
Effect of NEFAs on platelet maximum aggregation in platelets-in-plasma (n=12). Maximum
aggregation increased with addition of myristate (p=0.0006) but not octanoate. The presence
of 4 mol. eq. myristate and 100 µM Zn2+ increases maximum aggregation (n=9, p=0.0208).
The addition of the Zn2+ chelator TPEN reversed the effect of 4 molecular equivalent of
myristate. The data is represented as mean ± SD. Statistical significance is indicated by ns
where p>0.05, * where p<0.05, ** where p<0.01 and *** where p<0.001.
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Figure 4.
Figure 4. Effects of Zn2+ and NEFAs on fibrin clot parameters and on fibrin fibre
diameter in a purified system and effects relative to the parameter values in the absence
of Zn2+. Turbidimetric fibrin clotting and lysis assays were performed in buffer (50 mM Tris,
100 mM NaCl, pH 7.4) with a final concentration of 0.5 mg/mL (2.9 µM) fibrinogen, 100
µM HSA, 2.5 mM CaCl2, 0.05 U/mL thrombin, 39 ng/mL tPA, 3.12 µg/mL plasminogen, 0-
100 µM ZnCl2, and either 0 or 4 mol. eq. myristate (n=4). Fibrin clot parameters including
(A) maximum absorbance, (B) clot time and (C) lysis time were measured. Two-way
ANOVA followed by Sidak’s multiple comparisons test were used to analyse the data. All
three parameters were significantly increased in the presence of Zn2+ (p<0.0001, p<0.0001
and p<0.0001 respectively). Maximum absorbance increased in the presence of 4 mol. eq.
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myristate (p=0.0276), while clot time and lysis time were not significantly altered. The
parameter values relative to their values in the absence of Zn2+ were then calculated: (D)
maximum absorbance, (E) clot time and (F) lysis time. Two-way ANOVA followed by
Sidak’s multiple comparisons test were used to analyse the data. All three parameters were
significantly increased in the presence of Zn2+ (p<0.0001, p<0.0001 and p<0.0001
respectively). Maximum absorbance and clot time were increased in the presence of 4 mol.
eq. myristate (p=0.0455 and p=0.0194 respectively), while lysis time was not significantly
altered. (G) Fibrin fibre diameters from SEM experiments using a purified system with final
concentrations of 4.5 µM fibrinogen, 270 µM HSA, 2.5 mM CaCl2, 0.5 U/mL thrombin and
either 0 or 18 µM ZnCl2 (duplicates of clot, 5 images per samples, 50 fibres measured per
images). On the abscise axes, “0 µM Zn2+” refers to no added Zn2+ in the system. (H)
Representative pictures. Addition of Zn2+ significantly increased fibrin fibre diameter
(p<0.0001). The data is represented as mean ± SD. Statistical significance is indicated by ns
where p>0.05, * where p<0.05, ** where p<0.01 and *** where p<0.001.
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Figure 5.
Figure 5. Comparison of the plasma concentrations of NEFA, HSA and zinc and of the
clot formation and lysis parameters in plasma samples from patient with T2DM and
controls. Comparison of (A) plasma NEFA concentration (B) HSA concentration (C) total
plasma zinc concentration in the subjects with T2DM and the controls with t-test. (D)
Maximum absorbance and (E) clot time. Turbidimetric fibrin clotting and lysis assays were
performed in plasma samples with final concentration of plasma diluted 3-fold in buffer, 7.5
mM CaCl2, 0.03 U/mL thrombin and 0 or 20 µM available Zn2+ as ZnCl2 (concentration
calculated before the dilution of plasma) (n=54 for diabetes subjects and 18 for controls). (F)
Lysis time; the final concentrations used were plasma diluted 6-fold in buffer, 7.5 mM CaCl2,
0.03 U/mL thrombin, 20.8 ng/mL tPA and 0 or 20 µM available Zn2+ as ZnCl2 (concentration
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calculated before the dilution of plasma). The data is represented as mean ± SD. Two-way
ANOVA followed by Sidak multiple comparison tests were used to analyse the data.
Maximum absorbance and lysis time increased significantly in T2DM subjects compared to
controls (p<0.0001 and p=0.0448 respectively), but not in the presence of Zn2+. Clot time was
not significantly altered in the presence or absence of Zn2+ or when comparing the T2DM
group to the control group. (G) Positive correlation between BMI and NEFA levels
(p=0.0420). (H) Positive correlation between maximum absorbance and NEFA levels
(p=0.0310). Statistical significance is indicated with ns where p>0.05, * where p<0.05, **
where p<0.01 and *** where p<0.001.
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Figure 6.
Figure 6. Comparison of the fibrin fibre diameters in plasma samples from patient with
diabetes T2DM and controls. SEM experiments were performed using pooled-plasma
samples (n=6) from subjects with T2DM and controls with final concentrations of 9:2 plasma
in buffer (22.5 µL buffer in a total of 45 µL), 2.5 mM CaCl2, 0.5 U/mL thrombin and either 0
or 9 µM available Zn2+ as ZnCl2 (duplicates of clot, 5 images per sample, 50 fibres measured
per image). Representative pictures taken (A) in the absence of Zn2+ and (B) in the presence
of 9 µM available Zn2+. (C) Comparison of fibrin fibre diameter in clots formed from plasma
taken from subjects with T2DM and controls. Two-way ANOVA followed by Sidak multiple
comparison tests were used to analyse the data. Addition of Zn2+ significantly increased fibrin
fibre diameter and this effect was exacerbated in the presence of diabetes (p<0.0001 for
both). The data is represented as mean ± SD. Statistical significance is indicated with ns
where p>0.05, * where p<0.05, ** where p<0.01 and *** where p<0.001.
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Figure 7.
Figure 7. Comparison of the plasma concentration of major NEFA species in samples
from patient with diabetes T2DM and controls and associations with fibrin clot
parameters. A. Example of typical GC-MS chromatogram showing FAME separation. B.
Comparison of plasma concentrations of NEFAs between subjects with T2DM and controls.
The data is represented as mean ± SD. Statistical significance is indicated with ns where
p>0.05, * where p<0.05, ** where p<0.01 and *** where p<0.001. Plasma concentrations of
myristate (14:0), palmitate (16:0), linolenate (18:3), oleate (18:1c9), vaccinate (18:c11),
stearate (18:0), eicosapentaenoate (20:5) and arachidonate (20:4) were elevated in individuals
with T2DM (p=0.0010, p=0.0023, p=0.0003, p=0.0054, p=0.0029, p=0.0002, p=0.0092 and
p=0.0099 respectively). Concentrations of palmitoleate (16:1), linoleate (18:2), dihomo-γ-
linoleate (20:3) and docosahexanoate (22:6) were unchanged. C. Relationships between
maximum absorbance and the plasma concentrations of the major NEFA species. Plasma
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concentration of myristate, palmitate, oleate, vaccinate and stearate positively correlated with
maximum absorbance (p=0.0313, p=0.0202, p=0.0307, p= 0.0067, p=0.0184 respectively).
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Figure 8.
Figure 8. Zn2+ increases platelet aggregation and fibrin clot formation. Platelet activation
during coagulation triggers the release of Zn2+. In addition, NEFA binding to HSA disrupts
Zn2+ binding at site A. This will likely increase the available plasma Zn2+ concentration
resulting in an increase in platelets aggregation and influencing fibrin clot formation.
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