Effects of Fish Oil-derived N-3 Polyunsaturated Fatty Acids on the Generation and Functional Activities of Extracellular Vesicles
Phase: NA
Conditions: Extracellular Vesicles; Generation and Function
Sponsor: University of Reading

 
 
 
 
 
 
Fish Oil-derived N-3 Polyunsaturated Fatty Acids 
and Extracellular Vesicles 
(NCT03203512) 
 
Study Protocol 
Feb 2017 
 
 
 
 
 
 
 
 
 

Study objectives 
The main objectives of the study were divided into two strands (Figure 1). 
 
 
 
 
 
Strand 1: the influence of n-3 PUFA supplementation on the number and phenotype 
of total circulating EVs, and their fatty acid composition and coagulatory activities. 
Strand 2: the influence of n-3 PUFA on the production of EVs by platelets taken from 
subjects and generated in vitro as well as their fatty acid composition and coagulatory 
activities. 
 
Figure 1. Overview of the experimental work. Strand 1 focused on the numeration and 
functional analysis of circulating EVs; strand 2 focused on the numeration and functional 
analysis of platelet-derived EVs. EVs, extracellular vesicles; PDEVs, platelet-derived EVs. 

Study design 
The study was a randomised, double-blind, controlled crossover intervention. A total 
of 40 subjects aged between 40 and 70 years with moderate CVD risk (see criteria 
below) were recruited and randomly allocated to consume either fish oil or control 
(high-oleic safflower oil) in the first 12-week intervention period. After a 12-week 
washout, they then crossed over to the other intervention for a further 12 weeks. 
Random assignment of subjects for intervention order (“1” and “2”) was performed 
using online software (https://www.randomizer.org/). Fish oil and control capsules 
were of identical appearance and blinded by an individual not involved in the study. 
All investigators involved in the study remained blind until all sample and statistical 
analysis had been completed.   
The length of the intervention and washout periods was based on evidence that 
incorporation of n-3 PUFA in all cell types reaches a plateau after 12 weeks’ 
intervention and that a 12- week washout period is sufficient to avoid carryover 
effects (Walker et al., 2015).  
During the study, there were four intervention visits, which took place at the 
beginning and end of each 12-week intervention period (weeks 0, 12, 24 and 36). 
Before each study visit, subjects were asked to abstain from alcohol and strenuous 
exercise during the 24 hours prior to the study day. On each visit day, subjects were 
asked to come to the nutrition unit in an unfed state (fasted, not eating or drinking 
anything but water from 8 pm the night before). After taking height, weight and blood 
pressure measurements, a blood sample of approximately 100 ml was collected into 
vacutainer tubes containing 3.2% sodium citrate (Greiner Bio-One, Gloucestershire, 
UK) for EV-related analysis (details see below). After the first visit, subjects were asked 
to start to consume 6 capsules per day of either fish oil (Wiley's Finest Easy Swallow 
Minis, Canada), providing a total daily intake of 1.8 g n-3 PUFA, or high-oleic safflower 
oil (Wiley's Finest, Canada), providing 740mg oleic acid + 120mg linoleic acid per day. 
Subjects were advised to take capsules with breakfast, lunch and dinner (2 at each 
meal). Weeks 12-24 represented a washout period and then subjects crossed over to 
the other intervention product until week 36 (Figure 2).  

Subjects were asked to complete food frequency questionnaires at home before their 
first visit and again during each arm of the intervention study (weeks 0, 12, 24 and 36). 
These were used to assess their habitual diet and to confirm that they had low 
consumption of oily fish (less than one portion per month). 
 
 
 
Study methods 
Blood collection and processing 
On the visit day, subjects were asked to come to the Hugh Sinclair Unit of Human 
Nutrition in an unfed state (fasted, not eating or drinking anything but water from 8 
pm the night before). Venous blood samples were collected from fasted subjects into 
vacutainer tubes containing 3.2% sodium citrate (Greiner Bio-One, Gloucestershire, 
United Kingdom), and were kept at room temperature and processed within 30 
minutes after transfer into polypropylene tubes (Fisher Scientific, Loughborough, 
United Kingdom). Platelet-rich plasma (PRP) was prepared by centrifugation of whole 
blood at 175 x g for 15 minutes, with no brake at room temperature. Platelet-poor 
plasma (PPP) was obtained by centrifugation of PRP at 13,000 x g at room temperature 
for 2 minutes and removal of the supernatant from the platelet pellet. To obtain 
platelet-free plasma (PFP), blood  was centrifuged at 1500 x g for 15 minutes at room 
temperature  and the upper two-thirds were collected and centrifuged once again at 
13,000 x g for 2 minutes at room temperature, before collecting the upper three 
quarters from each tube as PFP. Freshly prepared PRP and PPP were used in the 
platelet aggregation assay. Some of the PFP was used for the enumeration and 
characterization of circulating total EVs, while the remainder was stored at − 80°C for 
Figure 2. Study timeline 

a n al ysis  of  t hr o m b o g e nic  acti vit y  of  P F P  (t hr o m bi n  g e n er ati o n  a n d  fi bri n  cl ot  
pr o p erti es) a n d of E Vs (t hr o m bi n g e n er ati o n).  
 
Is ol ati o n of P D E Vs ge n er at e d i n vitr o fr o m u nsti m ul at e d a n d sti m ul at e d pl at el ets 
T h e is ol ati o n pr oc ess f or P D E Vs g e n er at e d i n vitr o fr o m u nsti m ul at e d a n d sti m ul at e d 
(t hr o m bi n r ec e pt or acti v at or p e pti d e 6, T R A P-6) pl at el ets c o nsist e d of i) is ol ati o n of 
pl at el ets fr o m w h ol e bl o o d, ii) sti m ul ati o n or n ot of is ol at e d pl at el ets a n d iii) is o l ati o n 
of E Vs fr o m t h e s u p er n at a nts of sti m ul at e d a n d u nsti m ul at e d pl at el ets.  
 
T o is ol at e pl at el ets, P R P w as c e ntrif u g e d at 1, 0 0 0 x g, f or 1 0 mi n, wit h l o w br a k e, at 
r o o m t e m p er at ur e i n t h e pr es e nc e of pr ost acycli n ( P GI 2, 1 µ g/ ml; Si g m a-Al dric h, 
D ors er, U K), as d escri b e d b y Ar mstr o n g et al ( Ar mstr o n g et al., 2 0 1 7). T h e p ell et w as 
w as h e d t wic e t o o bt ai n w as h e d pl at el ets i n m o difi e d T yr o d e’s ( M T H) b uff er pr e p ar e d 
b y t h e s u p pl e m e nt ati o n of T yr o d e`s H E P E S st oc k (c o nt ai ni n g 1 3 4 m m ol/l N a Cl, 2. 9 
m m ol/l K Cl, 0. 3 4  m m ol/l N a 2 H P O 4, 1 2 m m ol/l N a H C O 3, 1 m m ol/l M g Cl 2 a n d 2 0 
m m ol/l H E P E S; P h 7. 4; filt er e d o n 0. 2 2 µ m) wit h 0. 1 % of gl uc os e ( Si g m a -Al dric h, D ors et, 
U K). Pl at el et c o nc e ntr ati o n i n t h e s us p e nsi o n w as c o u nt e d ( S ys m ex, U S A) a n d a dj ust e d 
t o 3x 1 0 8 pl at el ets/ ml wit h M T H b uff er. W as h e d pl at el ets at 3 x 1 0 8 pl at el ets/ ml w er e 
s u p pl e m e nt e d wit h 2 µ M of C a Cl 2 ( Si g m a-Al dric h, D ors et, U K) t h e n i m m e di at el y 
sti m ul at e d wit h eit h er 3 0  M of T R A P -6 (sti m ul at e d pl at el ets) or P B S ( u n -sti m ul at e d 
pl at el ets). Sti m ul at e d a n d u nsti m ul a t e d pl at el ets w er e i nc u b at e d at 3 7° C f or 2 h o urs 
i n  n o n-stirri n g  c o n diti o ns.  Pl at el ets  w er e  t h e n  r e m o v e d  b y  t w o  s e q u e nti al  
c e ntrif u g ati o ns at 1, 2 0 0 x g f or 1 0 mi n ut es. T h e u p p er 9 0 % of t h e s u p er n at a nt, w hic h 
c o nt ai ns t h e E Vs, w as c oll ect e d a n d p ell et e d b y c e ntrif u g ati o n at 1 5, 0 0 0 x g f or 3 0 
mi n ut es  at  4  ° C.  T h e  s u p er n at a nt  w as  disc ar d e d  a n d  t h e  p ell et  ric h  i n  P D E Vs  w as 
s us p e n d e d. Is ol at e d P D E Vs w er e p o ol e d i n a vi al t o e ns ur e t h eir h o m o g e n eit y a n d t h e n 
di vi d e d i nt o ali q u ots of 3 0 μl e ac h a n d st or e d at -8 0 ° C u ntil us e.  
 

Preparation of pooled vesicle free plasma (VFP) 
Venous blood samples were collected from three healthy, fasted subjects as described 
above to prepare pooled VFP. Whole blood was first centrifuged twice at 2,500 x g for 
15 min to remove blood cells and obtain a pooled plasma. Vesicles were removed from 
pooled plasma by ultracentrifugation at 20,000g for 1 hour at 4 °C, then the 
supernatant was collected, and the remaining pellets (EVs) was discarded. The vesicle 
poor plasma was ultracentrifuged at 100,000g for 1 hour at 4 °C, followed by filtration 
on 0.1 µm (filter info) four times. This filtered supernatant was considered VFP and 
stored in 1 ml aliquots at -80 °C until use. 
 
Isolation, enumeration and characterization of circulating EVs using nanoparticle 
tracking analysis (NTA) 
PFP was subjected to size exclusion chromatography (SEC) using Izon qEV columns 
(Izon Science Ltd, Oxford, United Kingdom) to isolate EVs prior to NTA. PBS was used 
to elute 10 sequential fractions of 0.5ml and fractions 7-9 were diluted with PBS to 
achieve the recommended concentration range of particles (1-10 x 108 vesicles/ml) 
(Gardiner et al., 2013, Vestad et al., 2017; Dragovic et al., 2011) for analysis on the 
NanoSight 300 (Malvern, Amesbury, United Kingdom). Data from 5 separate videos, 
each of 60 seconds’ duration, were analyzed using the NTA 3.20 instrument software, 
which identifies individual particles and estimates size based on the Stokes-Einstein 
Equation (van der Pol et al., 2010). A threshold of 70nm was set to ensure minimal 
interference by small lipoproteins. Total circulating EV numbers enumerated by NTA 
were defined as total EVs (TEVs). 
 
Enumeration and characterization of circulating EVs using flow cytometry 
For enumeration and phenotyping of circulating EVs by flow cytometry (BD FACSCanto 
II flow cytometer, BD Biosciences, Wokingham, United Kingdom), an initial EV size gate 
was set using non-fluorescent, silica ApogeeMix beads with diameters of 180nm, 240 
nm, 300nm, 590nm, 880nm, and 1300nm (Apogee flow systems, Hemel Hempstead, 
United Kingdom) and additionally, fluorescent latex beads with diameters of 110nm 

and 500nm.  The 240nm silica beads corresponded to the lowest reliable detection 
limit of the flow cytometer and any particles below this size (on SSC) were excluded 
to minimize background noise. In order to exclude platelets and cellular material, the 
upper detection gate would ideally be set at 1µm, but the closest available diameter 
of silica beads was 880 nm, so the gate was set just above this on scatter mode.  
 
The following fluorochrome-coupled antibodies and their corresponding isotypes 
were used for EV phenotyping: Annexin V conjugated to Allophycocyannin (APC) 
(Thermo Fisher Scientific, Basingstoke, United Kingdom) for phosphatidylserine (PS) 
positive EVs, anti-CD41 conjugated to Phycoerythrin (PE) (Diagnostica Stago LTD, 
Theale, United Kingdom) for platelet-derived EVs (PDEVs) and anti-CD105 conjugated 
to eFluor450 (Life Technologies LTD (Invitrogen division), Paisley, United Kingdom) for 
endothelial-derived EVs (EDEVs). PFP (5 µl) was incubated with FcR blocking reagent  
(5µl) (Miltenyi Biotec Ltd, Surrey, United Kingdom) and Annexin V buffer (10mM 
HEPES, 140mM NaCl, 2.5mM CaCl2, pH 7.40; Cambridge Bioscience, Cambridge, 
United Kingdom) for 15 minutes in the dark at room temperature, before incubation 
with antibodies and isotype-matched controls for a further 15 minutes in the dark at 
room temperature. Samples were then diluted with 200µl Annexin V buffer and 
analyzed by flow cytometry, as illustrated in Figure 3. EVs staining positive for Annexin 
V were classified as PS+EVs, those staining positive for Annexin V and CD41 were 
classified as PS+ platelet-derived EVs (PDEVs) and those staining positive for Annexin 
V and CD105 were classified as PS+ endothelium-derived EVs (EDEVs). Matching 
isotype controls (IgG1-PE and IgG1-eFlour450) were used as appropriate. Absolute 
numbers of vesicles were calculated using BD Trucount tubes (BD Biosciences, 
Wokingham, United Kingdom).  

 
 
 
 
 
For characterisation of PDEVs generated in vitro using flow cytometry, all antibodies 
and reagents were filtered using 100 nm syringe filters (Sartorius, UK) and 100 nm 
centrifugal devices (Sartorius, UK) and analysed for 1 minute to evaluate the level of 
background particles that fell in the EV gate before each analysis day. To measure the 
PS expression of PDEVs, samples were assessed for their ability to bind cell marker-
Fluorescence Triggering 
(APC) 
  
 
     
            
  
                  
Fluorescence Triggering 
(PB) 
 
                                       
     
  (C) 
  
(B) 
(A) 
Figure 3. Flow cytometric analysis of a typical sample of PFP labelled with Annexin V-APC, anti-
CD41-PE and anti-CD105-eFluor450. EVs were characterized as follows: (A) All Annexin-APC 
fluorescently labelled particles were identified as PS+EVs (in blue); (B) APC vs PE quadrant plot 
were set to identify both Annexin-APC and CD41-PE labelled particles as PDEVs (in red); (C) APC vs 
PB quadrant plot to identify both Annexin-APC and CD105-eFluor450 labelled particles as EDEVs 
(in yellow). AnnV, Annexin V; APC, Allophycocyanin; EDEVs, endothelial-derived extracellular 
vesicles; PB, Pacific Blue; PDEVs, platelet-derived extracellular vesicles; PE, phycoerythrin; PS, 
phosphatidylserine. 

specific monoclonal antibody anti CD41 conjugated to PE (Biocytex, Marseille, France) 
to detect platelet-derived EVs and Annexin V conjugated to APC (ThermoFisher, UK), 
which binds externalized PS residues. For this double staining, frozen PDEVs were 
thawed, then 2 µl of each PDEV sample was incubated with both anti CD41 and 
Annexin V (3µl each) for at least 30 minutes in the dark at room temperature. The 
incubation was followed by dilution with 70 µl of Annexin V-binding buffer (10mM 
HEPES, 140mM NaCl, 2.5mM CaCl2, pH 7.4) and samples were analysed by FCM. 
Events were collected with low flow rate for 1 minute on the threshold corresponding 
to the labelled fluorophore avoiding an event rate higher than 5,000 events/second. 
For anti CD41, sample incubation with its isotype control had lower fluorescence than 
the antibody on its own; therefore the background was set on the fluorescence 
produced by the antibody only. EV positive events were calculated, by subtracting the 
number of events obtained from the antibody mix on each experimental day from the 
absolute count of positive events of the labelled sample (Figure 4). Data was captured 
using FACSDiva Software version 6.1.3 and analysed using FlowJo version 10. 
 
 
 
 
 
 
Figure 4. Representative flow cytometry histograms with signal triggered in fluorescence mode 
showing mean fluorescence intensity (MFI) of the antibody background (red) and PDEVs positive 
events (blue). PE = CD41, APC = Annexin V. The number of positive events was calculated 
subtracting the events collected in blue minus events collected in red. 

Functional assays of PDEVs generated in vitro 
Measurement of clot formation induced by PDEVs  
The impact of PDEVs (before and after intervention) on fibrin clot formation in VFP 
was assessed in 96-well flat-bottom microplates (Greiner Bio-One, Stonehouse, 
Gloucestershire, UK) by a change in turbidity of the VFP at 405 nm (A405) every 30 
seconds, for 1 hour at 37°C using a Flex Station 3 plate reader (Molecular Devices, 
United State). Therefore, pooled PFP (positive control) and VFP (negative control), 
served as benchmarking in this assay. The assay was then set up by diluting 30% of 
VFP in clot buffer (10 mM Tris [pH 7.4] and 0.01% Tween 20) and 5 µg/ml protein 
concentration of PDEVs (final concentration) was then added. Fibrin clot formation 
was initiated by addition of 5 mM CaCl2 in a final volume of 100 µl of clotting mixture. 
Plates were read at 37 °C for 1 hour at 30 second intervals using a fluorescence plate 
reader (FlexStation 3, United State) at wavelength of 405 nm. Data were then analysed 
by an online application (https://drclongstaff.shinyapps.io/Clot_or_HaloCL/) to obtain 
relative parameters: time to 50% change, absorbance at 50% change, absorbance at 
peak, area under curve. 
 
Measurement of thrombin generation  
Thrombin formation was assessed using a commercially available, plate-based 
thrombin generation assay (Technothrombin TGA kit, Austria), which assesses a 
change in fluorescence as a result of cleavage of a fluorogenic substrate by thrombin 
over time upon activation of the clotting cascade by tissue factor. Two separate 
analyses were conducted: (i) determination of the effect of n-3 PUFA supplementation 
on thrombin generation in PFP from study samples relative to VFP and (ii) 
determination of the effects of in vitro-generated PDEVs or circulating EVs derived 
from study samples on thrombin generation in VFP. Prior to analysis, a thrombin 
calibration curve was constructed from dilutions of lyophilised Hepes-NaCl-buffer 
containing 0.5 % bovine serum albumin and ~ 1000 nM thrombin in buffer with BSA. 
A kinetic reading of a plate was initiated by additional of 50 µl of fluorogenic substrate 
solution containing fluorogenic substrate 1 mM Z-G-G-R-AMC and 15 mM CaCl2. 

Calibration curves were recorded at 37 °C for 10 minutes with 30 seconds intervals 
using the plate reader (FlexStation 3, United State) at 360 nm for excitation and at 460 
nm for emission. 
 
The methods for both approaches were based on the use of pooled VFP as a negative 
control to allow assessment of thrombin generation specifically resulting from the 
presence of EVs. For the first approach (i), 40 µl aliquots of either pre-thawed study 
sample PFP or pooled VFP or pooled PFP were added into the plate. For the second 
approach, PDEVs or circulating EVs (10 µl of EV suspensions at 5 µg protein/ml, final 
concentration) produced from either unstimulated or stimulated platelets (UP-EVs 
and SP-EVs, respectively) from intervention samples, or PBS (negative control) were 
added to 30 µl VFP. This was followed by addition of a 10 µl suspension of phospholipid 
micelles containing recombinant human tissue factor (TF) in Tris-Hepes-NaCl buffer 
(RCL), which was provided in the kit. Formation of thrombin was initiated by additional 
of 50 µl of fluorogenic substrate solution containing fluorogenic substrate 1 mM Z-G-
G-R-AMC and 15 mM CaCl2. Plates were immediately read at 37 °C for 1 hour at 1 min 
intervals using a fluorescence plate reader (FlexStation 3, United State) at excitation 
and emission wavelengths of 360 and 460 nm, respectively. All samples were 
measured in duplicate. Fluorescent intensity was detected by TGA Evaluation 
Software to calculate thrombin generation in samples. Data were then analysed by 
the TGA Evaluation Software manually to convert the unit of thrombin generation 
from RFU to nM and presented as three variables: lag time, time to the peak, the 
concentration of thrombin (nM), velocity-index and area under curve. 
 
Measurement of fibrinolysis by PDEVs  
The assessment of fibrinolysis induced by PDEVs added to VFP was performed in 96-
well flat-bottom microplates (Greiner Bio-One, Stonehouse, Gloucestershire, UK) by a 
change in turbidity of VFP at 405 nm (A405) every 30 seconds, for 8 hours at 37°C using 
a Flex Station 3 plate reader (Molecular Devices, United State. In this assay, pooled 
PFP (positive control) and VFP (negative control), therefore, served as benchmarking. 

The assay for fibrinolysis of PDEVs generated in vitro was set up by diluting 30% of VFP 
in clot buffer (10 mM Tris [pH 7.4] and 0.01% Tween 20) in the presence of tPA (100 
pM; Sigma-Aldrich, Dorset, UK) and 5 µg/ml protein concentration of PDEVs (final 
concentration) was then added.  Fibrin clot formation to lysis was initiated by addition 
of 5 mM CaCl2 in a final volume of 100 µl of clotting mixture. Plates were read at 37 °C 
for 8 hours at 30 second intervals using a fluorescence plate reader (FlexStation 3, 
United State) at wavelength of 405 nm. Data were then analysed by an online 
application (https://drclongstaff.shinyapps.io/Clot_or_HaloCL/) to obtain relative 
parameters: area under curve, time to 50% lysis from zero, absorbance at %50 lysis, 
time clotting to 50% lysis (time between 50% clotting and lysis), time peak to 50% lysis 
(time between the peak and 50% lysis).  
 
Measurement of in vitro thrombus formation under flow 
PDEVs prepared from the study samples were subjected to analysis of their effect on 
thrombus formation in whole blood under flow, a specialist technique conducted by 
Dr Joanne Mitchell from the School of Biological Sciences, University of Reading. The 
assay was performed by adding either buffer (control) or 30 µl of PDEVs generated 
from TRAP-6 stimulated platelets (final protein concentration of EVs: 5 µl/ml) into 
whole blood in modified Tyrode’s-HEPES buffer (134 mM NaCl, 2.9 mM KCL, 0.34 mM 
Na2HPO4, 20 mM HEPES, 1 mM MgCl2, pH 7.4). Cellix Vena8 Fluoro+ biochips with 
capillaries 0.01 cm high and 0.04 cm wide were coated with collagen (100 mg/ml) prior 
to thrombus formation studies. Whole citrated blood labelled with 3,3'-
Dihexyloxacarbocyanine Iodide (DiOC6) was flowed for 10 minutes over the collagen 
coated capillary chambers to form platelet thrombi at an arterial shear rate of 1000s-
1. Images were taken every 2-4 seconds using a Nikon A1R fluorescence confocal 
microscope at 20X magnification. Data were analysed using Fiji (Image J) by measuring 
the fluorescence intensity of the DiOC6 over time which corresponded to the increase 
in thrombus size. 
 

Assessment of platelet aggregation 
Freshly prepared PRP (40 µl) was added to appropriate wells of microplate containing 
six separate agonists (ADP, CRP-XL, EPI, TRAP-6, U46619 and AA) and the plate was 
tapped gently to ensure that the PRP reached the bottom. The plate was shaken at 
1,200 rpm at 37°C for 5 minutes using a plate shaker (Thermo-shaker, Grant 
Instruments, UK).PRP or PPP were then added to the relevant control wells (agonist 
free) in plate which was tapped sharply to remove any aggregates from the center of 
the wells. A plate-reader (Tecan Microplate Reader Spark, Switzerland) was used to 
determine the absorbance at 405nm. Absorbance data were converted to a percent 
aggregation by reference to the absorbance of PRP and PPP as 0% and 100% 
aggregation controls, respectively. Dose-response curves in response to each agonist 
were obtained and curves were fitted by a four-parameter logistic non-linear 
regression using Prism (Version 8.2, GraphPad Software, Inc., San Diego, CA). 
 
Clot growth and fibrinolysis 
Clot growth and fibrinolysis in samples of PFP was assessed using a thrombodynamics 
analyzer and thrombodynamics kit (HemaCore, Moscow, Russia). PFP (120µl) of PFP 
was thawed in a 37 °C water bath for 5 min. Plasma placed into a special plastic vial 
contained a lyophilized solution of protein-inhibitor and incubated for 15 minutes at 
37°C within the thermostat of the analyzer, following which 4nM of tissue-type 
plasminogen activator (tPA, final concentration) (Sigma-Aldrich, Dorset, UK) was 
added. This step was followed by the addition of a lyophilized solution of calcium salt. 
The sample was immediately transferred into an optically transparent cuvette with 
two thin channels, which were placed into the 37°C temperature-controlled chambers 
of the instrument. Finally, an activating insert, the end edges of which were covered 
with immobilized tissue factor to activate clotting, was gently placed fully into the 
cuvette. Growth and lysis of the fibrin clot was quantified using video microscopy 
software over a 60 minutes period (see Figure 5). 

 
 
 
 
 
 
 
 
 
 
 
Conventional cardiovascular risk markers assessment 
Anthropometric data included height, weight and body mass index (BMI), the latter 
recorded using the Tanita MC-780MA P (Tanita Europe BV, Netherlands). SBP and 
diastolic BP (DBP) were recorded as the average of three using an Omron M2 Upper 
Arm Blood Pressure Monitor (OMRON Healthcare Europe BV, United Kingdom). An 
overnight fasting blood sample of approximately 9ml was taken into a serum-
separating tube (Greiner Bio-One, Gloucestershire, United Kingdom) for the analysis 
Figure 5. Clot formation is activated by introducing the TF bearing surface to PFP and 
fibrinolysis was assessed by addition of 4 nM tPA. (A) Representative images of coagulation 
process and fibrinolysis were recorded over 60 min and all parameters were calculated by 
the thrombodynamics software; (B) Plot of fibrin clot growth versus time was constructed 
to calculate coagulation-related parameters. Lag time was calculated as the time from the 
beginning of the measurement until the beginning of the clot growth when the first 
significant levels of fibrin can be detected. The rate of clot growth and the initial rate of clot 
growth, which indicate the propagation stage of blood coagulation were calculated on the 
interval 15-25 minutes and 2-6 minutes after the beginning of clot growth. The clot size was 
measured at the 30th minute of clot growth. Clot density was measured as amount of light 
scattering from a fibrin clot; (C) Plot of fibrin clot intensity versus time was constructed to 
calculate fibrinolysis-related parameters. Lysis onset time was calculated as the time, when 
the light scattering intensity (green line) in the clot reach to 30% reduction from the 
beginning and the lysis progression was calculated as the linear rate of the light scattering 
intensity decrease as the percentage of the initial value in the following 5 min. 

of blood lipid and glucose concentrations. Serum-separating tubes were kept upright 
at room temperature for 30 minutes (and no more than 60 minutes) before 
centrifuging at 1700 x g for 15 minutes at room temperature. The plasma (160µl) was 
collected and analyzed by iLab (iLab 600 Clinical Chemistry System, Diamond 
Diagnostics, United States) for triacylglycerol (TAG), TC, HDL-C, and glucose 
concentrations using standard reagent kits (Werfen Limited, Warrington, United 
Kingdom). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Fish Oil-derived N-3 Polyunsaturated Fatty Acids 
and Extracellular Vesicles 
(NCT03203512) 
 
Statistical Analysis Plan 
Feb 2017 
 
 
 
 
 
 
 
 
 
 
 
 
 

Statistical analysis  
Data are expressed as mean ± SEM if normally distributed and as median with inter-
quartile range when not. The Kolmogorov-Smirnov test was applied to assess the 
normal distribution of continuous variables and all variables with non-normal 
distribution were log-transformed to achieve normal distribution before analysis. A 
general linear model with fixed factors of treatment and period was conducted to 
determine the differences in the effect of two treatments and two periods on 
biological characteristics and EVs parameters. A paired t-test comparing the baseline 
data (before fish oil and before control oil) was performed to check potential carry-
over effects. All statistical analyses were performed with SPSS Statistics version 25 and 
a p-value < 0.05 was considered statistically significant.