1

Supplemental Material to Huskens, Roest, Remijn et

al. “Strenuous exercise induces a hyperreactive

rebalanced haemostatic state that is more

pronounced in men” (Thromb Haemost 2016; 115.6)

Sample size calculation

The purpose of this study is to compare the coagulation status of healthy donors before and after

strenuous exercise (= participation at the Amstel Gold Race). Intitially, we considered the peak height

of the thrombin generation assay in whole blood as the most important parameter in our study. The

mean value of the peak is 128 nM (n=40) with SD of 40 nM. We expect to have at least an effect of

10%, which means that the sample size has to be 90 if tested 2-sided with a desired power of 85% and

a p-value of 0.05 (Calculated with Power and Sample Size Calculations version 3.12). A letter with

information about our study was send to all participants and 103 persons wanted to participate in our

study. On the day of the Amstel Gold Race, 94 participants presented themselves before the start of

the race and finally two were excluded because of age or not cycling the minimal distance (100 km). In

total 92 volunteers were tested.

Blood samples

Blood of the healthy participants was aseptically drawn in Vacutainer tubes (BD Vacutainer System)

just before the race (baseline) and immediately after the race (post-exercise). Serum was stored at -

80°C until further use. PRP was prepared by centrifuging the whole blood at 250 g for 15 min. PPP was

prepared by double centrifugation of the blood at 1,000 g for 10 min. The residual PPP was stored at -

80°C until further use. Cell counts in whole blood and PRP were performed with a Coulter Counter

analyzer (Beckman Coulter, Woerden, the Netherlands).

Reagents

2

PRP reagent (1 pmol/L of Tissue Factor (TF) and a minimal amount of phospholipids), PPP reagent (5

pmol/L of TF and phospholipids), PPP reagent low (1 pmol/L of TF and phospholipids), MP reagent

(phospholipids but no TF) and Thrombin Calibrator, purchased from Thrombinoscope BV (Maastricht,

the Netherlands), were reconstituted in 1 ml of deionized water. The FluCa-kit consisting of Fluo-

Substrate and Fluo-buffer was also purchased from Thrombinoscope BV. For thrombin generation in

whole blood we used rhodamine substrate (P2Rho), a gift from Diagnostica Stago (Asnières sur Seine,

France). Recombinant TF was purchased from Innovin (Dade-Behring, Marburg, Germany). The

calibrator, α2-macroglobulin-thrombin complex, used for whole blood thrombin generation was

prepared in house as described previously (1). BSA5 contains 20 mmol/L Hepes, 140 mmol/L NaCl,

0.02% NaN3 and 5 g/L bovine serum albumine (BSA) at pH 7.35. BSA60 contains 20 mmol/L Hepes,

0.02 % NaN3 and 60 g/L BSA at pH 7.35.

FITC-conjugated anti-fibrinogen and PE-conjugated anti-P-selectin were purchased from DAKO (F0111,

Dako, Glostrup, Denmark) and BD-Biosciences (555524; BD PharmingenTM, Franklin Lakes, NJ, USA),

respectively. Platelet agonists include the P2Y12 agonist ADP (01897, Sigma-Aldrich, Zwijndrecht, the

Netherlands), the protease activated receptor (PAR)-1 agonist thrombin receptor activator peptide

(SFLLRN, H-2936; Bachem, Weil am Rhein, Germany), the PAR-4 agonist AYPGKF (Netherlands Cancer

Institute, Amsterdam, the Netherlands) and the glycoprotein VI (GPVI) agonist collagen-related

peptide (CRP, a generous gift by Professor Farndale, university of Cambridge, UK).

Measurement of in vitro thrombin generation by means of CAT

The plasma CAT thrombin generation was measured in a mixture of 80 µL PPP or PRP and 20 µL of PPP

reagent, PPP reagent LOW, MP reagent or PRP reagent (1). In the calibration well, 20 µL of Thrombin

Calibrator was added to 80 µL of plasma. Immulon 2HB, 96-well plates were placed on a heating plate

to reach 37°C (10 min). At the start of the experiment, the fluorometer (Ascent reader,

Thermolabsystems OY, Helsinki, Finland) dispensed 20 µL of FluCa into the wells before reading. The

thrombin generation curves and their parameters were derived from the fluorescence curves using

3

the dedicated software provided with the CAT method (Thrombinoscope BV, Maastricht, the

Netherlands) (2).

Platelet activation test in whole blood

Briefly, 5 µL whole blood was added to 50 µL reaction mix, consisting of agonist (500 ng/mL collagen-

related peptide (CRP), 50 µmol/l thrombin receptor activator peptide (SFLLRN), 15 µmol/l ADP, 750

µmol/l AYPGKF (protease activated receptor (PAR)-4 agonist)) and 40 µl/ml FITC-conjugated anti-

fibrinogen, 40 µl/ml PE-conjugated anti-P-selectin in HEPES-buffered saline (HBS: 10 mmol/l HEPES,

150 mmol/l NaCl, 1 mmol/l MgSO4, 5 mmol/l KCL, pH 7.4). Flow cytometry was used to distinguish

between platelets and other cells on forward and sideward scatter pattern. Fluorescent intensity in

the PE gate was selected to determine P-selectin density, and fluorescent intensity in the FITC gate

was used to determine fibrinogen binding, which indicates αIIbβ3 activation. Results are expressed as

arbitrary units (AU).

4

Suppl. Table 1. Platelet reactivity at baseline and post-exercise

Agonist

No

agonist PAR-1 PAR-4 P2Y12 GPVI

P-selectin

expression

(AU)

Baseline median 27 4404 3549 1012 3839

IQR (21-31) (3625-5192) (2600-4332) (706-1418) (3112-4435)

Post-

exercise

median 29**** 5497**** 4901**** 1488**** 5276****

IQR (24-39) (4890-6171) (3815-5466) (1110-1873) (4212-6138)

αIIbβ3

activation

(AU)

Baseline median 42 809 524 663 877

IQR (27-47) (659-944) (331-711) (476-823) (641-1017)

Post-

exercise

median 50**** 1304**** 1060**** 1041**** 1477****

IQR (44-53) (1034-1510) (598-1322) (828-1278) (1091-1799)

Data are given as medians with interquartile ranges (n=53-60) and tested with the Wilcoxon matched-

pairs signed rank test. **** P<0.0001.

5

Suppl. Table 2. Cytokine concentration at baseline and after exercise

Baseline Post-exercise P-value

Cytokine (pg/ml)* median IQR median IQR

IL-1β 0.85 (0.61-1.35) 1.1 (0.77-1.55) NS

IL-1ra 59.6 (32.25-127.7) 69.38 (44.85-140.3) NS

IL-4 0.81 (0.26-1.41) 0.81 (0.43-1.31) NS

IL-5 3.3 (1.26-4.6) 2.79 (1.72-5.05) NS

IL-6 4.48 (2.84-7.20) 15.95 (10.91-20.29) <0.0001

IL-7 6.29 (4.42-6.91) 5.08 (3.43-7.41) 0.003

IL-8 7.32 (5.67-11.31) 8.81 (6.95-12.42) 0.02

IL-9 12.27 (7.59-24.86) 12.93 (8.29-23.13) NS

IL-10 11.81 (7.89-25.56) 13.49 (9.023-22.77) NS

IL-12 16.42 (8.50-42.2) 17.11 (7.35-40.83) 0.03

IL-13 26.38 (14.46-48.61) 22.15 (11.12-39.88) 0.0003

IL-17 19.79 (1.7-53.46) 22.73 (1.7-56.07) NS

Eotaxin 55.1 (46.61-73.91) 52.55 (42.57-66.29) <0.0001

FGF 43.88 (18.68-83.94) 43.88 (18.32-75.98) NS

G-CSF 93.71 (50.7-138.4) 52.5 (28.49-84.54) <0.0001

IFN-γ 19.93 (4.26-43.87) 23.43 (11.71-38.46) NS

IP-10 253.6 (208.8-347.6) 195.8 (164-276) <0.0001

MCP-1 38.9 (20.57-54.87) 68.84 (44.44-89.61) <0.0001

MIP-1α 5.34 (3.54-8.30) 5.16 (3.12-7.86) 0.0085

MIP-1b β 35.18 (28.32-43.03) 35.17 (28.15-45.37) NS

PDGF 59.57 (37.46-95.48) 117.4 (68.79-212.5) <0.0001

RANTES 4285 (3037-6295) 5518 (4237-7227) <0.0001

TNF-α 15.72 (9.74-29.95) 17.84 (11.66-29.71) NS

*The levels of IL-2, IL-15, GM-CSF and VEGF were below the detection limit. Data are listed as medians

with interquartile ranges (n=78) and tested with the Wilcoxon matched-pairs signed rank test.

6

0 5 1 0 1 5 2 0 2 5

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

T im e (m in )

Th

ro

mb

in (

nm

ol/

L)

P P P

n o a d d F V III 1 U /m l F V III 2 U /m l

F V III 5 U /m l F V III 1 0 U /m l

0 1 0 2 0 3 0 4 0

0

5 0

1 0 0

1 5 0

2 0 0

T im e (m in )

Th

ro

mb

in (

nm

ol/

L)

P R P

0 5 1 0 1 5 2 0 2 5

0

5 0

1 0 0

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T im e (m in )

Th

ro

mb

in (

nm

ol/

L)

w h o le b lo o d

Suppl. Figure 1. Effect of the exercise-induced increase of FVIII on thrombin generation. Thrombin

generation measured in PPP (A) and PRP (B) triggered with 1 pM of TF and in whole blood (C) triggered

with 0.5 pM of TF. Plasma and whole blood were spiked with or without different concentrations of

FVIII.

A

B

C

7

II

b

3 a

cti

va

tio

n (

AU

)

Baselin

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Po

st-

exerc

ise

Baselin

e

Po

st-

exerc

ise

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

*** ***

P A R -1

P-s

ele

cti

n e

xp

re

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ion

(A

U)

Baselin

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Po

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ise

Baselin

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ise

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2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

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***

P A R -1

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cti

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n (

AU

)

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Baselin

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P A R -4

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)

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***

P 2 Y 12

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(A

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G P V I

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G P V I

m e n w o m e n

8

Suppl. Figure 2. Effect of strenuous exercise on platelet reactivity in men and women after

adjustment for sample size, age and distance cycled. Thrombin receptor activator peptide (SFLLRN),

AYPGKF, ADP and collagen-related peptide (CRP) were used to activate platelets via Proteinase

Activated Receptor-1 (PAR-1) (A and B), PAR-4 (C and D), P2Y12 receptor (E and F) and glycoprotein VI

receptor (GPVI) (G and H), respectively. Scatter plots of αIIbβ3 activation (left) and P-selectin expression

(right) in men and women measured before and after the race are shown. To compare males and

females of the same age and distance cycled, a restricted analysis was performed on men below 40

years who have ridden 150 km or less (12 men with mean age 31 years and mean distance cycled 137

km were compared with 15 women with mean age 28 years and mean distance cycled 123 km). The

black bars represent medians with interquartile ranges and significance was tested with the Wilcoxon

matched-pairs signed rank test. *P<0.05, *** P<0.001, **** P<0.0001.

Suppl. References

1. Hemker HC, Giesen P, Al Dieri R, et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb. 2003; 33: 4-15. 2. Hemker HC, Kremers R. Data management in thrombin generation. Thromb Res. 2013; 131: 3-11.