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Institute of Physiological Chemistry, Medical University of Graz, Graz, Austria; 2Green Beat-Institute of Nutrient Research
and Sport Nutrition, Graz, Austria; and 3Department of Pediatrics, Medical University of Graz, Graz, Austria
Submitted 11 February 2013; accepted in final form 19 April 2013
Lamprecht M, Moussalli H, Ledinski G, Leschnik B, Schlagenhauf A, Koestenberger M, Polt G, Cvirn G. Effects of a single bout
of walking exercise on blood coagulation parameters in obese women.
J Appl Physiol 115: 57 63, 2013. First published April 25, 2013;
doi:10.1152/japplphysiol.00187.2013.Obesity is associated with increased prevalence of thromboembolic events. We aimed to investigate whether obese women might benefit from vigorous aerobic
exercise. Forty-two overweight and obese women performed a 30-min
walking exercise test (treadmill ergometer) at an intensity of 70% of
individual peak oxygen uptake. Blood samples were collected before
and immediately after exercise. Thrombelastometry and platelet function measurements were performed on whole blood. Standard coagulation times, thrombin generation curves, markers of thrombin generation,
fibrinolytic parameters, plasma levels of pro- and anticoagulatory factors,
and microparticle procoagulant activity were determined in platelet-poor
plasma samples. Thrombelastometry revealed a significant prolongation
of clot formation time (P 0.037) and a significant deceleration of
fibrin build up (alpha angle, P 0.034) after exercise. Calibrated
automated thrombography revealed a significant exercise-induced
decrease in endogenous thrombin potential (P 0.039). Moreover,
thrombin formation stopped earlier postexercise, reflected in shortened StartTail (P 0.046). Significantly elevated tissue-plasminogen
activator levels (P 0.001) indicate an exercise-induced activation of
the fibrinolytic system. White blood cell count increased significantly
from pre- to postexercise (P 0.045), indicating a mild exerciseinduced leukocytosis. The results of this study demonstrate that
vigorous aerobic exercise might be a suitable tool to protect obese
women from thrombotic events. We show that a single bout of
vigorous aerobic exercise is clearly associated with an activation of
the fibrinolytic system and a decreased readiness of the postexercise
samples to form a clot and to generate thrombin, the pivotal enzyme
of hemostasis.
coagulation; exercise; obese women; thrombelastometry; thrombin
generation
CARDIOVASCULAR DISEASE
57
58
Age, yr
BMI, kg/m2
Weight, kg
O2, ml
Peak V
O2, ml kg1 min1
Peak V
VT, ml
O2
VT, % of peak V
O2, ml
70% of peakV
O2, % of VT
70% of peakV
Pmax, km/h slope (%)
P683.1% peakVO2, km/h
Clinical chemistry:
Glucose, mmol/l
Hemoglobin, g/l
Iron, mol/l
Ferritin, g/l
Cholesterol, mmol/l
HDL, mmol/l
Triglycerides, mmol/l
Reference Range*
42 Women
41.1 3.8
34.4 4.2
92.0 12.1
2,198 147
25.8 3.6
1,952 145
88.8 7.5
1,539 128
78.9 8.3
6 7 (1.77)
6 0.59
3.96.1
115155
1129
18300
6.35
0.802.35
1.80
5.1 1.5
128 14
16.8 5.7
79.9 57.5
5.57 1.33
1.17 0.28
1.37 0.58
O2, oxygen
Data are presented as means SD. BMI, body mass index; V
uptake; VT, ventilatory threshold; HDL, high-density lipoprotein; Pmax, maximum
O2 expressed via speed.
performance; P683.1% peak VO2, performance at 68% peak V
*See Ref. 40.
women met the inclusion and exclusion criteria and were enrolled in
the study program.
Study design and time schedule. This was a controlled clinical
intervention study. All eligibility testing was finalized 4 wk prior to
the 30-min walking exercise test. On the morning of the exercise test
a standardized breakfast (23 h prior to exercise) was provided. The
women came to the laboratory to perform the 30-min walking exercise
O2. All subjects were
test with an intensity of 70% of individual peak V
checked by a physician before each exercise test. The walking tests
were scheduled between days 10 and 20 of the menstrual cycle.
Incremental exercise tests. As part of eligibility testing each
woman performed an incremental exercise test on a treadmill ergometer (QUASARmed, HP Cosmos Sports and Medical, Traunstein,
Germany) to check heart and circulatory functions and for determi O2. Subjects started at 3 km/h on the treadmill for 3
nation of peak V
min (0% slope). After 3 min, women walked at 3.2 km/h on the first
step for 1 min. Each minute we increased treadmill velocity 0.4 km/h
until 6 km/h. From 6 km/h on we did not increase speed anymore (to
ensure walking technique/coordination) but increased the slope 1%
each minute for women who could maintain performance. Last step
and exhaustion were achieved when subjects were no longer able to
maintain walking speed/performance. A standard electrocardiogram
was recorded during the entire test, which was supervised by a
physician. Blood pressure (RR) was measured at the beginning,
during, and the end of the test.
Respiratory gas exchange and heart rate monitoring. Respiratory
gas exchange (RGE) variables were measured throughout the incremental exercise tests using a breath-by-breath mode (Metalyzer 3B,
Cortex Biophysik, Leipzig, Germany). During these tests, subjects
O2, carbon dioxide output (V
CO2),
breathed through a facemask. V
minute ventilation (VE), breathing rate (BR), and tidal volume (VT)
were continuously obtained. Heart rate (HR) was monitored throughout the tests using a commercially available HR monitor (Polar
Vantage NV, Polar Electro Finland).
Thirty-minute walking exercise test. For the 30-min aerobic walking exercise tests individual walking speed was adjusted to 70% of
O2 for 30 min on the same treadmill ergometer. All
individual peak V
exercise tests were carried out 23 h after a standardized breakfast.
Lamprecht M et al.
time), time to peak (ttPeak), peak height (Peak), and endogenous thrombin potential (ETP), and the time point at which free thrombin has
disappeared (StartTail). Measurements were carried out in the presence of
5 pmol/l of tissue factor (TF) (final concentration).
Markers of thrombin generation. Plasma levels of prothrombin
fragment 12 (F 12) and thrombin-antithrombin complexes (TAT)
were determined using ELISA kits from Behring Diagnostics (Marburg, Germany).
Fibrinolytic values. Plasma levels of tissue-plasminogen activator
(t-PA) and plasminogen activator inhibitor-I (PAI-I) were determined
using the assays IMUBIND tPA ELISA kit and IMUBIND PAI-I
Plasma ELISA, respectively, from American Diagnostica (Pfungstadt,
Germany).
Procoagulatory factors. Plasma levels of prothrombin (FII), FVII,
and FVIII were determined on a BM/Hitachi 917 from Roche (Vienna, Austria). TF was determined by means of the assay ACTICHROME Tissue Factor ELISA from American Diagnostica.
Anticoagulatory factors. Plasma levels of tissue factor pathway
inhibitor (TFPI) were determined using the ACTICHROME Total
TFPI ELISA assay from American Diagnostica.
Microparticle procoagulant activity. Microparticle procoagulant
activity was determined using the functional assay ZYMUPHEN
MP-Activity from HYPHEN BioMed (Neuville, France) on a Microplate Scanning Spectrometer (Bio-Tek Instruments, Winooski, VT).
Plasma samples, supplemented with calcium, FXa and FIIa inhibitors were introduced to microplate wells coated with streptavidin
and biotinylated annexin V. Then FXa-FVa mixtures containing
calcium and purified prothrombin were introduced. Microparticles
bind to annexin V and expose their phospholipid surface, thus
allowing conversion of prothrombin to thrombin. Phospholipid
concentration is the limiting factor. The amount of thrombin
generation, reflecting the phospholipid concentration, is measured
via its specific activity on a chromogenic thrombin substrate.
Absorbance was measured at 405 nm.
Statistical analyses. All statistical analyses were performed using
SPSS for Windows software, version 19.0. Data are presented as
means SD. All data were adjusted for plasma volume changes as
described elsewhere (16). Statistical significance was set at P 0.05.
The Shapiro-Wilk test was used to determine normal distribution and
Levene test for homogeneity of variances. Pre- and postexercise data
were compared by paired Students t-test.
59
Lamprecht M et al.
Table 2. Coagulation values in WB samples prior and after the 30-min aerobic walking exercise test
Preexercise
(Mean SD)
Postexercise
(Mean SD)
6.7 1.8
4.1 0.3
12.6 0.8
38.3 1.8
239 56
7.5 1.9
4.2 0.4
12.9 0.8
39.0 1.8
257 64
382 86
142 31
60.9 3.6
63.2 6.2
398 83
165 47
61.6 4.2
59.8 7.7
322 55b
119 28b
61.3 5b
0.1
0.037
0.1
0.034
133.1 95.1
9.6 4.8
12.1 3.1
131.6 84.4
10.2 4.4
12.9 3.2
58.6 9.7c
8.4 2.0c
13.6 1.4c
0.1
0.1
0.1
12.5 3.9
41.7 16.9
1,853 415
12.8 4.1
38.1 14.0
2,002 512
14.9 2.5d
39.4 5.2d
1,380e
0.1
0.1
0.1
7.0
4.5
13.5
42
250
(4.310.0)a
(3.55.5)a
(1215)a
(3648)a
(125318)a
Significance
(Pre vs. Post)
0.045
0.1
0.072
0.077
0.1
WB, whole blood; WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin; Hct, hematocrit; CT, coagulation time; CFT, clot formation time; MCF,
maximum clot firmness; Pre, preexercise; Post, postexercise. See text for definition of alpha angle. aSee Ref. 21; bsee Ref. 11; csee Ref. 10; dsee Ref. 15; esee
Ref. 25. Boldface indicates significant difference, Pre vs. Post.
J Appl Physiol doi:10.1152/japplphysiol.00187.2013 www.jappl.org
60
Lamprecht M et al.
Table 3. Coagulation values in PPP samples prior and after the 30-min aerobic walking exercise test
Coagulation times
APTT, s
INR
Thrombin generation
Lag time, min
ETP, nmol l1 min1
Peak, nmol/l
Time to peak, min
Starttail, min
Markers of thrombin generation
F1 2, pmol/l
TAT, g/l
Fibrinolytic values
t-PA Ag, ng/ml
PAI-1 Ag, ng/ml
Procoagulatory factors
F II, %
F VII, %
F VIII, %
TF, pg/ml
MP procoagulant activity, nmol/l
Anticoagulatory factor
TFPI, g/l
Preexercise Mean SD
Postexercise Mean SD
Reference Range
35.2 4.0
1
36.1 4.7
1
3040
1
2.07 0.5
1,981 311
479 56
3.8 0.7
19.2 2.0
2.1 0.3
1,840 304
463 59
3.7 0.6
18.4 2.1
3.1 1.4a
1,879 284a
458 60a
0.1
0.039
0.1
0.1
0.046
247.9 97.0
6.26 10.7
268.2 107.0
3.1 1.2
69229b
1.04.1b
0.1
0.064
6.4 2.2
16.5 13.9
8.6 3.1
15.1 11.6
1.48.4c
340c
0.001
0.1
145.1 24.6
143.0 34.9
168
187 59
19.1 13.1
148.1 22.0
142.6 35.2
168
195 64
21.9 15.2
70120
70120
70120
9.2 2.8d
0.1
0.1
0.1
0.1
49.8 17.1
49.5 16.3
60180e
0.1
0.1
0.1
PPP, platelet-poor plasma; APTT, activated partial thromboplastin time; INR, international normalized ratio; ETP, endogenous thrombin potential; F 12,
prothrombin fragment 12; TAT, thrombin-antithrombin complex; t-PA, tissue-plasminogen activator; PAI-1, plasminogen activator inhibitor I; TF, tissue
factor; MP, microparticles; TFPI, tissue factor pathway inhibitor. aSee Ref. 17; baccording to Dade Behring (unpublished observations); csee Ref. 9; dsee Ref.
30; esee Ref. 3. Boldface indicates significant difference, Pre vs. Post.
J Appl Physiol doi:10.1152/japplphysiol.00187.2013 www.jappl.org
mentioned assumption, these women were in a hypercoagulable state compared with healthy, nonobese subjects. Preexercise plasma levels of F 12 were markedly higher than those
of healthy nonobese subjects, indicating elevated formation of
thrombin. Moreover, preexercise plasma levels of FII, FVII,
and FVIII were markedly higher than normal. Elevated plasma
levels of these three coagulation factors have been shown to be
associated with an increased readiness to form thrombin (4, 7,
22). Further evidence of a hypercoagulable state in these obese
women was the low level of TFPI. Low levels of TFPI have been
shown to have a procoagulant effect by facilitating the initiation of
thrombin generation (1, 4). Evidently, these obese women were
apparently in a hypercoagulable state prior to exercise due to a
markedly elevated procoagulant activity of microparticles compared with healthy young men (38). These results emphasize the
importance of an antithrombotic prophylaxis/treatment of obese
women.
The data show that regular vigorous exercise might be a
suitable tool to shift the hemostatic system of obese women
toward an anticoagulant state. 1) The fibrinolytic system was
significantly activated; and both the 2) clot-forming capacity as
well as the 3) thrombin-forming capacity were significantly
decreased in the postexercise samples.
1) Obesity-related atherothrombotic risk has been shown to
be associated with an impaired capability of endothelial cells to
release t-PA, the pivotal enzyme initiating the endogenous
fibrinolytic response (5, 40). The present study showed, in
accordance with others, significantly elevated t-PA levels in
the postexercise PPP samples (29, 36, 45). We conclude that
particularly obese women, known to be at an elevated risk for
thromboembolic complications, might benefit from the exercise-induced high plasma levels of t-PA known to enhance the
efficacy of endogenous fibrinolysis (23).
2) TEM measurements revealed an impaired clot-formation
capacity of the postexercise WB samples. CFTs were significantly prolonged and alpha angles were significantly lower in
the postexercise compared with the preexercise WB samples,
indicating impaired fibrin build up and cross-linking (37).
Presumably, this impaired clot formation is attributable, at least
in part, to the exercise-induced increase of t-PA. Several
groups have reported impaired clot development in the presence of increasing amounts of t-PA. Nielsen et al. (31) have
shown significantly decreased TTP (total thrombus generation)
in the presence of increasing amounts of t-PA. Ploppa et al.
(33) have shown significantly impaired clot formation in a
patient with a massive pulmonary embolism after administration of recombinant t-PA, and DAmico et al. (14) have shown
reduced MCFs and prolonged CTs in the presence of recombinant t-PA (1 g/ml).
3) CAT measurements revealed an impaired thrombin-forming capacity after vigorous exercise. Free thrombin was generated in significantly lower amounts (reflected by lower ETP
values) and disappeared significantly earlier (reflected by
shorter StartTails) in the postexercise PPP samples stimulated
by addition of TF. The underlying mechanisms leading to this
diminished capability of the postexercise PPP samples to
generate thrombin remain to be elucidated. The two most
reasonable explanations, decreased plasma levels of prothrombin and/or decreased plasma levels of TF, do not apply. It has
been shown that thrombin generation dose dependently decreases in the presence of decreasing amounts of prothrombin
Lamprecht M et al.
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DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: M.L. conception and design of research; M.L., H.M.,
G.L., B.L., A.S., M.K., G.P., and G.C. performed experiments; M.L., H.M.,
A.S., and M.K. analyzed data; M.L., H.M., G.P., and G.C. interpreted results
of experiments; M.L. and G.C. drafted manuscript; M.L., G.P., and G.C. edited
and revised manuscript; M.L., H.M., G.L., B.L., A.S., M.K., G.P., and G.C.
approved final version of manuscript; H.M., G.L., and B.L. prepared tables.
REFERENCES
1. Agledahl I, Brodin E, Svartberg J, Hansen JB. Impact of long-term
testosterone treatment on plasma levels of free TFPI and TF-induced
thrombin generation ex vivo in elderly men with low testosterone levels.
Thromb Haemost 102: 945950, 2009.
2. Andrew M, Schmidt B, Mitchell L, Paes B, Ofosu F. Thrombin
generation in newborn plasma is critically dependent on the concentration
of prothrombin. Thromb Haemost 19: 2730, 1990.
2a.Beltan E, Chalabi T, Tripette J, Chout R, Connes P. Coagulation
responses after a submaximal exercise in sickle cell trait carriers. Thromb
Res 127: 167169, 2011.
3. Broze GJ Jr, Girard TJ, Novotny WF. The lipoprotein associated
inhibitor. Prog Hemost Thromb 10: 24368, 1991.
4. Butenas S, vant Veer C, Mann KG. Normal thrombin generation.
Blood 94: 2169 2178, 1999.
5. Caballero AE. Endothelial dysfunction in obesity and insulin resistance:
a road to diabetes and heart disease. Obes Res 11: 1278 1289, 2003.
6. Cerneca E, Simeone R, Bruno G, Gombacci A. Coagulation parameters
in senior athletes practicing endurance sporting activity. J Sports Med
Phys Fitness 45: 576 579, 2005.
7. Christensen TD, Jensen C, Larsen TB, Christiansen K, Srensen B.
Thrombin generation and coagulation factor activities: evaluation and
comparison with the international normalized ratio. Blood Coagul Fibrinolysis 20: 358 365, 2009.
8. Commission of Experts of the German Society of Sports Medicine and
Prevention. Guidelines For Testing in Sports Medicine (German). Level
IV, March, 2002.
9. Cooper JA, Nagelkirk PR, Coughlin AM, Pivarnik JM, Womack CJ.
Temporal changes in t-PA and PAI-1 after maximal exercise. Med Sci
Sports Exerc 36: 1884 1887, 2004.
10. Cvirn G, Gallistl S, Koestenberger M, Kutschera J, Ferstl U, Kellner
J, Jurgens G, Gries A. Effects of beta2-glycoprotein-I on platelet aggregation in cord vs. adult whole blood. Platelets 18: 24 28, 2007.
11. Cvirn G, Gallistl S, Kutschera J, Wagner T, Ferstl U, Jurgens G,
Koestenberger M. Collagen/endogenous thrombin-induced platelet aggregation in whole blood samples. Blood Coagul Fibrinolysis 18: 585
588, 2007.
12. Cvirn G, Gallistl S, Leschnik B, Muntean W. Low tissue factor pathway
inhibitor (TFPI) together with low antithrombin allows sufficient thrombin
generation in neonates. J Thromb Haemost 1: 263268, 2003.
13. Cvirn G, Gallistl S, Muntean W. Alpha-2-macroglobulin inhibits the
anticoagulant action of activated protein C in cord and adult plasma.
Haemostasis 31: 111, 2001.
14. DAmico EA, Chamone DA, Bydlowski SP. In vitro effects of rt-PA on
platelet function and clot strength. Thromb Res 85: 519 522, 1997.
15. Department of Health. At Least Five a Week: Evidence on the Impact of
Physical Activity and Its Relation to Health, edited by the Department of
Health. London, UK, 2004.
16. Dill DB, Costill DL. Calculation of percentage changes in volumes of
blood, plasma, and red cells in dehydration. J Appl Physiol 73: 12651272,
1999.
17. Edwards RL, Rickles FR. The role of leukocytes in the activation of
blood coagulation. Semin Hematol 29: 202212, 1992.
18. El-Sayed MS, Sale C, Jones PG, Chester M. Blood haemostasis in
exercise and training. Med Sci Sports Exerc 32: 918 925, 2000.
19. Freidenreich DJ, Volek JS. Immune responses to resistance exercise.
Exerc Immunol Rev 18: 8 41, 2012.
20. Garber CE, Blissmer B, Deschenes MR, Franklin BA, Lamonte MJ,
Lee IM, Nieman DC, Swain DP. Quantity and quality of exercise for
developing and maintaining cardiorespiratory, musculoskeletal, and neuromotor fitness in apparently healthy adults: guidance for prescribing
exercise. Med Sci Sports Exerc 43: 1334 1339, 2011.
Lamprecht M et al.
Lamprecht M et al.
63
47. Van Veen JJ, Gatt A, Cooper PC, Kitchen S, Bowyer AE, Makris M.
Corn trypsin inhibitor in fluorogenic thrombin-generation measurements is
only necessary at low tissue factor concentrations and influences the
relationship between factor VIII coagulant activity and thrombogram
parameters. Blood Coagul Fibrinolysis 19: 183189, 2008.