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Tissue-type plasminogen activator and plasminogen activator inhibitor-1 after exercise: comparison to venous occlusion and DDAVP
Fibrinolysir (1990) 4. Supp: 2.105-107 63 Longman Group UK Ltd 1990
cm%-9499/90/0004-0105/$10.00
Tissue-type Plasminogen Activator and Plasminogen Activator Inhibitor-l after Exercise: Comparison to Venous Occlusion and DDAVP
N. Vene, M. Stegnar
SUMMARY.Nineteen healthy young men were subjected randomly to maximal exercise on treadmill, venous occlusion (VO) and infusion of DDAVP on three different mornings. After exercise t-PA antigen increased by 2.8 fold, after VO by 5.1 fold and after DDAVP by 2.4 fold (all PcO.01). PAIantigen did not change significantly after any of the stimuli. Therefore, increases in t-PA activity and decreases in PAI-1 activity observed after stimulation were attributed to releasedt-PA antigen only: increase in t-PA activity after exercise was 2.2 fold lower than that after VO and 1.8 fold higher than after DDAVP. No PAI- activity could be detected in 8/19 samples after exercise, in lYl9 samples alter VO and in 5/l9 after DDAVP. Positive correlations were observed for t-PA antigen release between the three stimuli (VO-DDAVP: rz0.78, PcO.01; exercise-DDAVP: rz0.53, P-zO.05;VO-exercise: r=0.30, not significant). It was concluded that maximal exercise as a stimulus of t-PA release is less effective than VO and similar to DDAVP. Divergent alterations of t-PA and PAI- antigens afker stimulation argue against a common regulation of their acute releasefrom tissue stores. KEYWORDS.t-PA. PAI-1. Physical exercise. Venous occlusion. DDAVP The observationthat exerciseincreasesblood fibrinolytic activity is much older than the observationon increased fibrinolytic activity after venous occlusion (VO) and DDAVP. In spite of this, physical exerciseis used less frequently as a stimulus for acute t-PA releaseprobably because it is difficult to standardize and carry out, specially in patients. Therefore, comparative data on t-PA releaseby different stimuli in the samesubjectsare scarcelW3. Due to recent methodologyfor PAI- antigen4 even less is known about its changesafter stimulation of fibrinolytic activity. So it seemedreasonableto study levels of t-PA and PAI- during physical exercise, in comparisonto VO and DDAVP, in order to establishthe magnitude of their changes and possible correlations betweenthem Additionally, the study was conducted in order to eludicate possible common mechanismsof acute t-PA and PAI- 1 responsesto stimulation. SUBJECTS AND METHODS Nineteen young men, 19 to 28 (24k3) years old were included in the study. In a randommanner 3 stimuli were performed on separatedays: exerciseas a continuoustest on a treadmill according to the Bruce protocol until maximal age-predictedheart rate was reached; 20 min N. Vene, M. Stegnar, University Clinical Centre, Tmovo Hospital of Internal Medicine, 6100 Ljubljana, Yugoslavia 105
VO was carried out at midpoint between systolic and diastolic blood pressure; DDAVP (0.4 pg/kg body weight) was injected intravenously over 10 min. Blood sampleswere taken before each stimulus, immediately after exercise,in the last minute of VO and 20 min after the start of DDAVP infusion. PAI- antigen was determined by ELISA using Tintelize PAI- Kit from Biopoo14.Methods for t-PA antigen, t-PA activity, global fibrinolytic activity with euglobulin clot lysis time (ECLT) and PAl-1 activity are describedelsewhere’. RESULTS No significant differencesin resting levels of fibrinolytic parameterswere found before eachtest. After exercise t-PA antigen increased by 2.8 fold comparedwith 5.1 fold increaseafter VO and 2.4 fold increaseafter DDAVP (Fig. 1). Since PAI- antigen did not change significantly after any of the three tests (exercise 9% decrease,VO 11% increase and DDAVP 8% decrease)(Fig. 2), changesin t-PA activity and in ECLT were attributed to increasesin t-PA antigen. ECLT increasedby 4.2 fold after exercise compared to 10.1 fold increaseafter VO and 6.0 fold increaseafter DDAVP. Increaseof t-PA activity after exercisewas 2.2 fold lower that after VO and 1.8 fold higher than after DDAVP. PAI- activity fell after stimulation and was
106
PA & PAT-1 After Exercise: Venous Occl. & DDAVP
E
w
DDAVP
60-
Fig. 1 t-PA antigen concentration before (b) and after (a) exercise (E), venous occlusion (VO) and DDAVP. Shaded bars represent medians.
DDAVP
E 60
50
40
30
20
1C
C Fig. 2 PAI-
b
a
b
a
b
a
antigen concentration before (b) and after (a) exercise (E), venous occlusion (VO) and DDAVP. Shaded bars represent medians.
undetectableafter exercise in 8 (42%), after VO in 15 (79%) and after DDAVP in 5 (26%) of the subjects. Correlation was found between an increase in t-PA antigen after VO and DDAVP (1=+0.78 P
RO.01) and between increase after VO and DDAVP (r-+0.30, not significant). Significant correlations were found between increases in global fibrinolytic activity (ECLT) and t-PA activity after all tests (from 1=+0.41to 1==+0.84, fromP
Fibrinolysis
DISCUSSION Our study has shown that maximal exercise in young men produced t-PA release which was comparableto t-PA release after DDAVP, but was approximately two times lower than t-PA releaseafter VO. Theserers;lts are in agreement with previous observations’ . No significant change in PAI- antigen level was found either after exercise or after the other two stimuli with an immunoassaywhich detects with equal sensitivity both free (active) PAI- and PAL1 complexed with t-PA and is thus measuringtotal PAI- in plasma.However, fall in PAI- activity after stimulation indicated that free PAIwas consumed by complexing with released t-PA antigen. Increase in t-PA antigen without concomitant change of PAI- antigen resulted in increasein plasma t-PA activity and global euglobulin activity. Divergent changes of both antigens (increase in t-PA and no change in PAI-1) observed after all three stimuli suggested that their levels in blood are presumably regulatedin different ways. Since the mechanismsof t-PA antigen release after various stimuli are mostly not understood, the explanation of the correlations betweenthe three stimuli observed in this study is highly speculative. However, these correlations m ight suggest that the level of basal t-PA production and release from the endothelial cells could be the common denominatorfor all stimuli, if the following hypotheseshold true: increasein t-PA durin VO results from local accumulation of basal t-PA f!, DDAVP acting via cerebral hormone increases t-PA proportionaly to basal t-PA release and increase after exercise results from flushing of basal t-PA into circulation from previously closed vessels in exercising muscles7. Significant correlations between increases in fibrinolytic activity after different sstsimuhhave already been pzbservedby some authors ’ , but not by the others ’ . However, these correlations were calculated mostly for global fibrinolytic activity and t-PA activity,
107
since measurementsof t-PA antigen were not performed in all of thesestudies.Therefore,they probably reflect to some degree correlations in t-PA release after stimulation and to some pm-stimulation PAT-1 levels influencing post-stimulationt-PA and global fibrinolytic activity. ACKNOWLEDGEMENTS This work was done within the framework of ECAT (European Contented Action on Thombosis and Disability) and was supported by Slovenian Research Community Grant No (33-0564326.
REFERENCES 1. Pmwse CV, Farrugia A, Boulton FER et al. 1984 A comparative study using immunological and biological assays of haemostatic responses to DDAVP infusion, venous occlusion and exercise in normal men. Thromb Haemostas 51: 110-l 14 2. Stegnar M, Keber I, Keber D, Petemel P 1987 Fibrinolytic potential in healthy men and patients with tbrombotic disease. Period Bio189: 95-104 3. Smith P, Dahl KH, Holst JK, Nilsen DWT, Opstad T, Andersen P 1986 Fibrinolytic activity following venous occlusion and intravenously admistered DDAVP in healthy subjects. Fibrinolysis, Eight international congress on fibrinolysis: 75 4. Declerek PJ, Alessi MC, Verstreken M, Kruithof EKO, Juhan-Vague I, Collen D 1988 Measurement of plasminogen activator inhibitor 1 in biologic fluids with murine monoclonal antibody-based enzyme-linked immunosorbent assay. Blood 71: 220-22s 5. Stegnar M, Keber D, Petemel P,Khift C 1988 Diminished release of tissue plasminogen activator during venous occlusion in manual workers. Fibrinolysis 2: 175-179 6. Keber D 1988 Mechanism of tissue plasminogen activator release during venous occlusion. Fibrinolysis 3 (Suppl2): 96-103. 7. Cash JD 1978 Control mechanism of activator release. In Davidson JF, Rowan R, Samama MM, Desnoyers PC (ed) Progress in chemical fibrinolysis and thrombolysis ~013, Raven Press, New York p 65-75 8. Nilsson IM 1977 Plasminogen activator release assays. Acta Clin Belgica 32: 391-397
cm%-9499/90/0004-0105/$10.00
Tissue-type Plasminogen Activator and Plasminogen Activator Inhibitor-l after Exercise: Comparison to Venous Occlusion and DDAVP
N. Vene, M. Stegnar
SUMMARY.Nineteen healthy young men were subjected randomly to maximal exercise on treadmill, venous occlusion (VO) and infusion of DDAVP on three different mornings. After exercise t-PA antigen increased by 2.8 fold, after VO by 5.1 fold and after DDAVP by 2.4 fold (all PcO.01). PAIantigen did not change significantly after any of the stimuli. Therefore, increases in t-PA activity and decreases in PAI-1 activity observed after stimulation were attributed to releasedt-PA antigen only: increase in t-PA activity after exercise was 2.2 fold lower than that after VO and 1.8 fold higher than after DDAVP. No PAI- activity could be detected in 8/19 samples after exercise, in lYl9 samples alter VO and in 5/l9 after DDAVP. Positive correlations were observed for t-PA antigen release between the three stimuli (VO-DDAVP: rz0.78, PcO.01; exercise-DDAVP: rz0.53, P-zO.05;VO-exercise: r=0.30, not significant). It was concluded that maximal exercise as a stimulus of t-PA release is less effective than VO and similar to DDAVP. Divergent alterations of t-PA and PAI- antigens afker stimulation argue against a common regulation of their acute releasefrom tissue stores. KEYWORDS.t-PA. PAI-1. Physical exercise. Venous occlusion. DDAVP The observationthat exerciseincreasesblood fibrinolytic activity is much older than the observationon increased fibrinolytic activity after venous occlusion (VO) and DDAVP. In spite of this, physical exerciseis used less frequently as a stimulus for acute t-PA releaseprobably because it is difficult to standardize and carry out, specially in patients. Therefore, comparative data on t-PA releaseby different stimuli in the samesubjectsare scarcelW3. Due to recent methodologyfor PAI- antigen4 even less is known about its changesafter stimulation of fibrinolytic activity. So it seemedreasonableto study levels of t-PA and PAI- during physical exercise, in comparisonto VO and DDAVP, in order to establishthe magnitude of their changes and possible correlations betweenthem Additionally, the study was conducted in order to eludicate possible common mechanismsof acute t-PA and PAI- 1 responsesto stimulation. SUBJECTS AND METHODS Nineteen young men, 19 to 28 (24k3) years old were included in the study. In a randommanner 3 stimuli were performed on separatedays: exerciseas a continuoustest on a treadmill according to the Bruce protocol until maximal age-predictedheart rate was reached; 20 min N. Vene, M. Stegnar, University Clinical Centre, Tmovo Hospital of Internal Medicine, 6100 Ljubljana, Yugoslavia 105
VO was carried out at midpoint between systolic and diastolic blood pressure; DDAVP (0.4 pg/kg body weight) was injected intravenously over 10 min. Blood sampleswere taken before each stimulus, immediately after exercise,in the last minute of VO and 20 min after the start of DDAVP infusion. PAI- antigen was determined by ELISA using Tintelize PAI- Kit from Biopoo14.Methods for t-PA antigen, t-PA activity, global fibrinolytic activity with euglobulin clot lysis time (ECLT) and PAl-1 activity are describedelsewhere’. RESULTS No significant differencesin resting levels of fibrinolytic parameterswere found before eachtest. After exercise t-PA antigen increased by 2.8 fold comparedwith 5.1 fold increaseafter VO and 2.4 fold increaseafter DDAVP (Fig. 1). Since PAI- antigen did not change significantly after any of the three tests (exercise 9% decrease,VO 11% increase and DDAVP 8% decrease)(Fig. 2), changesin t-PA activity and in ECLT were attributed to increasesin t-PA antigen. ECLT increasedby 4.2 fold after exercise compared to 10.1 fold increaseafter VO and 6.0 fold increaseafter DDAVP. Increaseof t-PA activity after exercisewas 2.2 fold lower that after VO and 1.8 fold higher than after DDAVP. PAI- activity fell after stimulation and was
106
PA & PAT-1 After Exercise: Venous Occl. & DDAVP
E
w
DDAVP
60-
Fig. 1 t-PA antigen concentration before (b) and after (a) exercise (E), venous occlusion (VO) and DDAVP. Shaded bars represent medians.
DDAVP
E 60
50
40
30
20
1C
C Fig. 2 PAI-
b
a
b
a
b
a
antigen concentration before (b) and after (a) exercise (E), venous occlusion (VO) and DDAVP. Shaded bars represent medians.
undetectableafter exercise in 8 (42%), after VO in 15 (79%) and after DDAVP in 5 (26%) of the subjects. Correlation was found between an increase in t-PA antigen after VO and DDAVP (1=+0.78 P
RO.01) and between increase after VO and DDAVP (r-+0.30, not significant). Significant correlations were found between increases in global fibrinolytic activity (ECLT) and t-PA activity after all tests (from 1=+0.41to 1==+0.84, fromP
Fibrinolysis
DISCUSSION Our study has shown that maximal exercise in young men produced t-PA release which was comparableto t-PA release after DDAVP, but was approximately two times lower than t-PA releaseafter VO. Theserers;lts are in agreement with previous observations’ . No significant change in PAI- antigen level was found either after exercise or after the other two stimuli with an immunoassaywhich detects with equal sensitivity both free (active) PAI- and PAL1 complexed with t-PA and is thus measuringtotal PAI- in plasma.However, fall in PAI- activity after stimulation indicated that free PAIwas consumed by complexing with released t-PA antigen. Increase in t-PA antigen without concomitant change of PAI- antigen resulted in increasein plasma t-PA activity and global euglobulin activity. Divergent changes of both antigens (increase in t-PA and no change in PAI-1) observed after all three stimuli suggested that their levels in blood are presumably regulatedin different ways. Since the mechanismsof t-PA antigen release after various stimuli are mostly not understood, the explanation of the correlations betweenthe three stimuli observed in this study is highly speculative. However, these correlations m ight suggest that the level of basal t-PA production and release from the endothelial cells could be the common denominatorfor all stimuli, if the following hypotheseshold true: increasein t-PA durin VO results from local accumulation of basal t-PA f!, DDAVP acting via cerebral hormone increases t-PA proportionaly to basal t-PA release and increase after exercise results from flushing of basal t-PA into circulation from previously closed vessels in exercising muscles7. Significant correlations between increases in fibrinolytic activity after different sstsimuhhave already been pzbservedby some authors ’ , but not by the others ’ . However, these correlations were calculated mostly for global fibrinolytic activity and t-PA activity,
107
since measurementsof t-PA antigen were not performed in all of thesestudies.Therefore,they probably reflect to some degree correlations in t-PA release after stimulation and to some pm-stimulation PAT-1 levels influencing post-stimulationt-PA and global fibrinolytic activity. ACKNOWLEDGEMENTS This work was done within the framework of ECAT (European Contented Action on Thombosis and Disability) and was supported by Slovenian Research Community Grant No (33-0564326.
REFERENCES 1. Pmwse CV, Farrugia A, Boulton FER et al. 1984 A comparative study using immunological and biological assays of haemostatic responses to DDAVP infusion, venous occlusion and exercise in normal men. Thromb Haemostas 51: 110-l 14 2. Stegnar M, Keber I, Keber D, Petemel P 1987 Fibrinolytic potential in healthy men and patients with tbrombotic disease. Period Bio189: 95-104 3. Smith P, Dahl KH, Holst JK, Nilsen DWT, Opstad T, Andersen P 1986 Fibrinolytic activity following venous occlusion and intravenously admistered DDAVP in healthy subjects. Fibrinolysis, Eight international congress on fibrinolysis: 75 4. Declerek PJ, Alessi MC, Verstreken M, Kruithof EKO, Juhan-Vague I, Collen D 1988 Measurement of plasminogen activator inhibitor 1 in biologic fluids with murine monoclonal antibody-based enzyme-linked immunosorbent assay. Blood 71: 220-22s 5. Stegnar M, Keber D, Petemel P,Khift C 1988 Diminished release of tissue plasminogen activator during venous occlusion in manual workers. Fibrinolysis 2: 175-179 6. Keber D 1988 Mechanism of tissue plasminogen activator release during venous occlusion. Fibrinolysis 3 (Suppl2): 96-103. 7. Cash JD 1978 Control mechanism of activator release. In Davidson JF, Rowan R, Samama MM, Desnoyers PC (ed) Progress in chemical fibrinolysis and thrombolysis ~013, Raven Press, New York p 65-75 8. Nilsson IM 1977 Plasminogen activator release assays. Acta Clin Belgica 32: 391-397