Correlation of in V i m and in Viva Decreased Fibrinolytic Activity Caused by Dexamethasone J. J. J. VAN GIEZEN AND J. W. C. M. JANSEN Solpay Duphar B.V: Lkprtment of Vmculrrr PhamacoloBy l?o.Box 900 NG1380 DA W M ~the,Nethwhnh INTRODUCTION The blood fibrinolytic activity is a reflection of anabolic and catabolic processes. Key mediators in the fibrinolytic process are tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1). It has been demonstrated that this process can be modulated by the synthetic glucocorticoid dexamethasone. This substance decreases the fibrinolytic activity measured in cultured medium of several cell types, ea.,rat pituitary cells,' bovine aortic endothelial cells,2 and H K rat hepatoma c e k 3 It appeared that the lowered fibrinolytic activity in cultured medium of H K cells is caused by a strong induction of PAI-1 synthesis while simultaneously a small induction of tPA synthesis was observed. In primary cultures of rat hepatocytes dexamethasone reduced the fibrinolytic activity not only by induction of PAI-1 synthesis but also by decreasing tPA synthesis.4 We showed that oral administration of dexamethasone to rats resulted in a lowered blood clot lysis and a reduced fibrinolytic activity in plasma.5 Based on these results we have studied in depth the effects of dexamethasone treatment in rats in order to elucidate the mechanism of the measured inhibition of the blood fibrinolytic activity.
EXPERIMENTAL PROCEDURES Dexamethasone was administered orally to rats (5 rats per p u p ) once daily for a period of 5 consecutive days in a dose range of 0. 3 to 3.0 mg/kg/day. This dose range inhibited the fibrinolytical activity as measured with the diluted blood clot lysis technique up to 4096.5 The PAI-1 level in plasmas of rats treated with dexamethasone or vehicle was measured with the spectrophotometric rate assay by titration of the plasmas with human tPA.6 Plasminogen activator activity in plasma was determined in 0.5 cm gel slices after SDS-PAGE electrophoresis. The gel slices were incubated firstly in a 2.5% Triton X-100 Tris/HCl buffer (0.1 M; pH = 7.4) fbr one hour at 37OC in order to remove SDS. PA activity was detected by measuring the extinction at 405 nm after addition of plas199
200
ANNALS NEW YORK ACADEMY OF SCIENCES
minogen, stimulator (CNBrfragments ofhuman fibrinogen) and the chromogenic substrate S2251. Because of SDS-’ton treatment of the gel slices it is possible to detect free plasminogen activator activity and plasminogen activator activity derived from its complex with inhibitor. The 1251-fibrincoated aorta loop method as described previously7was used to measure directly in PiPo the fibrinolytic activity. This model is based on the ability of the fibrinolytic system to remove a labeled fibrin layer coated to a poly-ethylene loop (i.d. 0.9 mm) which is placed in the abdominal aorta of rats. The rate of label disappearance from the loop in percentage per minute was used as a measure of the fibrinolytic activity of the blood.
RESULTS Dexamethasone treatment resulted in a dose-dependent increase of the PAI-1 level in plasma. Control plasma contained a PAI-1 level of 5.6 f 0.9 IU/ml which increased to 8.4 f 4.0, 13.0 f 1.8 and 15.3 f 2.6 IU/ml (fSD)for plasma from rats treated with resp. 0.3, 1.0, and 3.0 mgkg dexamethasone. The effects on plasma PA activity were determined by subjecting the plasmas to electrophoresis subsequently followed by measuring the PA activity in the gel. FIGURE1 shows a typical PA activity pattern after incubation of the gel slices for 18 h at 4 O C . It appeared that dexamethasone treatment reduced both the peak representing free tPA activity (Rf= 0.69) and the peak representing tPA activity in complex with PAI-1 (Rf= 0.31) by about 40%at a dose of 3 mgkg. In order to establish the RY vh measured effects of dexamethasone, in PiPo experiments were performed using the lZ5I-fibrincoated aorta loop. It appeared that the rate of label disappearance as a measure for the blood fibrinolytic activity, was inhibited
FIGURE 1. The extinction
0.00
pattern at 405 nm (E405)of PAgel slices after incubation in the presence of plasminogen, stimulator and S2251 (representative for three experiments).The plasminogen activator pattern of plasma obtained from vehicle(+) and dexamethasone(---+---) treated rats is shown. The Rf = 0.69 peak represents tPA activity and the Rf= 0.31 peak is derived from the tPA/PAI complex. The tPA activity in plasma of dexamethasone treated rats was inhibited by 39 f 11% compared to plasma from vehicle treated rats. The tPA/PAI activity was inhibited by 53 f 14% (aU values are mean f SEM; n = 3).
I
o.00
0.26
0.50
0.78
1.00
VAN
GIEZEN & JANSEN: DEXAMETHASONE AND PIBRINOLYSIS
201
by 32 f 7% (n = 3; fSEM) and 33 f 4%(n = 10; fSEM) owing to dexamethasone treatment at doses of resp. 1 and 3 mg/kg. This result correlates well with the increase in PAI-1 levels caused by these doses.
DISCUSSION AND CONCLUSION These results confirm previous observations that dexamethasone reduced the fibrinolytic activity in rats. Based on the lowered tPA activity measured a t Rf= 0.31 (complex peak) it is suggested that the tPA level in plasma from dexamethasone treated rats is reduced. Plasma titration experiments with tPA clearly showed that plasma PAI-1 levels were increased after dexamethasone treatment. Both effects may act together to diminish the fibrinolytic activity. The in Pivo measured inhibition of the fibrinolytic activity caused by dexamethasone correlates well with ex Pivo measured activities.
SUMMARY The effect of dexamethasone administration to rats was studied o n blood fibrinolytic activity. PAI-1 levels were dose-dependently enhanced by dexamethasone after a pretreatment period of 5 days while simultaneously a decreased tPA level was observed. with a specially developed fibriThese ex mV0 measured effects were confirmed in nolysis model. It is concluded that the in mV0 measured inhibition of the fibrinolytic activity caused by dexamethasone correlates well with ex Pivo measured activities. REFERENCES 1 . GRANELLI-PIPERNO, A. & E. REICH. 1988. J. Cell Biol. 97: 1029-1037. 2. LOSKUTOFF, D. J . , K. ROEGNER, L. A. ERICKSON, R R SCHLEEF,A. HUTTENLOCHEX, P. L. COLEMAN & T. D. GELEHRTER. 1986. Thrornb. Haernostasis 55: 8-11. T. D., R SZNYCER-LASUK, R ZEHEB& B. J. CWICKEL. 1987. Mol. Endo3. GELEHRTER, crinol. 1: 97-101. 4. HEATON, J . H., V. L. NEBES,L. G. ODELL,S. M. M o m s & T. D. GELEHRTER. 1989. Mol. Endocrind. 3: 185-192. 5. JANSEN, J. W. C. M. & J . J . J. VAN GIEZEN. 1989. Thrornb. Haernostasis 62: 475. J . H., E. MULLMRT, G. T. C. CHANG,C. KLUFI. & G. WIJNGAARDS. 1982. 6. VERHEIJEN, Thrornb. Haernostasis 48: 266-269. 7. JANSEN, J. W. C. M. 1986. Fibrinolysis (Suppl): 223.
EXPERIMENTAL PROCEDURES Dexamethasone was administered orally to rats (5 rats per p u p ) once daily for a period of 5 consecutive days in a dose range of 0. 3 to 3.0 mg/kg/day. This dose range inhibited the fibrinolytical activity as measured with the diluted blood clot lysis technique up to 4096.5 The PAI-1 level in plasmas of rats treated with dexamethasone or vehicle was measured with the spectrophotometric rate assay by titration of the plasmas with human tPA.6 Plasminogen activator activity in plasma was determined in 0.5 cm gel slices after SDS-PAGE electrophoresis. The gel slices were incubated firstly in a 2.5% Triton X-100 Tris/HCl buffer (0.1 M; pH = 7.4) fbr one hour at 37OC in order to remove SDS. PA activity was detected by measuring the extinction at 405 nm after addition of plas199
200
ANNALS NEW YORK ACADEMY OF SCIENCES
minogen, stimulator (CNBrfragments ofhuman fibrinogen) and the chromogenic substrate S2251. Because of SDS-’ton treatment of the gel slices it is possible to detect free plasminogen activator activity and plasminogen activator activity derived from its complex with inhibitor. The 1251-fibrincoated aorta loop method as described previously7was used to measure directly in PiPo the fibrinolytic activity. This model is based on the ability of the fibrinolytic system to remove a labeled fibrin layer coated to a poly-ethylene loop (i.d. 0.9 mm) which is placed in the abdominal aorta of rats. The rate of label disappearance from the loop in percentage per minute was used as a measure of the fibrinolytic activity of the blood.
RESULTS Dexamethasone treatment resulted in a dose-dependent increase of the PAI-1 level in plasma. Control plasma contained a PAI-1 level of 5.6 f 0.9 IU/ml which increased to 8.4 f 4.0, 13.0 f 1.8 and 15.3 f 2.6 IU/ml (fSD)for plasma from rats treated with resp. 0.3, 1.0, and 3.0 mgkg dexamethasone. The effects on plasma PA activity were determined by subjecting the plasmas to electrophoresis subsequently followed by measuring the PA activity in the gel. FIGURE1 shows a typical PA activity pattern after incubation of the gel slices for 18 h at 4 O C . It appeared that dexamethasone treatment reduced both the peak representing free tPA activity (Rf= 0.69) and the peak representing tPA activity in complex with PAI-1 (Rf= 0.31) by about 40%at a dose of 3 mgkg. In order to establish the RY vh measured effects of dexamethasone, in PiPo experiments were performed using the lZ5I-fibrincoated aorta loop. It appeared that the rate of label disappearance as a measure for the blood fibrinolytic activity, was inhibited
FIGURE 1. The extinction
0.00
pattern at 405 nm (E405)of PAgel slices after incubation in the presence of plasminogen, stimulator and S2251 (representative for three experiments).The plasminogen activator pattern of plasma obtained from vehicle(+) and dexamethasone(---+---) treated rats is shown. The Rf = 0.69 peak represents tPA activity and the Rf= 0.31 peak is derived from the tPA/PAI complex. The tPA activity in plasma of dexamethasone treated rats was inhibited by 39 f 11% compared to plasma from vehicle treated rats. The tPA/PAI activity was inhibited by 53 f 14% (aU values are mean f SEM; n = 3).
I
o.00
0.26
0.50
0.78
1.00
VAN
GIEZEN & JANSEN: DEXAMETHASONE AND PIBRINOLYSIS
201
by 32 f 7% (n = 3; fSEM) and 33 f 4%(n = 10; fSEM) owing to dexamethasone treatment at doses of resp. 1 and 3 mg/kg. This result correlates well with the increase in PAI-1 levels caused by these doses.
DISCUSSION AND CONCLUSION These results confirm previous observations that dexamethasone reduced the fibrinolytic activity in rats. Based on the lowered tPA activity measured a t Rf= 0.31 (complex peak) it is suggested that the tPA level in plasma from dexamethasone treated rats is reduced. Plasma titration experiments with tPA clearly showed that plasma PAI-1 levels were increased after dexamethasone treatment. Both effects may act together to diminish the fibrinolytic activity. The in Pivo measured inhibition of the fibrinolytic activity caused by dexamethasone correlates well with ex Pivo measured activities.
SUMMARY The effect of dexamethasone administration to rats was studied o n blood fibrinolytic activity. PAI-1 levels were dose-dependently enhanced by dexamethasone after a pretreatment period of 5 days while simultaneously a decreased tPA level was observed. with a specially developed fibriThese ex mV0 measured effects were confirmed in nolysis model. It is concluded that the in mV0 measured inhibition of the fibrinolytic activity caused by dexamethasone correlates well with ex Pivo measured activities. REFERENCES 1 . GRANELLI-PIPERNO, A. & E. REICH. 1988. J. Cell Biol. 97: 1029-1037. 2. LOSKUTOFF, D. J . , K. ROEGNER, L. A. ERICKSON, R R SCHLEEF,A. HUTTENLOCHEX, P. L. COLEMAN & T. D. GELEHRTER. 1986. Thrornb. Haernostasis 55: 8-11. T. D., R SZNYCER-LASUK, R ZEHEB& B. J. CWICKEL. 1987. Mol. Endo3. GELEHRTER, crinol. 1: 97-101. 4. HEATON, J . H., V. L. NEBES,L. G. ODELL,S. M. M o m s & T. D. GELEHRTER. 1989. Mol. Endocrind. 3: 185-192. 5. JANSEN, J. W. C. M. & J . J . J. VAN GIEZEN. 1989. Thrornb. Haernostasis 62: 475. J . H., E. MULLMRT, G. T. C. CHANG,C. KLUFI. & G. WIJNGAARDS. 1982. 6. VERHEIJEN, Thrornb. Haernostasis 48: 266-269. 7. JANSEN, J. W. C. M. 1986. Fibrinolysis (Suppl): 223.
