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The effect of prostacyclin infusion on tissue plasminogen activator
THROMBOSIS RESEARCH 46; 741-745, 1987 0049-3848/87 $3.00 t .OO Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.
BRIEF
COMMUNICATION
THE EFFECT OF PROSTACYCLIN INFUSION ON TISSUE PLASMINOGEN ACTIVATOR. K Winther", K Snorrason***$B Knudsen*, and S Medgyesi . The Coagula$ion Laboratory, Department of Clinical Chemistry and Department of Plastic Surgery**, Rigshospitalet, Copenhagen, Denmark. (Received
29.10.1986; Accepted in original form 7.1.1987
by Editor B. Wiman) (Received in final form by Executive Editorial Office 13.3.1987)
INTRODUCTION Intravenous infusion of prostacyclin (PGI2) inhibits platelet aggregation and elevates the intracellular concentration of cyclic-AMP in platelets in vitro and in vivo (1,2). The infusion of prostacyclin also enhances the fibrinolytic activity measured as shortening of the euglobulin clot lysis time (ECLT) and results in spontaneously healing and relief of pain in patients suffering from ulceration of the lower limb (3,410 The ECLT is dependent on the conversion .ofplasminogen to plasmin regulated among other factors by the tissue plasminogen activator (t-PA) from endothelial cells. In vitro investigation shows that addition of cyclic-AMP to human endothelial cells in culture enhances production of t-PA after incubation for 24 h (5). This report aims to investigate the in vivo effect of i.v. prostacyclin infusion on ECLT as well as t-PA production. MATERIALS AND METHODS The patient presented in this report was a 54 years old female suffering from leg ulcers treated with prostacyclin infusion on three occasions in order to heal the ulcerations (3). Pre-dose testing showed normal ECLT and t-PA activity before and after standardized venous stasis, without indications of t-PA inhibitor. Key
Words:
Prostacyclin, activator.
fibrinolysis, tissue
741
plasminogen
742
PROSTACYCLIN, FIBRINOLYSIS, t-PA
Vol. 46, No. 5
Methods: During the study the patient was placed in the supine position. Infusions of prostacyclin (epoprostenol FLOLAN, Wellcome) were started each time between 9.00 and 11.00 a.m. with an initial dose rate of 2.5 ng/kg body weight/minute. After 60 minutes the infusion rate was increased to 5.0 ng/kg body weight/minute for one hour and continued for the following 22 hours at 7.5 ng/kg body weight/minute. The treatment was stopped after a total infusion period of 24 hours. No infusion was performed the following 18 days. Laboratory tests: Prior to prostacyclin infusion and after 1, 5 and 24 hours and again after 3, 7, 12 and 15 days, the following biochemical parameters were measured: Plasma 6~~c'-~G~~ al ha (a stable metabolite of prostacyclin), ECLT -. Plasma 6-keto-PGFl was measured as described by Parry using a double anti o y procedure (61, ECLT was akpga measured using conventional methods. All reagents for the t-PA assay were produced as described ;;ng;9"" (7). Tissue plasminogen activator was used in it's chain form with a specific activity of about 200,000 IU/mg when compared to an international standard (WHO International Laboratory for Biological Standards, Holly Hill, London, UK). Solubilized des A fibrin was prepared by adding bathroxobin (10 pl, 250 Bu/ml) to fibrinogen (5 ml, 6 FM fibrinogen). After 6 hours incubation at 20°C the performed gel was dissolved in 5 ml 7 M urea. With minimum stasis 4.5 ml of venous blood was drawn into tubes, each containing 0.5 ml sodium citrate 3.13 %. For determination of t-PA the plasma was acidified immediately after separation of the blood cells by adding an equal volume of 1 M sodium acetate buffer, pH 3.9 and pH was then adjusted to 4.1. The samples were snap frozen and stored at -8OOC until analyzed for t-PA activity. The acidified plasma samples were diluted l/50 - l/100 with 0.05 mol/l tris-HCl buffer, pH 8.8, containing 0.10 mol/l NaCl and 0.1 g/l triton X-100. An equal volume of this buffer containing 0.1 mg plasminogen/ml (KABI, Sweden) and 0.6 mmol/l chromogenic substrate (S-2251) (KABI, Sweden) was added, followed by addition of solubilized fibrin (final concentration 0.07 g/l) as stimulator. After incubation overnight (18 h) at 20°C the absorbance was measured at 405 nm. RESULTS The infusion of prostacyclin resulted in a 280% increase in the plasma 6-keto-PGFl alpha level after 60 minutes, a 520% increase after 5 hours and a 730% increase after 24 hours of treatment. Three days after termination of infusion the plasma level of 6-keto-PGFl alpha had reached pre-treatment values (Fig. 1).
743
PROSTACYCLIN, FIBRINOLYSIS, t-PA
Vol. 46, No. 5
Parallel to the increase in plasma 6-keto-PGFl level a decrease in ECLT of 154 minutes was observed aft%p 91 hours of infusion. Following termination of treatment the ECLT level slowly increased with approximately 30 min per week the following 3 weeks (Fig. 2). In agreement with the decrease in ECLT, indicating an enhanced fibrinolytic activity, the t-PA concentration increased from 0,023 to 0,072 IU/ml 24 hours after infusion. One week after treatment the plasma t-PA had returned to pre-treatment levels (Fig. 2).
0
l
2
5
24h
w-c3
7
12
15days
Figure 1. The squared area (left panel) shows the infusion of prostacycline during 24 hours. The right panel shows the level of plasma 6-keto-PGFl ilpha after 1, 5 and 24 hours of prostaagain 3, 7, 12 and 15 days after treatcycline infusion an ment (one of three representative experiments).
PROSTACYCLIN, FIBRINOLYSIS, t-PA
744
Vol. 46, No. 5
.O.lO -i -0.08 ‘= H .0.06
2 L
-0.04
.O.O2
0 -
.
I-----
1
5
INFUSION
OF
12
24h
3
7
T-
15 days
PROSTACYCLINE -
Figure 2. Left panel (x) shows the euglobulin clot lysis time (ECLT) before, during and after the infusion of prostacyclin. The right panel (01 shows the t-PA concentration before, during and after treatment with prostacyclin (one of three representative experiments). Some spontaneous healing of the leg ulcer was observed after each of the three treatments. After two weeks, however, the beneficial effect disappeared and the ulcer started to progress again. DISCUSSION No increase in t-PA was observed after l-5 hours of prostacycline infusion, although, a 280% and 730% increase in plasma 6-keto-PGFl al ha level was observed. This might indicate that the effeca of prostacyclin is not related to an increased liberation of pre-formed t-PA from the endothelial cells as observed during exercise or after venous compression (7). The present data suggests that infusion of prostacyclin enhances the fibrinolytic activity by increasing the production of tissue plasminogen activator (t-PA). This hypothesis fits very well with the observation that the addition of cyclic-AMP to cultureq endothelial cells increases the production of t-PA two-threefold if incubation is continued for 24 hours. (5) Several reports indicate that prostacyclin infusion elevates cyclic-AMP in various cell types (1,2,8). Our data might reflect an increased synthesis by endothelial cells of tissue plasminogen activator secondary to an enhanced cyclic-AMP production induced by prostacyclin infusion.
Vol. 46, No. 5
PROSTACYCLIN, FIBRINOLYSIS, t-PA
745
In conclusion: the infusion of prostacyclin has earlier been reported to shorten the ECLT. This study confirms these data and suggests that the shortening of the ECLT is related to an enhanced production of tissue plasminogen activator. ACKNOWLEDGEMENTS We are very grateful to Miss Janne Olsen for excellent technical assistance and to Miss Ingrid Kryhlmand for typing the manuscript. REFERENCES 1.
TATESON, J. B., MONCADA, S., and VANE, J. R. Effects of Prostacyclin (PGX) on cyclic-AMP concentrations in human platelets. Prostaglandins, 2, 389-395, 1977.
2.
DATA, J. L., MOLONY, B. A, MEINZINGER, M. M., and GORMAN, R. R. Intravenous infusion of prostacyclin sodium in man: Clinical effects and influence on platelet adenosine diphosphate sensivity and adenosine 3':5'-cyclic monophosphate levels. Circulation, 64, 4-11, 1981.
3.
GRYGLEWSKI, R. J. Prostacyclin-Experimental and Clinical in Prostaglandin, Thromboxane and Approach. Advances Leukotriene Research, 11, 457-461, 1983.
4.
KAY, S., and NANCARROW, J.D. Spontaneous healing and relief of pain in a patient with intractable vasculitic ulcuation of the lower limb following an intravenous prostacyclin. Br. J. Piast. Surg., 37, infusion of 175-178, 1984.
5.
KOOISTA, T., VERHEIJEN, J. H., CHANG, G. T. G., van den BERG, A. P., van HINSBERGH, RIJKEN, D. C., van ZONNEVELD, A. J., PANNEKOEK, H., and KLUFT, C. Regulation of tissue-type plasminogen activator (t-PA) production by (EC) in culture. Thrombos. endothelial human cells Haemost., 54(l), 192(P1133), 1985.
6.
PARRY, M. J. Effects of thromboxane synthetase inhibitors on arachidonic acid methabolism and platelet behaviour. Br. J. Clin. Pharmacol., l5, 235-295, 1983.
7.
WIMAN, B., MELLBRING, G., and RANBY, M. Plasminogen activator release during venous stasis and exercise as determined by a new specific assay. Clinica Chemica Acta, 127, 279-288, 1983.
8.
GRYGLEWSKI, R. J., NIZANKOWSKA, E., SZCZEKLIK, A., NIZANKOWSKA, R., MUSIAL, J. Pulmonary and antiplatelet effects of intravenously and inhaled prostacyclin in man. Prostaglandins, 16, 654-660 1978.
BRIEF
COMMUNICATION
THE EFFECT OF PROSTACYCLIN INFUSION ON TISSUE PLASMINOGEN ACTIVATOR. K Winther", K Snorrason***$B Knudsen*, and S Medgyesi . The Coagula$ion Laboratory, Department of Clinical Chemistry and Department of Plastic Surgery**, Rigshospitalet, Copenhagen, Denmark. (Received
29.10.1986; Accepted in original form 7.1.1987
by Editor B. Wiman) (Received in final form by Executive Editorial Office 13.3.1987)
INTRODUCTION Intravenous infusion of prostacyclin (PGI2) inhibits platelet aggregation and elevates the intracellular concentration of cyclic-AMP in platelets in vitro and in vivo (1,2). The infusion of prostacyclin also enhances the fibrinolytic activity measured as shortening of the euglobulin clot lysis time (ECLT) and results in spontaneously healing and relief of pain in patients suffering from ulceration of the lower limb (3,410 The ECLT is dependent on the conversion .ofplasminogen to plasmin regulated among other factors by the tissue plasminogen activator (t-PA) from endothelial cells. In vitro investigation shows that addition of cyclic-AMP to human endothelial cells in culture enhances production of t-PA after incubation for 24 h (5). This report aims to investigate the in vivo effect of i.v. prostacyclin infusion on ECLT as well as t-PA production. MATERIALS AND METHODS The patient presented in this report was a 54 years old female suffering from leg ulcers treated with prostacyclin infusion on three occasions in order to heal the ulcerations (3). Pre-dose testing showed normal ECLT and t-PA activity before and after standardized venous stasis, without indications of t-PA inhibitor. Key
Words:
Prostacyclin, activator.
fibrinolysis, tissue
741
plasminogen
742
PROSTACYCLIN, FIBRINOLYSIS, t-PA
Vol. 46, No. 5
Methods: During the study the patient was placed in the supine position. Infusions of prostacyclin (epoprostenol FLOLAN, Wellcome) were started each time between 9.00 and 11.00 a.m. with an initial dose rate of 2.5 ng/kg body weight/minute. After 60 minutes the infusion rate was increased to 5.0 ng/kg body weight/minute for one hour and continued for the following 22 hours at 7.5 ng/kg body weight/minute. The treatment was stopped after a total infusion period of 24 hours. No infusion was performed the following 18 days. Laboratory tests: Prior to prostacyclin infusion and after 1, 5 and 24 hours and again after 3, 7, 12 and 15 days, the following biochemical parameters were measured: Plasma 6~~c'-~G~~ al ha (a stable metabolite of prostacyclin), ECLT -. Plasma 6-keto-PGFl was measured as described by Parry using a double anti o y procedure (61, ECLT was akpga measured using conventional methods. All reagents for the t-PA assay were produced as described ;;ng;9"" (7). Tissue plasminogen activator was used in it's chain form with a specific activity of about 200,000 IU/mg when compared to an international standard (WHO International Laboratory for Biological Standards, Holly Hill, London, UK). Solubilized des A fibrin was prepared by adding bathroxobin (10 pl, 250 Bu/ml) to fibrinogen (5 ml, 6 FM fibrinogen). After 6 hours incubation at 20°C the performed gel was dissolved in 5 ml 7 M urea. With minimum stasis 4.5 ml of venous blood was drawn into tubes, each containing 0.5 ml sodium citrate 3.13 %. For determination of t-PA the plasma was acidified immediately after separation of the blood cells by adding an equal volume of 1 M sodium acetate buffer, pH 3.9 and pH was then adjusted to 4.1. The samples were snap frozen and stored at -8OOC until analyzed for t-PA activity. The acidified plasma samples were diluted l/50 - l/100 with 0.05 mol/l tris-HCl buffer, pH 8.8, containing 0.10 mol/l NaCl and 0.1 g/l triton X-100. An equal volume of this buffer containing 0.1 mg plasminogen/ml (KABI, Sweden) and 0.6 mmol/l chromogenic substrate (S-2251) (KABI, Sweden) was added, followed by addition of solubilized fibrin (final concentration 0.07 g/l) as stimulator. After incubation overnight (18 h) at 20°C the absorbance was measured at 405 nm. RESULTS The infusion of prostacyclin resulted in a 280% increase in the plasma 6-keto-PGFl alpha level after 60 minutes, a 520% increase after 5 hours and a 730% increase after 24 hours of treatment. Three days after termination of infusion the plasma level of 6-keto-PGFl alpha had reached pre-treatment values (Fig. 1).
743
PROSTACYCLIN, FIBRINOLYSIS, t-PA
Vol. 46, No. 5
Parallel to the increase in plasma 6-keto-PGFl level a decrease in ECLT of 154 minutes was observed aft%p 91 hours of infusion. Following termination of treatment the ECLT level slowly increased with approximately 30 min per week the following 3 weeks (Fig. 2). In agreement with the decrease in ECLT, indicating an enhanced fibrinolytic activity, the t-PA concentration increased from 0,023 to 0,072 IU/ml 24 hours after infusion. One week after treatment the plasma t-PA had returned to pre-treatment levels (Fig. 2).
0
l
2
5
24h
w-c3
7
12
15days
Figure 1. The squared area (left panel) shows the infusion of prostacycline during 24 hours. The right panel shows the level of plasma 6-keto-PGFl ilpha after 1, 5 and 24 hours of prostaagain 3, 7, 12 and 15 days after treatcycline infusion an ment (one of three representative experiments).
PROSTACYCLIN, FIBRINOLYSIS, t-PA
744
Vol. 46, No. 5
.O.lO -i -0.08 ‘= H .0.06
2 L
-0.04
.O.O2
0 -
.
I-----
1
5
INFUSION
OF
12
24h
3
7
T-
15 days
PROSTACYCLINE -
Figure 2. Left panel (x) shows the euglobulin clot lysis time (ECLT) before, during and after the infusion of prostacyclin. The right panel (01 shows the t-PA concentration before, during and after treatment with prostacyclin (one of three representative experiments). Some spontaneous healing of the leg ulcer was observed after each of the three treatments. After two weeks, however, the beneficial effect disappeared and the ulcer started to progress again. DISCUSSION No increase in t-PA was observed after l-5 hours of prostacycline infusion, although, a 280% and 730% increase in plasma 6-keto-PGFl al ha level was observed. This might indicate that the effeca of prostacyclin is not related to an increased liberation of pre-formed t-PA from the endothelial cells as observed during exercise or after venous compression (7). The present data suggests that infusion of prostacyclin enhances the fibrinolytic activity by increasing the production of tissue plasminogen activator (t-PA). This hypothesis fits very well with the observation that the addition of cyclic-AMP to cultureq endothelial cells increases the production of t-PA two-threefold if incubation is continued for 24 hours. (5) Several reports indicate that prostacyclin infusion elevates cyclic-AMP in various cell types (1,2,8). Our data might reflect an increased synthesis by endothelial cells of tissue plasminogen activator secondary to an enhanced cyclic-AMP production induced by prostacyclin infusion.
Vol. 46, No. 5
PROSTACYCLIN, FIBRINOLYSIS, t-PA
745
In conclusion: the infusion of prostacyclin has earlier been reported to shorten the ECLT. This study confirms these data and suggests that the shortening of the ECLT is related to an enhanced production of tissue plasminogen activator. ACKNOWLEDGEMENTS We are very grateful to Miss Janne Olsen for excellent technical assistance and to Miss Ingrid Kryhlmand for typing the manuscript. REFERENCES 1.
TATESON, J. B., MONCADA, S., and VANE, J. R. Effects of Prostacyclin (PGX) on cyclic-AMP concentrations in human platelets. Prostaglandins, 2, 389-395, 1977.
2.
DATA, J. L., MOLONY, B. A, MEINZINGER, M. M., and GORMAN, R. R. Intravenous infusion of prostacyclin sodium in man: Clinical effects and influence on platelet adenosine diphosphate sensivity and adenosine 3':5'-cyclic monophosphate levels. Circulation, 64, 4-11, 1981.
3.
GRYGLEWSKI, R. J. Prostacyclin-Experimental and Clinical in Prostaglandin, Thromboxane and Approach. Advances Leukotriene Research, 11, 457-461, 1983.
4.
KAY, S., and NANCARROW, J.D. Spontaneous healing and relief of pain in a patient with intractable vasculitic ulcuation of the lower limb following an intravenous prostacyclin. Br. J. Piast. Surg., 37, infusion of 175-178, 1984.
5.
KOOISTA, T., VERHEIJEN, J. H., CHANG, G. T. G., van den BERG, A. P., van HINSBERGH, RIJKEN, D. C., van ZONNEVELD, A. J., PANNEKOEK, H., and KLUFT, C. Regulation of tissue-type plasminogen activator (t-PA) production by (EC) in culture. Thrombos. endothelial human cells Haemost., 54(l), 192(P1133), 1985.
6.
PARRY, M. J. Effects of thromboxane synthetase inhibitors on arachidonic acid methabolism and platelet behaviour. Br. J. Clin. Pharmacol., l5, 235-295, 1983.
7.
WIMAN, B., MELLBRING, G., and RANBY, M. Plasminogen activator release during venous stasis and exercise as determined by a new specific assay. Clinica Chemica Acta, 127, 279-288, 1983.
8.
GRYGLEWSKI, R. J., NIZANKOWSKA, E., SZCZEKLIK, A., NIZANKOWSKA, R., MUSIAL, J. Pulmonary and antiplatelet effects of intravenously and inhaled prostacyclin in man. Prostaglandins, 16, 654-660 1978.