55
Cuncer Letters, 53 (1990) 55-69
Elsevier Scientific Publishers
Ireland Ltd
Fibrinolytic activity Guerin epithelioma R. Tomasiuk,
of rat
plasma
during
development
of
M. Mirowski and R. Wierzbicki
Medical Academy in Lodz, Institute of Environmental Muszynskiego 1, YO- 151 Lode (Poland)
Research
and Bioanalysis,
Department
of Biochemistry,
ul.
(Received 2 August 1989) (Revision received 22 March 1990) (Accepted 24 May 1990)
Summary
Introduction
Fibrinolytic activity in the blood of rats during the deuelopment of Guerin epithelioma was studied. It was measured by means of radiometric method, based on the amount of plasmin degradation products released from ‘251-fibrin, as well as by means of amidolytic technique with the use of Chromozym PL.
Neoplastic diseases are often accompanied by disorders in the fibrinolytic system. Lowered fibrinolytic activity was observed in the blood of patients with lung and colorectum carcinomas and other malignant diseases [3,19,22]. There appeared, however, reports describing the opposite phenomenon. In some patients with neoplastic diseases increased fibrinolytic activity was detected [7]. Also, the increase of plasmin degradation products of fibrinogen in the plasma was reported [1,7,23,24]. Neoplastic transformation of the cells usually results in the increased activity of plasminogen activator. Such elevated activity was detected in a number of human and experimental cancers [2,4,8,15]. The secretion of plasminogen activator in neoplastic cells is quicker than in normal cells. Mouse and rat mammary cancers in cultures release plasminogen activator about 100-times faster than normal tissues [ 14,201. The dependence of fibrinolytic activity dn the phase of the disease might be one of the causes of observed discrepancy in the fibrinolytic activity. However, it is difficult ot examine such dependency in humans. The mechanism of the involvement of neoplastic factors in human fibrinolysis is also difficult to study, as there are many interfering factors which by
During the initial phase of epithelioma deoelopment the fibrinolytic activity of plasma, determined afier inactiuating plasma proteinase inhibitors,
increases. It also increases in fraction. Simultaneously, the content of fibrintogen) degradation products (FDP) increases in the blood. During the stage
the euglobulin
of the intensive deuelopment of neoplastic disease fibrinolytic activity as well as plasminogen actiuator activity become inhibited, whereas the concentration of FDP retains the level observed in healthy animals. Znhibition of fibrinolytic activity in the later phase of the disease coincides with the appearance of low-molecular weight antifibrinolytic factor in the blood of rats loaded with epithelioma.
Keywords: fibrinolytic Guerin epithelioma. Correspondence
activity;
rat; plasma;
to: R. Wierzbicki.
0 1990 Elsevier Scientific Publishers 0304-3835/90/$03.50 Published and Printed in Ireland
Ireland Ltd
56
themselves can modify the fibrinolytic system. Investigation of tumor-bearing animals is therefore useful and may provide some insight into the problem mentioned above. The aim of our experiments was to study the fibrinolytic activity in rats during development of Guerin epithelioma, a fast-growing experimental tumor with high metastatic ability.
inactivate the plasma proteinase inhibitors. These inhibitors were alternatively eliminated by chromatography on Lys-Sepharose [ 10). In this case the adsorbed fraction was eluted with 1.5 M NaCl in 0.05 M Tris-HCI buffer (pH 8.0), containing 0.05% Tween-80 and tested by amidolytic method. isolating the euglobulin fraction
Materials and methods Reagents
Plasminogen and human fibrinogen were from Kabi (Sweden). Lys.-Sepharose was purchased from Pharmacia (Sweden). Labelling of human fibrogen with Nalz51 was performed with chloramine method [9] after previous purification according to the method of Laki 113). Thrombin (400 NIH) was obtained from Serum and Vaccine Factory (Poland). Human urokinase was from Calbiochem (U.S.A.). Chromozym PL was from Behringer Mannheim (F.R.G.). Materials
The experiments were performed on female Wistar rats fed with standard LSM diet of an average body weight of 100 g (age h 8 weeks). Fragments of Guerin epithelioma (Z3 mm in diameter, Q 0.15 g) were implanted subcutaneously onto 3 places on the dorsal region. The implanted epithelioma originated from rat uterus cancer is characteristic of quick development (the max, time of survival is w 50 days) and high ability of metastasis mainly in lymphatic nodes (at about 80% of animals taken into consideration in this paper). Occasionally metastases to other tissues like lungs, liver and pancreas were observed. Blood was taken from the portal vein into 3.8% sodium citrate so&ion (9 : 1) and plasma was prepared by centrifugation for 10 min at 3000 x g (4OC). Prior to the determination of the fibrinolytic activity the plasma was acidified according to Mussoni et al. [18] to
Cold distilled water (10 ml) was added to 0.5 ml of plasma. The samples were put into ice bath and 0.15 ml of 1% acetic acid were added. After stirring, the mixture was left for 10 min. The samples were then centrifuged for 10 min at 3000 x g (4OC). The supernatant was removed whereas the precipitate was dissolved in 0.5 M Palitsch phosphate buffer (pH 7.6), containing 0.2 M H,BO,, 0.05 M Na,B,O, x 10 H,O and 0.05 M NaCl. Fibrinolytic activity was estimated by measuring the radioactivity of the plasmin degradation products of [1251]-fibrinas we have recently described (251. Plasmin was generated from endogenous plasma plasminogen by plasma plasminogen activator or by plasminogen activator present in the euglobulin fraction. The sample contained 120 ~1 of the plasma acidified according to Mussoni et al. [18] which corresponded to 20 ~1 of undiluted plasma of 50 ~1 of euglobulin fraction. Amidolytic activity was determined with the use of Chromozym PL. The amount of pnitroaniline liberated from the substrate by plasmin, generated from plasminogen by plasminogen activator, was measured. The assay mixture (total volume 500 ~1) in 0.05 M TrisHCI buffer (pH 7.4), with 0.11 M NaCl containing plasminogen (50 L(g) and source of piasminogen activator (up to 75 ~1) was preincubated during 30 min at 37OC and after the reaction was inhibited by 50% CH,COOH (50 ~1) absorbancy was measured at 405 nm. Molar absorption coefficient for p-nitroaniline = 9905. Determination of the fibri(nogen) degradation products was performed with the use of latex Welcome FDP Kit.
57
Statistics 20
Results were expressed as arithmetical mean & S.D. Statistical analysis was performed with Student’s t-test.
g = s
Results
10
n LL
The fibrinolytic activity of the plasma, measured after inactivating the plasma proteinase inhibitors, between 11 and 13 days following implantation displays observable, tumor though statistically insignificant increase. In the initial period of the disease (up to 1 lth day), we also observed statistically significant increase of the fibrinolytic activity in plasma euglobulin fraction (Fig. 1). The increased fibrinolytic activity was accompanied by the elevated level of plasmin degradation products of fibrin(ogen) in the rat serum (from 5th to 15th days). Later, the content of FDP, similar to the fibrinolytic activity of euglobulin fraction, reaches its normal values (Fig. 1, Fig. 2). Simultaneously, the fibrinolytic activity of
10
0
20 Days
30
40
Fig. 2. The content of plasmin degradation products of fibrin/fibrinogen (FDP) in the serum of rats during the development of the epithelioma. (Mean values k S.D., n = 5-6). The content of FDP in the serum of control rats was 1.5 f 0.4 pg/ml (n = 12). *Differences statistically significant at P < 0.05. -U-UPrimary tumor, -m-m- metastasis.
the plasma is reduced and during the intensive growth of the tumor (from 26th day) it drastically decreases in a significant way. The decrease of firbinolytic activity in the rat plasma during the intensive growth of epithelioma is also confirmed by determination of the plasma plasminogen activity with the use of Chromozym PL, a substrate specific for plasmin. Plasminogen activation dependent on this activator is inhibited during the later period of tumor growth (Table 1). Table 1.
0
10
20 Days
30
40
Fig. 1. Fibrinolytic activity of the plasma (n = 4) - 0 0 - and euglobulin fraction (n = 5) -O-O- in rats during the development of epithelioma. The values represent the arithmetical mean k S.D. The plasma fibrinolytic activity of control rats (100%) corresponds to 4.9 + 0.7% of [Ylfibrin lysis/20 pl of plasma (n = 10). In the case of euglobulin fraction the control values (100%) correspond to 6.3 + 1.4% of [‘251]fibrin lysis/50 pl of plasma (n = 5). *Differences statistically significant at P < 0.05.
Plasminogen activator in plasma (a) and plasma protein fraction adsorbed on Lys-Sepharose (b) in rats during the development of epithelioma. The results are expressed as pmol of p-nitroaniline/min per ml of plasma (mean values from 2-4 results; N.D.- not determined). Control
Days after tumor implantation 9
0.17’ n=3 0.14b
0.27
n=3
n=
n=
16
26
33
N.D.
N.D.
0.01 n=2
0.04
0.03 n=2 0.05
n=2
n=2
4
0.23 4
58
Discussion There are few data concerning the fibrinolytic activity of the animals with neoplastic diseases. In mice bearing Lewis lung carcinoma the increased content of fibrinogen, as well as shortening of its half-life and temporary increase of FDP content in the blood [Zl] were detected. In dogs with different neoplastic disease, apart from the disorders in fibrinogen content, shortening of the euglobulin lysis time was frequently reported [16]. The elevated fibrinogen content was also detected in mice loaded with fibrosarcoma [6] and with JW sarcoma [5]. In the case of the last mentioned disease, prolonged fibrinogen survival time was accompanied by depressed fibrinolysis. Lowered fibrinolytic activity was noticed in rats with leukemia [II]. Also in the blood of rats loaded with Guerin epithelioma, an increased content of antyplasmins and lowered fibrinolytic activity of euglobulins were reported [26]. We may suppose that the increase of fibrinolytic activity in the plasma of rats bearing epithelioma observed in the initial phase of the tumor development can result from the increased content of plasminogen activator in the blood. The results obtained with the use of Chromozym PL as well as the fact that the examined epithelioma contains plasminogen activator (unpublished data) confirm such assumption. However, during the intensive growth of the primary tumor and the formation of new metastatic foci the fibrinolytic activity of the plasma becomes inhibited. Similarly, additional antifibrinolytic factor appears in the plasma of rats loaded with epithelioma [25]. As we assume it is a low-molecular weight, thermo- and acid-stable plasmin inhibitor, present in epithelioma [ 171. It seems possible that this inhibitor is responsible for the decrease of fibrinolytic activity observed in the later phase of the disease, by inhibiting plasmin and/or plasminogen activator. The dependence of plasma fibrinolytic activity on the stage of epithelioma development, described in this study, allows to suppose that similar relationship exists in humans with neo-
plastic disease. Different response of the fibrinolytic system of the patients may, at least to some extent, be explained by the fact that the samples were probably collected in different stages of the disease. There arises a question, whether disorders in fibrinolytic system may play any role in the development and spreading of neoplastic disease. The correlation between the increased fibrinolytic activity and the invasiveness was observed, among other, in the case of lung and mammary cancers [8,15]. The participation of plasminogen activators in spreading of the neoplastic diseases is confirmed by stimulation of the growth and the formation of metastases under the influence of urokinase in rabbits with V, cancer [ 121. The invasiveness of the neoplastic disease can also be facilitated by the hydrolysis of the fibrin surrounding the tumor, the process induced by the locally generated plasmin. Lowered fibrinolytic activity of the blood which results in the formation of the fibrin deposits on the other hand may be one of the factors leading to new metastatic foci. Acknowledgement We are grateful to Miss A. Jurek M.A. for her assistance in preparation of the manuscript. References Anstey, J.T. and Blythe, J.G. (1978) Fibrin degradation products and the diagnosis of ovarian carcinoma. J. Obstet. Gvnecol., 52.605-608. Bruesch, M.R., Johnson, G.L., Palackdhary, C.S., Weber, N.J. and Carl, P.L. (1983) Plasminogen activator in normal and tumor-bearing mice. Int. J. Cancer, 32, 121 -126. Brugardas, A., Elias, E.G., Takita, H., Mink, J.B., Mittelman, A. and Ambrus, J.L. (1973) Blood coagulation and fibrinolysis in patients with carcinoma of the lung. J. Med., 4,96. Camiolo, S.M., Markus, G., Englander, L.S., Sinta, N.R., Hobika, G.H. and Kohga, S. (1984) Plasminogen activator content and secretion in explants of neoplastic and benign human prostate tissues, Cancer Res., 44, 311318. Chmielewska, J., Poggi, A., Mussoni, L., Donati M.B. and Gerattini, S. (1980) Blood coagulation changes in JW
59
6
7
8
9
10
11 12
13
14
15
sarcoma, a new metastasizing tumor in mice. Eur. J. Cancer, 16, 1399-1407. Delaini, F., Giavazzi, R., De Bellis Vim, G., Alessandri, G., Manytovani, A. and Donati, M.B. (1981) Tumour sublines with different metastatic capacity induced similar blood coagulation changes in the host. Br. J. Cancer, 43, 100-104. Dube, B., Khanna, MS. and Kulkarni, B.J. (1976) Blood fibrinolytic activity in cancer patients. Indian J. Pathol. Microbial., 19, 155-160. Evers, J.L., Patel, J. and Madeja, J. (1982) Plasminogen activator activity and composition in human breast cancer. Cancer Res., 42,219-226. Greenwood, F.C., Hunter, W.M. and Glover, J.S. (1963) The preparation of ‘3%labelled human growth hormone of high specific radioactivity. Biochem. J., 89, 114-123. Gyzander,.E., Ericksson, E. and Teger-Nilsson, A.C. (1984) A sensitive assay for tissue plasminogen activator activity in plasma, using adsorption on lysine-Sepharose. Thromb. Res., 35,547-558. Hilgard, P. (1977) Coagulation studies in the BNML, rat leukemia. LeukemiaRes., 1, 175. Kodama, Y. and Tanaka, K. (1978) Effects of urokinase on growth and metastasis of rabbit V, carcinoma. Gann, 69,9-15. Laki, K. (1951) The polmerization of proteins: the action of thrombin and fibrinogen. Arch. Biochem. Biophys., 32, 317. Lopez, M., Reich, E. and Ossowski, L. (1983) Modulation of plasminogen activator in rodent mammary tumors by hormones and other effecters. Cancer Res., 43, 54675477. Markus, G., Jakita, H., Camiolo, S., Evers, J.L. and Hobika, G. (1980) Content and characterization of plasminogen activator in human lung tumors and normal lung tissue. Cancer
Res., 40.841-848.
16
Madawell, B.R., Feldman, B.F. and O’Neill, S. (1980) Coagulation abnormalities in dogs with neoplastic disease. Thromb. Haemost.. 44.35-38.
17
Mirowski, M. and Wierzbicki, R. (1989) Low molecuIar weight fibrinolytic inhibitor from Guerin epithelioma. Cancer Lett., 47,57-62. Mussoni, J., Raczke, E., Chmielewska, J., Donati, M.D. and Latallo, Z.S. (1979) Plasminogen assay in rabbit, rat and mouse plasma using chromogenic substrate S-2251. Thromb. Res., 15,341-349. Newstead, G.L., Griffiths, J.D. and Salsbury, A.J. (1976) Fibrinolytic activity in carcinomas of the colorectum. Surg. Gynecol. Obst., 143.61-64. Ossowski, L., Biegel, D. and Reich, E. (1979) Mammary plasminogen activator. Correlation with involution, hor-
18
19
20
21
22 23 24
25
26
monal modulation and comparison neoplastic tissue. Cell, 16, 929-940. Poggi, A., Polentarutti, N., Donati, (1977) Blood coagulation changes
between
normal
and
M.B. and Garattini, S. in mice bearing Lewis
lung carcinoma, a metastasizing tumor. Cancer 273-277. Rennie, J.A. and Ogston, D. (1975) Fibrinolytic malignant disease. J. Clin. Pathol., 28.872-874.
Res., 37, activity in
Sufrin, G., Mink, J. and Fitzpatrick, J. (1978) Coagulation factors in renal adenocarcinoma. J. Ural., 119, 727-730. Sun, N.C.J., McAfee, W.M., Hum, G.J. and Weiner, J.M. (1979) Hemostatic abnormalities in malignancy, a prospective study of one hundred eight patients. Part I. Coagulation studies. Am. J. Clin. Pathol., 71, 10-16. Wienbicki, R. and Mirowski, M. (1988) Low molecular weight fibrinolytic inhibitor in plasma of rats bearing Guerin epithelioma. Thromb. Res., 49,623-628. Zurawski, P., Zagorska, M., Grygoruk, M. and Worowski, K. (1985) The factors of fibrinolytic enzyme system in Guerin epithelioma (in Polish). Acta Haematol. Pol., 3-4, 151-157.
Cuncer Letters, 53 (1990) 55-69
Elsevier Scientific Publishers
Ireland Ltd
Fibrinolytic activity Guerin epithelioma R. Tomasiuk,
of rat
plasma
during
development
of
M. Mirowski and R. Wierzbicki
Medical Academy in Lodz, Institute of Environmental Muszynskiego 1, YO- 151 Lode (Poland)
Research
and Bioanalysis,
Department
of Biochemistry,
ul.
(Received 2 August 1989) (Revision received 22 March 1990) (Accepted 24 May 1990)
Summary
Introduction
Fibrinolytic activity in the blood of rats during the deuelopment of Guerin epithelioma was studied. It was measured by means of radiometric method, based on the amount of plasmin degradation products released from ‘251-fibrin, as well as by means of amidolytic technique with the use of Chromozym PL.
Neoplastic diseases are often accompanied by disorders in the fibrinolytic system. Lowered fibrinolytic activity was observed in the blood of patients with lung and colorectum carcinomas and other malignant diseases [3,19,22]. There appeared, however, reports describing the opposite phenomenon. In some patients with neoplastic diseases increased fibrinolytic activity was detected [7]. Also, the increase of plasmin degradation products of fibrinogen in the plasma was reported [1,7,23,24]. Neoplastic transformation of the cells usually results in the increased activity of plasminogen activator. Such elevated activity was detected in a number of human and experimental cancers [2,4,8,15]. The secretion of plasminogen activator in neoplastic cells is quicker than in normal cells. Mouse and rat mammary cancers in cultures release plasminogen activator about 100-times faster than normal tissues [ 14,201. The dependence of fibrinolytic activity dn the phase of the disease might be one of the causes of observed discrepancy in the fibrinolytic activity. However, it is difficult ot examine such dependency in humans. The mechanism of the involvement of neoplastic factors in human fibrinolysis is also difficult to study, as there are many interfering factors which by
During the initial phase of epithelioma deoelopment the fibrinolytic activity of plasma, determined afier inactiuating plasma proteinase inhibitors,
increases. It also increases in fraction. Simultaneously, the content of fibrintogen) degradation products (FDP) increases in the blood. During the stage
the euglobulin
of the intensive deuelopment of neoplastic disease fibrinolytic activity as well as plasminogen actiuator activity become inhibited, whereas the concentration of FDP retains the level observed in healthy animals. Znhibition of fibrinolytic activity in the later phase of the disease coincides with the appearance of low-molecular weight antifibrinolytic factor in the blood of rats loaded with epithelioma.
Keywords: fibrinolytic Guerin epithelioma. Correspondence
activity;
rat; plasma;
to: R. Wierzbicki.
0 1990 Elsevier Scientific Publishers 0304-3835/90/$03.50 Published and Printed in Ireland
Ireland Ltd
56
themselves can modify the fibrinolytic system. Investigation of tumor-bearing animals is therefore useful and may provide some insight into the problem mentioned above. The aim of our experiments was to study the fibrinolytic activity in rats during development of Guerin epithelioma, a fast-growing experimental tumor with high metastatic ability.
inactivate the plasma proteinase inhibitors. These inhibitors were alternatively eliminated by chromatography on Lys-Sepharose [ 10). In this case the adsorbed fraction was eluted with 1.5 M NaCl in 0.05 M Tris-HCI buffer (pH 8.0), containing 0.05% Tween-80 and tested by amidolytic method. isolating the euglobulin fraction
Materials and methods Reagents
Plasminogen and human fibrinogen were from Kabi (Sweden). Lys.-Sepharose was purchased from Pharmacia (Sweden). Labelling of human fibrogen with Nalz51 was performed with chloramine method [9] after previous purification according to the method of Laki 113). Thrombin (400 NIH) was obtained from Serum and Vaccine Factory (Poland). Human urokinase was from Calbiochem (U.S.A.). Chromozym PL was from Behringer Mannheim (F.R.G.). Materials
The experiments were performed on female Wistar rats fed with standard LSM diet of an average body weight of 100 g (age h 8 weeks). Fragments of Guerin epithelioma (Z3 mm in diameter, Q 0.15 g) were implanted subcutaneously onto 3 places on the dorsal region. The implanted epithelioma originated from rat uterus cancer is characteristic of quick development (the max, time of survival is w 50 days) and high ability of metastasis mainly in lymphatic nodes (at about 80% of animals taken into consideration in this paper). Occasionally metastases to other tissues like lungs, liver and pancreas were observed. Blood was taken from the portal vein into 3.8% sodium citrate so&ion (9 : 1) and plasma was prepared by centrifugation for 10 min at 3000 x g (4OC). Prior to the determination of the fibrinolytic activity the plasma was acidified according to Mussoni et al. [18] to
Cold distilled water (10 ml) was added to 0.5 ml of plasma. The samples were put into ice bath and 0.15 ml of 1% acetic acid were added. After stirring, the mixture was left for 10 min. The samples were then centrifuged for 10 min at 3000 x g (4OC). The supernatant was removed whereas the precipitate was dissolved in 0.5 M Palitsch phosphate buffer (pH 7.6), containing 0.2 M H,BO,, 0.05 M Na,B,O, x 10 H,O and 0.05 M NaCl. Fibrinolytic activity was estimated by measuring the radioactivity of the plasmin degradation products of [1251]-fibrinas we have recently described (251. Plasmin was generated from endogenous plasma plasminogen by plasma plasminogen activator or by plasminogen activator present in the euglobulin fraction. The sample contained 120 ~1 of the plasma acidified according to Mussoni et al. [18] which corresponded to 20 ~1 of undiluted plasma of 50 ~1 of euglobulin fraction. Amidolytic activity was determined with the use of Chromozym PL. The amount of pnitroaniline liberated from the substrate by plasmin, generated from plasminogen by plasminogen activator, was measured. The assay mixture (total volume 500 ~1) in 0.05 M TrisHCI buffer (pH 7.4), with 0.11 M NaCl containing plasminogen (50 L(g) and source of piasminogen activator (up to 75 ~1) was preincubated during 30 min at 37OC and after the reaction was inhibited by 50% CH,COOH (50 ~1) absorbancy was measured at 405 nm. Molar absorption coefficient for p-nitroaniline = 9905. Determination of the fibri(nogen) degradation products was performed with the use of latex Welcome FDP Kit.
57
Statistics 20
Results were expressed as arithmetical mean & S.D. Statistical analysis was performed with Student’s t-test.
g = s
Results
10
n LL
The fibrinolytic activity of the plasma, measured after inactivating the plasma proteinase inhibitors, between 11 and 13 days following implantation displays observable, tumor though statistically insignificant increase. In the initial period of the disease (up to 1 lth day), we also observed statistically significant increase of the fibrinolytic activity in plasma euglobulin fraction (Fig. 1). The increased fibrinolytic activity was accompanied by the elevated level of plasmin degradation products of fibrin(ogen) in the rat serum (from 5th to 15th days). Later, the content of FDP, similar to the fibrinolytic activity of euglobulin fraction, reaches its normal values (Fig. 1, Fig. 2). Simultaneously, the fibrinolytic activity of
10
0
20 Days
30
40
Fig. 2. The content of plasmin degradation products of fibrin/fibrinogen (FDP) in the serum of rats during the development of the epithelioma. (Mean values k S.D., n = 5-6). The content of FDP in the serum of control rats was 1.5 f 0.4 pg/ml (n = 12). *Differences statistically significant at P < 0.05. -U-UPrimary tumor, -m-m- metastasis.
the plasma is reduced and during the intensive growth of the tumor (from 26th day) it drastically decreases in a significant way. The decrease of firbinolytic activity in the rat plasma during the intensive growth of epithelioma is also confirmed by determination of the plasma plasminogen activity with the use of Chromozym PL, a substrate specific for plasmin. Plasminogen activation dependent on this activator is inhibited during the later period of tumor growth (Table 1). Table 1.
0
10
20 Days
30
40
Fig. 1. Fibrinolytic activity of the plasma (n = 4) - 0 0 - and euglobulin fraction (n = 5) -O-O- in rats during the development of epithelioma. The values represent the arithmetical mean k S.D. The plasma fibrinolytic activity of control rats (100%) corresponds to 4.9 + 0.7% of [Ylfibrin lysis/20 pl of plasma (n = 10). In the case of euglobulin fraction the control values (100%) correspond to 6.3 + 1.4% of [‘251]fibrin lysis/50 pl of plasma (n = 5). *Differences statistically significant at P < 0.05.
Plasminogen activator in plasma (a) and plasma protein fraction adsorbed on Lys-Sepharose (b) in rats during the development of epithelioma. The results are expressed as pmol of p-nitroaniline/min per ml of plasma (mean values from 2-4 results; N.D.- not determined). Control
Days after tumor implantation 9
0.17’ n=3 0.14b
0.27
n=3
n=
n=
16
26
33
N.D.
N.D.
0.01 n=2
0.04
0.03 n=2 0.05
n=2
n=2
4
0.23 4
58
Discussion There are few data concerning the fibrinolytic activity of the animals with neoplastic diseases. In mice bearing Lewis lung carcinoma the increased content of fibrinogen, as well as shortening of its half-life and temporary increase of FDP content in the blood [Zl] were detected. In dogs with different neoplastic disease, apart from the disorders in fibrinogen content, shortening of the euglobulin lysis time was frequently reported [16]. The elevated fibrinogen content was also detected in mice loaded with fibrosarcoma [6] and with JW sarcoma [5]. In the case of the last mentioned disease, prolonged fibrinogen survival time was accompanied by depressed fibrinolysis. Lowered fibrinolytic activity was noticed in rats with leukemia [II]. Also in the blood of rats loaded with Guerin epithelioma, an increased content of antyplasmins and lowered fibrinolytic activity of euglobulins were reported [26]. We may suppose that the increase of fibrinolytic activity in the plasma of rats bearing epithelioma observed in the initial phase of the tumor development can result from the increased content of plasminogen activator in the blood. The results obtained with the use of Chromozym PL as well as the fact that the examined epithelioma contains plasminogen activator (unpublished data) confirm such assumption. However, during the intensive growth of the primary tumor and the formation of new metastatic foci the fibrinolytic activity of the plasma becomes inhibited. Similarly, additional antifibrinolytic factor appears in the plasma of rats loaded with epithelioma [25]. As we assume it is a low-molecular weight, thermo- and acid-stable plasmin inhibitor, present in epithelioma [ 171. It seems possible that this inhibitor is responsible for the decrease of fibrinolytic activity observed in the later phase of the disease, by inhibiting plasmin and/or plasminogen activator. The dependence of plasma fibrinolytic activity on the stage of epithelioma development, described in this study, allows to suppose that similar relationship exists in humans with neo-
plastic disease. Different response of the fibrinolytic system of the patients may, at least to some extent, be explained by the fact that the samples were probably collected in different stages of the disease. There arises a question, whether disorders in fibrinolytic system may play any role in the development and spreading of neoplastic disease. The correlation between the increased fibrinolytic activity and the invasiveness was observed, among other, in the case of lung and mammary cancers [8,15]. The participation of plasminogen activators in spreading of the neoplastic diseases is confirmed by stimulation of the growth and the formation of metastases under the influence of urokinase in rabbits with V, cancer [ 121. The invasiveness of the neoplastic disease can also be facilitated by the hydrolysis of the fibrin surrounding the tumor, the process induced by the locally generated plasmin. Lowered fibrinolytic activity of the blood which results in the formation of the fibrin deposits on the other hand may be one of the factors leading to new metastatic foci. Acknowledgement We are grateful to Miss A. Jurek M.A. for her assistance in preparation of the manuscript. References Anstey, J.T. and Blythe, J.G. (1978) Fibrin degradation products and the diagnosis of ovarian carcinoma. J. Obstet. Gvnecol., 52.605-608. Bruesch, M.R., Johnson, G.L., Palackdhary, C.S., Weber, N.J. and Carl, P.L. (1983) Plasminogen activator in normal and tumor-bearing mice. Int. J. Cancer, 32, 121 -126. Brugardas, A., Elias, E.G., Takita, H., Mink, J.B., Mittelman, A. and Ambrus, J.L. (1973) Blood coagulation and fibrinolysis in patients with carcinoma of the lung. J. Med., 4,96. Camiolo, S.M., Markus, G., Englander, L.S., Sinta, N.R., Hobika, G.H. and Kohga, S. (1984) Plasminogen activator content and secretion in explants of neoplastic and benign human prostate tissues, Cancer Res., 44, 311318. Chmielewska, J., Poggi, A., Mussoni, L., Donati M.B. and Gerattini, S. (1980) Blood coagulation changes in JW
59
6
7
8
9
10
11 12
13
14
15
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