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Thromboplastic activity of human ovarian tumours
THROMBOSIS RESEARCH Printed in the United States
Vol.
6, PP. 307-313, 1975 Pergamon Press, Inc.
THROMBOPLASTIC ACTIVITY OF HUMAN OVARIAN TUMOURS Lars Svanberg
From the Coagulation Laboratory and the Department of Gynecology and Obstetrics, Allmanna Sjukhuset, University of Lund, Malmo, Sweden (Received 14.11.1974; in revised form 1.2.1975. Accepted by Editor P. Olsson)
ABSTRACT The thromboplastic activity in human ovarian tumours was found to be significantly higher (p ~0.01 resp. p
INTRODUCTION Tissue thromboplastin is present in all human tissues, particularly brain and lung. Several investigations of the relative thromboplastic activity in various organs and in different animal and human species have been published (1, 2, 31, but only few concerning that of neoplastic tissues. Thus O'Meara and Thornes (8) found it to be many times higher than in normal tissue. Svanberg et al. (10) found high fibrinolytic activity in ovarian tumours to vary more closely with the degree of vascularity than with that of malignancy. But, as far as we know, no reports are available on the thromboplastic activity in ovarian tumours. This paper concerns the thromboplastic activity of benign as well as of malignant human ovarian tumours. 307
THROMBOPLASTIC ACTIVITY
308
MATERIAL AND METHODS Tissues Eighteen ovarian tumours, 5 benign, 10 primarily malignant and 3 ovarian metastases, were obtained at laparotomy. The specimens were immediately placed in sterile physiological saline and solid parts carefully dissected. Samples were weighed and kept in sealed vials at -2OOC until studied. 5 normal ovaries from agematched patients operated upon because of non-malignant diseases were prepared in the same way and served as controls.
Assay of tissue thromboplastin (4) Human platelet-poor plasma from 8 healthy volunteers was used for coagulation assays. The plasma was stored in siliconized glass tubes at -200~ until used. Saline-barbital buffer. This buffer consisted of 0.05 M sodium barbital in 0.1 M NaCl at pH 7.75 (total ionic strength 0.15). Gelatine-barbital buffer. This consisted of saline-barbital buffer with 0.25 % gelatine. CaC12-buffer solution. This was a mixture of 3 volumes of 0.05 M CaC12 with 2 volumes of saline-barbital buffer. The final CaC12 concentration was 0.03 M and the total ionic strength 0.15. Thromboplastin. Rabbit thromboplastin (Simplastin, WarnerChilcott, General Diagn. Div.) was used. The content of a vial was suspended in 6 ml gelatine-barbital buffer and 4 ml of a 0.25 % aqueous gelatine solution to obtain a stable suspension, which was distributed among vials and stored at -2OOC until used (always within 2 weeks). Tissue samples. A Potter-Elvehjem homogenizer, cooled in ice water, was used for preparing the tissue suspension with 0.9 ml gelatine-barbital buffer per 100 mg tissue (about 300 mg tissue was used). The tissue was homogenized for 30 minutes to complete cell destruction. The resulting 10 % suspension was stored at -2OOC overnight and passed through a cotton plug to remove coarse particles. The intermediate freezing and the addition of gelatine facilitated the preparation of uniform suspensions, necessary for reproducible results. For assays serial dilutions were prepared
vo1.6,~0.4
309
THROMBOPLASTIC ACTIVITY
POOLED OVARIAN
200-
NORMAL TISSUE
100-
50-
SIMPLASTIN
1030-
-
= MALIGNANT
OVARIAN
TUMOURS
A
60 CLOTTING
liL0
lie0
$60
TISSUE
DILUTION
TIME
stc
POOLED OVARIAN
200-
NORMAL TISSUE
SIMPLASTIN
20-
10
~___----I '/lo
= BENIGN = OVARIAN I ‘120
FIG. 1 A and B.
1 ‘/lo
OVARIAN
TUMOURS
METASTASES I
I
‘/SO
l/l60
B TISSUE
DILUTION
Clotting time in logarithmic scale with increasing tissue dilution. Benign and malignant ovarian tumours, metastases and normal ovarian tissue.
THROMBOPLASTIC
310
ACTIVITY
Vo1.6,No.4
bdith gelatine-barbital buffer. Assay system. The clotting mixture contained 0.2 ml titrated human plasma, 0.1 ml gelatine-barbital buffer and 0.1 ml thromboplastin suspension in unsiliconized glass tubes. The mixture was preheated for 3 minutes in a water bath at 37OC. The coagulation was initiated with 0.2 ml of the CaC12 solution and the coagulation time was determined. Assays were performed in duplicate.
RSSULTS The thromboplastic activity in ovarian tumurs,
normal
ovaries and Simplastin yielded linear curves in a double-logarithmic plot (Fig. 1 A and B). The results of assays performed in duplicate were largely identical. The curves are based on the mean of each pair of determination. Reanalyses of aliquots of the same samples one week later gave practically the same results. The method therefore appeared reliable. The thromboplastic activity in pooled normal ovaries was low, only 0.3 % of the Simplastin activity. Also the ovarian tumours had low activities, but they were significantly higher than in the normal ovaries. There was no difference in activity between benign and malignant ovarian tumours or ovarian metastases (Fig. 1 A and B; Table I and II).
DISCUSSION The growth and spread of malignant tumours is believed to vary with the thromboplastic and fibrinolytic activity. Thus, O'lvleara(6, 7) extracted a thermostable, water-soluble thromboplastic factor from human tumour tissue and found its activity to be related to the amount of fibrin deposits in the tissue, which was thought to be a suitable stroma for tumour cell invasion. Thornes (11) found this factor to convert fibrinogen to fibrin in the same way as thrombin. Others (9) feel that the deposition of fibrin is rather a reaction to the tumour. Holyoke et al. (5) showed in vitro that trauma increases the release of thromboplastic substances from both normal and tumour tissue and claimed that thromboplastic activity is not a unique feature
THROMBOPLASTIC
TABLE
ACTIVITY
131.
I
Classification of the ovarian tumours according to International Federation of Gynaecology and Obstetrics (F.I.G.O.) A. Non-neoplastic tumours
n= n=
B. Endometriotic tumours
1 1
C. Primary neoplastic tumours I. Neoplasm with endocrine significanceX
n = 1
II.Neoplasm with no endocrine significance Epithelial tumours: 1. serous (1A) serous (lCjx 2. mutinous
(2A)
n=
2
n=
4
n=l
mutinous (2CJx
n=
1
3. endometroidx
n=
2
4. concomitantx
n=
2
from gastric carcinomaX
n=
1
from uterine carcinomax
n=
1
from pancreatic carcinomax
n=
1
D. Ovarian metastases
x = malignant
TABLE
II
Thromboplastic activity (mean clotting time in set) in benign and malignant ovarian tumours and normal ovaries. Dilution 1:lO Tissue
Mean
S.D.
Malignant tumours Metastases Benign tumours
57 48 59
14 7 15
Normal ovaries
91
13
Stat. signif. diff.x p
x) significantly different from that in normal ovaries
312
THROMBOPLASTIC ACTIVITY
vo1.6,~0.4
of malignant cells. This postulation is corroborated by the present study, in which the thromboplastic activity of both benign and malignant ovarian tumours was found to be low, but significantly higher than that of normal ovaries. Fibrin degradation products (FDP) were often found in the blood in patients with malignant, but not in those with benign, ovarian tumours, a finding that proved to be of diagnostic value (12). FDP was thought to be due not only to the fibrinolytic activity of the tumours, but also to its thromboplastic activity, whose purpose was to promote the formation of fibrin. No difference in the fibrinolytic activity has been found between benign and malignant ovarian tumours (10). A difference in the occurrence of thromboplastic substances were therefore assumed. But the findings in the present investigation clearly refuted this assumption. The explanation should probably therefore be sought in the infiltrative growth of the malignant tumours with increased escape of thromboplastic substances, fibrinolytic activators or locally formed FDP into the bloodstream.
ACKNOWLEDGEMENT This investigation was supported by grants fran the Swedish Medical Research Council (B75-19X-87-llA), the Medical Faculty, University of Lund, and Tore Nilson's Fund for Medical Research.
REFERENCES 1. ASTRUP,T. Assay and content of tissue thromboplastin in different organs. Thromb. Diath. haemorrh.: 14., 401. 1965.. 2. ASTRUP,T., ALBRECHTSEN,O.K., CLAASSEN,M., and RASMUSSEN,J.
Thromboplastic and fibrinolytic activity of the human aorta. Circ. yes.: 7., 969. 1959. 3. GLAS,P., and ASTRUP,T. Fibrinolytic and thromboplastic activity of normal human heart valves. Amer. Heart J.: 76:4., 504. 1968. 4. GLAS,P., and ASTRUP,T. Thromboplastin and plasminogen
activator in tissues of the rabbit. Amer. J. Physiol.: 219:4., 1140. 1970.
vol.6,~0.4
THROMBOPUSTIC
ACTIVITY
313
5. HOLYOKE,E.D., FRANK,A.L., and WEISS,L. Tumour thromboplastin activity in vitro. Int. J. Cancer: 9., 258. 1972. 6. o'MEARA,R.A.Q. Coagulative properties of cancers. Irish J. Med. Sci.: 394., 474. 1958. 7. O'MEARA,R.A.Q. Fibrin formation and tumour growth. Thromb. Diath. haemorrh.: Suppl. 28., 137. 1968. 8. O'MEARA,R.A.Q., and THORNES,R.D. Some properties of the cancer coagulative factor. Irish J. Med. Sci.: 423., 106. 1961. 9. PETERSON,H.-I. Experimental studies on fibrinolysis in growth and spread of tumour. Acta chir. stand.: Suppl. 394., 1968. 10. SVANBERG,L., LINEU,F., PANDOLFI,M., and ASTEDT,B. Plasminogen activators in ovarian tumours. Acta path. microbial. stand.: 1974 (in press). 11. THORNES,R.D. Fibrinogen and the interstitial behaviour of cancer. In: Endogenous Factors Influencing Host-Tumor Balance pp 255-66. Ed. Wissler,R.W., Dao,T.L. and Wood,S. Jr. The University of Chicago Press, Chicago 1967. 12. ASTEDT,B., SVANBERG,L., and NILSSON,I.M. Fibrin degradation products and ovarian tumours. Brit. med. J. 4., 458. 1971.
Vol.
6, PP. 307-313, 1975 Pergamon Press, Inc.
THROMBOPLASTIC ACTIVITY OF HUMAN OVARIAN TUMOURS Lars Svanberg
From the Coagulation Laboratory and the Department of Gynecology and Obstetrics, Allmanna Sjukhuset, University of Lund, Malmo, Sweden (Received 14.11.1974; in revised form 1.2.1975. Accepted by Editor P. Olsson)
ABSTRACT The thromboplastic activity in human ovarian tumours was found to be significantly higher (p ~0.01 resp. p
INTRODUCTION Tissue thromboplastin is present in all human tissues, particularly brain and lung. Several investigations of the relative thromboplastic activity in various organs and in different animal and human species have been published (1, 2, 31, but only few concerning that of neoplastic tissues. Thus O'Meara and Thornes (8) found it to be many times higher than in normal tissue. Svanberg et al. (10) found high fibrinolytic activity in ovarian tumours to vary more closely with the degree of vascularity than with that of malignancy. But, as far as we know, no reports are available on the thromboplastic activity in ovarian tumours. This paper concerns the thromboplastic activity of benign as well as of malignant human ovarian tumours. 307
THROMBOPLASTIC ACTIVITY
308
MATERIAL AND METHODS Tissues Eighteen ovarian tumours, 5 benign, 10 primarily malignant and 3 ovarian metastases, were obtained at laparotomy. The specimens were immediately placed in sterile physiological saline and solid parts carefully dissected. Samples were weighed and kept in sealed vials at -2OOC until studied. 5 normal ovaries from agematched patients operated upon because of non-malignant diseases were prepared in the same way and served as controls.
Assay of tissue thromboplastin (4) Human platelet-poor plasma from 8 healthy volunteers was used for coagulation assays. The plasma was stored in siliconized glass tubes at -200~ until used. Saline-barbital buffer. This buffer consisted of 0.05 M sodium barbital in 0.1 M NaCl at pH 7.75 (total ionic strength 0.15). Gelatine-barbital buffer. This consisted of saline-barbital buffer with 0.25 % gelatine. CaC12-buffer solution. This was a mixture of 3 volumes of 0.05 M CaC12 with 2 volumes of saline-barbital buffer. The final CaC12 concentration was 0.03 M and the total ionic strength 0.15. Thromboplastin. Rabbit thromboplastin (Simplastin, WarnerChilcott, General Diagn. Div.) was used. The content of a vial was suspended in 6 ml gelatine-barbital buffer and 4 ml of a 0.25 % aqueous gelatine solution to obtain a stable suspension, which was distributed among vials and stored at -2OOC until used (always within 2 weeks). Tissue samples. A Potter-Elvehjem homogenizer, cooled in ice water, was used for preparing the tissue suspension with 0.9 ml gelatine-barbital buffer per 100 mg tissue (about 300 mg tissue was used). The tissue was homogenized for 30 minutes to complete cell destruction. The resulting 10 % suspension was stored at -2OOC overnight and passed through a cotton plug to remove coarse particles. The intermediate freezing and the addition of gelatine facilitated the preparation of uniform suspensions, necessary for reproducible results. For assays serial dilutions were prepared
vo1.6,~0.4
309
THROMBOPLASTIC ACTIVITY
POOLED OVARIAN
200-
NORMAL TISSUE
100-
50-
SIMPLASTIN
1030-
-
= MALIGNANT
OVARIAN
TUMOURS
A
60 CLOTTING
liL0
lie0
$60
TISSUE
DILUTION
TIME
stc
POOLED OVARIAN
200-
NORMAL TISSUE
SIMPLASTIN
20-
10
~___----I '/lo
= BENIGN = OVARIAN I ‘120
FIG. 1 A and B.
1 ‘/lo
OVARIAN
TUMOURS
METASTASES I
I
‘/SO
l/l60
B TISSUE
DILUTION
Clotting time in logarithmic scale with increasing tissue dilution. Benign and malignant ovarian tumours, metastases and normal ovarian tissue.
THROMBOPLASTIC
310
ACTIVITY
Vo1.6,No.4
bdith gelatine-barbital buffer. Assay system. The clotting mixture contained 0.2 ml titrated human plasma, 0.1 ml gelatine-barbital buffer and 0.1 ml thromboplastin suspension in unsiliconized glass tubes. The mixture was preheated for 3 minutes in a water bath at 37OC. The coagulation was initiated with 0.2 ml of the CaC12 solution and the coagulation time was determined. Assays were performed in duplicate.
RSSULTS The thromboplastic activity in ovarian tumurs,
normal
ovaries and Simplastin yielded linear curves in a double-logarithmic plot (Fig. 1 A and B). The results of assays performed in duplicate were largely identical. The curves are based on the mean of each pair of determination. Reanalyses of aliquots of the same samples one week later gave practically the same results. The method therefore appeared reliable. The thromboplastic activity in pooled normal ovaries was low, only 0.3 % of the Simplastin activity. Also the ovarian tumours had low activities, but they were significantly higher than in the normal ovaries. There was no difference in activity between benign and malignant ovarian tumours or ovarian metastases (Fig. 1 A and B; Table I and II).
DISCUSSION The growth and spread of malignant tumours is believed to vary with the thromboplastic and fibrinolytic activity. Thus, O'lvleara(6, 7) extracted a thermostable, water-soluble thromboplastic factor from human tumour tissue and found its activity to be related to the amount of fibrin deposits in the tissue, which was thought to be a suitable stroma for tumour cell invasion. Thornes (11) found this factor to convert fibrinogen to fibrin in the same way as thrombin. Others (9) feel that the deposition of fibrin is rather a reaction to the tumour. Holyoke et al. (5) showed in vitro that trauma increases the release of thromboplastic substances from both normal and tumour tissue and claimed that thromboplastic activity is not a unique feature
THROMBOPLASTIC
TABLE
ACTIVITY
131.
I
Classification of the ovarian tumours according to International Federation of Gynaecology and Obstetrics (F.I.G.O.) A. Non-neoplastic tumours
n= n=
B. Endometriotic tumours
1 1
C. Primary neoplastic tumours I. Neoplasm with endocrine significanceX
n = 1
II.Neoplasm with no endocrine significance Epithelial tumours: 1. serous (1A) serous (lCjx 2. mutinous
(2A)
n=
2
n=
4
n=l
mutinous (2CJx
n=
1
3. endometroidx
n=
2
4. concomitantx
n=
2
from gastric carcinomaX
n=
1
from uterine carcinomax
n=
1
from pancreatic carcinomax
n=
1
D. Ovarian metastases
x = malignant
TABLE
II
Thromboplastic activity (mean clotting time in set) in benign and malignant ovarian tumours and normal ovaries. Dilution 1:lO Tissue
Mean
S.D.
Malignant tumours Metastases Benign tumours
57 48 59
14 7 15
Normal ovaries
91
13
Stat. signif. diff.x p
x) significantly different from that in normal ovaries
312
THROMBOPLASTIC ACTIVITY
vo1.6,~0.4
of malignant cells. This postulation is corroborated by the present study, in which the thromboplastic activity of both benign and malignant ovarian tumours was found to be low, but significantly higher than that of normal ovaries. Fibrin degradation products (FDP) were often found in the blood in patients with malignant, but not in those with benign, ovarian tumours, a finding that proved to be of diagnostic value (12). FDP was thought to be due not only to the fibrinolytic activity of the tumours, but also to its thromboplastic activity, whose purpose was to promote the formation of fibrin. No difference in the fibrinolytic activity has been found between benign and malignant ovarian tumours (10). A difference in the occurrence of thromboplastic substances were therefore assumed. But the findings in the present investigation clearly refuted this assumption. The explanation should probably therefore be sought in the infiltrative growth of the malignant tumours with increased escape of thromboplastic substances, fibrinolytic activators or locally formed FDP into the bloodstream.
ACKNOWLEDGEMENT This investigation was supported by grants fran the Swedish Medical Research Council (B75-19X-87-llA), the Medical Faculty, University of Lund, and Tore Nilson's Fund for Medical Research.
REFERENCES 1. ASTRUP,T. Assay and content of tissue thromboplastin in different organs. Thromb. Diath. haemorrh.: 14., 401. 1965.. 2. ASTRUP,T., ALBRECHTSEN,O.K., CLAASSEN,M., and RASMUSSEN,J.
Thromboplastic and fibrinolytic activity of the human aorta. Circ. yes.: 7., 969. 1959. 3. GLAS,P., and ASTRUP,T. Fibrinolytic and thromboplastic activity of normal human heart valves. Amer. Heart J.: 76:4., 504. 1968. 4. GLAS,P., and ASTRUP,T. Thromboplastin and plasminogen
activator in tissues of the rabbit. Amer. J. Physiol.: 219:4., 1140. 1970.
vol.6,~0.4
THROMBOPUSTIC
ACTIVITY
313
5. HOLYOKE,E.D., FRANK,A.L., and WEISS,L. Tumour thromboplastin activity in vitro. Int. J. Cancer: 9., 258. 1972. 6. o'MEARA,R.A.Q. Coagulative properties of cancers. Irish J. Med. Sci.: 394., 474. 1958. 7. O'MEARA,R.A.Q. Fibrin formation and tumour growth. Thromb. Diath. haemorrh.: Suppl. 28., 137. 1968. 8. O'MEARA,R.A.Q., and THORNES,R.D. Some properties of the cancer coagulative factor. Irish J. Med. Sci.: 423., 106. 1961. 9. PETERSON,H.-I. Experimental studies on fibrinolysis in growth and spread of tumour. Acta chir. stand.: Suppl. 394., 1968. 10. SVANBERG,L., LINEU,F., PANDOLFI,M., and ASTEDT,B. Plasminogen activators in ovarian tumours. Acta path. microbial. stand.: 1974 (in press). 11. THORNES,R.D. Fibrinogen and the interstitial behaviour of cancer. In: Endogenous Factors Influencing Host-Tumor Balance pp 255-66. Ed. Wissler,R.W., Dao,T.L. and Wood,S. Jr. The University of Chicago Press, Chicago 1967. 12. ASTEDT,B., SVANBERG,L., and NILSSON,I.M. Fibrin degradation products and ovarian tumours. Brit. med. J. 4., 458. 1971.