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Blood coagulation abnormalities in Fischer strain rats bearing tumors
Life Sciences, Vol. 40, pp. 2523-2529 Printed in the U.S.A.
Pergamon Journals
BLOOD COAGULATIONABNORMALITIES IN FISCHER STRAIN RATS BEARING TUMORS M. Nakane, D.E. Morgan, G.R. Matyas, D.M. Morr6, and D.J. Morr6, Departments of Medicinal Chemistry and Foods and Nutrition Purdue University, West Lafayette, IN 47907
(Received in final form April 16, 1987)
Summar~
This study reports an unusual subpopulation of rats bearing transplanted tumors approximately 50% of the total and apparently unrelated to the presence of metastases, that exhibited shortened bleeding times despite reduced platelet numbers and/or fibrinogen levels. The remaining rats exhibited the expected inverse relationships between bleeding time and platelet numbers and/or fibrinogen level. Tumors were hepatomas and squamous cell carcinomas i n i t i a l l y induced in the Fischer strain of rats and carried by passage through tissue culture or syngenic recipient animals. Abnormalities in b l o o d coagulation are associated with clinical malignancy ( I ) . However, the relationships among abnormalities of coagulation and tumor presence are poorly understood and have been reported to vary widely (2). For example, Madewall et al. (13) reported that 25% of tumor-bearing dogs showed hypofibrinogenemia and 25% of the dogs showed hyperfibrinogenemia with approximately half of the animals being within the normal range. This variation may even extend to individual patients where a trend in one direction may reverse and the opposite trend will be established (19). In previous work from our laboratory, lipid-associated sialic acid of the sera was employed to detect and monitor tumor burden in rodents (10, 11), dogs (9), horses (12), and man (10). In these studies, we noted unusual but variable coagulation responses, some of which might have been related to tumor presence. In the present study, several fundamental factors of blood coagulation were monitored and correlated with tumor presence, size and growth rate in rats implanted with transplantable hepatocellular or squamous cell carcinomas. Bleeding time, platelet numbers, fibrinogen levels, and calcium levels were monitored as well as were prothrombin time as a measure of the extrinsic coagulation system and activated partial thromboplastin time (APTT) as a measure of the intrinsic coagulation system (5). The findings point to patterns of blood coagulation abnormalities related to tumor presence but with no apparent single commonality with respect to platelet numbers or fibrinogen levels. Reprint requests:
to Dr. D. J. Morr~ 0024-3205/87 $3,00 + .00 Copyright (c) 1987 Pergamon Journals Ind.
2524
Blood Coagulation in Rats With Tumors
Vol. 40, No. 26, 1987
Materials and Methods Male rats, 3-5 weeks old (30-60, Fischer) were obtained from Harlan Industries, Indianapolis, IN. Transplantable rat tumors were established lines of hepatocellular and squamous cell carcinomas (11) derived i n i t i a l l y by feeding a diet containing the chemical carcinogen 2-acetylaminofluorene. Hepatomas designated RL and squamous cell carcinomas designated JT were passaged in syngeneic recipient rats. Hepatomas designated as H strain were a line with metastatic potential derived from an RL tumor by passage through tissue culture (11). The RJ strains were squamous cell carcinomas derived from JT tumors in a similar manner. Tumors were transplanted by subcutaneous implantation of tumor (106 cells) into the lateral abdominal wall of rats under anesthesia. R a t s bearing tumors were sacrificed from 2 to 19 weeks (average of 5 weeks) after transplantation when tumors had grown to a sufficient size to be palpable. At the time of sacrifice, tumors were e~cised and their length and width measured in cm. Growth rate is given in cm~/week (see Fig. I). Ten normal rats and 50 rats bearing tumors were included in the study. Bleeding time was determined prior to animal sacrifice by a modification of the Duke method (20) in which 5 mm of the t a i l was snipped with a sharp scissors. Then the cut end of the t a i l was pressed against a piece of f i l t e r paper at 1 min intervals until bleeding ceased. The time at which bleeding ceased was designated as the bleeding time. Platelet numbers were counted with the aid of a hemacytometer and light microscope (20) using blood obtained by t a i l bleeding. Fibrinogen levels were obtained by a modification of the procedure of Ratnoff and Menzie (17). Blood was removed by cardiac puncture and c i t r a t e was added to obtain a plasma sample. Then thrombin (bovine topical) was added to form a f i b r i n clot and heated after addition of sodium hydroxide. Proteins released from the clot were estimated from the absorbance at 650 nm upon addition of Folin-Ciocalteu's phenol reagent. The standard curve was prepared using known concentrations of tyrosine.
Calcium was measured in. plasma (22) using a computerized atomic absorption spectrophotometer (Perkin-Elmer Model 5000). Prothrombin time and activated partial thromboplastin time were measured using a Becton-Dickinson Fibrosystem fibrometer. Data were analyzed by the least squares method and linear regression. Best f i t exponential curves were determined comparing results from only those animals designated as groups II and I I I in Figs. 2 and 3. The correlation coefficients between the experimental values and the computer-generated exponential functions represented by the data are given in the figures. Results Rats bearing a tumor of high growth rate (more than 0.4 cm2/week) had significantly shorter bleeding times (Fig. IA) and reduced platelet numbers (Fig. IB) compared to control rats and an average tendency for fibrinogen levels higher than normal (Fig. IC) but unchanged prothrombin times (Fig. ID). Calcium levels were within the normal range (Fig. IE). Approximately 50% of the animals showed metastases but there were no changes in blood parameters related to the presence of metastases. In order to analyze the relationships observed between bleeding time and platelet number (Fig. 2) and between bleeding time and fibrinogen level (Fig.3), the results from all animals were presented to show three groups of rats irrespective of tumor type, growth rate or metastatic incidence.
Vol. 40, No. 26, 1987
5o
Blood Coagulation
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2526
Blood Coagulation
in Rats With Tumors
Vol. 40, No. 26, 1987
Approximately 50% of the tumor-bearing rats analyzed with bleeding times shorter than normal also had reduced p l a t e l e t numbers (Fig. 2) and/or decreased fibrinogen levels (Fig. 3). These rats were designated as Group I in Figs. 2 and 3. The remaining rats showed the expected inverse correlations for p l a t e l e t number (r = -0.92) and for fibrinogen level (r : -0.74) and were designated as Groups II and I I I with Group II showing elevated p l a t e l e t numbers and/or fibrinogen levels and Group I I I showing reduced p l a t e l e t numbers and/or fibrinogen levels r e l a t i v e to control animals. There was no c o r r e l a t i o n between p l a t e l e t numbers and fibrinogen levels (r = -0.09) nor did plasma calcium levels correlate with bleeding time (r = -0.08). The numbers of animals are not the same for all data units since only bleeding times were f
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BLEEDING TIME (MIN) FIG. 2 P l a t e l e t number as a function of bleeding time. The c h a r a c t e r i s t i c response patterns resulted in three groups of animals. Group I = Bleeding times shorter than normal (27.6 min) and p l a t e l e t numbers lower than normal (13.4 X 10~/ml). Group I I = P l a t e l e t numbers greater than normal but with a shortened bleeding time. Group I I I = Prolonged bleeding time and with reduced number of platelets. • = H strain hepatomas. • = RT hepatomas. A= JT squamous cell carcinomas. • = WJ squamous cell carcinomas. Each group was approximately equally represented by tumor type, growth rate and metastatic incidence.
Vol. 40, No. 26, 1987
Blood Coagulation
in Rats With Tumors
2527
measured on all animals. The average prothrombin time which measures factors I I , V I I , and X was unchanged by tumor presence as was activated p a r t i a l thromboplastin time which measures p r i n c i p a l l y factors of the i n t r i n s i c pathway (20.3 ± 1.6 sec for I0 normal animals compared to 24.3 ± 10.2 sec for 8 animals bearing tumors). Response to tumor presence did not vary s i g n i f i c a n t l y comparing several d i f f e r e n t tumor strains.
Discussion Thrombic emboli and hemorrhage are often observed in patients with cancer, although embolism and hemorrhage are at opposite ends of the blood coagulation spectrum. In in vitro studies where tumor cells were injected into the t a i l vein of rats, abnormal coagulation (14) and plasma clot lysis (18) resulted from the presence of cells in circulation. Furthermore, feedback mechanisms involved in the coagulation system also may be influenced. Cooper et al. (4) proposed three states of blood coagulation abnormalities related to tumor presence: I) the decompensated state in which platelet number, fibrinogen level and some other factors decreased; 2) the compensated state in which several potentially depressed factors were normal; and 3) the over / w
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Fibrinogin levels as a function of bleeding time. As with p l a t e l e t numbers (Fig. 2), three groups of animals are depicted. Group I = Bleeding times shorter than normal (27.6 min) and fibrinogen levels lower than normal (130.7 mg/100 ml). Group I I = Fibrinogen levels higher than normal but with a shortened bleeding time. Group I I I = Prolonged bleeding time and with reduced fibrinogen l e v e l s . Symbols are as for Fig. 2. The c o r r e l a t i o n l i n e excludes those animals with values less than normal plus one standard deviation.
2528
Blood Coagulation in Rats With Tumors
compensated state excess of normal.
Vol. 40, No. 26, 1987
in which one or more coagulation factors were present in
In animal studies, decreased platelet numbers have been reported in mice (3, 15) and rats (7). Decreased p l a t e l e t numbers could be due to the aggregation of platelets (6) or the consumption of platelets within the tumor tissues (21). However, Poggi et al. (3, 15) suggested that thrombocytopenia was caused by an impaired production rather than an enhanced destruction, which might explain the consistency of reduced p l a t e l e t numbers since the p o s s i b i l i t y of a negative feedback on platelet production would be excluded. Tumor cells reportedly form a f i b r i n clot around themselves (15), and enhance f i b r i n o l y s i s (16, 23) but, in dogs, Madewall et al. (13) observed increased fibrinogen in 25% of the animals and a decreased fibrinogen in another 25% of the animals. The f i b r i n consumption resulting from the enhancement of f i b r i n formation and f i b r i n o l y s i s could be compensated, and frequently overcompensated, as described by Cooper et al. (4). In fact, Chmielewska et al. (3) reported impaired f i b r i n o l y s i s with increased fibrinogen levels in mice bearing JW sarcomas. Thus, potential compensation mechanisms for the control of f i b r i n production and impaired f i b r i n o l y s i s might h a v e contributed to the variations of fibrinogen levels in our observations. The measurement of blood c l o t t i n g or bleeding time has been ignored in most studies of the abnormalities of blood coagulation related to tumor presence. Our findings show that rats bearing tumors of high growth rates have shortened bleeding times compared to normal and low p l a t e l e t numbers without s i g n i f i c a n t changes in other coagulation parameters. Some of these rats also had low fibrinogen levels associated with shortened bleeding times. The findings suggest that .one or more coagulation factors, yet unidentified, are overproduced in response to the presence of tumors. One of the possible factors is phospholipid, which has been reported to be increased in sera of animals bearing tumors (8). Values of phospholipid 1.2- to 3-times normal (80-120 nmoles/ml phospholipid phosphorous) were observed (8) in rats bearing the RL tumors used in the present investigation. Acknowledgements We thank Dorothy A. Werderitsh for technical assistance Pharmaceutical Company Ltd., Tokyo, for financial support.
and Mochida
References I. 2. 3. 4. 5.
6. 7. 8.
J.L. AMBRUS, C.M., AMBRUS, I.B. MINK and J.W. PICKERN. J. Medicine 6, 61-64 (1975). M. BROWN. Cancer Res. 33, 1217-1224 (1973) J. CHMIELEWSKA, A. POGGI, L., MUSSONI, M.B., DONATI and S. GARATTINI. Eur. J. Cancer 16, 1399-1407 (1980). H.A. COOPER, E.J. BOWIE, C.A. OWENJr. Mayo Clin Proc. 49, 654 (1974). M.D. DONATI, L. POGGI, G. MUSSANI, G. DE GAETANOand S. GARATTINI. In: Cancer Invasion and Metastasis: Biologic mechanisms and therapy, ed., S.B. Day, W.P., Laird Myers, P. Stansly, S. Garattini and M.G. Lewis. Progress in Cancer Research and Therapy 5, 151-180 (1977). P. HILGARD. Br. J. Cancer 28, 429-435 (1973). P. HILGARD, R. HOHAGE, W. SCHMITT. Br. J. Haematol. 24, 245-254 (1973). T.M. KLOPPEL. Doctoral Thesis, Purdue University (1979).
Vol. 40, No. 26, 1987
9. 10. 11. 12. 13. 14. 15. 16.
17. 18. 19. 20. 21. 22. 23.
Blood Coagulation
in Rats With Tumors
2529
T.M. KLOPPEL, C.P. FRANZ, D.J. MORRCand R.C. RICHARDSON. Amer. J. Vet. Res. 39, 1313-1327 (1978). T.M. KLOPPEL, T.W. KEENAN, M.J. FREEMAN and D.J. MORRO. Proc.Natl.Acad. Sci. USA 74, 3011-3013 (1977). T.M. KLOPPEL and D.J. MORRE. J. Natl. Cancer Inst. 64, 1401-1411 (1977) T.M. KLOPPEL, R. RICHARDSON, D.S. TRAVER and D.J. MORRO. Amer. J. Vet. Res. 42, 1829-1830 (1981). B.R. MADEWALL, B.F. FELDMAN and S. O'NEILL. Throb. Haemost. 44, 35-38 (1980). R.A.Q. O'MEARE. Irish J. Med. Sci. 394, 474-479 (1980). A.N. POGGI, N. POLENTARUTTI, M.B. DONATI, G. DE GAETANOand S. GARATTINI. Cancer Res. 37, 272-277 (1977). J.P. QUIGLEY, L. OSSOWSKI and E. REICH. J. Bio. Chem. 249, 4306-4311 (1974). O:D. RATNOFF and C. MENZIE. In: Blood, Coagulation, Hemorrhage and Thrombosis, ed. L.M. Tocantins and L.A. Kazal, Grune & S t r a t t e n , New York, 224-226 (1964). E. REICH. Fed. Proc. 32, 2174-2175 (1973). G.H. SACK, J. LEVIN and W.R. BELL. Medicine 56, 1-37 (1977). C.E. SEIVERD. Hematology for Medical Technologists, Lea & Febiger, Philadelphia, 840 pp (1972). S.J. SLICHTER and L.A. HARKER. Ann. N.Y. Acad. Sci. 230, 252-261 (1974). F.W. SUNDERMANand J.E. CARROLL. Am. J. Clin. Path. 43, 302-310 (1965). J. UNKELESS, K. DANO, G.M. KELLERMAN and E. REICH. J. Biol. Chem. 249, 4295-4305 (1974).
Pergamon Journals
BLOOD COAGULATIONABNORMALITIES IN FISCHER STRAIN RATS BEARING TUMORS M. Nakane, D.E. Morgan, G.R. Matyas, D.M. Morr6, and D.J. Morr6, Departments of Medicinal Chemistry and Foods and Nutrition Purdue University, West Lafayette, IN 47907
(Received in final form April 16, 1987)
Summar~
This study reports an unusual subpopulation of rats bearing transplanted tumors approximately 50% of the total and apparently unrelated to the presence of metastases, that exhibited shortened bleeding times despite reduced platelet numbers and/or fibrinogen levels. The remaining rats exhibited the expected inverse relationships between bleeding time and platelet numbers and/or fibrinogen level. Tumors were hepatomas and squamous cell carcinomas i n i t i a l l y induced in the Fischer strain of rats and carried by passage through tissue culture or syngenic recipient animals. Abnormalities in b l o o d coagulation are associated with clinical malignancy ( I ) . However, the relationships among abnormalities of coagulation and tumor presence are poorly understood and have been reported to vary widely (2). For example, Madewall et al. (13) reported that 25% of tumor-bearing dogs showed hypofibrinogenemia and 25% of the dogs showed hyperfibrinogenemia with approximately half of the animals being within the normal range. This variation may even extend to individual patients where a trend in one direction may reverse and the opposite trend will be established (19). In previous work from our laboratory, lipid-associated sialic acid of the sera was employed to detect and monitor tumor burden in rodents (10, 11), dogs (9), horses (12), and man (10). In these studies, we noted unusual but variable coagulation responses, some of which might have been related to tumor presence. In the present study, several fundamental factors of blood coagulation were monitored and correlated with tumor presence, size and growth rate in rats implanted with transplantable hepatocellular or squamous cell carcinomas. Bleeding time, platelet numbers, fibrinogen levels, and calcium levels were monitored as well as were prothrombin time as a measure of the extrinsic coagulation system and activated partial thromboplastin time (APTT) as a measure of the intrinsic coagulation system (5). The findings point to patterns of blood coagulation abnormalities related to tumor presence but with no apparent single commonality with respect to platelet numbers or fibrinogen levels. Reprint requests:
to Dr. D. J. Morr~ 0024-3205/87 $3,00 + .00 Copyright (c) 1987 Pergamon Journals Ind.
2524
Blood Coagulation in Rats With Tumors
Vol. 40, No. 26, 1987
Materials and Methods Male rats, 3-5 weeks old (30-60, Fischer) were obtained from Harlan Industries, Indianapolis, IN. Transplantable rat tumors were established lines of hepatocellular and squamous cell carcinomas (11) derived i n i t i a l l y by feeding a diet containing the chemical carcinogen 2-acetylaminofluorene. Hepatomas designated RL and squamous cell carcinomas designated JT were passaged in syngeneic recipient rats. Hepatomas designated as H strain were a line with metastatic potential derived from an RL tumor by passage through tissue culture (11). The RJ strains were squamous cell carcinomas derived from JT tumors in a similar manner. Tumors were transplanted by subcutaneous implantation of tumor (106 cells) into the lateral abdominal wall of rats under anesthesia. R a t s bearing tumors were sacrificed from 2 to 19 weeks (average of 5 weeks) after transplantation when tumors had grown to a sufficient size to be palpable. At the time of sacrifice, tumors were e~cised and their length and width measured in cm. Growth rate is given in cm~/week (see Fig. I). Ten normal rats and 50 rats bearing tumors were included in the study. Bleeding time was determined prior to animal sacrifice by a modification of the Duke method (20) in which 5 mm of the t a i l was snipped with a sharp scissors. Then the cut end of the t a i l was pressed against a piece of f i l t e r paper at 1 min intervals until bleeding ceased. The time at which bleeding ceased was designated as the bleeding time. Platelet numbers were counted with the aid of a hemacytometer and light microscope (20) using blood obtained by t a i l bleeding. Fibrinogen levels were obtained by a modification of the procedure of Ratnoff and Menzie (17). Blood was removed by cardiac puncture and c i t r a t e was added to obtain a plasma sample. Then thrombin (bovine topical) was added to form a f i b r i n clot and heated after addition of sodium hydroxide. Proteins released from the clot were estimated from the absorbance at 650 nm upon addition of Folin-Ciocalteu's phenol reagent. The standard curve was prepared using known concentrations of tyrosine.
Calcium was measured in. plasma (22) using a computerized atomic absorption spectrophotometer (Perkin-Elmer Model 5000). Prothrombin time and activated partial thromboplastin time were measured using a Becton-Dickinson Fibrosystem fibrometer. Data were analyzed by the least squares method and linear regression. Best f i t exponential curves were determined comparing results from only those animals designated as groups II and I I I in Figs. 2 and 3. The correlation coefficients between the experimental values and the computer-generated exponential functions represented by the data are given in the figures. Results Rats bearing a tumor of high growth rate (more than 0.4 cm2/week) had significantly shorter bleeding times (Fig. IA) and reduced platelet numbers (Fig. IB) compared to control rats and an average tendency for fibrinogen levels higher than normal (Fig. IC) but unchanged prothrombin times (Fig. ID). Calcium levels were within the normal range (Fig. IE). Approximately 50% of the animals showed metastases but there were no changes in blood parameters related to the presence of metastases. In order to analyze the relationships observed between bleeding time and platelet number (Fig. 2) and between bleeding time and fibrinogen level (Fig.3), the results from all animals were presented to show three groups of rats irrespective of tumor type, growth rate or metastatic incidence.
Vol. 40, No. 26, 1987
5o
Blood Coagulation
A
~ ~ 200
30
m ~ 1oo 12
O
~20
m
~ 10
15
2525
3oo
E 40
o
in Rats With Tumors
~
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N L M H FIG. ] Blood coagulation factors related to tumor growth rate. Approximately half the tumors included in each growth rate group was H-strain hepatomas with the remainder divided equally between RL hepatomas and squamous cell carcinomas (WJ and JT). Since averages by category did not d i f f e r from overall averages, final averages only are presented. The numbers above the bars give total animals averaged (e.g. the number 23 for medium growth rate tumors of Fig. 1A was derived from 12 rats bearing H-strain hepatomas, 6 rats bearing R-strain hepatomas and 5 rats bearing squamous cell carcinomas). N = range of control animals. L = low growth rate; rats bearing tumors that grew less than 0.2 cm~/week. M = medium growth rate; rats bearing tumors growing 0.2 cm2/week or more but less than 0.4 cm~/week. H= high growth rate; 0.4 cm2 or more/ week. Numbers above each bar indicate the number of animals. Deviations shown are standard deviations.
2526
Blood Coagulation
in Rats With Tumors
Vol. 40, No. 26, 1987
Approximately 50% of the tumor-bearing rats analyzed with bleeding times shorter than normal also had reduced p l a t e l e t numbers (Fig. 2) and/or decreased fibrinogen levels (Fig. 3). These rats were designated as Group I in Figs. 2 and 3. The remaining rats showed the expected inverse correlations for p l a t e l e t number (r = -0.92) and for fibrinogen level (r : -0.74) and were designated as Groups II and I I I with Group II showing elevated p l a t e l e t numbers and/or fibrinogen levels and Group I I I showing reduced p l a t e l e t numbers and/or fibrinogen levels r e l a t i v e to control animals. There was no c o r r e l a t i o n between p l a t e l e t numbers and fibrinogen levels (r = -0.09) nor did plasma calcium levels correlate with bleeding time (r = -0.08). The numbers of animals are not the same for all data units since only bleeding times were f
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BLEEDING TIME (MIN) FIG. 2 P l a t e l e t number as a function of bleeding time. The c h a r a c t e r i s t i c response patterns resulted in three groups of animals. Group I = Bleeding times shorter than normal (27.6 min) and p l a t e l e t numbers lower than normal (13.4 X 10~/ml). Group I I = P l a t e l e t numbers greater than normal but with a shortened bleeding time. Group I I I = Prolonged bleeding time and with reduced number of platelets. • = H strain hepatomas. • = RT hepatomas. A= JT squamous cell carcinomas. • = WJ squamous cell carcinomas. Each group was approximately equally represented by tumor type, growth rate and metastatic incidence.
Vol. 40, No. 26, 1987
Blood Coagulation
in Rats With Tumors
2527
measured on all animals. The average prothrombin time which measures factors I I , V I I , and X was unchanged by tumor presence as was activated p a r t i a l thromboplastin time which measures p r i n c i p a l l y factors of the i n t r i n s i c pathway (20.3 ± 1.6 sec for I0 normal animals compared to 24.3 ± 10.2 sec for 8 animals bearing tumors). Response to tumor presence did not vary s i g n i f i c a n t l y comparing several d i f f e r e n t tumor strains.
Discussion Thrombic emboli and hemorrhage are often observed in patients with cancer, although embolism and hemorrhage are at opposite ends of the blood coagulation spectrum. In in vitro studies where tumor cells were injected into the t a i l vein of rats, abnormal coagulation (14) and plasma clot lysis (18) resulted from the presence of cells in circulation. Furthermore, feedback mechanisms involved in the coagulation system also may be influenced. Cooper et al. (4) proposed three states of blood coagulation abnormalities related to tumor presence: I) the decompensated state in which platelet number, fibrinogen level and some other factors decreased; 2) the compensated state in which several potentially depressed factors were normal; and 3) the over / w
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40 50 60 70 80 BLEEDING TIME (MIN) FIG.
I
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3
Fibrinogin levels as a function of bleeding time. As with p l a t e l e t numbers (Fig. 2), three groups of animals are depicted. Group I = Bleeding times shorter than normal (27.6 min) and fibrinogen levels lower than normal (130.7 mg/100 ml). Group I I = Fibrinogen levels higher than normal but with a shortened bleeding time. Group I I I = Prolonged bleeding time and with reduced fibrinogen l e v e l s . Symbols are as for Fig. 2. The c o r r e l a t i o n l i n e excludes those animals with values less than normal plus one standard deviation.
2528
Blood Coagulation in Rats With Tumors
compensated state excess of normal.
Vol. 40, No. 26, 1987
in which one or more coagulation factors were present in
In animal studies, decreased platelet numbers have been reported in mice (3, 15) and rats (7). Decreased p l a t e l e t numbers could be due to the aggregation of platelets (6) or the consumption of platelets within the tumor tissues (21). However, Poggi et al. (3, 15) suggested that thrombocytopenia was caused by an impaired production rather than an enhanced destruction, which might explain the consistency of reduced p l a t e l e t numbers since the p o s s i b i l i t y of a negative feedback on platelet production would be excluded. Tumor cells reportedly form a f i b r i n clot around themselves (15), and enhance f i b r i n o l y s i s (16, 23) but, in dogs, Madewall et al. (13) observed increased fibrinogen in 25% of the animals and a decreased fibrinogen in another 25% of the animals. The f i b r i n consumption resulting from the enhancement of f i b r i n formation and f i b r i n o l y s i s could be compensated, and frequently overcompensated, as described by Cooper et al. (4). In fact, Chmielewska et al. (3) reported impaired f i b r i n o l y s i s with increased fibrinogen levels in mice bearing JW sarcomas. Thus, potential compensation mechanisms for the control of f i b r i n production and impaired f i b r i n o l y s i s might h a v e contributed to the variations of fibrinogen levels in our observations. The measurement of blood c l o t t i n g or bleeding time has been ignored in most studies of the abnormalities of blood coagulation related to tumor presence. Our findings show that rats bearing tumors of high growth rates have shortened bleeding times compared to normal and low p l a t e l e t numbers without s i g n i f i c a n t changes in other coagulation parameters. Some of these rats also had low fibrinogen levels associated with shortened bleeding times. The findings suggest that .one or more coagulation factors, yet unidentified, are overproduced in response to the presence of tumors. One of the possible factors is phospholipid, which has been reported to be increased in sera of animals bearing tumors (8). Values of phospholipid 1.2- to 3-times normal (80-120 nmoles/ml phospholipid phosphorous) were observed (8) in rats bearing the RL tumors used in the present investigation. Acknowledgements We thank Dorothy A. Werderitsh for technical assistance Pharmaceutical Company Ltd., Tokyo, for financial support.
and Mochida
References I. 2. 3. 4. 5.
6. 7. 8.
J.L. AMBRUS, C.M., AMBRUS, I.B. MINK and J.W. PICKERN. J. Medicine 6, 61-64 (1975). M. BROWN. Cancer Res. 33, 1217-1224 (1973) J. CHMIELEWSKA, A. POGGI, L., MUSSONI, M.B., DONATI and S. GARATTINI. Eur. J. Cancer 16, 1399-1407 (1980). H.A. COOPER, E.J. BOWIE, C.A. OWENJr. Mayo Clin Proc. 49, 654 (1974). M.D. DONATI, L. POGGI, G. MUSSANI, G. DE GAETANOand S. GARATTINI. In: Cancer Invasion and Metastasis: Biologic mechanisms and therapy, ed., S.B. Day, W.P., Laird Myers, P. Stansly, S. Garattini and M.G. Lewis. Progress in Cancer Research and Therapy 5, 151-180 (1977). P. HILGARD. Br. J. Cancer 28, 429-435 (1973). P. HILGARD, R. HOHAGE, W. SCHMITT. Br. J. Haematol. 24, 245-254 (1973). T.M. KLOPPEL. Doctoral Thesis, Purdue University (1979).
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