- Email: [email protected]
fibrinogen in the mink
Animal Reproduction Science, 16 (1988) 135-144
135
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Breeding Season and Pregnancy-Associated Increase in P l a s m a Levels of Fibrin/Fibrinogen in the Mink J. HAU', O.M. POULSEN 2, A. BRANDT 3 and S. ALEXANDERSEN 4
1Laboratory Animal Unit, Department of Veterinary Pathology, Royal Veterinary and Agricultural University, Copenhagen, Bi~lowsvej13, 1870 Frederiksberg C (Denmark) 2Institute of Obstetrics and Gynaecology, University of Odense (Denmark) 3National Institute of Animal Science, Research in Fur Animals, TroUesminde, Roskildevej 48H, 3400 Hillerod (Denmark) 4National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana (U.S.A.) (Accepted 7 October 1987)
ABSTRACT Hau, J., Poulsen, O.M., Brandt, A. and Alexandersen, S., 1988. Breeding season and pregnancyassociated increase in plasma levels of fibrin/fibrinogen in the mink. Anim. Reprod. Sci., 16: 135-144. Antisera against a mink plasma protein complex which cross reacted immunologically with human fibrinogen were produced. During the breeding season extremely high plasma levels of fibrinogen/fibrin were recorded in female mink using rocket immunoelectrophoresis. During the subsequent gestation period the levels gradually decreased to reach non-breeding season levels at term. The mink fibrin/fibrinogen molecules were found to exist mainly in large aggregates with a molecular weight around 630 kD. The molecules were found to bind heparin and concanavalin A.
INTRODUCTION
The breeding season of the mink is approximately 4 weeks in March in the Northern hemisphere, and during the mating season the female usually exhibits oestrus two or three times. Ovulation is normally induced through mating, and, like many other mammalian species, the mink has delayed implantation. The length of gestation in the mink varies from 40 to 75 days, and gestational period is usually presented as the number of days between the last mating and parturition. During the first half of the gestational period it is extremely difficult to diagnose pregnancy in mink. From mid-April it is possible to judge pregnancy in the majority of animals. It is, however, of economic interest to obtain a method for distinguishing between pregnant and non-pregnant females as early as the 0378-4320/88/$03.50
© 1988 Elsevier Science Publishers B.V.
136 end of March when the pelt still has a profitable value. From a reproductive viewpoint it is of minor importance to diagnose pregnancy since it is not possible to repeat mating after a negative diagnosis until the next year (Venge, 1973). Pregnancy-related quantitative and qualitative changes in serum proteins have been studied for many years, and pregnancy-associated serum proteins which increase markedly in concentration in the maternal circulation during pregnancy have been detected in many species (Hau, 1986). As early as 1971, a new pregnancy-associated y-mobile serum protein was described in the mink (Larsen et al., 1971 ), but, unfortunately antisera against this protein are no longer available (Larsen, personal communication). With the exception of afetoprotein (AFP), which has been demonstrated in all species examined (Gitlin, 1975 ), most of the so-called new pregnancy-associated serum/plasma proteins possess extensive species specificity with respect to antigenic determinants. Other pregnancy proteins may have a limited number of epitopes in common, and analogies between mammalian species may be demonstrated using antisera raised in phylogenetically remote species, e.g. the hen (Hau et al., 1980b; 1981b). In plasma or serum of the pregnant mink only one pregnancy protein, namely AFP, may be demonstrated using a number of available antisera against pregnancy proteins of other species (results not shown ), and the aim of the present study was to examine pregnancy-associated changes in the plasma proteins of the mink. MATERIALSAND METHODS
Production of antisera Pooled heparinized plasma of 5 pregnant female mink was separated by size chromatography using Sephacryl sepharose (Pharmacia) packed in an LKB column kept at 20 °C and equilibrated with 0.05 M sodium phosphate buffer. The flow rate was 2 ml cm -2 h -1, and fractions of 5 ml were collected. The separation profile measured continuously at 280 nm consisted of three major peaks. The fractions from each of these were separately pooled, dialyzed against isotonic saline and concentrated to 2 mg ml- 1, using a cut off of 10 kD. A series of rabbits were immunized using a previously described procedure (Hau et al., 1978). Following four immunizations, the two rabbits immunized with the high molecular weight fraction of the separation had produced antibodies against fibrin/fibrinogen - antibodies which remained in the antiserum preparations following solid phase absorption with male heparinized plasma. The solid phase absorption was performed by chromatography of the antiserum using divinylsulfone activated agarose (Kem-En-Tec, Denmark) which
137 was coupled with male mink plasma proteins in the following manner. The gel was washed on a Biichner funnel with three volumes of 0.5 M phosphate buffer pH 7.2. Five ml pooled male mink plasma was mixed with 8.5 g gel. The mixture was rotated overnight at room temperature. The mixture was washed in 10 volumes 1 M NaC1 and remaining active groups blocked with 1 volume 0.1 M ethanolamine pH 8.0 overnight (rotation). The gel was then packed in a Pharmacia K/9 column and washed with five volumes 1 M NaC1, five volumes phosphate buffer pH 7.2, five volumes acetate buffer pH 4.0 and finally five volumes phosphate buffer pH 7.2. All buffers contained 1 mM NAN3. Antiserum samples of 20 ml were chromatographed, and the unbound fraction of proteins was monospecific rabbit anti-fibrin/fibrinogen/fibrinogen degradation products (FDP) as monitored by crossed immunoelectrophoresis using the antiserum preparation at a concentration of 1:5 in the second dimension gel. Size chromatography of plasma from pregnant mink was also performed in Sephadex G-100 and Sepharose 4B (Pharmacia) using a similar column-buffer system. The Sepharose 4B column was calibrated for molecular weight estimation using antisera (Dakopatts) against the following marker proteins: human IgG (150 kD), human fibrinogen (340 kD) and human IgM (971 kD).
Blood samples Small blood samples of mink were obtained by cutting a toe nail too close and collecting the blood in Na-heparinized haematocrit tubes. Larger samples were obtained by heart puncture of animals anaesthetized with pentobarbital (50 mg/kg) injected intraperitoneally. The blood samples were centrifuged (15 000 g for 15 min) immediately after they were drawn, and the plasma was stored at - 2 0 ° C until analysis. Serial blood samplings from 12 females were begun on 6 March and lasted until 18 March. In this period samples were taken every second day. Following 18 March samples were taken every week, until one week following delivery. The animals gave birth between 29 April and 13 May.
Electroimmunoassays Crossed immunoelectrophoresis (Hau et al., 1978), crossed concanavalin A affino immunoelectrophoresis (Hau et al., 1980a), crossed heparin affino immunoelectrophoresis (Westergaard et al., 1983), quantitative rocket immunoelectrophoresis (Hau et al., 1978) and rocket line immunoelectrophoresis (Hau et al., 1981a) were performed as previously described.
138
Statistics Differences between means were evaluated by the Student's t-test. RESULTS
Immunological cross-reaction with human fibrinogen Immunological cross-reaction between the mink protein against which antibodies had been raised and human fibrinogen was demonstrated by crossed immunoelectrophoreses with intermediary gels containing rabbit anti-human fibrinogen antibodies (Dakopatts Code A080). The antiserum against human fibrinogen completely precipitated the fibrinogen in the mink plasma used as the antigen. This resulted in a shift of the precipitate towards the first dimension gel (Fig. 1).
Analysis of antisera Following solid phase absorption of the antisera from the rabbits immunized with the high molecular weight plasma protein fraction, antibodies against fibrin/fibrinogen/FDP remained. Fig. 2 shows two crossed immunoelectrophoreses with (a) non -pregnant female plasma and (b) pregnant female plasma as antigen. Only pregnant females had fibrin/fibrinogen/FDP levels above the detection limits of this assay. The precipitate demonstrated a large extent of heterogeneity, and, regardless of the distance of first dimension migration, the various discrete populations of molecules with immunoreactivity could not be separated electrophoretically.
Levels of fibrin/fibrinogen/FDP in the circulation of female mink The levels of fibrin/fibrinogen/FDP were measured by rocket immunoelectrophoresis in 130 heparin plasma samples from 12 female mink during the breeding season and during pregnancy. The sensitivity of the assay was approximately 2 #g/ml. The between assay coefficients of variation were 4.7 +_0.1% (n = 15). Measurements within each week were pooled, and levels were very high during the breeding season. Around the time of implantation (5-6 weeks prior to the parturition) the levels decreased to close to the detection limit at parturition (Fig. 3 ). Compared to week 0 before parturition, levels were significantly increased at week 2 ( P < 0.01 ) and weeks 3, 4, 5, 6, 7, 8 and 9 (P<0.001).
139
a
b
3 i
~ -
~
2
Fig. 1. Crossed immunoelectrophoresis with antibody-containing intermediate gel, (1) first dimension gel, (2) first antibody-containing gel, and (3) reference antibody-containing gel containing the anti-mink fibrin/fibrinogen/FDP antibody preparation h5 (v/v). The antigen was plasma of pregnant mink and in (a) the first antibody-containing gel contained no antibodies, and in (b) the first antibody-containing gel contained rabbit anti-human fibrinogen (Dakopatts) 1:50 (v/v).
a il~ ~ili~!~:i! ~ I
Fig. 2. Crossed immunoelectrophoresis of 5/11 samples of plasma from (a) non-pregnant female mink and (b) pregnant mink. The second dimension gel contained the anti-mink fibrin/fibrinogen/FDP antibody preparation h20 (v/v).
Molecular weight estimation
U s i n g a c a l i b r a t e d c o l u m n p a c k e d w i t h S e p h a r o s e 4B a m o l e c u l a r weight o f 630 kD was e s t i m a t e d . N o molecules with f i b r i n / f i b r i n o g e n / F D P epitopes p r e s e n t in m i n k p l a s m a were f o u n d to have m o l e c u l a r weights smaller t h a n 150 kD b e c a u s e all t h e i m m u n o r e a c t i v e molecules were f o u n d in t h e void volu m e following gel f i l t r a t i o n of m i n k p l a s m a in S e p h a d e x G-100 with a n exclusion limit o f 150 kD.
140 2C • Au/m~"/'. 10-2 18
\
-+ls. dev.
\
16 14
i
12
I
10
\
8 6 4
2
9
,
,
•
,
8
7
6
5
.
3
2
1
o
Weeks before parturition Fig. 3. Rocket immunoelectrophoresis measurements of mink fibrin/fibrinogen/FDP in 130 heparin plasma samples from 12 female mink during breeding season and gestation. One hundred Arbitrary Units (AU)/ml refers to the concentration of protein in a pool of heparin plasma from late pregnant mink (n = 10). Values represent means +__1 SD.
Fig. 4. Crossed affino immunoelectrophoresis with anti-mink fibrin/fibrinogen/FDP antibody preparation, 1:20 (v/v). The antigen was heparin plasma of pregnant mink (a) without Con A and (b) with 500 #g Con A/cm 2 in the first dimension gel.
Interaction with heparin Incorporation of increasing amounts of heparin in the first dimension of crossed immunoelectrophoresis resulted in increasing migration velocity of the fibrin/fibrinogen complex. The following amounts of heparin/ml agarose were
141 employed: 0, 20 i.u., 50 i.u., 100 i.u. and 200 i.u., and the corresponding distances of the major components were: 4 mm, 9 mm, 10 mm, 11 mm and 15 mm. Interaction with concanavalin A
Increasing amounts of concanavalin A (Con A) in the first dimension gel in crossed immunoelectrophoreses resulted in increased retardation in migration velocity of fibrin/fibrinogen. At concentrations above 400 ]~g/cm2 in the first dimension gel, the fibrin/fibrinogen molecules were completely precipitated by Con A and not visible in the second dimension gel (Fig. 4). DISCUSSION The fibrin/fibrinogen/FDP molecules measured in the present assay were all found to be larger than 150 kD. The average molecular weight of the majority of molecules was 630 kD. This demonstrated that the assay measured an unknown mixture of soluble fibrin, fibrinogen and the FDP's fragment X (240 kD ) and - Y ( 155 kD), whereas fragment D (83 kD ) and E (50 kD ), if present at all, must have been circulating in very low amounts. Since fibrin, in the presence of plasmin, is degraded to fragment X, which in turn is degraded to fragment Y and - D ( 1:1 ) it seems unlikely that fragment Y is present when - D is not. This suggests that fibrinolysis is at a low level in female mink and not above the detection limit of the assay during the breeding season and pregnancy, and that the present assay predominantly measured soluble fibrin and high molecular weight fibrinogen (HMW). Moreover the molecular weight analyses indicate that the majority of the fibrinogen epitopes found in pregnant female mink plasma are localized as HMW fibrinogen and soluble fibrin. HMW fibrinogen-fibrin has also been described in women, the molecular weights of the complexes being between 500 and 1000 kD. HMW fibrinogen in the human has also been demonstrated in other situations such as acute glomerulonephritis, thromboembolism after operation and oral contraceptive use (Ozanne et al., 1983). Quantitative clot-weight assays are still widely used for fibrinogen quantification and yield results comparable to immunodiffusion methods (Reid and Onwuameze, 1984). However, the rocket immunoelectrophoresis is generally accepted to be more sensitive and rapid than immunodiffusion, and, in the present context, it is an advantage to use micro methods which require appreciably smaller volumes of plasma. The mating system employed in the present study was homoandry with three mating periods (1 + 9 + 1 ) to ensure maximal fertility. Mating induces ovulation, but, in mink, the time between ovulation and implantation varies due to
142 delayed implantation. The females implanted around April, i.e. week - 5 prior to parturition, at which time the concentration of fibrinogen was decreased from its very high levels in the maternal circulation during the breeding season. Around a week before parturition the levels were very low, quickly approaching the extremely low levels of males and females outside the breeding season. During human pregnancy, levels of circulating FDP and fibrinogen which increase until term have been extensively demonstrated (Maki et al., 1980; Paternoster et al., 1980; Hellgren and Blomb~ck, 1981; Mutoh et al., 1982; Persson et al., 1982; Ozanne et al., 1983; Whitfield et al., 1983; Nakamura et al., 1984; Stifling et al., 1984). Thrombin-mediated fibrin generation during human pregnancy has been demonstrated by measuring increasing levels of fibrinopeptide A (FPA) which is cleaved from fibrinogen in the process of fibrin generation (Wallmo et al., 1984; Weiner et al., 1984). Increasing levels of plasma soluble fibrin and FDP, but fairly constant fibrinogen levels, were measured by Thorburn and coworkers (1982), suggesting a degree of haemostatic activation. It is generally agreed upon that human pregnancy constitutes a hypercoagulable condition, whereas the fibrinogen levels and viscosity are lower in fetal plasma (Foley et al., 1978). In cows, moderately higher maternal plasma levels of fibrinogen in the last month of gestation have been reported (Ek, 1972; Gentry et al., 1979), and, in the pregnant dog, a two- or three-fold increase in fibrinogen concentration is seen during gestation (Gentry et al., 1981 ). By contrast, an apparent lack of increase in fibrinogen concentrations towards the end of the gestation period was observed in pregnant sows, which exhibited an increase at parturition only (Liptrap et al., 1984). No changes in plasma fibrinogen levels were observed throughout the gestation period of rabbits (Muller-Berghaus et al., 1978), and the present study of concentration changes in maternal plasma of mink makes it apparent that major differences in the fibrinogen profile during pregnancy exist between species. Like human fetal plasma, fetal lamb plasma fibrinogen concentration is much lower than that of maternal plasma (Kisker et al., 1981 ). Further studies are needed to reveal when the plasma levels begin to increase to reach the very high levels seen in female mink during the breeding season and to elucidate the biological significance of these findings. Heparin is a heterogenous glycosaminoglycan with anticoagulant activity, and controversy exists as to whether heparin binds fibrinogen or not (McKay and Laurell, 1980). The present study demonstrates that mink fibrinogen binds to heparin during electrophoresis, and this is manifested by an increased migration velocity due to the high negative charge of heparin. The complete precipitation by Con A demonstrates that the fibrin/fibrinogen molecules presumably have more than one binding site for Con A and are glycosylated (Bog-Hansen and Hau, 1983). Twenty percent of total circulating proteins in plasma has been observed to bind to immobilized heparin (Teisner et al., 1983), and the affinity of mink
143
fibrinogen for heparin as well as Con A suggests selective procedures for the isolation and purification of this molecule. ACKNOWLEDGEMENT
This study was kindly financed by Dansk Pelsdyravlerforenings Forskningsfond, and the authors thank Mrs. Regina Lund for skilful technical assistance.
REFERENCES Bog-Hansen, T.C. and Hau, J., 1983. Glycoproteins and glycopeptides. J. Chromatogr. Libr., 18B: 219-252. Ek, N., 1972. The quantitative determination of fibrinogen in normal bovine plasma and in cows with inflammatory conditions. Acta Vet. Scand., 13: 175-184. Foley M.E., Isherwood, D.M. and McNicol, G.P., 1978. Viscosity, haematocrit, fibrinogen and plasma proteins in maternal and cord blood. Br. J. Obstet. Gynaecol., 85: 500-504. Gentry, P.A., Liptrap, R.M. and Black, W.D., 1979. Changes in blood coagulation profiles of dairy cows during pregnancy and in heifer calves after hormone treatment. Can. J. Anim. Sci., 59 (3): 503-510. Gentry, P.A. and Liptrap, R.M., 1981. Influence of progesterone and pregnancy on canine fibrinogen values. J. Small Anim. Pract., 22: 185-194. Gitlin, D., 1975. Normal biology of ~-fetoprotein. Ann. N.Y. Acad. Sci., 259: 7-16. Hau, J. (Editor), 1986. Pregnancy Proteins in Animals. Walter de Gruyter, Berlin, NY. Hau, J., Svendsen, P.E., Teisner, B. and Svehag, S.-E., 1978. Studies of pregnancy-associated murine serum proteins. J. Reprod. Fertil., 54: 239-243. Hau, J., Svendsen, P., Teisner, B. and Thomsen Pedersen, G., 1980a. Immunochemical characterization and serum concentrations of pregnancy-associated serum proteins in mice. J. Reprod. Fertil., 58: 389-393. Hau, J., Westergaard, J.G., Svendsen, P., Bach, A. and Teisner, B., 1980b. Comparison of the pregnancy-associated murine protein-2 and human pregnancy-specific fll-glycoprotein. J. Reprod. Fertil., 60: 115-119. Hau, J., Svendsen, P., Teisner, B. and Brandt, J., 1981a. Pregnancy-associated murine protein 3 (PAMP -3 ): Characterization and serum levels. Biol. Reprod., 24:163-169. Hau, J., Westergaard, J.G., Svendsen, P., Bach, A. and Teisner, B., 1981b. Comparison of pregnancy-associated murine protein-1 and human pregnancy zone protein. J. Reprod. Immunol., 3: 341-349. Hellgren, M. and Blomb~ick, M., 1981. Studies on blood coagulation and fibrinolysis in pregnancy, during delivery and in the puerperium. Gynecol. Obstet. Invest., 12: 141-154. Kisker, C.T., Robillard, J.E. and Clark, W.R., 1981. Development of blood coagulation - A fetal lamb model. Pediatr. Res., 15: 1045-1050. Larsen, A.E., Porter, D.D. and Porter, H.G., 1971. Pregnancy-associated serum protein in mink. Proc. Soc. Exp. Biol. Med., 136(2): 430-32. Liptrap, R.M. and Gentry, P.A., 1984. Interrelationships between blood coagulation factors and plasma hormone levels in the periparturient sow. Can. J. Anim. Sci., 64: 655-665. Maki, M., Soga, K. and Seki, H., 1980. Fibrinolytic activity during pregnancy. Tohoku J. Exp. Med., 132: 349-354. McKay, E.J. and Laurell, C.-B., 1980. The interaction of heparin with plasma proteins. Demon-
144
stration of different binding sites for antithrombin III complexes and antithrombin III. J. Lab. Clin. Med., 95 (1): 69-80. Muller-Berghaus, G., MoeUer, R.M. and Mahn, I., 1978. Fibrinogen turnover in pregnant rabbits during the first and last thirds of gestation. Am. J. Obstet. Gynecol., 131: 655-660. Mutoh, S., Maki, M. and Takahashi, S.,1982. Cold insoluble globulin. Studies on coagulationfibrinolysis system and cold insoluble globulin in normal pregnancy. Acta Obstet. Gynaecol. Jpn. (Engl. Ed.), 34(3): 369-377. Nakamura, Y., Abbott, R., Takeshita, T., Nishimura, K. and Shinagawa, S., 1984. Fibrinogen and its fraction during pregnancy and puerperium. Biol. Res. Pregnancy, 5 (3): 110-112. Ozanne, P., Linderkamp, 0., Miller, F.C. and Meiselman, H.J., 1983. Erythrocyte aggregation during normal pregnancy. Am. J. Obstet. Gynecol., 147 (5): 576-583. Paternoster, D,, Grella, P., Canova, P., Maggino, T., Valente, S. and Menighetti, M., 1980. Modifications of coagulation factors during normal and pathological pregnancies. Clin. Exp. Obstet. Gynecol., 7 (2): 112-115. Persson, B., Holmberg, L., Astedt, B. and Nilsson, I., 1982. Coagulation and fibrinolysis in pregnancy complicated by intra uterine growth retardation. Acta Obstet. Gynecol. Scand., 61 (5): 455-459. Reid, H.L. and Onwuameze, I.C., 1984. Correlation of a clot-weight and radial immunodiffusion method for estimation of plasma fibrinogen concentration. Trop. Geogr. Med., 36: 13-16. Stirling, Y., Woolf, L., North, W.R.S., Seghatchian, M.J. and Meade, T.W., 1984. Haemostasis in normal pregnancy. Thromb. Haemostas. (Stuttgart), 52 (2): 176-182. Teisner, B., Davey, M.W. and Grudzinskas, J.G., 1983. Interaction between heparin and plasma proteins analysed by crossed immunoelectrophoresis and affinity chromatography. Clin. Chim. Acta, 127: 413-417. Thorburn, J., Drummond, M.M., Whigham, K.A., Lowe, G.D.O., Forbes, C.D., Prentice, C.R.M. and Whitfield, C.R., 1982. Blood viscosity and haemostatic factors in late pregnancy, preeclampsia and fetal growth retardation. Br. J. Obstet. Gynaecol., 89: 117-122. Venge, 0., 1973. Reproduction in the Mink. R. Vet. Agric. Univ. Yearbook, Copenhagen, Denmark. pp. 95-146. WaUmo, L., Karlsson, K. and Teger-Nilsson, A.-C., 1984. Fibrinopeptide A and intravascular coagulation in normotensive and hypertensive pregnancy and parturition. Acta Obstet. Gynecol. Scand., 63: 637-640. Weiner, C.P., Kwann, H., Hauck, W.W., Duboe, F.J., Paul, M. and Wallemark, C.-B., 1984. Fibrin generation in normal pregnancy. Obstet. Gynecol., 64 (1): 46-48. Westergaard, J.G., Hau, J., Teisner, B. and Grudzinskas, J.G., 1983. Specific and reversible interaction between pregnancy-associated plasma protein A and heparin. Placenta, 4: 13-18. Whitfield, L., Lele, A.S. and Levy, G., 1983. Effect of pregnancy on the relationship between concentration and anti coagulant action of heparin. Clin. Pharmacol. Ther., 34 (1): 23-28.
135
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Breeding Season and Pregnancy-Associated Increase in P l a s m a Levels of Fibrin/Fibrinogen in the Mink J. HAU', O.M. POULSEN 2, A. BRANDT 3 and S. ALEXANDERSEN 4
1Laboratory Animal Unit, Department of Veterinary Pathology, Royal Veterinary and Agricultural University, Copenhagen, Bi~lowsvej13, 1870 Frederiksberg C (Denmark) 2Institute of Obstetrics and Gynaecology, University of Odense (Denmark) 3National Institute of Animal Science, Research in Fur Animals, TroUesminde, Roskildevej 48H, 3400 Hillerod (Denmark) 4National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana (U.S.A.) (Accepted 7 October 1987)
ABSTRACT Hau, J., Poulsen, O.M., Brandt, A. and Alexandersen, S., 1988. Breeding season and pregnancyassociated increase in plasma levels of fibrin/fibrinogen in the mink. Anim. Reprod. Sci., 16: 135-144. Antisera against a mink plasma protein complex which cross reacted immunologically with human fibrinogen were produced. During the breeding season extremely high plasma levels of fibrinogen/fibrin were recorded in female mink using rocket immunoelectrophoresis. During the subsequent gestation period the levels gradually decreased to reach non-breeding season levels at term. The mink fibrin/fibrinogen molecules were found to exist mainly in large aggregates with a molecular weight around 630 kD. The molecules were found to bind heparin and concanavalin A.
INTRODUCTION
The breeding season of the mink is approximately 4 weeks in March in the Northern hemisphere, and during the mating season the female usually exhibits oestrus two or three times. Ovulation is normally induced through mating, and, like many other mammalian species, the mink has delayed implantation. The length of gestation in the mink varies from 40 to 75 days, and gestational period is usually presented as the number of days between the last mating and parturition. During the first half of the gestational period it is extremely difficult to diagnose pregnancy in mink. From mid-April it is possible to judge pregnancy in the majority of animals. It is, however, of economic interest to obtain a method for distinguishing between pregnant and non-pregnant females as early as the 0378-4320/88/$03.50
© 1988 Elsevier Science Publishers B.V.
136 end of March when the pelt still has a profitable value. From a reproductive viewpoint it is of minor importance to diagnose pregnancy since it is not possible to repeat mating after a negative diagnosis until the next year (Venge, 1973). Pregnancy-related quantitative and qualitative changes in serum proteins have been studied for many years, and pregnancy-associated serum proteins which increase markedly in concentration in the maternal circulation during pregnancy have been detected in many species (Hau, 1986). As early as 1971, a new pregnancy-associated y-mobile serum protein was described in the mink (Larsen et al., 1971 ), but, unfortunately antisera against this protein are no longer available (Larsen, personal communication). With the exception of afetoprotein (AFP), which has been demonstrated in all species examined (Gitlin, 1975 ), most of the so-called new pregnancy-associated serum/plasma proteins possess extensive species specificity with respect to antigenic determinants. Other pregnancy proteins may have a limited number of epitopes in common, and analogies between mammalian species may be demonstrated using antisera raised in phylogenetically remote species, e.g. the hen (Hau et al., 1980b; 1981b). In plasma or serum of the pregnant mink only one pregnancy protein, namely AFP, may be demonstrated using a number of available antisera against pregnancy proteins of other species (results not shown ), and the aim of the present study was to examine pregnancy-associated changes in the plasma proteins of the mink. MATERIALSAND METHODS
Production of antisera Pooled heparinized plasma of 5 pregnant female mink was separated by size chromatography using Sephacryl sepharose (Pharmacia) packed in an LKB column kept at 20 °C and equilibrated with 0.05 M sodium phosphate buffer. The flow rate was 2 ml cm -2 h -1, and fractions of 5 ml were collected. The separation profile measured continuously at 280 nm consisted of three major peaks. The fractions from each of these were separately pooled, dialyzed against isotonic saline and concentrated to 2 mg ml- 1, using a cut off of 10 kD. A series of rabbits were immunized using a previously described procedure (Hau et al., 1978). Following four immunizations, the two rabbits immunized with the high molecular weight fraction of the separation had produced antibodies against fibrin/fibrinogen - antibodies which remained in the antiserum preparations following solid phase absorption with male heparinized plasma. The solid phase absorption was performed by chromatography of the antiserum using divinylsulfone activated agarose (Kem-En-Tec, Denmark) which
137 was coupled with male mink plasma proteins in the following manner. The gel was washed on a Biichner funnel with three volumes of 0.5 M phosphate buffer pH 7.2. Five ml pooled male mink plasma was mixed with 8.5 g gel. The mixture was rotated overnight at room temperature. The mixture was washed in 10 volumes 1 M NaC1 and remaining active groups blocked with 1 volume 0.1 M ethanolamine pH 8.0 overnight (rotation). The gel was then packed in a Pharmacia K/9 column and washed with five volumes 1 M NaC1, five volumes phosphate buffer pH 7.2, five volumes acetate buffer pH 4.0 and finally five volumes phosphate buffer pH 7.2. All buffers contained 1 mM NAN3. Antiserum samples of 20 ml were chromatographed, and the unbound fraction of proteins was monospecific rabbit anti-fibrin/fibrinogen/fibrinogen degradation products (FDP) as monitored by crossed immunoelectrophoresis using the antiserum preparation at a concentration of 1:5 in the second dimension gel. Size chromatography of plasma from pregnant mink was also performed in Sephadex G-100 and Sepharose 4B (Pharmacia) using a similar column-buffer system. The Sepharose 4B column was calibrated for molecular weight estimation using antisera (Dakopatts) against the following marker proteins: human IgG (150 kD), human fibrinogen (340 kD) and human IgM (971 kD).
Blood samples Small blood samples of mink were obtained by cutting a toe nail too close and collecting the blood in Na-heparinized haematocrit tubes. Larger samples were obtained by heart puncture of animals anaesthetized with pentobarbital (50 mg/kg) injected intraperitoneally. The blood samples were centrifuged (15 000 g for 15 min) immediately after they were drawn, and the plasma was stored at - 2 0 ° C until analysis. Serial blood samplings from 12 females were begun on 6 March and lasted until 18 March. In this period samples were taken every second day. Following 18 March samples were taken every week, until one week following delivery. The animals gave birth between 29 April and 13 May.
Electroimmunoassays Crossed immunoelectrophoresis (Hau et al., 1978), crossed concanavalin A affino immunoelectrophoresis (Hau et al., 1980a), crossed heparin affino immunoelectrophoresis (Westergaard et al., 1983), quantitative rocket immunoelectrophoresis (Hau et al., 1978) and rocket line immunoelectrophoresis (Hau et al., 1981a) were performed as previously described.
138
Statistics Differences between means were evaluated by the Student's t-test. RESULTS
Immunological cross-reaction with human fibrinogen Immunological cross-reaction between the mink protein against which antibodies had been raised and human fibrinogen was demonstrated by crossed immunoelectrophoreses with intermediary gels containing rabbit anti-human fibrinogen antibodies (Dakopatts Code A080). The antiserum against human fibrinogen completely precipitated the fibrinogen in the mink plasma used as the antigen. This resulted in a shift of the precipitate towards the first dimension gel (Fig. 1).
Analysis of antisera Following solid phase absorption of the antisera from the rabbits immunized with the high molecular weight plasma protein fraction, antibodies against fibrin/fibrinogen/FDP remained. Fig. 2 shows two crossed immunoelectrophoreses with (a) non -pregnant female plasma and (b) pregnant female plasma as antigen. Only pregnant females had fibrin/fibrinogen/FDP levels above the detection limits of this assay. The precipitate demonstrated a large extent of heterogeneity, and, regardless of the distance of first dimension migration, the various discrete populations of molecules with immunoreactivity could not be separated electrophoretically.
Levels of fibrin/fibrinogen/FDP in the circulation of female mink The levels of fibrin/fibrinogen/FDP were measured by rocket immunoelectrophoresis in 130 heparin plasma samples from 12 female mink during the breeding season and during pregnancy. The sensitivity of the assay was approximately 2 #g/ml. The between assay coefficients of variation were 4.7 +_0.1% (n = 15). Measurements within each week were pooled, and levels were very high during the breeding season. Around the time of implantation (5-6 weeks prior to the parturition) the levels decreased to close to the detection limit at parturition (Fig. 3 ). Compared to week 0 before parturition, levels were significantly increased at week 2 ( P < 0.01 ) and weeks 3, 4, 5, 6, 7, 8 and 9 (P<0.001).
139
a
b
3 i
~ -
~
2
Fig. 1. Crossed immunoelectrophoresis with antibody-containing intermediate gel, (1) first dimension gel, (2) first antibody-containing gel, and (3) reference antibody-containing gel containing the anti-mink fibrin/fibrinogen/FDP antibody preparation h5 (v/v). The antigen was plasma of pregnant mink and in (a) the first antibody-containing gel contained no antibodies, and in (b) the first antibody-containing gel contained rabbit anti-human fibrinogen (Dakopatts) 1:50 (v/v).
a il~ ~ili~!~:i! ~ I
Fig. 2. Crossed immunoelectrophoresis of 5/11 samples of plasma from (a) non-pregnant female mink and (b) pregnant mink. The second dimension gel contained the anti-mink fibrin/fibrinogen/FDP antibody preparation h20 (v/v).
Molecular weight estimation
U s i n g a c a l i b r a t e d c o l u m n p a c k e d w i t h S e p h a r o s e 4B a m o l e c u l a r weight o f 630 kD was e s t i m a t e d . N o molecules with f i b r i n / f i b r i n o g e n / F D P epitopes p r e s e n t in m i n k p l a s m a were f o u n d to have m o l e c u l a r weights smaller t h a n 150 kD b e c a u s e all t h e i m m u n o r e a c t i v e molecules were f o u n d in t h e void volu m e following gel f i l t r a t i o n of m i n k p l a s m a in S e p h a d e x G-100 with a n exclusion limit o f 150 kD.
140 2C • Au/m~"/'. 10-2 18
\
-+ls. dev.
\
16 14
i
12
I
10
\
8 6 4
2
9
,
,
•
,
8
7
6
5
.
3
2
1
o
Weeks before parturition Fig. 3. Rocket immunoelectrophoresis measurements of mink fibrin/fibrinogen/FDP in 130 heparin plasma samples from 12 female mink during breeding season and gestation. One hundred Arbitrary Units (AU)/ml refers to the concentration of protein in a pool of heparin plasma from late pregnant mink (n = 10). Values represent means +__1 SD.
Fig. 4. Crossed affino immunoelectrophoresis with anti-mink fibrin/fibrinogen/FDP antibody preparation, 1:20 (v/v). The antigen was heparin plasma of pregnant mink (a) without Con A and (b) with 500 #g Con A/cm 2 in the first dimension gel.
Interaction with heparin Incorporation of increasing amounts of heparin in the first dimension of crossed immunoelectrophoresis resulted in increasing migration velocity of the fibrin/fibrinogen complex. The following amounts of heparin/ml agarose were
141 employed: 0, 20 i.u., 50 i.u., 100 i.u. and 200 i.u., and the corresponding distances of the major components were: 4 mm, 9 mm, 10 mm, 11 mm and 15 mm. Interaction with concanavalin A
Increasing amounts of concanavalin A (Con A) in the first dimension gel in crossed immunoelectrophoreses resulted in increased retardation in migration velocity of fibrin/fibrinogen. At concentrations above 400 ]~g/cm2 in the first dimension gel, the fibrin/fibrinogen molecules were completely precipitated by Con A and not visible in the second dimension gel (Fig. 4). DISCUSSION The fibrin/fibrinogen/FDP molecules measured in the present assay were all found to be larger than 150 kD. The average molecular weight of the majority of molecules was 630 kD. This demonstrated that the assay measured an unknown mixture of soluble fibrin, fibrinogen and the FDP's fragment X (240 kD ) and - Y ( 155 kD), whereas fragment D (83 kD ) and E (50 kD ), if present at all, must have been circulating in very low amounts. Since fibrin, in the presence of plasmin, is degraded to fragment X, which in turn is degraded to fragment Y and - D ( 1:1 ) it seems unlikely that fragment Y is present when - D is not. This suggests that fibrinolysis is at a low level in female mink and not above the detection limit of the assay during the breeding season and pregnancy, and that the present assay predominantly measured soluble fibrin and high molecular weight fibrinogen (HMW). Moreover the molecular weight analyses indicate that the majority of the fibrinogen epitopes found in pregnant female mink plasma are localized as HMW fibrinogen and soluble fibrin. HMW fibrinogen-fibrin has also been described in women, the molecular weights of the complexes being between 500 and 1000 kD. HMW fibrinogen in the human has also been demonstrated in other situations such as acute glomerulonephritis, thromboembolism after operation and oral contraceptive use (Ozanne et al., 1983). Quantitative clot-weight assays are still widely used for fibrinogen quantification and yield results comparable to immunodiffusion methods (Reid and Onwuameze, 1984). However, the rocket immunoelectrophoresis is generally accepted to be more sensitive and rapid than immunodiffusion, and, in the present context, it is an advantage to use micro methods which require appreciably smaller volumes of plasma. The mating system employed in the present study was homoandry with three mating periods (1 + 9 + 1 ) to ensure maximal fertility. Mating induces ovulation, but, in mink, the time between ovulation and implantation varies due to
142 delayed implantation. The females implanted around April, i.e. week - 5 prior to parturition, at which time the concentration of fibrinogen was decreased from its very high levels in the maternal circulation during the breeding season. Around a week before parturition the levels were very low, quickly approaching the extremely low levels of males and females outside the breeding season. During human pregnancy, levels of circulating FDP and fibrinogen which increase until term have been extensively demonstrated (Maki et al., 1980; Paternoster et al., 1980; Hellgren and Blomb~ck, 1981; Mutoh et al., 1982; Persson et al., 1982; Ozanne et al., 1983; Whitfield et al., 1983; Nakamura et al., 1984; Stifling et al., 1984). Thrombin-mediated fibrin generation during human pregnancy has been demonstrated by measuring increasing levels of fibrinopeptide A (FPA) which is cleaved from fibrinogen in the process of fibrin generation (Wallmo et al., 1984; Weiner et al., 1984). Increasing levels of plasma soluble fibrin and FDP, but fairly constant fibrinogen levels, were measured by Thorburn and coworkers (1982), suggesting a degree of haemostatic activation. It is generally agreed upon that human pregnancy constitutes a hypercoagulable condition, whereas the fibrinogen levels and viscosity are lower in fetal plasma (Foley et al., 1978). In cows, moderately higher maternal plasma levels of fibrinogen in the last month of gestation have been reported (Ek, 1972; Gentry et al., 1979), and, in the pregnant dog, a two- or three-fold increase in fibrinogen concentration is seen during gestation (Gentry et al., 1981 ). By contrast, an apparent lack of increase in fibrinogen concentrations towards the end of the gestation period was observed in pregnant sows, which exhibited an increase at parturition only (Liptrap et al., 1984). No changes in plasma fibrinogen levels were observed throughout the gestation period of rabbits (Muller-Berghaus et al., 1978), and the present study of concentration changes in maternal plasma of mink makes it apparent that major differences in the fibrinogen profile during pregnancy exist between species. Like human fetal plasma, fetal lamb plasma fibrinogen concentration is much lower than that of maternal plasma (Kisker et al., 1981 ). Further studies are needed to reveal when the plasma levels begin to increase to reach the very high levels seen in female mink during the breeding season and to elucidate the biological significance of these findings. Heparin is a heterogenous glycosaminoglycan with anticoagulant activity, and controversy exists as to whether heparin binds fibrinogen or not (McKay and Laurell, 1980). The present study demonstrates that mink fibrinogen binds to heparin during electrophoresis, and this is manifested by an increased migration velocity due to the high negative charge of heparin. The complete precipitation by Con A demonstrates that the fibrin/fibrinogen molecules presumably have more than one binding site for Con A and are glycosylated (Bog-Hansen and Hau, 1983). Twenty percent of total circulating proteins in plasma has been observed to bind to immobilized heparin (Teisner et al., 1983), and the affinity of mink
143
fibrinogen for heparin as well as Con A suggests selective procedures for the isolation and purification of this molecule. ACKNOWLEDGEMENT
This study was kindly financed by Dansk Pelsdyravlerforenings Forskningsfond, and the authors thank Mrs. Regina Lund for skilful technical assistance.
REFERENCES Bog-Hansen, T.C. and Hau, J., 1983. Glycoproteins and glycopeptides. J. Chromatogr. Libr., 18B: 219-252. Ek, N., 1972. The quantitative determination of fibrinogen in normal bovine plasma and in cows with inflammatory conditions. Acta Vet. Scand., 13: 175-184. Foley M.E., Isherwood, D.M. and McNicol, G.P., 1978. Viscosity, haematocrit, fibrinogen and plasma proteins in maternal and cord blood. Br. J. Obstet. Gynaecol., 85: 500-504. Gentry, P.A., Liptrap, R.M. and Black, W.D., 1979. Changes in blood coagulation profiles of dairy cows during pregnancy and in heifer calves after hormone treatment. Can. J. Anim. Sci., 59 (3): 503-510. Gentry, P.A. and Liptrap, R.M., 1981. Influence of progesterone and pregnancy on canine fibrinogen values. J. Small Anim. Pract., 22: 185-194. Gitlin, D., 1975. Normal biology of ~-fetoprotein. Ann. N.Y. Acad. Sci., 259: 7-16. Hau, J. (Editor), 1986. Pregnancy Proteins in Animals. Walter de Gruyter, Berlin, NY. Hau, J., Svendsen, P.E., Teisner, B. and Svehag, S.-E., 1978. Studies of pregnancy-associated murine serum proteins. J. Reprod. Fertil., 54: 239-243. Hau, J., Svendsen, P., Teisner, B. and Thomsen Pedersen, G., 1980a. Immunochemical characterization and serum concentrations of pregnancy-associated serum proteins in mice. J. Reprod. Fertil., 58: 389-393. Hau, J., Westergaard, J.G., Svendsen, P., Bach, A. and Teisner, B., 1980b. Comparison of the pregnancy-associated murine protein-2 and human pregnancy-specific fll-glycoprotein. J. Reprod. Fertil., 60: 115-119. Hau, J., Svendsen, P., Teisner, B. and Brandt, J., 1981a. Pregnancy-associated murine protein 3 (PAMP -3 ): Characterization and serum levels. Biol. Reprod., 24:163-169. Hau, J., Westergaard, J.G., Svendsen, P., Bach, A. and Teisner, B., 1981b. Comparison of pregnancy-associated murine protein-1 and human pregnancy zone protein. J. Reprod. Immunol., 3: 341-349. Hellgren, M. and Blomb~ick, M., 1981. Studies on blood coagulation and fibrinolysis in pregnancy, during delivery and in the puerperium. Gynecol. Obstet. Invest., 12: 141-154. Kisker, C.T., Robillard, J.E. and Clark, W.R., 1981. Development of blood coagulation - A fetal lamb model. Pediatr. Res., 15: 1045-1050. Larsen, A.E., Porter, D.D. and Porter, H.G., 1971. Pregnancy-associated serum protein in mink. Proc. Soc. Exp. Biol. Med., 136(2): 430-32. Liptrap, R.M. and Gentry, P.A., 1984. Interrelationships between blood coagulation factors and plasma hormone levels in the periparturient sow. Can. J. Anim. Sci., 64: 655-665. Maki, M., Soga, K. and Seki, H., 1980. Fibrinolytic activity during pregnancy. Tohoku J. Exp. Med., 132: 349-354. McKay, E.J. and Laurell, C.-B., 1980. The interaction of heparin with plasma proteins. Demon-
144
stration of different binding sites for antithrombin III complexes and antithrombin III. J. Lab. Clin. Med., 95 (1): 69-80. Muller-Berghaus, G., MoeUer, R.M. and Mahn, I., 1978. Fibrinogen turnover in pregnant rabbits during the first and last thirds of gestation. Am. J. Obstet. Gynecol., 131: 655-660. Mutoh, S., Maki, M. and Takahashi, S.,1982. Cold insoluble globulin. Studies on coagulationfibrinolysis system and cold insoluble globulin in normal pregnancy. Acta Obstet. Gynaecol. Jpn. (Engl. Ed.), 34(3): 369-377. Nakamura, Y., Abbott, R., Takeshita, T., Nishimura, K. and Shinagawa, S., 1984. Fibrinogen and its fraction during pregnancy and puerperium. Biol. Res. Pregnancy, 5 (3): 110-112. Ozanne, P., Linderkamp, 0., Miller, F.C. and Meiselman, H.J., 1983. Erythrocyte aggregation during normal pregnancy. Am. J. Obstet. Gynecol., 147 (5): 576-583. Paternoster, D,, Grella, P., Canova, P., Maggino, T., Valente, S. and Menighetti, M., 1980. Modifications of coagulation factors during normal and pathological pregnancies. Clin. Exp. Obstet. Gynecol., 7 (2): 112-115. Persson, B., Holmberg, L., Astedt, B. and Nilsson, I., 1982. Coagulation and fibrinolysis in pregnancy complicated by intra uterine growth retardation. Acta Obstet. Gynecol. Scand., 61 (5): 455-459. Reid, H.L. and Onwuameze, I.C., 1984. Correlation of a clot-weight and radial immunodiffusion method for estimation of plasma fibrinogen concentration. Trop. Geogr. Med., 36: 13-16. Stirling, Y., Woolf, L., North, W.R.S., Seghatchian, M.J. and Meade, T.W., 1984. Haemostasis in normal pregnancy. Thromb. Haemostas. (Stuttgart), 52 (2): 176-182. Teisner, B., Davey, M.W. and Grudzinskas, J.G., 1983. Interaction between heparin and plasma proteins analysed by crossed immunoelectrophoresis and affinity chromatography. Clin. Chim. Acta, 127: 413-417. Thorburn, J., Drummond, M.M., Whigham, K.A., Lowe, G.D.O., Forbes, C.D., Prentice, C.R.M. and Whitfield, C.R., 1982. Blood viscosity and haemostatic factors in late pregnancy, preeclampsia and fetal growth retardation. Br. J. Obstet. Gynaecol., 89: 117-122. Venge, 0., 1973. Reproduction in the Mink. R. Vet. Agric. Univ. Yearbook, Copenhagen, Denmark. pp. 95-146. WaUmo, L., Karlsson, K. and Teger-Nilsson, A.-C., 1984. Fibrinopeptide A and intravascular coagulation in normotensive and hypertensive pregnancy and parturition. Acta Obstet. Gynecol. Scand., 63: 637-640. Weiner, C.P., Kwann, H., Hauck, W.W., Duboe, F.J., Paul, M. and Wallemark, C.-B., 1984. Fibrin generation in normal pregnancy. Obstet. Gynecol., 64 (1): 46-48. Westergaard, J.G., Hau, J., Teisner, B. and Grudzinskas, J.G., 1983. Specific and reversible interaction between pregnancy-associated plasma protein A and heparin. Placenta, 4: 13-18. Whitfield, L., Lele, A.S. and Levy, G., 1983. Effect of pregnancy on the relationship between concentration and anti coagulant action of heparin. Clin. Pharmacol. Ther., 34 (1): 23-28.