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Immunolocalization of factor V in adult and fetal rat liver
THROMBOSIS RESEARCH 40; 807-816, 1985 0049-3848/85 $3.00 t .OO Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserired.'
ItMJNOLOCALIZaTION OF FACTOR V IN ADULT AND FETAL RAT LIVER. MAEZORANAM., INSEW
CORNILLON B.,
Unit6
BELLEVILLE J.,
37, 18 avenue
PAUL J.,
Doyen L&pine 69500
ELOY R. BRON
France
(Received 26.5.1985; Accepted in original form 2.9.1985 by Editor B. Vargaftig)
ABSTRACT
V was performed in adult and Immunolocalization of the factor fetal rat liver by means of an indirect peroxidase labelling. This could be done owing to the production in our laboratory of a monoantiserum anti rat factor V. In all the cases (perfused specific and non-perfused adult liver and fetal one) the observation of the has revealed an intense circular or granular labelling sections into all the hepatocytes whatever was their localization in the endothelial cells seemed to be negative hepatic lobule. Hepatic for factor V and this aspect of our results was discussed.
INTRODUCTION Factor
V is a high molecular weight single chain glycoprotein. Its actithe conversion (factor Va) serves as a nonenzymatic co-factor for of prothrombin to thrombin by factor Xa on the surface of endothelial cells and platelets (l-6) and also accelerates the activation of protein C by thrombin (7,8). Megakaryocytes synthesize the factor V present in ocgranules of the platelets (9). However, the cell type or types responsible for the synthesis of circulating plasma factor V have not been clearly demonstrated. Hepatic synthesis and/or storage of plasma factor V has been mainly suggested by means of indirect studies : clinical observations of acute hepatic deficiencies (10, 11) and organ perfusions (12-14). Whether these processes take place in hepatocytee and/or in liver endothelial vascular cells remains unclear, al though the synthesis of factor V had been reported in a human hepatocellular carcinoma cell line (Hep 62) (15) able to synthesize a wide range of proteins usually secreted by untransformed hepatocytee : albumin, fibrinogen, prothrombin and antithrombin III (16).
vated form
Key words labelling
: Factor
V - adult
and fetal
rat
807
liver
- indirect
immunoperoxidase
808
IMMUNOLOCALIZATION OF FV IN LIVER
On the other hand, is aleo capable of
recent factor
work on bovine
aorta
euggests
Vol. 40, No. 6 that
the endothelium
V synthesis (6,171. The present study was performed to define the cellular type or types reeponeible of the synthesis and/or storage of factor V in fetal and normal adult rat liver by means of a monoepecific antiserum anti rat factor V.
Liver specimens and tieeue preparation : - adult and anesthesia,
(16 daye) full-blood fixed livers : after N&nbutal was quickly removed, cut in small fragments and fixed 4’C in 4% parafomaldehyde in phosphate buffer (NaH2
fetal
the liver hours at
for four PO /K2HPO > 0.1 M, pH : 7.4. Then, the fragments were washed in the same bu4fer 0.'iM at 4'C overnight and underwent three new rinses with fresh buffer 1 hour each. Finally, they were classically embedded in paraffin. Sections 3um thick were prepared. Fetal liver was proceeeed in the same manner. - adult blood-free fixed liver : the liver was perfused "in situ" under constant pressure. After Nembutal anesthesia, the abdomen of an adult Wiatar Rat (average body weight 300 g) was opened and 200 IU of heparin needle con(Kabivitrum) were injected Into a peritoneal vein. A butterfly nected to the perfusion flask containing 150 ml of prewarmed (4O'C) HAM F 12 medium (so as to obtain 37'C at the level of the needle) wae then inserted into the portal vein and maintained by a ligature. When the cannula wae well inserted, the circuit was opened and the suprahepatic veina were quickly aectioned. A constant preeeure (120 mm Hg) was maintained in the flask. During the experiment, the pressure wae continuously monitored by an electronic pressure head, followed on the screen of a pressure recorder and readjusted if necessary. The aim of this kind of perfusion was to exclude the blood present In the liver and to preserve the histological structure of the organ as far as possible, particularly that of the vaecular endothelial cells. The liver was then treated as described above. Purification of rat factor V Buffers used are - Buffer - Buffer - Buffer - Buffer
: A B C D
: : : :
Tris/HCL 50 mM, benzamidine 5 mM (pH 7.5) Buffer A, MgCl 10 n&i,DFP 1mM Buffer A conta ning CaCl 10 mM Buffer A, NaN3z5mM, DFP ZmM , aminocaproIc acid 50 mM
Uninterrupted purification wae performed at 4'C during 36 h. Thawed rat plasma was mixed with an aluminlum hydroxide euspeneion (final concentration 1 : 40 After stirring, the sample waa centrifuged. A eolution of (NH41 w/v>. S04/NH40H 3.5 M, pH 7.5 wae added to the eupernatant eo as to obtain a fina1 concentration of 1.05 M. After stirring and centrifugation, the eupernatant was collected and the pellet washed once again. The two eupernatante were pooled and the ammonium sulfate concentration was adjusted to 1.95 M. After shaking, the mixture was centrifuged and the pellet wae washed with the 1.95 M anrPonium sulfate solution. The resulting pellet dissolved in buffer B was applied on a Matrex gel green A column. The gel wae washed with 150 ml buffer B containing NaCl 0.45 M and eluted with 100 ml of buffer B containing NaCl 1.1 M. The fractions were collected, pooled and precipitated with ammonium sulfate (final concentration 2M). After centrifugation of the precipitate, the pellet resuspended in 2 ml buffer D was applied on a Sepharose 6B column eluted with buffer D at a flowrate of 22.5 ml/hour.
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IMMUNOLOCALIZATION OF FV IN LIVER
809
The fractions (4 ml) containing factor V activity were pooled and layed down on a WGA Ultrogel column (6.5 x 1.2 cm). After washing with buffer A, factor V was eluted with buffer A containing 30 mM N-acetyl-glucosamine. This fraction was applied on a DEAE trisacryl column. The gel was washed with buffer A and eluted with a linear NaCl gradient from 0 to 0.4 M (60 ml) in buffer C. The fractions containing factor V were concentrated by ultrafiltration in an Amicon U.F. cell fitted with an YM 30 membrane. The electrophoresis on polyacrylamide gradient gel (PAA 4/30) was carried out for 5 hours at 150 V in Tris 40 mM sodium acetate 20 mM and SDS 1 2. (pH 7.4). Gels were then electrophoretically stained with Coomassie Brilliant Blue R250 and destained. Production of factor V antibody Rabbits were immunized with the final product : firstly, a subcutaneous injection with complete Freund'e adjuvant was given followed by three boosters injections with incomplete Freund's adjuvant every ten days. IgG were isolated frao antiserum by the IBF's method (18). Factor V deficient fraction for adsorption After Sepharose chromatography of the purification scheme, factor V containing fractions were pooled, concentrated on YM 30 membrane and rinsed with Trie 50 ml4pli 7.5 to eliminate protease inhibitions. Ten units of RVV-VAE (Russell viper venom factor V activation enzyme) were added for an incubation during two hours at 37-C. Then, the sample was applied to the Sepharose 6B column under the same conditions as mentioned in the preceding section and fractions identical with these collected in the previous filtration were pooled. At this time, they were devoid of factor V activity. Adsorption of the anti-factor V - IgG Proteins of the factor V deficient fraction were coupled to Act-Ultrogel AC A22 (IBF) and were uaed to adsorb antifactor V IgG (48 h at 4°C). Iuanunoelectrophoresis The experiments were performed at 4'C on standard microscope slides overlaid with 3 ml of 1 X agarose in 0.06 M barbital/O.05 sodium acetate buffer at pB 8.6. Samples of 5 ~1 (total plasma or purified factor V) were applied and a current of 8 mA per slide was maintained for 1 hour. A trough was then filled with antisera for a 48 h diffusion. After washing, slides were stained with Coomassie Brilliant Blue. Only one precipitation band was observed when this electrophoresis was performed between purified factor V and the antifactor V antibody. Irmnuneand non-immune sera Except for the antigen (rat factor V> and the monospecific primary antiserum both purified in our laboratory as mentioned above, the preparations used were commercial ones : Normal Swine serum (Nordic), Normal Rabbit serum (Dako). Peroxidase-conjugated swine IgG anti Rabbit IgG (Dako). Antisera and normal sera were diluted in phosphate buffer (PBS). All the operations were carried out at room temperature (except for the primary antiserum : 4'C) and in a humid chamber.
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IMMUNOLOCALIZATION OF FV IN LIVER
Vol. 40, No. 6
Indirect immunoperoxidase Two changes of xylene 1 ?in ? each ; two changes of absolute ethanol 1 min each ; 95' ethanol 1 min ; 70' ethanol 1 min ; two changes of PBS 5 min each ; inhibition of endogenoue peroxidasee (H 02 3 % in absolute methanol) 30 min ; ? ; primary three changes of PBS 5 min each ; norma1 swine serum (1:lO) 20 ?in monoepecific antiserum anti rat factor V (1:lOO) 24 h at 4'C ; three changes PBS 5 ?in ? each ; secondary antiserum (peroxidase-conjugated swine IgG anti rabbit IgG 1 : 30) 30 min ; three changes PBS 5 min each ; diaminobenzidine LEfidf202 5 min (19) ; two changes PBS 5 min each 0 0 1 % in PBS only few n with Mayer's hemalum ; rinse quickly in distilled water ; counterstaf4 ? ; dehydrate and coverelip with (20 seconds) ; rinse in running tap water 5 ?in Canada Balsam ; the sections were then observed on a Reichert microscope (Polyvar). Controls Indirect immunoperoxidase specificity controls were the following : a) "in vitro", previous absorption of the monospecific primary antiserum with purified antigen before incubation with the section (inhibition control) (Ab/Ag (1 : 9) with Ab 1/50e). b) Peroxidase-conjugated secondary antiserum alone c) Normal rabbit serum in place of the primary antiserum diluted l/lOe d) DAB-H202 alone RESULTS . Blood-free and full blood adult fixed liver The peroxidase labelling specific for factor V was found in all hepatocytes (Fig. 1 et 2). This labelling did not depend on the cell localization with regard to blood vessels referring to the well-known structure of the Kiernan liver lobule. Differences in the labelling intensity were still observed from one cell to the other. Endothelial cells of the main blood vessels showed no labelling. However, a quite final answer with regard to the real lack of labelling in these cells will be obtained only by electron microscopic studies. In some parts of the section, sinusoids showed a positive staining but whether this labelling was present into the endothelial cells or on their surface remains obscure. In inhibition control, the positive staining found into hepatocytes had completely disappeared and thus, the hepatocellular staining for factor V was specific. On the other hand, einusoIde still exhibited a positive reaction which even spread on the whole section and was amplified as compared to the antiserum used alone. This was further increased in the case of the full-blood liver, drastically in the case of the blood-free one (Fig. 3). An unexpected labelling also appeared in the endothelial cells of the main blood vessels. This labelling was not specific for factor V. Normal rabbit serum control was perfectly negative (Fig. 4). . Fetal liver (stage 16 days) At this stage, hepatocytes formed only one third of the whole liver cell population, the two other parts representing mainly hematopoietic cells. Once again, all hepatocytes were positive for factor V (Fig.5). The stem blood line cells did not show any labelling, and nor did the endothelial cells of the few blood vessels found at this stage of development. Inhibition showed the same results as these mentioned above : total disappearance of the labelling present in the hepatocytee and appearance of an intense labelling at the surface or within the endothelial cells of the main blood vessels (~ig.61. All the controls for fetal or adult liver were perfectly negative for factor V.
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IMMUNOLOCALIZATIOM OF FV IN LIVER
811
Pig. 2 adult fixed liver. Fig. 2 : full blood adult fixed liver. Fig. 1 : blood-free See the very good present in all hepatocytes (arrows). Factor V Is clearly preservation of the endothellal cells after perfusion (E, fig. 1). (x 1125).
812
IMMUNOLOCALIZATION OF FV IN LIVER
Pig.
Adult liver for factor
Vol. 40, No. 6
4
controls : Fig. 3 : inhibition V. Note the positive amplified
control reaction
: hepatocytes exhibited
(arrows). Fig. 4 : normal rabbit serum control. (x 1125).
are by
negative
ainueoide
Vol. 40, No. 6
IMMUNOLOCALIZATION OF FV IN LIVER
Pig.
6
Fetal liver. Fig. 5 : factor V is present in the hepatocytes (arrows). Hm : hematopoIetfc cells. Bv : blood vessel. M : megakaryocyte-Like cell. Fig. 6 : inhibition control. Note the intense labelllng at the surface or within endothelial cells (arrows). (x 1125).
813
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IMMUNOLOCALIZATION OF FV IN LIVER
Vol. 40, No. 6
DISCUSSION Although not uniformly stained, all the hepatocytes were clearly positive for factor V. We had never observed a preferential topography of the labelled hepatocytes around the centrilobular vein as did Giddings et al. (20). Different experimental conditions could account for such discrepancies : 1) Giddings et al. used human tissue obtained by autopsy or after surgical sampling ; in this case, the fatty infiltration occurring after autopsy could account for the concentrated labelling of the hepatocytes around the central vein in view of the pronounced affinity of factor V for phospholipids, 2) they worked on frozen sections. Unlike Giddings and his coworkers, we did not obthe main blood serve specific immunostaining into the endothelial cells of vessels nor in the blood capillaries since the fine lining sometimes observed on the endothelium did not disappear during inhibition. Although recent studies had demonstrated factor V synthesis in cultured endothelial cells of the bovine aorta (6,17), Rodgers et al. (6) showed that the ability of the endothelial cells to activate prothrombin (dependent on factor V among other factors) varied according to their localization. In the bovine species, the labelling was of decreasing intensity as follows : aortic cells)))umbilical venous cells)) pulmonary venous cells. Thus, it is not unlikely that all endothelial cells are probably not able to synthesize factor V. This might explain the lack of labelling in the endothelial vascular liver cells. In another unpublished preliminary publication on the rat aorta we reported the same phenomena, i.e. a fine border of labelling on the endothelium which did not disappear during inhibition. The presence of a nonspecific immunostaining in sinusoIda liver capillaries, its amplification and appearance in the main blood vessel endothelial cells during inhibition might result from a non-specific binding of the anti-rat factor V antibody on the receptors of the endothelial cells through the Fc fragment of the antibody molecule (21). During the contact of the tissue section with the primary antiserum, the affinity of the antibody for the antigen through the Fab fragment would be more important than for the endothelial receptors through Fc fragment. Thus, the labelling was observed especially into the hepatocytes and affected the binding of the antigen and the Fab fragment of the antibody molecule. Furthermore, a more important affinity of Fc fragment may exist for the receptors of the endothelial cells of the liver capillaries than for those of the main vascular blood vessels. In the case of inhibition, Fab fragments of the antibodies were saturated by the antigen and so the inhibition was total into the hepatocytes. On the other hand, Fc fragments remained available and all the immunocomplexes (antigen-antibody) formed were susceptible to bind to the receptors of the endothelial cells. The immunoetaining became more and more prominent and extended to the whole section even reaching endothelial cells of the main blood vessels. In the case of the blood-free fixed liver, the amplification of the labelling was particularly pronounced. It cannot be precluded that the organ washing allowed a number of Fc endothelial receptors to be released. This hypothesis will be tested next in our laboratory owing to the obtention of the F(ab'J2 fragment of anti rat factor V. If this hypothesis is correct, then the use of the F(ab') fragment would prevent this nonspecific immunostaining. The 2 presence of a contaminating antibody in our antiserum does not explain the amplification and the generalization of the labelling during inhibition. Furthermore, the immunoelectrophoresis of factor V against rabbit anti rat factor V showed only one band (see Materials and Methods).
Vol. 40, No. 6
815
IMMUNOLOCALIZATION OF FV IN LIVER
V is present very early in the Our results demonstrate that the factor fetal rat hepatocytes (16 days old fetuses). This confirms our knowledge about the earliest coagulation human fetal coagulation. Fibrinogen and factor V are life and so, factors, which appear as soon as 11 or 12 weeks (22) of fetal fetal blood showed very early its ability to coagulate (23). Furthermore, we demonstrate a real synthesis and not a maternal factor V storage since coagulation factors do not cross the placental barrier (24).
ACRNON-LEDGNJD?TS :
This work was supported
by Grant-INSElW
: CRL 82.5.028
REFERENCES 1 MANN, K.G., NESHEIM, M.E., HIBBARD, L.S. and TRACY, P.B. The role of factor of the prothrombinase complex. Ann. N.Y. Acad. Sci. 370V in the assembly 378, 1981. 2 MILETICH, J.P., JACKSON, C.M. and MAJERUS, P.W. Properties of the factor Xa binding site on human platelets. J. Biol. Chem. 253, 6908, 1978. 3 TUSZYNSKI, G.P., MAUCO, G.P., KOSKY, A, SCHICK, P.K. and WALSH, P.N. The platelet cytoskeleton contains elements of the prothrombinase complex. J. Biol. Chem. -259, 6947, 1984. 4 TUSZYNSKI, G.P. and WALSH, P.N. Factor V : a platelet ted protein. Haematologia l7, 67, 1984. 5 WABSH, P.N. Activation of coagulation Haematologia l.7, 57, 1984.
cytoskeletal
associa-
factors on the surface of platelets.
6 RODGERS, G.M., and SHUMAN M.A. Prothrombin thelial cells by factor Xa and calcium. 1983.
is activated on vascular endoProc. Natl. Acad. Sci. 80, 7001,
7 MARUYAMA, I., SALEM, H-H., and MAJERUS P.W. Coagulation factor Va binds to human umbilical vein endothelial cells and accelerates protein C activation. J. Clin. Invest. 74, 224, 1984. 8 SALEM, H.H., BROZE, G.J., MILETICH, J.P. and MAJERUS P.W. Human coagulation factor Va is a cofactor for the activation of protein C. Proc. Natl. Acad. Sci. 80, 1584, 1983. 9 CHIU, C., SCHICK, P., and COLMAN R.W. Biosynthesis of coagulation by megakaryocytes. Fed. Proc. 3, 1994 (abstr.), 1983. 10 DEUTSCH, disease.
E. Blood coagulation 2, 69, 1965.
changes
11 WALLS, W.D., and LASOWSKY M.S. The Gastroenterology. 60_, 108, 1971.
in liver disease. Progress
hemostatic
defect
factor V
in liver
of liver disease.
12 MATII, R., AMBRUS, J.L., SOKAL, J.E. and MINK, I. Production of members of the blood coagulation and fibrynolysin systems by the isolated perfused liver. Proc. Sot. Exp. Biol. Med. 116, 69, 1964. 13 OLSON, J.P., MILLER, L.L., and TROUP S.B. Synthesis of clotting factors by the isolated perfused Rat liver. J. Clin. Invest. 42, 690, 1966.
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14 OWEN, C.A. jr. and BOWIE E.J.W. Generation of coagulation factors V, XI and XII by the isolated Rat liver. Haemostasis 6_, 205, 1977. 15 WILSON, D.B., SALEM, H.H., MRUK, J.S., MARUYAMA, I, and MAJERUS, P.W. Biosynthesis of coagulation factor V by a human hepatocellular carcinoma cell line. J. Clin. Invest. 73, 654, 1984. 16 FAIR, D.S. and BAHNAK, B.R. Human hepatoma cells secrete single chain factor X, prothrombin and antithrombin III. -_ Blood 64, 194-204, 1984. 17 CERVENY, T.J., FASS, D.N. and MANN, K.G. Synthesis of coagulation factor V by cultured aortic endothelium. -Blood 63, 1467, 1984. 18 SAINT-BLANCARD, J., KIRZIN, J.M., RIBERON, P., PETIT, F.. FOURCART, J., GIROT, P. and -BOSCHETTI, E. Affinity, chromatography and related tech: niques. Eds. GRIBNAU, T.C., VISSER, J. and NIVAR, R.J.F. Elsevier Amsterdam
19 GFMIAM, R.C. and KARNOWSKY M.J.
The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney : ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-302, 1966.
20 GIDDINGS, J.C., SHEARN, S.A.M. and BLOOM, A.L. The immunological localization of factor V in human tissue. Br. J. Haematol. 29, 57-65, 1975. 21 HANSSON, G.K., STARKEBAUM, G.A., BENDITT, E.P. and SCHWARTZ, S.M. Fcmediated binding of IgG to vimentin-type intermediate filaments in vascular endothelial cells. Proc. Natl. Acad. Sci. 8l, 3103-3107, 1984. 22 HATHAWAY, W.E. and BONNAR, J. Physiology of coagulation in the fetus and newborn infant. Perinatal coagulation Eds. Hathaway W.E. and Bonnar J. Griineet Stratton. 53-80, 19 . 23 ZILLIACUS, H., OTTELIN, A.M. and MATTSSON, T. Blood totting and fibrinolysis in human fetuses. Biol. Neonate lC, 108-112, 1966. 24 CADE, J.F, HIRSH, J. and MARTIN, M. Placental barrier to coagulation factors : its relevance to the coagulation defect at birth and to haemorrhage in the newborn. Br. Med.?, 2, 281-283,1969.
ItMJNOLOCALIZaTION OF FACTOR V IN ADULT AND FETAL RAT LIVER. MAEZORANAM., INSEW
CORNILLON B.,
Unit6
BELLEVILLE J.,
37, 18 avenue
PAUL J.,
Doyen L&pine 69500
ELOY R. BRON
France
(Received 26.5.1985; Accepted in original form 2.9.1985 by Editor B. Vargaftig)
ABSTRACT
V was performed in adult and Immunolocalization of the factor fetal rat liver by means of an indirect peroxidase labelling. This could be done owing to the production in our laboratory of a monoantiserum anti rat factor V. In all the cases (perfused specific and non-perfused adult liver and fetal one) the observation of the has revealed an intense circular or granular labelling sections into all the hepatocytes whatever was their localization in the endothelial cells seemed to be negative hepatic lobule. Hepatic for factor V and this aspect of our results was discussed.
INTRODUCTION Factor
V is a high molecular weight single chain glycoprotein. Its actithe conversion (factor Va) serves as a nonenzymatic co-factor for of prothrombin to thrombin by factor Xa on the surface of endothelial cells and platelets (l-6) and also accelerates the activation of protein C by thrombin (7,8). Megakaryocytes synthesize the factor V present in ocgranules of the platelets (9). However, the cell type or types responsible for the synthesis of circulating plasma factor V have not been clearly demonstrated. Hepatic synthesis and/or storage of plasma factor V has been mainly suggested by means of indirect studies : clinical observations of acute hepatic deficiencies (10, 11) and organ perfusions (12-14). Whether these processes take place in hepatocytee and/or in liver endothelial vascular cells remains unclear, al though the synthesis of factor V had been reported in a human hepatocellular carcinoma cell line (Hep 62) (15) able to synthesize a wide range of proteins usually secreted by untransformed hepatocytee : albumin, fibrinogen, prothrombin and antithrombin III (16).
vated form
Key words labelling
: Factor
V - adult
and fetal
rat
807
liver
- indirect
immunoperoxidase
808
IMMUNOLOCALIZATION OF FV IN LIVER
On the other hand, is aleo capable of
recent factor
work on bovine
aorta
euggests
Vol. 40, No. 6 that
the endothelium
V synthesis (6,171. The present study was performed to define the cellular type or types reeponeible of the synthesis and/or storage of factor V in fetal and normal adult rat liver by means of a monoepecific antiserum anti rat factor V.
Liver specimens and tieeue preparation : - adult and anesthesia,
(16 daye) full-blood fixed livers : after N&nbutal was quickly removed, cut in small fragments and fixed 4’C in 4% parafomaldehyde in phosphate buffer (NaH2
fetal
the liver hours at
for four PO /K2HPO > 0.1 M, pH : 7.4. Then, the fragments were washed in the same bu4fer 0.'iM at 4'C overnight and underwent three new rinses with fresh buffer 1 hour each. Finally, they were classically embedded in paraffin. Sections 3um thick were prepared. Fetal liver was proceeeed in the same manner. - adult blood-free fixed liver : the liver was perfused "in situ" under constant pressure. After Nembutal anesthesia, the abdomen of an adult Wiatar Rat (average body weight 300 g) was opened and 200 IU of heparin needle con(Kabivitrum) were injected Into a peritoneal vein. A butterfly nected to the perfusion flask containing 150 ml of prewarmed (4O'C) HAM F 12 medium (so as to obtain 37'C at the level of the needle) wae then inserted into the portal vein and maintained by a ligature. When the cannula wae well inserted, the circuit was opened and the suprahepatic veina were quickly aectioned. A constant preeeure (120 mm Hg) was maintained in the flask. During the experiment, the pressure wae continuously monitored by an electronic pressure head, followed on the screen of a pressure recorder and readjusted if necessary. The aim of this kind of perfusion was to exclude the blood present In the liver and to preserve the histological structure of the organ as far as possible, particularly that of the vaecular endothelial cells. The liver was then treated as described above. Purification of rat factor V Buffers used are - Buffer - Buffer - Buffer - Buffer
: A B C D
: : : :
Tris/HCL 50 mM, benzamidine 5 mM (pH 7.5) Buffer A, MgCl 10 n&i,DFP 1mM Buffer A conta ning CaCl 10 mM Buffer A, NaN3z5mM, DFP ZmM , aminocaproIc acid 50 mM
Uninterrupted purification wae performed at 4'C during 36 h. Thawed rat plasma was mixed with an aluminlum hydroxide euspeneion (final concentration 1 : 40 After stirring, the sample waa centrifuged. A eolution of (NH41 w/v>. S04/NH40H 3.5 M, pH 7.5 wae added to the eupernatant eo as to obtain a fina1 concentration of 1.05 M. After stirring and centrifugation, the eupernatant was collected and the pellet washed once again. The two eupernatante were pooled and the ammonium sulfate concentration was adjusted to 1.95 M. After shaking, the mixture was centrifuged and the pellet wae washed with the 1.95 M anrPonium sulfate solution. The resulting pellet dissolved in buffer B was applied on a Matrex gel green A column. The gel wae washed with 150 ml buffer B containing NaCl 0.45 M and eluted with 100 ml of buffer B containing NaCl 1.1 M. The fractions were collected, pooled and precipitated with ammonium sulfate (final concentration 2M). After centrifugation of the precipitate, the pellet resuspended in 2 ml buffer D was applied on a Sepharose 6B column eluted with buffer D at a flowrate of 22.5 ml/hour.
Vol. 40, No. 6
IMMUNOLOCALIZATION OF FV IN LIVER
809
The fractions (4 ml) containing factor V activity were pooled and layed down on a WGA Ultrogel column (6.5 x 1.2 cm). After washing with buffer A, factor V was eluted with buffer A containing 30 mM N-acetyl-glucosamine. This fraction was applied on a DEAE trisacryl column. The gel was washed with buffer A and eluted with a linear NaCl gradient from 0 to 0.4 M (60 ml) in buffer C. The fractions containing factor V were concentrated by ultrafiltration in an Amicon U.F. cell fitted with an YM 30 membrane. The electrophoresis on polyacrylamide gradient gel (PAA 4/30) was carried out for 5 hours at 150 V in Tris 40 mM sodium acetate 20 mM and SDS 1 2. (pH 7.4). Gels were then electrophoretically stained with Coomassie Brilliant Blue R250 and destained. Production of factor V antibody Rabbits were immunized with the final product : firstly, a subcutaneous injection with complete Freund'e adjuvant was given followed by three boosters injections with incomplete Freund's adjuvant every ten days. IgG were isolated frao antiserum by the IBF's method (18). Factor V deficient fraction for adsorption After Sepharose chromatography of the purification scheme, factor V containing fractions were pooled, concentrated on YM 30 membrane and rinsed with Trie 50 ml4pli 7.5 to eliminate protease inhibitions. Ten units of RVV-VAE (Russell viper venom factor V activation enzyme) were added for an incubation during two hours at 37-C. Then, the sample was applied to the Sepharose 6B column under the same conditions as mentioned in the preceding section and fractions identical with these collected in the previous filtration were pooled. At this time, they were devoid of factor V activity. Adsorption of the anti-factor V - IgG Proteins of the factor V deficient fraction were coupled to Act-Ultrogel AC A22 (IBF) and were uaed to adsorb antifactor V IgG (48 h at 4°C). Iuanunoelectrophoresis The experiments were performed at 4'C on standard microscope slides overlaid with 3 ml of 1 X agarose in 0.06 M barbital/O.05 sodium acetate buffer at pB 8.6. Samples of 5 ~1 (total plasma or purified factor V) were applied and a current of 8 mA per slide was maintained for 1 hour. A trough was then filled with antisera for a 48 h diffusion. After washing, slides were stained with Coomassie Brilliant Blue. Only one precipitation band was observed when this electrophoresis was performed between purified factor V and the antifactor V antibody. Irmnuneand non-immune sera Except for the antigen (rat factor V> and the monospecific primary antiserum both purified in our laboratory as mentioned above, the preparations used were commercial ones : Normal Swine serum (Nordic), Normal Rabbit serum (Dako). Peroxidase-conjugated swine IgG anti Rabbit IgG (Dako). Antisera and normal sera were diluted in phosphate buffer (PBS). All the operations were carried out at room temperature (except for the primary antiserum : 4'C) and in a humid chamber.
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Indirect immunoperoxidase Two changes of xylene 1 ?in ? each ; two changes of absolute ethanol 1 min each ; 95' ethanol 1 min ; 70' ethanol 1 min ; two changes of PBS 5 min each ; inhibition of endogenoue peroxidasee (H 02 3 % in absolute methanol) 30 min ; ? ; primary three changes of PBS 5 min each ; norma1 swine serum (1:lO) 20 ?in monoepecific antiserum anti rat factor V (1:lOO) 24 h at 4'C ; three changes PBS 5 ?in ? each ; secondary antiserum (peroxidase-conjugated swine IgG anti rabbit IgG 1 : 30) 30 min ; three changes PBS 5 min each ; diaminobenzidine LEfidf202 5 min (19) ; two changes PBS 5 min each 0 0 1 % in PBS only few n with Mayer's hemalum ; rinse quickly in distilled water ; counterstaf4 ? ; dehydrate and coverelip with (20 seconds) ; rinse in running tap water 5 ?in Canada Balsam ; the sections were then observed on a Reichert microscope (Polyvar). Controls Indirect immunoperoxidase specificity controls were the following : a) "in vitro", previous absorption of the monospecific primary antiserum with purified antigen before incubation with the section (inhibition control) (Ab/Ag (1 : 9) with Ab 1/50e). b) Peroxidase-conjugated secondary antiserum alone c) Normal rabbit serum in place of the primary antiserum diluted l/lOe d) DAB-H202 alone RESULTS . Blood-free and full blood adult fixed liver The peroxidase labelling specific for factor V was found in all hepatocytes (Fig. 1 et 2). This labelling did not depend on the cell localization with regard to blood vessels referring to the well-known structure of the Kiernan liver lobule. Differences in the labelling intensity were still observed from one cell to the other. Endothelial cells of the main blood vessels showed no labelling. However, a quite final answer with regard to the real lack of labelling in these cells will be obtained only by electron microscopic studies. In some parts of the section, sinusoids showed a positive staining but whether this labelling was present into the endothelial cells or on their surface remains obscure. In inhibition control, the positive staining found into hepatocytes had completely disappeared and thus, the hepatocellular staining for factor V was specific. On the other hand, einusoIde still exhibited a positive reaction which even spread on the whole section and was amplified as compared to the antiserum used alone. This was further increased in the case of the full-blood liver, drastically in the case of the blood-free one (Fig. 3). An unexpected labelling also appeared in the endothelial cells of the main blood vessels. This labelling was not specific for factor V. Normal rabbit serum control was perfectly negative (Fig. 4). . Fetal liver (stage 16 days) At this stage, hepatocytes formed only one third of the whole liver cell population, the two other parts representing mainly hematopoietic cells. Once again, all hepatocytes were positive for factor V (Fig.5). The stem blood line cells did not show any labelling, and nor did the endothelial cells of the few blood vessels found at this stage of development. Inhibition showed the same results as these mentioned above : total disappearance of the labelling present in the hepatocytee and appearance of an intense labelling at the surface or within the endothelial cells of the main blood vessels (~ig.61. All the controls for fetal or adult liver were perfectly negative for factor V.
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Pig. 2 adult fixed liver. Fig. 2 : full blood adult fixed liver. Fig. 1 : blood-free See the very good present in all hepatocytes (arrows). Factor V Is clearly preservation of the endothellal cells after perfusion (E, fig. 1). (x 1125).
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Pig.
Adult liver for factor
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4
controls : Fig. 3 : inhibition V. Note the positive amplified
control reaction
: hepatocytes exhibited
(arrows). Fig. 4 : normal rabbit serum control. (x 1125).
are by
negative
ainueoide
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IMMUNOLOCALIZATION OF FV IN LIVER
Pig.
6
Fetal liver. Fig. 5 : factor V is present in the hepatocytes (arrows). Hm : hematopoIetfc cells. Bv : blood vessel. M : megakaryocyte-Like cell. Fig. 6 : inhibition control. Note the intense labelllng at the surface or within endothelial cells (arrows). (x 1125).
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DISCUSSION Although not uniformly stained, all the hepatocytes were clearly positive for factor V. We had never observed a preferential topography of the labelled hepatocytes around the centrilobular vein as did Giddings et al. (20). Different experimental conditions could account for such discrepancies : 1) Giddings et al. used human tissue obtained by autopsy or after surgical sampling ; in this case, the fatty infiltration occurring after autopsy could account for the concentrated labelling of the hepatocytes around the central vein in view of the pronounced affinity of factor V for phospholipids, 2) they worked on frozen sections. Unlike Giddings and his coworkers, we did not obthe main blood serve specific immunostaining into the endothelial cells of vessels nor in the blood capillaries since the fine lining sometimes observed on the endothelium did not disappear during inhibition. Although recent studies had demonstrated factor V synthesis in cultured endothelial cells of the bovine aorta (6,17), Rodgers et al. (6) showed that the ability of the endothelial cells to activate prothrombin (dependent on factor V among other factors) varied according to their localization. In the bovine species, the labelling was of decreasing intensity as follows : aortic cells)))umbilical venous cells)) pulmonary venous cells. Thus, it is not unlikely that all endothelial cells are probably not able to synthesize factor V. This might explain the lack of labelling in the endothelial vascular liver cells. In another unpublished preliminary publication on the rat aorta we reported the same phenomena, i.e. a fine border of labelling on the endothelium which did not disappear during inhibition. The presence of a nonspecific immunostaining in sinusoIda liver capillaries, its amplification and appearance in the main blood vessel endothelial cells during inhibition might result from a non-specific binding of the anti-rat factor V antibody on the receptors of the endothelial cells through the Fc fragment of the antibody molecule (21). During the contact of the tissue section with the primary antiserum, the affinity of the antibody for the antigen through the Fab fragment would be more important than for the endothelial receptors through Fc fragment. Thus, the labelling was observed especially into the hepatocytes and affected the binding of the antigen and the Fab fragment of the antibody molecule. Furthermore, a more important affinity of Fc fragment may exist for the receptors of the endothelial cells of the liver capillaries than for those of the main vascular blood vessels. In the case of inhibition, Fab fragments of the antibodies were saturated by the antigen and so the inhibition was total into the hepatocytes. On the other hand, Fc fragments remained available and all the immunocomplexes (antigen-antibody) formed were susceptible to bind to the receptors of the endothelial cells. The immunoetaining became more and more prominent and extended to the whole section even reaching endothelial cells of the main blood vessels. In the case of the blood-free fixed liver, the amplification of the labelling was particularly pronounced. It cannot be precluded that the organ washing allowed a number of Fc endothelial receptors to be released. This hypothesis will be tested next in our laboratory owing to the obtention of the F(ab'J2 fragment of anti rat factor V. If this hypothesis is correct, then the use of the F(ab') fragment would prevent this nonspecific immunostaining. The 2 presence of a contaminating antibody in our antiserum does not explain the amplification and the generalization of the labelling during inhibition. Furthermore, the immunoelectrophoresis of factor V against rabbit anti rat factor V showed only one band (see Materials and Methods).
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V is present very early in the Our results demonstrate that the factor fetal rat hepatocytes (16 days old fetuses). This confirms our knowledge about the earliest coagulation human fetal coagulation. Fibrinogen and factor V are life and so, factors, which appear as soon as 11 or 12 weeks (22) of fetal fetal blood showed very early its ability to coagulate (23). Furthermore, we demonstrate a real synthesis and not a maternal factor V storage since coagulation factors do not cross the placental barrier (24).
ACRNON-LEDGNJD?TS :
This work was supported
by Grant-INSElW
: CRL 82.5.028
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