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Antithrombin III synthesis in rat liver parenchymal cells
THROMBOSIS RESEARCH 30 ; 369-375t 1983 0049-3848/83/100369 -07$03 .00/0 Printed in the USA . All rights reserved . Copyright (c) 1983 Pergamon Press Ltd .
ANTITHROMBIN III SYNTHESIS IN RAT LIVER PARENCHYMAL CELLS
M . Leonlt M . Aiach 2 t E . Coezy 3 t J .-Y . Guennec 4 t J .-N . Fiessingerl . 1 . Centre Claude-Bernard de recherche sur lea maladies vasculaires (Pr E . Housset) and 2 . Laboratoire d'hdmostase (Pr M . Leclerc) H6pital Broussais - 96t rue Didot - 75674 PARIS CEDEX 14 (France) . 3 . INSERM U 36 - 17t rue du Fer-d-Moulin - 75005 PARIS (France) 4 . Laboratoire des produits biomedicaux - CEA - 30200 BAGNOLS SUR CEZE (France) (Received 20 .1 .1983 ; Accepted in revised form 21 .2 .1983 by Editor U . Abildgaard) ABSTRACT We have previously demonstrated by immunoperoxydase the presence of immunoreactive antithrombin III (AT III) in rat hepatocytes . We now present direct evidence that rat hepatocytes in culture synthesize AT III like immunoreactive material : 35 S-methionine was added to the culture medium and incubated with hepatocytes . After incubationt AT III was immunologically characterized in the medium . We found significant amounts of 35 8-AT III among the radioactive proteins synthesized and secreted by the cells .
INTRODUCTION Several observations and experimental results over the past years provide indirect evidences that the liver is a major site for antithrombin III (AT III) biosynthesis . In hepatic diseases for examplet it has been shown that AT III levels in plasma are decreased (1) . Owens showed that the perfused rat liver is able to release AT III and that this release is inhibited by puromycint an inhibitor of protein synthesis (2) . The cellular localization of AT III in the liver has been investigated by several authors by the use of immunohistochemical techniques . Conflicting results have been obtained . Watadat by classic immunofluorescencet observed the presence of AT III in human hepatocytes (3) . Leet on the other handt did not find any AT III in this cell type by immunoperoxydase (4) . We recently reported that AT III is detectable in rat hepatocytes by immunoperoxydase (5) . Furthermoret in electron microscopyt AT III was located in the Golgi apparatus (6) . These latter observations suggest that AT III is synthesized by these cells . There is however no direct evidence reported so far that hepatocytes actually synthesize AT III . We therefore decided to study the incorporation by rat hepatocytes in culture of radiolabelled aminoacid into proteins and to characterize in this system the biosynthesis of AT III . Key-words
: Rat hepatocytes - Antithrombin III - Antithrombin III synthesis .
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MATERIALS AND METHODS Preparation of AT III and anti AT III antibodies AT III was purified from rat plasma as previously described . Specific antibodies were raised in the rabbit (5) . These antibodies were purified by immunoadsorption on an AT III- AH Sepharose 4B (Pharmacia - Bois-d'Arcy France) column . After purificationt antibodies were also coupled to AN Sepharose 4B . This couplaget as well as the preparation of AT III - AH Sepharose 4Bt was made according to the procedure described by Clement (7) . Isolation of hepatocytes The livers of adult male Wistar rats (200-250 g) were perfused first for 10 min at a flow rate of 35 ml/min with 0 .01 M Hepes buffer pH 7 .65 containing 0 .14 M NaClt 0 .005 M KC1t 0 .001 M Na2HP0 4 and then for an additional 20 min with 0 .01 M Hepes pH 7 .65t 0 .05 M CaCl2t 0 .025 % collagenase (Boehringer Mannheim - FRG) . The livers were removedt minced in 10 ml Modified Eagle Medium (MEM) (Gibco - Paisley - UK) containing 2 g/l bovine serum albumin (BSA) . The liver parenchyma was dissociated with a spatula . After dissociationt the cell suspension was filtered through sterile gauzes and 100 ml of 0 .01 M Hepes pH 7 .65 and 50 ml MEM both containing 2 g/1 BSA were added . The suspension was allowed to sediment for 25 min and then centrifuged at 50 g for 50 s . The pellet was resuspended in Hepes buffer containing 2 g/1 BSA then centrifuged again at 50 g for 50 s . This procedure was repeated three times . The washed pellet was resuspended in the incubation medium as described below . The viability of the cells preparation was checked by determining the percentage of cells excluding 0 .4 % Trypan blue . Hepatocytes were identified by morphological criteria . From a single livert an average of 200 x 10 6 hepatocytes was obtained . Hepatocytes culture The hepatocytes were seeded at approximately 16 x 10 6 cells/flask in 75 cm2 flasks and incubated for 6 hours at 37 ° C in 5 % C02 95 % air with 8 ml of MEM supplemented with penicillin (50 U/ml)t streptomycin (50 U/ml) and 10 % fetal calf serum (Gibco) . During this periodt the cells attached to the flasks . The medium was then replaced with 14 ml fresh MEM containing hydrocortisone (50 mg/1) and the cells were further incubated for 48 hours . An aliquot of 500 pl of incubation medium was taken at 5t 15t 24t and 48 hours incubation for the determination of AT III . Simultaneouslyt 2 flasks were cultured in 14 ml of methionine free MEM (Gibco)t supplemented as described beforet but also containing 500 pCi of 35 S-methionine (CEA - Gif-sur-Yvette France) specific activity 1t100 Ci/mMol . Aliquots of 500 pi were taken at 5t 15t 24t and 48 hours for the determination of AT III and for the study of 35S -methionine incorporation . Each sample of incubated medium was dialyzed one hour versus distilled watert then 24 hours versus 0 .1 M phosphate pH 7 .4 . The media were collected after 48 hours from the flasks incubated with 35S -methioninet pooledt dialyzedt and used for the immunological characterization of AT III . Measurement of protein synthesis The amount of radioactivity incorporated into the proteins was quantified by TCA precipitation . For this purposet 100 VI of BSA I g/l and then 100 VI of 10 % cold trichloracetic acid (TCA) were added to 25 pl of medium . The precipitate was washed four fines with I ml of cold 5 % TCA . Five hundred microliters of 0 .1 N NaOR was added to solubilize the precipitate and the mixture was counted in a 3 liquid scintillation counter .
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Determination and characterization of AT III - AT III was measured in the culture media by direct radioimmunoassay (RIA) (5) . - The incorporation of radioactivity into AT III was followed by immunoprecipitation : 10 pl of specific anti AT III serum was incubated with 10 ul of the culture medium samples for 24 hours at 4 ° C in 500 pl of 0 .05 M phosphate buffer pH 7 .4 . The AT III -anti AT III complexes were then adsorbed on Staphylococcal protein A Pansorbin (Calbiochem - La Jolla - CA - USA) by adding 30 pl of the 10 % w/v commercial suspension . After centrifugationt the pellets were washed three times with 3 ml of phosphate buffer containing 2 % w/v polyethylene glycol (PEG) and solubilized with 500 ul of 0 .1 N NaOH . The radioactivity in the solubilized precipitateswas counted . Samples were treated in parallel with 10 pl of rabbit preimmune serum instead of specific anti AT III serum . - The medium collected after 48 hours incubation with the cellstcontaining high amounts of radioactive proteins was used to purify AT III by affinity chromatography . For this purposet the dialyzed medium was applied to the anti AT III antibodies sepharose column previously equilibrated with 0 .1 M phosphate buffer pH 7 .4 . After washing the column with this buffert proteins were eluted with 0 .1 M glycine pH 2 .8 . Fractions of 1 .8 ml were collected and immediately neutralized with 0 .2 ml 1 M K2HP0 4 . The protein content was determined by quantifying absorbance at 280 nm . The fractions containing significant amounts of proteins were pooled and concentrated . This material was submitted to crossed immunoelectrophoresis (CIE) (5) . After migrationt the proteins were stained with Coomassie brilliant blue and the distribution of radioactivity in the gel was determined by autoradiography on Kodak X Ray film NS 2T with the help of fluorograph EN 3 Hance (NEN - Boston - MA - USA) . Exposure time was 15 days .
RESULTS Synthesis of proteins by hepatocytes The cells incubated with 35 S-methionine released radioactivity into the medium . The amount of radioactivity present in the TCA precipitable material increased linearly with time between 0 and 20 hours of incubation as shown on figure 1 . The cultured hepatocytes therefore incorporated 35 S-methionine into the proteins secreted in the medium . Synthesis of immunoreactive AT III Figure 2 shows the results of AT III measurement at different incubation t imes . AT III levels were determined by RIA in aliquots taken from seven different flasks . Five flasks were cultured in regular MEM and two flasks in methionine deficient MEM supplemented with 35 S-methionine . As shown on the figuret AT III levels in medium increased linearly with time for up to 24 hours . The hepatocytes therefore secrete AT III in the medium . Incorporation of radioactivity in AT III immunoreactive material Che amounts of radioactivity incorporated into AT III were determined in the protein secreted by immunoprecipitation with anti AT III antibodies as described in the "methods" section . The radioactivity precipitated by the preimmune serum was substracted from that precipitated by the specific anti AT III serum . The difference was taken as an estimation of the radioactivity
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specifically incorporated into AT III . The results are shown on figure 3 . AT III was found to represent about I % of the proteins secreted by the cells . When the levels of radioactive AT III at different times were compared to the correspondant results of AT III determination by RIAt a strong correlation between both determinationsof AT III was found (r = 0 .94 ; p < 0 .001) .
5
15
24
48 1 nb.tiu tW (b .t.)
FIGURE I Time dependent incorporation of 35 S-methionine into protein by cultured rat hepatocytes
9 753-
a
5
I5
24
48 (kn 1n.b .u .t t1.. .)
FIGURE 2 AT III levels measured by RIA in culture medium
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as laevbvtlvv Ums (boars)
FIGURE 3 Immunoprecipitation by anti AT III serum of 3 S-methionine labelled proteins
Characterization of radioactive AT III synthesized and released by hepatocytes Immunological analysis of the material eluted from the anti AT III sepharose column was performed on C IE . AT III secreted by hepatocytes was compared to plasma AT III and for this purpose CIE was performed with plasma or purified medium AT III either in the absence or in the presence of heparin . The results are shown on figure 4 . It can be shown that the radioactivity and the AT III antigen present in the protein fraction purified by immunoadsorption from the culture medium have identical electrophoretic mobilities . In the absence of heparint AT III secreted in the medium has the same electrophoretic mobility as plasma AT III . In the presence of heparin however both the radioactivity and the immunoreactive AT III are separated into two peaks . One of the peaks has the same mobility than plasma AT III whereas the other has a slower mobility .
DISCUSSION Our study shows that cultured rat hepatocytes synthesize and release a protein sharing common antigenic determinants with plasma AT III . The antiserum used to detect and characterize AT III in culture medium is highly specifict as previously reported (5) . On crossed immunoelectrophoresist two different forms of immunoreactive AT III however were characterized in the proteins secreted by hepatocytes . When CIE was performed in the presence of heparint one behaved like plasma AT III and the other had a slower mobility . The nature of this second form of AT III found in culture medium is unknownt but several hypothesis can be made : hepatocytes synthesize only AT III indentical to plasma AT III but the protein is degradated during immunoadsorptiont resulting in a lower electrophoretic mobility ; AT III can bind after its secretion proteases present in the fetal calf serum added to the culture medium and the complexes AT III proteases migrate slower than native AT III ; finallyt it can be proposed that hepatocytes synthesize a protein structurally but not immunologically different from plasma AT III .
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AT III
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AT III
FIGURE 4 Crossed immunoelectrophoresis of AT III purified from the culture medium by affinity chromatography . The gels are stained for protein (I) and the distribution of radioactivity is determined by autoradiography (II) . I in the absence of heparin . 2 in the presence of heparin (10 U/ml) . P : normal plasma .
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The release of AT III by hepatocytes is linear for up to 20 hours and then the rate of secretion decreases . This decrease can probably be explained by the occurence of changes in the functional capacities of the cell kept in culture . The hepatocytes used in this study are indeed surviving cells no more able to grow or multiplicate . Our data demonstrate synthesis of AT III by hepatocytes . Todayt it seems likely that the hepatocyte is not the only one cell type able to synthesize this protein . Indeedt Chan recently demonstrated that human endothelial cells in culture synthesize immunoreactive AT III (8) . This AT III however seems not to be released in the culture medium . Therefore we assume that the endothelial cell is not responsible for the synthesis of circulating AT III . It is possible that hepatocytes synthesize circulating AT III and that endothelial cells synthesize AT III only active in situ . Both AT III could together contribute to the protection of the vascular wall against thrombosis . ACKNOWLEDGMENTS We wish to thank Jose Deguingand for her help in preparing the manuscript .
REFERENCES 1 . HEDNERt U .t NILSSONt I .M . Antithrombin III in a clinical material . Thrombos . Res . 3t 631-641t 1973 . 2 . OWENSt M .R .t MILLERt L .C . Net biosynthesis of antithrombin III by the isolated rat liver perfused for 12-24 hours . Biochim . Biophys . Acta 627t 3039t 1980 . 3 . WATADAt M .t NAGAKAWAt M .t KITANIt T .t OKAJIMAt Y .t MAEDAt Y .t DRANOt S .t IJICHIt H . Identification of the AT III synthesizing hepatocytes by immunofluorescent technique . Thromb . Haemostas . 46t 284t 1981 . 4 . LEEt AKYt CHANt V .t CHANt T .K . The identification and localization of antithrombin III in human tissues . Thrombos . Res . 14t 209-217t 1979 . 5 . LEONt M .t MACHt M .t GUENNECt J .-Y .t JARNETt J .t GIROTt R .t FIESSINGERt J .-N .t JAUBERTt F . Antithrombin III in rat hepatocytes . Thrombos . Res . 28t 115-123t 1982 . 6 . GIROTt R .t JAUBERTt F .t LEONt M .t BELLONt B .t AIACHt M .t JOSSOt F . Albumint fibrinogent prothrombin and antithrombin III variations in bloodt urines and liver in rat nephritic syndrome (Heymann nephritis) . Thromb . Haemoatas .t in press . 7 . CLEMENTtL .T .t YAMASHITAtU .t SHEVACHtE .M . Characterization of an antiserum to Guinea pig antithrombin III (AT III) . I . Reactivity with Guinea pig T lymphocytes . J . IrmrtunoZ . 127t 1220-1227t 1981 . 8 . CHANt T .K .t CHANt V . Antithrombin IIIt the major modulator of intravascular coagulation is synthesized by human endothelial cells . Thromb . Haemostas . 46t 504-506t 1981 .
ANTITHROMBIN III SYNTHESIS IN RAT LIVER PARENCHYMAL CELLS
M . Leonlt M . Aiach 2 t E . Coezy 3 t J .-Y . Guennec 4 t J .-N . Fiessingerl . 1 . Centre Claude-Bernard de recherche sur lea maladies vasculaires (Pr E . Housset) and 2 . Laboratoire d'hdmostase (Pr M . Leclerc) H6pital Broussais - 96t rue Didot - 75674 PARIS CEDEX 14 (France) . 3 . INSERM U 36 - 17t rue du Fer-d-Moulin - 75005 PARIS (France) 4 . Laboratoire des produits biomedicaux - CEA - 30200 BAGNOLS SUR CEZE (France) (Received 20 .1 .1983 ; Accepted in revised form 21 .2 .1983 by Editor U . Abildgaard) ABSTRACT We have previously demonstrated by immunoperoxydase the presence of immunoreactive antithrombin III (AT III) in rat hepatocytes . We now present direct evidence that rat hepatocytes in culture synthesize AT III like immunoreactive material : 35 S-methionine was added to the culture medium and incubated with hepatocytes . After incubationt AT III was immunologically characterized in the medium . We found significant amounts of 35 8-AT III among the radioactive proteins synthesized and secreted by the cells .
INTRODUCTION Several observations and experimental results over the past years provide indirect evidences that the liver is a major site for antithrombin III (AT III) biosynthesis . In hepatic diseases for examplet it has been shown that AT III levels in plasma are decreased (1) . Owens showed that the perfused rat liver is able to release AT III and that this release is inhibited by puromycint an inhibitor of protein synthesis (2) . The cellular localization of AT III in the liver has been investigated by several authors by the use of immunohistochemical techniques . Conflicting results have been obtained . Watadat by classic immunofluorescencet observed the presence of AT III in human hepatocytes (3) . Leet on the other handt did not find any AT III in this cell type by immunoperoxydase (4) . We recently reported that AT III is detectable in rat hepatocytes by immunoperoxydase (5) . Furthermoret in electron microscopyt AT III was located in the Golgi apparatus (6) . These latter observations suggest that AT III is synthesized by these cells . There is however no direct evidence reported so far that hepatocytes actually synthesize AT III . We therefore decided to study the incorporation by rat hepatocytes in culture of radiolabelled aminoacid into proteins and to characterize in this system the biosynthesis of AT III . Key-words
: Rat hepatocytes - Antithrombin III - Antithrombin III synthesis .
369
370
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MATERIALS AND METHODS Preparation of AT III and anti AT III antibodies AT III was purified from rat plasma as previously described . Specific antibodies were raised in the rabbit (5) . These antibodies were purified by immunoadsorption on an AT III- AH Sepharose 4B (Pharmacia - Bois-d'Arcy France) column . After purificationt antibodies were also coupled to AN Sepharose 4B . This couplaget as well as the preparation of AT III - AH Sepharose 4Bt was made according to the procedure described by Clement (7) . Isolation of hepatocytes The livers of adult male Wistar rats (200-250 g) were perfused first for 10 min at a flow rate of 35 ml/min with 0 .01 M Hepes buffer pH 7 .65 containing 0 .14 M NaClt 0 .005 M KC1t 0 .001 M Na2HP0 4 and then for an additional 20 min with 0 .01 M Hepes pH 7 .65t 0 .05 M CaCl2t 0 .025 % collagenase (Boehringer Mannheim - FRG) . The livers were removedt minced in 10 ml Modified Eagle Medium (MEM) (Gibco - Paisley - UK) containing 2 g/l bovine serum albumin (BSA) . The liver parenchyma was dissociated with a spatula . After dissociationt the cell suspension was filtered through sterile gauzes and 100 ml of 0 .01 M Hepes pH 7 .65 and 50 ml MEM both containing 2 g/1 BSA were added . The suspension was allowed to sediment for 25 min and then centrifuged at 50 g for 50 s . The pellet was resuspended in Hepes buffer containing 2 g/1 BSA then centrifuged again at 50 g for 50 s . This procedure was repeated three times . The washed pellet was resuspended in the incubation medium as described below . The viability of the cells preparation was checked by determining the percentage of cells excluding 0 .4 % Trypan blue . Hepatocytes were identified by morphological criteria . From a single livert an average of 200 x 10 6 hepatocytes was obtained . Hepatocytes culture The hepatocytes were seeded at approximately 16 x 10 6 cells/flask in 75 cm2 flasks and incubated for 6 hours at 37 ° C in 5 % C02 95 % air with 8 ml of MEM supplemented with penicillin (50 U/ml)t streptomycin (50 U/ml) and 10 % fetal calf serum (Gibco) . During this periodt the cells attached to the flasks . The medium was then replaced with 14 ml fresh MEM containing hydrocortisone (50 mg/1) and the cells were further incubated for 48 hours . An aliquot of 500 pl of incubation medium was taken at 5t 15t 24t and 48 hours incubation for the determination of AT III . Simultaneouslyt 2 flasks were cultured in 14 ml of methionine free MEM (Gibco)t supplemented as described beforet but also containing 500 pCi of 35 S-methionine (CEA - Gif-sur-Yvette France) specific activity 1t100 Ci/mMol . Aliquots of 500 pi were taken at 5t 15t 24t and 48 hours for the determination of AT III and for the study of 35S -methionine incorporation . Each sample of incubated medium was dialyzed one hour versus distilled watert then 24 hours versus 0 .1 M phosphate pH 7 .4 . The media were collected after 48 hours from the flasks incubated with 35S -methioninet pooledt dialyzedt and used for the immunological characterization of AT III . Measurement of protein synthesis The amount of radioactivity incorporated into the proteins was quantified by TCA precipitation . For this purposet 100 VI of BSA I g/l and then 100 VI of 10 % cold trichloracetic acid (TCA) were added to 25 pl of medium . The precipitate was washed four fines with I ml of cold 5 % TCA . Five hundred microliters of 0 .1 N NaOR was added to solubilize the precipitate and the mixture was counted in a 3 liquid scintillation counter .
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Determination and characterization of AT III - AT III was measured in the culture media by direct radioimmunoassay (RIA) (5) . - The incorporation of radioactivity into AT III was followed by immunoprecipitation : 10 pl of specific anti AT III serum was incubated with 10 ul of the culture medium samples for 24 hours at 4 ° C in 500 pl of 0 .05 M phosphate buffer pH 7 .4 . The AT III -anti AT III complexes were then adsorbed on Staphylococcal protein A Pansorbin (Calbiochem - La Jolla - CA - USA) by adding 30 pl of the 10 % w/v commercial suspension . After centrifugationt the pellets were washed three times with 3 ml of phosphate buffer containing 2 % w/v polyethylene glycol (PEG) and solubilized with 500 ul of 0 .1 N NaOH . The radioactivity in the solubilized precipitateswas counted . Samples were treated in parallel with 10 pl of rabbit preimmune serum instead of specific anti AT III serum . - The medium collected after 48 hours incubation with the cellstcontaining high amounts of radioactive proteins was used to purify AT III by affinity chromatography . For this purposet the dialyzed medium was applied to the anti AT III antibodies sepharose column previously equilibrated with 0 .1 M phosphate buffer pH 7 .4 . After washing the column with this buffert proteins were eluted with 0 .1 M glycine pH 2 .8 . Fractions of 1 .8 ml were collected and immediately neutralized with 0 .2 ml 1 M K2HP0 4 . The protein content was determined by quantifying absorbance at 280 nm . The fractions containing significant amounts of proteins were pooled and concentrated . This material was submitted to crossed immunoelectrophoresis (CIE) (5) . After migrationt the proteins were stained with Coomassie brilliant blue and the distribution of radioactivity in the gel was determined by autoradiography on Kodak X Ray film NS 2T with the help of fluorograph EN 3 Hance (NEN - Boston - MA - USA) . Exposure time was 15 days .
RESULTS Synthesis of proteins by hepatocytes The cells incubated with 35 S-methionine released radioactivity into the medium . The amount of radioactivity present in the TCA precipitable material increased linearly with time between 0 and 20 hours of incubation as shown on figure 1 . The cultured hepatocytes therefore incorporated 35 S-methionine into the proteins secreted in the medium . Synthesis of immunoreactive AT III Figure 2 shows the results of AT III measurement at different incubation t imes . AT III levels were determined by RIA in aliquots taken from seven different flasks . Five flasks were cultured in regular MEM and two flasks in methionine deficient MEM supplemented with 35 S-methionine . As shown on the figuret AT III levels in medium increased linearly with time for up to 24 hours . The hepatocytes therefore secrete AT III in the medium . Incorporation of radioactivity in AT III immunoreactive material Che amounts of radioactivity incorporated into AT III were determined in the protein secreted by immunoprecipitation with anti AT III antibodies as described in the "methods" section . The radioactivity precipitated by the preimmune serum was substracted from that precipitated by the specific anti AT III serum . The difference was taken as an estimation of the radioactivity
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specifically incorporated into AT III . The results are shown on figure 3 . AT III was found to represent about I % of the proteins secreted by the cells . When the levels of radioactive AT III at different times were compared to the correspondant results of AT III determination by RIAt a strong correlation between both determinationsof AT III was found (r = 0 .94 ; p < 0 .001) .
5
15
24
48 1 nb.tiu tW (b .t.)
FIGURE I Time dependent incorporation of 35 S-methionine into protein by cultured rat hepatocytes
9 753-
a
5
I5
24
48 (kn 1n.b .u .t t1.. .)
FIGURE 2 AT III levels measured by RIA in culture medium
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as laevbvtlvv Ums (boars)
FIGURE 3 Immunoprecipitation by anti AT III serum of 3 S-methionine labelled proteins
Characterization of radioactive AT III synthesized and released by hepatocytes Immunological analysis of the material eluted from the anti AT III sepharose column was performed on C IE . AT III secreted by hepatocytes was compared to plasma AT III and for this purpose CIE was performed with plasma or purified medium AT III either in the absence or in the presence of heparin . The results are shown on figure 4 . It can be shown that the radioactivity and the AT III antigen present in the protein fraction purified by immunoadsorption from the culture medium have identical electrophoretic mobilities . In the absence of heparint AT III secreted in the medium has the same electrophoretic mobility as plasma AT III . In the presence of heparin however both the radioactivity and the immunoreactive AT III are separated into two peaks . One of the peaks has the same mobility than plasma AT III whereas the other has a slower mobility .
DISCUSSION Our study shows that cultured rat hepatocytes synthesize and release a protein sharing common antigenic determinants with plasma AT III . The antiserum used to detect and characterize AT III in culture medium is highly specifict as previously reported (5) . On crossed immunoelectrophoresist two different forms of immunoreactive AT III however were characterized in the proteins secreted by hepatocytes . When CIE was performed in the presence of heparint one behaved like plasma AT III and the other had a slower mobility . The nature of this second form of AT III found in culture medium is unknownt but several hypothesis can be made : hepatocytes synthesize only AT III indentical to plasma AT III but the protein is degradated during immunoadsorptiont resulting in a lower electrophoretic mobility ; AT III can bind after its secretion proteases present in the fetal calf serum added to the culture medium and the complexes AT III proteases migrate slower than native AT III ; finallyt it can be proposed that hepatocytes synthesize a protein structurally but not immunologically different from plasma AT III .
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AT III
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AT III
FIGURE 4 Crossed immunoelectrophoresis of AT III purified from the culture medium by affinity chromatography . The gels are stained for protein (I) and the distribution of radioactivity is determined by autoradiography (II) . I in the absence of heparin . 2 in the presence of heparin (10 U/ml) . P : normal plasma .
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The release of AT III by hepatocytes is linear for up to 20 hours and then the rate of secretion decreases . This decrease can probably be explained by the occurence of changes in the functional capacities of the cell kept in culture . The hepatocytes used in this study are indeed surviving cells no more able to grow or multiplicate . Our data demonstrate synthesis of AT III by hepatocytes . Todayt it seems likely that the hepatocyte is not the only one cell type able to synthesize this protein . Indeedt Chan recently demonstrated that human endothelial cells in culture synthesize immunoreactive AT III (8) . This AT III however seems not to be released in the culture medium . Therefore we assume that the endothelial cell is not responsible for the synthesis of circulating AT III . It is possible that hepatocytes synthesize circulating AT III and that endothelial cells synthesize AT III only active in situ . Both AT III could together contribute to the protection of the vascular wall against thrombosis . ACKNOWLEDGMENTS We wish to thank Jose Deguingand for her help in preparing the manuscript .
REFERENCES 1 . HEDNERt U .t NILSSONt I .M . Antithrombin III in a clinical material . Thrombos . Res . 3t 631-641t 1973 . 2 . OWENSt M .R .t MILLERt L .C . Net biosynthesis of antithrombin III by the isolated rat liver perfused for 12-24 hours . Biochim . Biophys . Acta 627t 3039t 1980 . 3 . WATADAt M .t NAGAKAWAt M .t KITANIt T .t OKAJIMAt Y .t MAEDAt Y .t DRANOt S .t IJICHIt H . Identification of the AT III synthesizing hepatocytes by immunofluorescent technique . Thromb . Haemostas . 46t 284t 1981 . 4 . LEEt AKYt CHANt V .t CHANt T .K . The identification and localization of antithrombin III in human tissues . Thrombos . Res . 14t 209-217t 1979 . 5 . LEONt M .t MACHt M .t GUENNECt J .-Y .t JARNETt J .t GIROTt R .t FIESSINGERt J .-N .t JAUBERTt F . Antithrombin III in rat hepatocytes . Thrombos . Res . 28t 115-123t 1982 . 6 . GIROTt R .t JAUBERTt F .t LEONt M .t BELLONt B .t AIACHt M .t JOSSOt F . Albumint fibrinogent prothrombin and antithrombin III variations in bloodt urines and liver in rat nephritic syndrome (Heymann nephritis) . Thromb . Haemoatas .t in press . 7 . CLEMENTtL .T .t YAMASHITAtU .t SHEVACHtE .M . Characterization of an antiserum to Guinea pig antithrombin III (AT III) . I . Reactivity with Guinea pig T lymphocytes . J . IrmrtunoZ . 127t 1220-1227t 1981 . 8 . CHANt T .K .t CHANt V . Antithrombin IIIt the major modulator of intravascular coagulation is synthesized by human endothelial cells . Thromb . Haemostas . 46t 504-506t 1981 .