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Factor V in human vascular endothelium and in endothelial cells in culture
THROMBOSIS RESEARCH 44; 829-835, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.
FACTOR V IN HUMAN VASCULAR ENDOTHELIUM AND IN ENDOTHELIAL CELLS IN CULTURE
J.C.Giddings, A.L.Jarvis and S.Hogg Department of Haematology, University of Wales College of Medicine, Heath Park, Cardiff. UK (Received 9.5.1986; Accepted in revised form 2.9.1986 by Editor J.R. O'Brien)
ABSTRACT
Human blood vessels and human umbilical vein endothelial cells in culture were examined for the presence and synthesis of factor V. Factor V antigen was detected on the luminal surface of blood vessels washed with buffers containing calcium but was absent from segments of the same vessels perfused with buffers containing EDTA. Very low levels of endogenous factor V antigen were found in endothelial cells in culture but these cells did not synthesise factor V in sufficient quantities to be detected by the present methods. Factor V activity was not detected in any of the present cell preparations.
INTRODUCTION Several studies over many years have indicated that hepatocytes megakaryocytes and platelets synthesise or store coagulation factor V. In addition, we have previously localised factor V antigen on human vascular endothelium using imnunohistological methods (1). More recently, Maztorana et al (2) studied adult and foetal rat tissue and largely confirmed our previous human data. In particular, hepatic parenchjimalcells but not hepatic endothelial cells illustrated a characteristic pattern of imnunostaining for factor V antigen. Positive staining wag noted also with some rat vascular endothelium but in these instances control experiments suggested that the results may have been due to nor?--.$pecific reactions. KEY WORDS:
Factor V, Vascular Endothelium, Endothelial cells
829
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Other workers have recently investigated factor V in bovine tissues. Rodgers and Shuman (3) demonstrated that endogenous, endothelial factor V contributed to the activation of prothrombin on the luminal surface of bovine blood vessels and Cerveny et al (4) observed that bovine aortic endothelial cells in culture synthesised both factor V antigen and factor V activity. In contrast, Maruyama et al (5) detected only low levels of endogenous factor V in human umbilical vein endothelial cells using highly sensitive radiometric methods. These workers showed that exogenous factor Va bound to these cells in culture and accelerated the activation of protein C by thrombin-thrombomodulincomplexes. The present study extends our previous observations on human vascular tissue. Intact blood vessels were examined for factor V antigen using immunofluorescence histology and human umbilical vein cells in culture were examined for their ability to synthesise factor V using functional coagulation methods and sensitive enzyme linked immunoassays. MATERIALS AND METHODS Adult Human Blood Vessels. Various adult human blood vessels were identified and collected at autopsy within 24 hours of death. For some experiments segments of blood vessels were obtained from fresh surgical specimens excised for other clinical purposes. All samples were processed immediately after collection. Isolation and Culture of Endothelial Cells. Human umbilical cords were obtained at delivery in a local maternity hospital department. Undamaged lengths of cord were selected, sealed in plastic envelopes and processed within three hours of collection. Endothelial cells were harvested by intraluminal collagenase digestion and cultured in medium 199 (Gibco) supplemented with 20% fetal calf serum as previously described (6). In outline, cells were recovered from the unbilical veins after treatment with 0.1% (w/v) collagenase for 10 minutes at 37OC. Cells were cultuted in an humidified atmosphere of 5% CO / 95% air at approximately 2X10 per ml of medium. Penicillin, 200 unitP per ml, and streptomycin, 200 ng per ml, were added as antibiotics, amphotericin 8, 5 Pg per ml as fungicide, and the medium was enriched with 2mM glutamine. Recovery of Cells and Extraction for Analysis Endothelial cells were harvested from culture flasks by gentle rubbing with a polypropylene rod and centrifuged at 100 xg for 15 minutes at room temperature. The cells were washed by suspension in fresh culture medium and recentrifuged. The cells were resuspended in approximately O.lml fresh culture medium and counted in a haemocytometer. The final suspension was frozen in liquid nitrogen and rapidly thawed at 37°C three times before analysis. Freshly isolated endothelial cells were obtained from the lumen of blood vessels by scraping with a sterile scalpel blade. The cells were washed and disrupted by freezing and thawing as described for cultured cells. Cell free culture medium was analysed after approximately ten fold concentration as described below. Incorporation of Radiolabelled aminoacid Studies of de novo protein synthesis were performed by adding
Vol. 44, No. 6
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radiolabelled methionine to culture medium and examining incorporation of the label into irnnunoprecipitatesby autoradiography as previously described (6). Medium was obtained from confluent cultures and was examined imnediately after collection and after concentration approximately 10 fold by ultrafiltration with an Amicon XM50 membrane. In'munofluorescenceHistology Frozen sections of unfixed tissue and endothelia1 cells at various stages of culture were stained by the indirect imnun0 fluorescence method described by Shearn et al (7). In some experiments segments of adult human blood vessels were perfused with phosphate buffered saline, pH 7.2, (PBS) in the presence of 2mM calcium chloride or in the presence of 2mM disodium ethylenediamine tetra-acetic acid before histology. Antiserum to Factor V Antigen (VAg) Antibody to factor V antigen was produced by irrmunisingrabbits with factor V purified from human plasma as described by Kane and Majerus (8). The rabbit serum was inactivated and adsorbed as described by Giddings et al (1). The final product specifically neutralised factor V activity in coagulation assays and gave a single arc against normal plasma and factor V concentrate in imnunoelectrophoresistests. No immunoprecipitin reactions were observed against plasma from a patient known to have severe, hereditary factor V deficiency. Imnunoglobulin fractions of the antiserum were prepared by chromatography on DEAE Sephacel (Pharmacia) following a minor modification of the method described by Mollison (9), and were labelled with horse radish peroxidase as described by Nakane and Kawaoi (10). In some experiments factor V antiserum kindly provided by Dakopatts (Dako Ltd., High Wycombe, Bucks) was used. Enzyme Linked Imnunoassays (ELISA)
ELISA were established in 96-well microtitre plates by minor modifications of standard procedures. Plates were coated overnight at 4OC with unlabelled immunoglobulin fractions of the factor V antibody diluted 1 in 1000 in bicarbonate buffer, pH 9.6. The plates were washed and antigen containing samples added. Incubation was continued at room temperature for 2 hours. After repeated washing, peroxidase labelled antibody was added and the plates again incubated at room temperature for a further two hours. The peroxidase label was detected using orthophenylenediamine as substrate. Absorbance was quantitated at 492rm in a Multiskan spectrophotometric plate reader (Flow Laboratories). Immunodiffusion and Immunoelectrophoresis Qualitative analyses of factor V antigen were performed by conventional imnunodiffusion and two dimensional crossed immunoelectrophoresis (11,12). Quantitative electrophoresis was performed by the rocket technique (13) using pooled human plasma as reference. Autoradiography Two dimensional crossed immunoelectrophoresis plates of culture medium containing radiolabelled methionine were incubated in contact with Kodak Ortho G film for two to three days at -86OC in a Kodak X-Omatic image
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intensifying cassette. The films were developed and examined using conventional radiographic equipment. Other methods will be described in the relevant parts of the text. RESULTS Immunofluorescence
FIG.l. Immunofluorescence of human splenic artery. a) blood vessel perfused with buffer containing calcium chloride before examination for factor V antigen. b) segment of the same blood vessel perfused with calcium-free buffer and examined as in (a). c) segment of the same blood vessel prepared as in (b) and examined for von Willebrand factor antigen. Immunofluorescence histology of human blood vessels treated with factor V antiserum is illustrated in figure 1. The luminal surface of all blood vessels examined showed positive fluorescence for factor V antigen (Figure la). The fluorescence pattern was not as consistent and was not as intense, however, as that observed usually in blood vessels examined for von Willebrand factor antigen (Figure lc). Moreover, factor V antigen was not demonstrated in segments of blood vessels examined after perfusion in calcium-free buffer (Figure lb) although it was found in sections of the same tissue perfused with buffer containing calcium. von Willebrand factor antigen was detected in all sections irrespective of the presence or absence of calcium.
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FACTOR V IN ENDOTHELIAL CELLS
Factor V antigen was not detected b imnunofluorescence in any preparations of endothelial cells in cuT ture. Quantitation of Factor V Activity and Factor V Antigen Table 1. Factor V Activity, Factor V Antigen and von Willebrand Factor Antigen (units per dl) in Endothelial Cell Extracts and in Supernatant Culture Medium after 9 Days Incubation.
-vc
vWFAg
!!!I9
Cell extracts
0.018 f 0.005
9.2 f 3.55
Culture supernatants
7.5 f 3.99
Basal culture medium without cells
co.01
Results (units per dl) are the mean f S.D. of 7 experiments. Confluent cell density was approximately 1X105 per cm2 culture surface.
Measurements of factor V activity and factor V antigen in endothelial cells and in culture supernatants are given in Table 1. Very low levels of factor V antigen were detected in cell extracts by the ELISA method. In each instance measurements were slightly higher than the minimum level of antigen detected in normal human plasma (0.01 units per dl) by this technique. Factor V antigen was not detected in culture supernatants, however, and factor V activity was not found in any of the present preparations. For comparison Table 1 also shows the levels of von Willebrand factor antigen (vWFAg, ELISA method) in these experiments. As expected, relatively large amounts of this material were measured both in cell extracts and in culture supernatants. Synthesis of Factor V Antigen Examination of cell extracts and culture supernatants by immunodiffusion and crossed itwnunoelectrophoresisfailed to demonstrate the presence of factor V antigen. In addition, autoradiography of cell products after culture in the presence of radiolabelled methionine did not reveal synthesis of factor V antigen. DISCUSSION The current data extend our previous immunohistological studies on the tissue localisation of human factor V antigen and are largely compatible
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with the results of Maruyama et al (5). These workers showed that low levels of endogenous factor V were present on human umbilical vein endothelial cells and that additional factor V bound to these cells in culture. They proposed that physiological concentrations of bound factor V (or factor Va) accelerated the activation of protein C by thrombomodulin on the endothelial surface in the presence of calcium ions. In the present study we confirmed that factor V antigen was present on intact human vascular endothelium and demonstrated that this was readily removed by perfusion of the blood vessels with calcium-free buffer. We were not able to detect factor V antigen on monolayers of endothelial cells in culture by imnunofluorescence but we measured very low levels of the protein by ELISA in extracts of these cells. The origin of the cellular factor V in these experiments remains unclear at present. Previous studies by Cerveny et al (4) demonstrated synthesis of factor V by bovine aortic endothelial cells in culture but we were not able to confirm this using our human umbilical vein endothelial cells. It is possible that the present methods were not sufficiently sensitive to detect synthesis of factor V. Cellular production of von Willebrand factor antigen (and of other proteins including fibronectin - data not shown) was readily observed, however. It is also possible that human endothelial cells might not have the same capacity as bovine endothelial cells to synthesise factor V. Differences between bovine and human factor V are well recognised and cells from different species might vary in their ability to synthesise protein. In addition, endothelial cells from various sites, even in the same species, might subserve different functions. For example, Rodgers and Shuman (3) suggested that arterial endothelial cells had a greater capacity to activate prothrombin than similar cells from veins and Giddings et al (6) demonstrated that whilst porcine umbilical vein endothelial cells could be shown readily to synthesise von Willebrand factor antigen the same was not true for porcine aortic endothelial cells. Furthermore, Mazzorana et al (2) localised factor V antigen in rat hepatocytes but failed to identify the material specifically in endothelium in this species. In the study of Maruyama et al (5) factor V might have been absorbed from the human serum used in their culture medium. In the present study, however, fetal calf serum was used and the antibody used in the ELISA did not cross react with bovine factor V antigen. It is possible that factor V was absorbed from umbilical cord blood by endothelial cells prior to culture and experiments are continuing on passaged cells from a variety of blood vessels in an attempt to define more clearly the origin of the cellular material. Nevertheless, the current findings emphasised that factor V binds to human vascular endothelial cell surfaces. The data supported the concept that bound factor V participates in physiological coagulation reactions at this site. ACKNOWLEDGEMENTS. This work was supported by grants from the Medical Research Council. REFERENCES. 1.
GIDDINGS, J.C., SHEARN, S.A.M. and BLOOM, A.L. The immunological localisation of factor V in hunan tissue. Brit. J. Haem. 29, 57-65, 1975
2.
MAZZORANA, M., CORNILLON, B., BELLEVILLE, J. and ELOY, R. Immunolocalisation of factor V in adult and fetal rat liver. Thrombosis. jQ. 9, 807-816, 1985.
Vol. 44, No. 6
FACTOR V IN ENDOTHELIAL CELLS
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3.
RODGERS G.M., and SHUMAN, M.A. Prothrombin is activated on vascular endothelial cells by factor Xa and calcium. Proc. Natl. Acad. Sci. USA. 8D_ 7001-7005, 1983.
4.
CERVENY, T.J., FASS, D.N. and MANN, K.G. Synthesis of coagulation factor V by cultured aortic endothelium. Blood.63, 1467-1474, 1984.
5.
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, 224-230, 1984.
6.
GIDDINGS, J.C., JARVIS, A.L. and BLOOM, A.L. Differential localisation and synthesis of porcine factor VIII related antigen (VIIIR:AG) in vascular endothelium and in endothelial cells in culture. Thrombosis Res. 29 299-312, 1983. --)
7.
SHEARN, S.A.M., PEAKE, I.R., GIDDINGS, J.C., HUMPHRYS, J. and BLOOM, A.L. The characterisation and synthesis of antigens related to factor VIII in vascular endothelium. Thrombosis Res. 11, 43-56, 1977.
8.
KANE, W.H. and MAJERUS, P.W. Purification and characterisation of human coagulation factor V. J. Biol. Chem.256, 1002-1007, 1981.
9.
MOLLISON, P.L. Separation of immunoglobulins by DEAE-cellulose fractionation. In: Blood Transfusion in Clinical Medicine. 7th. Edition. Blackwell Scientific Publications, Oxford, p791, 1983.
10. NAKANE, P.K and KAWAOI, A. Peroxidase-labelled antibody. A new method of conjugation. J. Histochem. and Cytochem.2, 1084-1091, 1974. 11. OUCHTERLONY, 0. Diffusion-in-gel methods for immunological analysis. II. Proqress in Allergy,&, 30-154, 1962. 12. CLARK, H.G.M. and FREEMAN, T. Quantitative immunoelectrophoresis of human serum proteins. Clin. Sci., 35, 403-414, 1968. 13. LAURELL, C.B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem., 15, 45-52, 1966.
FACTOR V IN HUMAN VASCULAR ENDOTHELIUM AND IN ENDOTHELIAL CELLS IN CULTURE
J.C.Giddings, A.L.Jarvis and S.Hogg Department of Haematology, University of Wales College of Medicine, Heath Park, Cardiff. UK (Received 9.5.1986; Accepted in revised form 2.9.1986 by Editor J.R. O'Brien)
ABSTRACT
Human blood vessels and human umbilical vein endothelial cells in culture were examined for the presence and synthesis of factor V. Factor V antigen was detected on the luminal surface of blood vessels washed with buffers containing calcium but was absent from segments of the same vessels perfused with buffers containing EDTA. Very low levels of endogenous factor V antigen were found in endothelial cells in culture but these cells did not synthesise factor V in sufficient quantities to be detected by the present methods. Factor V activity was not detected in any of the present cell preparations.
INTRODUCTION Several studies over many years have indicated that hepatocytes megakaryocytes and platelets synthesise or store coagulation factor V. In addition, we have previously localised factor V antigen on human vascular endothelium using imnunohistological methods (1). More recently, Maztorana et al (2) studied adult and foetal rat tissue and largely confirmed our previous human data. In particular, hepatic parenchjimalcells but not hepatic endothelial cells illustrated a characteristic pattern of imnunostaining for factor V antigen. Positive staining wag noted also with some rat vascular endothelium but in these instances control experiments suggested that the results may have been due to nor?--.$pecific reactions. KEY WORDS:
Factor V, Vascular Endothelium, Endothelial cells
829
830
FACTOR V IN ENDOTHELIAL CELLS
Vol. 44, No. 6
Other workers have recently investigated factor V in bovine tissues. Rodgers and Shuman (3) demonstrated that endogenous, endothelial factor V contributed to the activation of prothrombin on the luminal surface of bovine blood vessels and Cerveny et al (4) observed that bovine aortic endothelial cells in culture synthesised both factor V antigen and factor V activity. In contrast, Maruyama et al (5) detected only low levels of endogenous factor V in human umbilical vein endothelial cells using highly sensitive radiometric methods. These workers showed that exogenous factor Va bound to these cells in culture and accelerated the activation of protein C by thrombin-thrombomodulincomplexes. The present study extends our previous observations on human vascular tissue. Intact blood vessels were examined for factor V antigen using immunofluorescence histology and human umbilical vein cells in culture were examined for their ability to synthesise factor V using functional coagulation methods and sensitive enzyme linked immunoassays. MATERIALS AND METHODS Adult Human Blood Vessels. Various adult human blood vessels were identified and collected at autopsy within 24 hours of death. For some experiments segments of blood vessels were obtained from fresh surgical specimens excised for other clinical purposes. All samples were processed immediately after collection. Isolation and Culture of Endothelial Cells. Human umbilical cords were obtained at delivery in a local maternity hospital department. Undamaged lengths of cord were selected, sealed in plastic envelopes and processed within three hours of collection. Endothelial cells were harvested by intraluminal collagenase digestion and cultured in medium 199 (Gibco) supplemented with 20% fetal calf serum as previously described (6). In outline, cells were recovered from the unbilical veins after treatment with 0.1% (w/v) collagenase for 10 minutes at 37OC. Cells were cultuted in an humidified atmosphere of 5% CO / 95% air at approximately 2X10 per ml of medium. Penicillin, 200 unitP per ml, and streptomycin, 200 ng per ml, were added as antibiotics, amphotericin 8, 5 Pg per ml as fungicide, and the medium was enriched with 2mM glutamine. Recovery of Cells and Extraction for Analysis Endothelial cells were harvested from culture flasks by gentle rubbing with a polypropylene rod and centrifuged at 100 xg for 15 minutes at room temperature. The cells were washed by suspension in fresh culture medium and recentrifuged. The cells were resuspended in approximately O.lml fresh culture medium and counted in a haemocytometer. The final suspension was frozen in liquid nitrogen and rapidly thawed at 37°C three times before analysis. Freshly isolated endothelial cells were obtained from the lumen of blood vessels by scraping with a sterile scalpel blade. The cells were washed and disrupted by freezing and thawing as described for cultured cells. Cell free culture medium was analysed after approximately ten fold concentration as described below. Incorporation of Radiolabelled aminoacid Studies of de novo protein synthesis were performed by adding
Vol. 44, No. 6
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radiolabelled methionine to culture medium and examining incorporation of the label into irnnunoprecipitatesby autoradiography as previously described (6). Medium was obtained from confluent cultures and was examined imnediately after collection and after concentration approximately 10 fold by ultrafiltration with an Amicon XM50 membrane. In'munofluorescenceHistology Frozen sections of unfixed tissue and endothelia1 cells at various stages of culture were stained by the indirect imnun0 fluorescence method described by Shearn et al (7). In some experiments segments of adult human blood vessels were perfused with phosphate buffered saline, pH 7.2, (PBS) in the presence of 2mM calcium chloride or in the presence of 2mM disodium ethylenediamine tetra-acetic acid before histology. Antiserum to Factor V Antigen (VAg) Antibody to factor V antigen was produced by irrmunisingrabbits with factor V purified from human plasma as described by Kane and Majerus (8). The rabbit serum was inactivated and adsorbed as described by Giddings et al (1). The final product specifically neutralised factor V activity in coagulation assays and gave a single arc against normal plasma and factor V concentrate in imnunoelectrophoresistests. No immunoprecipitin reactions were observed against plasma from a patient known to have severe, hereditary factor V deficiency. Imnunoglobulin fractions of the antiserum were prepared by chromatography on DEAE Sephacel (Pharmacia) following a minor modification of the method described by Mollison (9), and were labelled with horse radish peroxidase as described by Nakane and Kawaoi (10). In some experiments factor V antiserum kindly provided by Dakopatts (Dako Ltd., High Wycombe, Bucks) was used. Enzyme Linked Imnunoassays (ELISA)
ELISA were established in 96-well microtitre plates by minor modifications of standard procedures. Plates were coated overnight at 4OC with unlabelled immunoglobulin fractions of the factor V antibody diluted 1 in 1000 in bicarbonate buffer, pH 9.6. The plates were washed and antigen containing samples added. Incubation was continued at room temperature for 2 hours. After repeated washing, peroxidase labelled antibody was added and the plates again incubated at room temperature for a further two hours. The peroxidase label was detected using orthophenylenediamine as substrate. Absorbance was quantitated at 492rm in a Multiskan spectrophotometric plate reader (Flow Laboratories). Immunodiffusion and Immunoelectrophoresis Qualitative analyses of factor V antigen were performed by conventional imnunodiffusion and two dimensional crossed immunoelectrophoresis (11,12). Quantitative electrophoresis was performed by the rocket technique (13) using pooled human plasma as reference. Autoradiography Two dimensional crossed immunoelectrophoresis plates of culture medium containing radiolabelled methionine were incubated in contact with Kodak Ortho G film for two to three days at -86OC in a Kodak X-Omatic image
832
FACTOR V IN ENDOTHELIAL CELLS
Vol. 44, No. 6
intensifying cassette. The films were developed and examined using conventional radiographic equipment. Other methods will be described in the relevant parts of the text. RESULTS Immunofluorescence
FIG.l. Immunofluorescence of human splenic artery. a) blood vessel perfused with buffer containing calcium chloride before examination for factor V antigen. b) segment of the same blood vessel perfused with calcium-free buffer and examined as in (a). c) segment of the same blood vessel prepared as in (b) and examined for von Willebrand factor antigen. Immunofluorescence histology of human blood vessels treated with factor V antiserum is illustrated in figure 1. The luminal surface of all blood vessels examined showed positive fluorescence for factor V antigen (Figure la). The fluorescence pattern was not as consistent and was not as intense, however, as that observed usually in blood vessels examined for von Willebrand factor antigen (Figure lc). Moreover, factor V antigen was not demonstrated in segments of blood vessels examined after perfusion in calcium-free buffer (Figure lb) although it was found in sections of the same tissue perfused with buffer containing calcium. von Willebrand factor antigen was detected in all sections irrespective of the presence or absence of calcium.
Vol. 44, No. 6
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FACTOR V IN ENDOTHELIAL CELLS
Factor V antigen was not detected b imnunofluorescence in any preparations of endothelial cells in cuT ture. Quantitation of Factor V Activity and Factor V Antigen Table 1. Factor V Activity, Factor V Antigen and von Willebrand Factor Antigen (units per dl) in Endothelial Cell Extracts and in Supernatant Culture Medium after 9 Days Incubation.
-vc
vWFAg
!!!I9
Cell extracts
0.018 f 0.005
9.2 f 3.55
Culture supernatants
7.5 f 3.99
Basal culture medium without cells
co.01
Results (units per dl) are the mean f S.D. of 7 experiments. Confluent cell density was approximately 1X105 per cm2 culture surface.
Measurements of factor V activity and factor V antigen in endothelial cells and in culture supernatants are given in Table 1. Very low levels of factor V antigen were detected in cell extracts by the ELISA method. In each instance measurements were slightly higher than the minimum level of antigen detected in normal human plasma (0.01 units per dl) by this technique. Factor V antigen was not detected in culture supernatants, however, and factor V activity was not found in any of the present preparations. For comparison Table 1 also shows the levels of von Willebrand factor antigen (vWFAg, ELISA method) in these experiments. As expected, relatively large amounts of this material were measured both in cell extracts and in culture supernatants. Synthesis of Factor V Antigen Examination of cell extracts and culture supernatants by immunodiffusion and crossed itwnunoelectrophoresisfailed to demonstrate the presence of factor V antigen. In addition, autoradiography of cell products after culture in the presence of radiolabelled methionine did not reveal synthesis of factor V antigen. DISCUSSION The current data extend our previous immunohistological studies on the tissue localisation of human factor V antigen and are largely compatible
834
FACTOR V IN ENDOTHELIAL CELLS
Vol. 44, No. 6
with the results of Maruyama et al (5). These workers showed that low levels of endogenous factor V were present on human umbilical vein endothelial cells and that additional factor V bound to these cells in culture. They proposed that physiological concentrations of bound factor V (or factor Va) accelerated the activation of protein C by thrombomodulin on the endothelial surface in the presence of calcium ions. In the present study we confirmed that factor V antigen was present on intact human vascular endothelium and demonstrated that this was readily removed by perfusion of the blood vessels with calcium-free buffer. We were not able to detect factor V antigen on monolayers of endothelial cells in culture by imnunofluorescence but we measured very low levels of the protein by ELISA in extracts of these cells. The origin of the cellular factor V in these experiments remains unclear at present. Previous studies by Cerveny et al (4) demonstrated synthesis of factor V by bovine aortic endothelial cells in culture but we were not able to confirm this using our human umbilical vein endothelial cells. It is possible that the present methods were not sufficiently sensitive to detect synthesis of factor V. Cellular production of von Willebrand factor antigen (and of other proteins including fibronectin - data not shown) was readily observed, however. It is also possible that human endothelial cells might not have the same capacity as bovine endothelial cells to synthesise factor V. Differences between bovine and human factor V are well recognised and cells from different species might vary in their ability to synthesise protein. In addition, endothelial cells from various sites, even in the same species, might subserve different functions. For example, Rodgers and Shuman (3) suggested that arterial endothelial cells had a greater capacity to activate prothrombin than similar cells from veins and Giddings et al (6) demonstrated that whilst porcine umbilical vein endothelial cells could be shown readily to synthesise von Willebrand factor antigen the same was not true for porcine aortic endothelial cells. Furthermore, Mazzorana et al (2) localised factor V antigen in rat hepatocytes but failed to identify the material specifically in endothelium in this species. In the study of Maruyama et al (5) factor V might have been absorbed from the human serum used in their culture medium. In the present study, however, fetal calf serum was used and the antibody used in the ELISA did not cross react with bovine factor V antigen. It is possible that factor V was absorbed from umbilical cord blood by endothelial cells prior to culture and experiments are continuing on passaged cells from a variety of blood vessels in an attempt to define more clearly the origin of the cellular material. Nevertheless, the current findings emphasised that factor V binds to human vascular endothelial cell surfaces. The data supported the concept that bound factor V participates in physiological coagulation reactions at this site. ACKNOWLEDGEMENTS. This work was supported by grants from the Medical Research Council. REFERENCES. 1.
GIDDINGS, J.C., SHEARN, S.A.M. and BLOOM, A.L. The immunological localisation of factor V in hunan tissue. Brit. J. Haem. 29, 57-65, 1975
2.
MAZZORANA, M., CORNILLON, B., BELLEVILLE, J. and ELOY, R. Immunolocalisation of factor V in adult and fetal rat liver. Thrombosis. jQ. 9, 807-816, 1985.
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835
3.
RODGERS G.M., and SHUMAN, M.A. Prothrombin is activated on vascular endothelial cells by factor Xa and calcium. Proc. Natl. Acad. Sci. USA. 8D_ 7001-7005, 1983.
4.
CERVENY, T.J., FASS, D.N. and MANN, K.G. Synthesis of coagulation factor V by cultured aortic endothelium. Blood.63, 1467-1474, 1984.
5.
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, 224-230, 1984.
6.
GIDDINGS, J.C., JARVIS, A.L. and BLOOM, A.L. Differential localisation and synthesis of porcine factor VIII related antigen (VIIIR:AG) in vascular endothelium and in endothelial cells in culture. Thrombosis Res. 29 299-312, 1983. --)
7.
SHEARN, S.A.M., PEAKE, I.R., GIDDINGS, J.C., HUMPHRYS, J. and BLOOM, A.L. The characterisation and synthesis of antigens related to factor VIII in vascular endothelium. Thrombosis Res. 11, 43-56, 1977.
8.
KANE, W.H. and MAJERUS, P.W. Purification and characterisation of human coagulation factor V. J. Biol. Chem.256, 1002-1007, 1981.
9.
MOLLISON, P.L. Separation of immunoglobulins by DEAE-cellulose fractionation. In: Blood Transfusion in Clinical Medicine. 7th. Edition. Blackwell Scientific Publications, Oxford, p791, 1983.
10. NAKANE, P.K and KAWAOI, A. Peroxidase-labelled antibody. A new method of conjugation. J. Histochem. and Cytochem.2, 1084-1091, 1974. 11. OUCHTERLONY, 0. Diffusion-in-gel methods for immunological analysis. II. Proqress in Allergy,&, 30-154, 1962. 12. CLARK, H.G.M. and FREEMAN, T. Quantitative immunoelectrophoresis of human serum proteins. Clin. Sci., 35, 403-414, 1968. 13. LAURELL, C.B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem., 15, 45-52, 1966.