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Effects of ticlopidine and dexamethasone on fibronectin and factor VIII-related antigen synthesis by cultured endothelial cells
THROMBOSTS RESEARCH, Supplement TV; 69-73, 1983 0049-3848183 $3.00 + .OO Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
EFFECTS OF TICLOPIDINE AND DEXAMETHASONE ON FIBRONECTIN AND FACTOR VIII-RELATED ANTIGEN SYNTHESIS BY CULTURED ENDOTHELIAL CELLS BRIEF
COMMUNICATION
F. Piovella, 'J.C. Giddings, P. Almasio, M.M. Ricetti, 'J.E. Thomas Istituto di Clinica Medica I0 Univgrsity of Pavia, 27100 Pavia, Italy Welsh National School of Medicine and University of Wales, Cardiff, U.K.
INTRODUCTION Ticlopidine, f-(2-chlorobenzyl)-4,5,6,7 tetrahydrothieno(3,2_c)pyridine hydrochloride, when given orally, causes marked prolongation in the bleeding time (1). This effect appears to be unrelated to the inhibition of platelet function or coagulation. Corticosteroids block the ticlopidine effect & vivo and restore bleeding time towards normal (1). The explanation for these effects is unclear. We found that dexamethasone causes a dramatic increase in fibronectin matrix formation by cultured human endothelial cells (2). This increase may be related to the improved haemostasis commonly seen after steroid administration in non-responding thrombocytopaenias. In contrast, we found in preliminary studies that ticlopidine inhibited the formation of fibronectin both intra- and extracellularly by endothelial cells in culture (3). This suggests that ticlopidine may interfere with the synthesis and/or release of extracellular matrix components from intracellular granules and promote bleeding. To study these effects in more detail, we examined the relative effects of ticlopidine and dexamethasone on fibronectin and factor VIII-related antigen (VIIIR:Ag) synthesis by cultured human endothelial cells derived from umbilical cord veins. METHODS Endothelial cells derived from human umbilical cord veins were grown in medium 199 with Earl's salts, supplemented with 20% foetal calf serum, 0.22% NaHCO 2 mM L-glutamine on glass coverslips in 35mm Petri dishes. Dishes were !incubated in 5% C02-95% air atmosphere, saturated with water vapor at 37Oc (4). After three hours incubation, dishes were washed and fresh medium containing increasing concentrations of ticlopidine (30-150 uM), dexamethasone (0.01-0.1 uM) or equal volumes of the suspending vehicle was
Key Words:
Ticlopidine, Dexamethasone, Fibronectin, VIII Levels
69
70
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Suppl.
IV, 1983
added. Cells were then incubated for 48 hours. The culture media were then decanted into separate tubes and the amount of fibronectin and VIIIR:Ag were determined using an enzyme-linked immunoassay (ELISA 1. The glass coverslips were washed twice with calcium and magnesium-free phosphate buffered saline, fixed briefly and the cell-associated fibronectin and VIIIR:Ag were detected by indirect double staining immunofluorescence (53. The antiseras were produced by animal immunization with partially purified antigens followed by extensive absorption to remove contaminating antibodies (6) or were purchased from commercial suppliers. We utilized a rabbit anti-human VIIIR:Ag antiserum, a goat anti-human fibronectin antiserum, a rhodamine-con jugated (TRITC) swine anti-rabbit gammaglobulin antiserum (Dako Corp.) and a fluorescein-conjugated (FITC) swine anti-goat gammaglobulin antiserum (Nordic Immunological Lab). RESULTS Both fibronectin Changes in Cell-Associated Fibronectin and VIIIR:Ag. filaments and punctate VIIIR:Ag granular staining were detectable in control cell preparations. The intensity of staining was unaffected by either In the presence of ticlopidine, fibronectin staining was suspending vehicle. markedly reduced and in some cases non-detectable and VIIIR:Ag granules were The opposite effects were seen with dexamethasone, increased (Figure lc,d). and VIIIR:Ag staining was namely fibronectin staining, was increased In addition, the formation of extracellular decreased (Figure la,b). fibrillar matrix was more rapid in these cells (data not shown). The results in Changes in Media-Associated Fibronectin and VIIIR:Ag. the supernatant media were parallel with the results observed in the cells. Thus, control levels of fibronectin were decreased 75% in the presence of 150 uM of ticlopidine and increased 56% in the presence of 0.1 uM of In contrast, VIIIR:Ag levels tended to dexamethasone, p < 0.001 (Table 1). The changes be increased with ticlopidine and decreased with dexamethasone. and may be related to the with ticlopidine, however, were not significant increased variation after drug treatment. TABLE 1 Effect
of 150 uM Ticlopidine and 0.1 uM Dexamethasone on Fibronectin (ug/ml) and VIIIR:Ag (U/d11 Release Mean 2 SEM, n=7
Control Fibronectin (ug/ml)
0.187 + 0.015
VIIIR :Ag (U/dl)
2.0 -+ 0.2
* p <
0.001
Dexametha sone
Ticlopidine 0.050
+ 0.011
2.4 2 0.5
*
0.293 2 c.019* 1.4 -+
9.1*
TICLOPIDINE EFFECT ON FSBRONECTIN
Suppl. IV, 1983
a
d
c
FIG. 1 Indirect combined immunofluorescence on human endothelial cells grown in the presence of 0.1 uM dexamethasone or 150 uM ticlopidine: a) Immunofluoroescence for fibronectin (FITC); b) Immunofluorescence for VIIIR:Ag (TRITC). Same field as (a) c) Immunofluorescence for fibronectin (FITC). d) Immunofluorescence for VIIIR:Ag (TRITC). Same field as (c)
72
TICLOPIDINE EFFECT ON FIBRONECTIM
Suppl. IV, 1083
CONCLUSIONS This study confirms our previous observations that ticlopidine influences on endothelial cell morphology and on fibronectin expression (31, as well as cell culture-produced subendothelium (7) can both be related to the decrease of fibronectin synthesis and/or release. If fibronectin is a mediator of platelet adhesion to subendothelium and of platelet/platelet interactions (at least in the earlier stages of attachment), a reduction of its availability could affect some of the platelet function as well as endothelial cell repair and may subsequently influence the bleeding time. These findings, in association with the well documented inhibitory action on platelet aggregability may better explain the effects of this drug in the haemostatic mechanisms. Thus its double-target mode of action may be important in preventing the occurrence of vascular thrombosis. Our study also confirms that dexamethasone has the opposite effect on endothelial cells. These experiments do not define the mechanism by which corticosteroids prevent or reverse endothelial alterations during ticlopidine treatment. Corticosteroids have been shown to stabilize cell membrane (8). They can also interact with RNA synthesis (9). The fact that fibronectin production is increased when VIIIR:Ag is decreased seems to suggest the presence of a relationship, i.e. an interdependence, between these two proteins in endothelial cell metabolism. These alterations in the synthesis of components which are present either in plasma or in subendothelium may be important and may explain in part the prolongation of bleeding associated with the use of ticlopidine and its modulation by corticosteroids. REFERENCES 1. THEBAULT, J., BLATRIX, C., BLANCHARD, J., and PANAK, E. A possible method to control prolongations of bleeding time under antiplatelet therapy with ticlopidine. Thromb. Haemostas. 48, 6-8, 1982. 2.
PIOVELLA, F., CIDDINGS, J.C., RICETTI, M.M., ALMASIO, P., and ASCARI, E. The effect of dexamethasone on formation of intra- and eXtraCellUlar fibronectin matrix by human endothelial cells in culture. bematOlOgiCa 67, 58-63, 1982.
3.
PIOVELLA, F., RICETTI, M.M., ALMASIO, P., SAMADEN, A., SEMINO, G., and ASCARI, E. The effect of ticlopidine on human endothelial cells in culture. Thromb. Res. submitted for publication.
4.
PIOVELLA, F., RICETTI, M.M., ALMASIO, P., CASTAGNOLA, C., PESENTI CAMPAGNONI, M., GALLOTTI, P., FEOLI, F.R., and ASCARI, E. in Characterization and synthesis of some factor VIII-related PrOPertieS cultured human endothelial cells. Haematologica 64, 714-725, 1979.
5.
PIOVELLA, F., GIDDINGS, J.C., RICETTI, M.M., and THOMAS, J.E. Factor VIII related antigen and fibronectin in cultured endothelial cells in the presence of dexamethasone and ticlopidine. Haemostasis (abstract) 12, 23, 1982.
6.
GIDDINGS, J.C., BROOKES, L.R., PIOVELLA, R., and 3LOOM, A.L. Immunohistological comparison of platelet factor 4 (PFQ), fibronectin (Fn) and factor VIII related antigen (VIIIR:Ag) in human platelet granules. Brit. H. Haemat. 52, 79-88, 1982.
Suppl. IV, 1983
TICLOPIDINEEFFECT ON FIBRONECTIN
73
7. PIOVELLA, F., PIOVELLA,L., ALMASIO, P., RICETTI,M.M., and ASCARI, E. The effect of ticlopidine on cultured endothelial CellS: Possible influence on extracellular matrix formation. Thromb. Haemostas. (abstract)46, 404, 1981. 8. BALLARD, P.L., and TOMKINS, G.M. Hormone induced modificationof the cell surface. Nature 224, 344-345,1969. 9. FAIN, J.N., KOVACEV, V.P., and SCOW, R.O. Effect of growth hormone and dexamethasone on lipolysis and metabolism in isolated fat cells of the rat. J. Biol. Chem. 240, 3522-3529,1965.
EFFECTS OF TICLOPIDINE AND DEXAMETHASONE ON FIBRONECTIN AND FACTOR VIII-RELATED ANTIGEN SYNTHESIS BY CULTURED ENDOTHELIAL CELLS BRIEF
COMMUNICATION
F. Piovella, 'J.C. Giddings, P. Almasio, M.M. Ricetti, 'J.E. Thomas Istituto di Clinica Medica I0 Univgrsity of Pavia, 27100 Pavia, Italy Welsh National School of Medicine and University of Wales, Cardiff, U.K.
INTRODUCTION Ticlopidine, f-(2-chlorobenzyl)-4,5,6,7 tetrahydrothieno(3,2_c)pyridine hydrochloride, when given orally, causes marked prolongation in the bleeding time (1). This effect appears to be unrelated to the inhibition of platelet function or coagulation. Corticosteroids block the ticlopidine effect & vivo and restore bleeding time towards normal (1). The explanation for these effects is unclear. We found that dexamethasone causes a dramatic increase in fibronectin matrix formation by cultured human endothelial cells (2). This increase may be related to the improved haemostasis commonly seen after steroid administration in non-responding thrombocytopaenias. In contrast, we found in preliminary studies that ticlopidine inhibited the formation of fibronectin both intra- and extracellularly by endothelial cells in culture (3). This suggests that ticlopidine may interfere with the synthesis and/or release of extracellular matrix components from intracellular granules and promote bleeding. To study these effects in more detail, we examined the relative effects of ticlopidine and dexamethasone on fibronectin and factor VIII-related antigen (VIIIR:Ag) synthesis by cultured human endothelial cells derived from umbilical cord veins. METHODS Endothelial cells derived from human umbilical cord veins were grown in medium 199 with Earl's salts, supplemented with 20% foetal calf serum, 0.22% NaHCO 2 mM L-glutamine on glass coverslips in 35mm Petri dishes. Dishes were !incubated in 5% C02-95% air atmosphere, saturated with water vapor at 37Oc (4). After three hours incubation, dishes were washed and fresh medium containing increasing concentrations of ticlopidine (30-150 uM), dexamethasone (0.01-0.1 uM) or equal volumes of the suspending vehicle was
Key Words:
Ticlopidine, Dexamethasone, Fibronectin, VIII Levels
69
70
TICLOPIDINE EFFECTON FIBRONECTIR
Suppl.
IV, 1983
added. Cells were then incubated for 48 hours. The culture media were then decanted into separate tubes and the amount of fibronectin and VIIIR:Ag were determined using an enzyme-linked immunoassay (ELISA 1. The glass coverslips were washed twice with calcium and magnesium-free phosphate buffered saline, fixed briefly and the cell-associated fibronectin and VIIIR:Ag were detected by indirect double staining immunofluorescence (53. The antiseras were produced by animal immunization with partially purified antigens followed by extensive absorption to remove contaminating antibodies (6) or were purchased from commercial suppliers. We utilized a rabbit anti-human VIIIR:Ag antiserum, a goat anti-human fibronectin antiserum, a rhodamine-con jugated (TRITC) swine anti-rabbit gammaglobulin antiserum (Dako Corp.) and a fluorescein-conjugated (FITC) swine anti-goat gammaglobulin antiserum (Nordic Immunological Lab). RESULTS Both fibronectin Changes in Cell-Associated Fibronectin and VIIIR:Ag. filaments and punctate VIIIR:Ag granular staining were detectable in control cell preparations. The intensity of staining was unaffected by either In the presence of ticlopidine, fibronectin staining was suspending vehicle. markedly reduced and in some cases non-detectable and VIIIR:Ag granules were The opposite effects were seen with dexamethasone, increased (Figure lc,d). and VIIIR:Ag staining was namely fibronectin staining, was increased In addition, the formation of extracellular decreased (Figure la,b). fibrillar matrix was more rapid in these cells (data not shown). The results in Changes in Media-Associated Fibronectin and VIIIR:Ag. the supernatant media were parallel with the results observed in the cells. Thus, control levels of fibronectin were decreased 75% in the presence of 150 uM of ticlopidine and increased 56% in the presence of 0.1 uM of In contrast, VIIIR:Ag levels tended to dexamethasone, p < 0.001 (Table 1). The changes be increased with ticlopidine and decreased with dexamethasone. and may be related to the with ticlopidine, however, were not significant increased variation after drug treatment. TABLE 1 Effect
of 150 uM Ticlopidine and 0.1 uM Dexamethasone on Fibronectin (ug/ml) and VIIIR:Ag (U/d11 Release Mean 2 SEM, n=7
Control Fibronectin (ug/ml)
0.187 + 0.015
VIIIR :Ag (U/dl)
2.0 -+ 0.2
* p <
0.001
Dexametha sone
Ticlopidine 0.050
+ 0.011
2.4 2 0.5
*
0.293 2 c.019* 1.4 -+
9.1*
TICLOPIDINE EFFECT ON FSBRONECTIN
Suppl. IV, 1983
a
d
c
FIG. 1 Indirect combined immunofluorescence on human endothelial cells grown in the presence of 0.1 uM dexamethasone or 150 uM ticlopidine: a) Immunofluoroescence for fibronectin (FITC); b) Immunofluorescence for VIIIR:Ag (TRITC). Same field as (a) c) Immunofluorescence for fibronectin (FITC). d) Immunofluorescence for VIIIR:Ag (TRITC). Same field as (c)
72
TICLOPIDINE EFFECT ON FIBRONECTIM
Suppl. IV, 1083
CONCLUSIONS This study confirms our previous observations that ticlopidine influences on endothelial cell morphology and on fibronectin expression (31, as well as cell culture-produced subendothelium (7) can both be related to the decrease of fibronectin synthesis and/or release. If fibronectin is a mediator of platelet adhesion to subendothelium and of platelet/platelet interactions (at least in the earlier stages of attachment), a reduction of its availability could affect some of the platelet function as well as endothelial cell repair and may subsequently influence the bleeding time. These findings, in association with the well documented inhibitory action on platelet aggregability may better explain the effects of this drug in the haemostatic mechanisms. Thus its double-target mode of action may be important in preventing the occurrence of vascular thrombosis. Our study also confirms that dexamethasone has the opposite effect on endothelial cells. These experiments do not define the mechanism by which corticosteroids prevent or reverse endothelial alterations during ticlopidine treatment. Corticosteroids have been shown to stabilize cell membrane (8). They can also interact with RNA synthesis (9). The fact that fibronectin production is increased when VIIIR:Ag is decreased seems to suggest the presence of a relationship, i.e. an interdependence, between these two proteins in endothelial cell metabolism. These alterations in the synthesis of components which are present either in plasma or in subendothelium may be important and may explain in part the prolongation of bleeding associated with the use of ticlopidine and its modulation by corticosteroids. REFERENCES 1. THEBAULT, J., BLATRIX, C., BLANCHARD, J., and PANAK, E. A possible method to control prolongations of bleeding time under antiplatelet therapy with ticlopidine. Thromb. Haemostas. 48, 6-8, 1982. 2.
PIOVELLA, F., CIDDINGS, J.C., RICETTI, M.M., ALMASIO, P., and ASCARI, E. The effect of dexamethasone on formation of intra- and eXtraCellUlar fibronectin matrix by human endothelial cells in culture. bematOlOgiCa 67, 58-63, 1982.
3.
PIOVELLA, F., RICETTI, M.M., ALMASIO, P., SAMADEN, A., SEMINO, G., and ASCARI, E. The effect of ticlopidine on human endothelial cells in culture. Thromb. Res. submitted for publication.
4.
PIOVELLA, F., RICETTI, M.M., ALMASIO, P., CASTAGNOLA, C., PESENTI CAMPAGNONI, M., GALLOTTI, P., FEOLI, F.R., and ASCARI, E. in Characterization and synthesis of some factor VIII-related PrOPertieS cultured human endothelial cells. Haematologica 64, 714-725, 1979.
5.
PIOVELLA, F., GIDDINGS, J.C., RICETTI, M.M., and THOMAS, J.E. Factor VIII related antigen and fibronectin in cultured endothelial cells in the presence of dexamethasone and ticlopidine. Haemostasis (abstract) 12, 23, 1982.
6.
GIDDINGS, J.C., BROOKES, L.R., PIOVELLA, R., and 3LOOM, A.L. Immunohistological comparison of platelet factor 4 (PFQ), fibronectin (Fn) and factor VIII related antigen (VIIIR:Ag) in human platelet granules. Brit. H. Haemat. 52, 79-88, 1982.
Suppl. IV, 1983
TICLOPIDINEEFFECT ON FIBRONECTIN
73
7. PIOVELLA, F., PIOVELLA,L., ALMASIO, P., RICETTI,M.M., and ASCARI, E. The effect of ticlopidine on cultured endothelial CellS: Possible influence on extracellular matrix formation. Thromb. Haemostas. (abstract)46, 404, 1981. 8. BALLARD, P.L., and TOMKINS, G.M. Hormone induced modificationof the cell surface. Nature 224, 344-345,1969. 9. FAIN, J.N., KOVACEV, V.P., and SCOW, R.O. Effect of growth hormone and dexamethasone on lipolysis and metabolism in isolated fat cells of the rat. J. Biol. Chem. 240, 3522-3529,1965.