- Email: [email protected]
Decreased inducibility of tissue factor activity on human umbilical vein endothelial cells cultured with endothelial cell growth factor and heparin
THROMBOSISRESEARCH50; 339-344,1988 0049-3848/88 $3.00 t .OO Printed in the USA. Copyright (c) 1988 Pergamon Press plc.
All rights reserved.
BRIEF COMMUNICATION DECREASED INDUCIBILITY OF TISSUE FACTOR ACTIVITY ON HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS CULTURED WITH ENDOTHELIAL CELL GROWTH FACTOR AND HEPARIN
Fanny E. Almus, L. Vijaya Mohan Rao, and Samuel I. Rapaport* Departments of Medicine and Pathology, University of California, San Diego Medical Center (HBllK), San Diego, California 92103, U.S.A. (Received 12.11.1987; Accepted in original form 12.2.1988 by Editor J.H. Griffin)
INTRODUCTION Tissue factor (TF) is an apoprotein/phospholipid complex found constitutively on the surface membrane of some cells, such as glial cells or trophoblasts, and expressed on the surface membrane of other cells after stimulation. Unperturbed cultured vascular endothelial cells do not possess surface membrane TF activity but develop such activity to a variable degree after exposure to perturbing stimuli such as thrombin, phorbol, endotoxin or interleukin 1. Conditions of culture are known to affect many responses of endothelial cells (1). We report herein that growing primary cultures of human umbilical vein endothelial cells (HUVEC) with endothelial cell growth factor (ECGF) and heparin impairs the ability of monolayers of HUVEC to express surface membrane TF after exposure to thrombin or phorbol myristate acetate.
Key words:
tissue factor, human endothelium, growth factors, heparin
*TO whom correspondence should be addressed. 339
340
TISSUE FACTOR AND ECGF-HEPARIN
Vol. 50, No. 2
MATERIALS AM) METHODS Cell Culture: Human umbilical vein endothelial cells were cultured according to the method of Jaffe et al. (2) with slight modifications. Cells were suspended in RPM1 1640 (Whittaker Bioproducts, Walkersville, MD) supplemented with L-glutamine 200mM, 1 mL/lOOmL media, the usual antibiotics, and 20% v/v fetal calf serum (Irvine Scientific, Santa Ana, CA) an adjusted to pH 7.4. Cells were plated at a density of 2 x 10 !! cells/ml/dish in 35 mm Falcon 3001 (Becton Dickinson, CA) Petri dishes coated with fibronectin (Boehringer Mannheim, Indianapolis, IND), 40 ug/dish. After incubation for 24 hr at 37OC in humidified 5% CO2 atmosphere, the media was discarded, the cells were washed in phosphate buffered saline (PBS), pH 7.4 and new RPM1 media was added. The new media differed from the initial media in containing 14% v/v human serum (inactivated at 56'C for 30 min) instead of fetal calf serum. It was either not supplemented further or supplemented in one of the following ways: (1) with heparin (Organon, W. Orange, NJ), 90 ug/mL of media; (2) with ECGF (Meloy, Springfield, VA), 20 ug/mL of media; or (3) with these concentrations of both heparin and ECGF. Incubation was continued as described above and media were replaced every 2 days. Cells grown in media containing both ECGF and heparin usually reached confluence in 5 days; cells grown in the absence of both ECGF and heparin usually reached confluence in 7 days. Cells were characterized as endothelial cells by morphological criteria (uniform polygonal shape) and, on by immunofluorescent demonstration of von two occasions, Willebrand antigen. Purified Clotting Proteins. Human factors VII, IX, and X were purified as described elsewhere (3, 4). Tritiated sialyl factors IX and X were prepared by the general technique of Van Lenten and Ashwell with slight modifications as described ea ier from this laboraiory (5). Spec'fic radioactivity wa#: for ? H-factor IX, 3.2 x 10 CPM/mg; for 5 H-factor X, 2.0 x 10 CPM/mg. Factor VIIa: Purified factor VII at a concentration of loo-150 ug/mL was incubated with factor Xa at a ratio of 5O:l (wt/wt) in the presence of cephalin, 0.52 mM, and calcium, 5 mM, for 20 minutes at 37'C. The reaction was terminated by adding EDTA to a final concentration of 10 mM. Factor VIIa was stored at -8OOC in small aliquots. Reconstituted purified human TF and soat anti-TF u were prepared as described earlier (6). TF Induction: Only primary cultures were used in these studies. Confluent cell monolayers, (approximately 6 x lo5 viable cells per dish) were washed three times with PBS. For induction of TF with thrombin, human alpha thrombin (gift of Dr. J. W. Fenton, NY State Dept. of Health, Albany NY) was added in a final concentration of 2.5 U/mLto RPM1 containing 0.5 % bovine serum albumin, (Fraction V, ICN Immunobiologicals, Lisle, IL). For induction of TF with phorbol, Phorbol 12-myristate 13-acetate (Sigma, St. Louis MO) was added in a final concentration of 100 ng/mL to RPM1 containing 20% v/v human serum that had been heat inactivated at 56'C for 1 hr. One mL of either thrombincontaining or phorbol-containing medium was added to dishes
Vol. 50, No. 2
TISSUE FACTOR AND ECGF-HEPARIN
341
containing confluent monolayers that had been grown in media differing as described above in its added supplements. After incubation for 6 hrs at 37O C, the media were then decanted and the monolayers were washed 3 times with a buffer containing 0.01 M Hepes, 0.14 M NaCl, 0.004 M KCl, and 0.011 M glucose, pH 7.45 (buffer A). Then, 1 ml of buffer A containing 1 mg/mL bovine serum albumin and 5mM calcium (buffer B) was added and the TF activity of the monolayers was measured. Measurement of TF Activitv. Surface membrane TF activity was measured as the abilit of a monolayer to support activation peptide release from Y H-factor X or 3H-factor IX when one of these substrates was added with factor VIIa to the overlying buffer B. The concentrations of purified clott'ng factors in eaction mixtures were: factor VIIa, 0.5 ug/mL, 3 H-factor X or 5H-factor IX, 5 ug/mL. The activation peptide release assays were performed as described earlier (5). Multiple aliquots were sampled over 2 hours and the initial linear portion of progress curves of activation were used to calculate activation rates. One hundred per cent activation of factor IX or factor X, as determined by radioactivity profiles on reduced and nonreduced SDS gels, yielded approximately 35 to 37% trichloroacetic acid soluble counts. Two controls were performed with each experiment: a negative control with a monolayer not treated with thrombin or phorbol; and a positive control in which reconstituted purified human brain TF (0.5 ng/mL) was used instead of an HUVEC monolayer. This concentration of purified TF yielded rates of factor X and factor IX activation approximating the average rates obtained when treated monolayers of HUVEC not grown with ECGF and heparin were used as the source of TF. Procoagulant activity was verified as TF in two ways: first, showing that activation of the substrates required the by presence of F.VIIa; and second, by showing that adding a specific goat anti-human tissue factor IgG (6) abolished activation of the substrates. RESULTS m
DISCUSSION
Batches of HUVEC grown from different pools of cord cells differed in their ability to develop surface TF activity after treatment with thrombin or phorbol. Factor X activation at 1 hour varied from 11% to 32% with monolayers grown in the absence of ECGF and heparin from 12 different pools of cord cells. However, duplicate dishes of cells prepared in the same way from the same pool of cord cells gave closely similar rates of factor X Within primary cultures from the same cord cell activation. pool, we consistently found that monolayers grown in the absence of ECGF and heparin possessed greater surface TF activity after treatment with thrombin or phorbol than did monolayers grown with ECGF and heparin. The data from experiments, usually performed in duplicate, from primary cultures prepared from the 12 different pools of cord cells are summarized in Table I. The statistical significance of these data, as evaluated by a paired t test, is as follows: for phorbol treated-HUVEC and F. X as and F. X as substrate, p <0.05: for thrombin-treated-HUVEC
342
Vol. 50, No. 2
TISSUE FACTOR AND ECGF:HEPARIN
substrate, p < 0.001; for thrombin-treated HUVEC and F. IX as substrate, 0.05 < p
Phorbol Thrombin
Assay Substrate
+
Reaction rate nM/h
+ +
F.X 2.6 f 1.4, F.X 13.6 f 6.3, + + F.X 2.6 f 1.4, + F.X 16.7 2 6.4, + + F.IX 0.7 _+ 0.4, + F.IX 4.1 _+ 2.3, Group mean !:SD, n = number of primary cultures
n=4 n= 4 n=8 n=8 n= 4 n= 4
These data provide substantial evidence that HUVEC grown in RPM1 1640 in the presence of ECGF-heparin generate less measurable surface TF activity after thrombin or phorbol induction than do HUVEC grown in their absence. Similar results (data not shown) were obtained in several experiments in which the cells were cultured in Medium 199 (Cell Culture Facility, UC San Diego). Further experiments were carried out to investigate whether the impaired inducibility of TF expression in cells grown with both ECGF and heparin would also be observed in cells grown with ECGF alone or heparin alone. Rates of factor X activation calculated from duplicate dishes from monolayers cultured from four different cord pools and treated with thrombin to induce TF expression are summarized in Table II. F.X Activation
Table II (nM/hr) Observed with HUVEC Cultured Different Additives
Primary Culture ECGF/Heparin A B* C D
183:: 1.0 1.7
Additive ECGF Heparin 4.5 25.2 3;:: 4.2 6.2 9.4 7.7
with
Nothing 8.0 34.3 1:::
*The unusually high TF activity observed with HUVEC monolayers cultured from cord cell pool B illustrates strikingly the inherent and unpredictable variability of TF expression that we observed with primary cultures grown from different pools of cord cells.
Vol. 50, No. 2
TISSUE FACTOR AND ECGF-HEPARIN
343
One notes that growing cells in the presence of heparin alone had little effect upon the ability of the cells to express TF activity. Growing the cells with ECGF alone partially diminished inducibility of surface membrane TF activity; however, growing the cells with both ECGF and heparin was required for maximum suppression of TF activity after exposure of HUVEC to thrombin. These data suggest that ECGF is the material responsible for altering the ability of cultured endothelial cells to express surface TF activity after exposure to phorbol or thrombin, since heparin is known to interact structurally with ECGF to induce a conformational change in the polypeptide that stabilizes or increases its biological activity (7). In supplemental experiments, HUVEC monolayers grown without ECGF and heparin were treated with thrombin to induce expression of TF. Then ECGF 20 ug/mL, was add d to the overlying buffer B and, after 20 min, factor VIIa and 5H-F.X were added to initiate factor X activation. The added ECGF had no effect upon the rate of activation of F.X. In other experiments, to be reported elsewhere in detail, adding 1 U/mL of heparin to reaction mixtures overlying treated HUVEC monolayers was found to increase, rath r than decrease , rates of activation of both 3Hfactor IX and 3 H-factor X. Thus, it appears that neither ECGF nor heparin impedes the activity of TF once it is present on the surface membrane of HUVEC. The data support the hypothesis that HUVEC grown with ECGF and heparin have reduced surface TF activity after treatment withthrombin or phorbolbecause they synthesize less TF apoprotein. However, direct evidence for this hypothesis is still lacking. Since treatment with either thrombin or phorbol of HUVEC cultured with ECGF and heparin yielded diminished surface membrane TF activity, we suspect that decreased TF activity would also be found with other perturbing stimuli, e.g., interleukin 1 or endotoxin. Why culturing HUVEC with ECGF and heparin should lead to decreased responsiveness is unknown. Evidence is growing that noxious stimuli may induce an endothelial cell response to injury in which the surface membrane acquires TF activity, thrombomodulin surface membrane sites are down regulated (81, and the surface membrane acquires properties that cause adhesion of leukocytes (9). Whether culturing HUVEC with ECGF and heparin affects responses other than the generation of surface membrane TF activity remains to be determined. In any event, it would appear that although ECGF-heparin accelerates proliferation of HUVEC in culture, its use has a counterbalancing disadvantage when HUVEC are grown for studies of the TF pathway of coagulation on endothelium. ACKNOWLEDGEMENT We thank Dr. J. W. Fenton for the gift of alpha thrombin and Ms. Angela Wakeham for preparation of the manuscript. This research was supported by NIH grant HL27234. Dr. Fanny E. Almus was supported by NIH Hematology Training Grant HL07107-12.
344
TISSUE FACTOR AND ECGF-HEPARIN
Vol. 50, No. 2
REFERENCES 1.
BALCONI, G., DEJANA, E. Cultivation of endothelial cells: limitations and perspectives. Med. Biol. 64: 231-245, 1986.
2.
JAFFE, E.A., NACHMAN, R.L., BECKER, C.G. and MINICK, C.R. Culture of human endothelialcells derived from umbilical veins. J. Clin. Invest. 52: 2745-2756, 1973.
3.
RAO, L.V.M., BAJAJ, S.P. Purification of human factor VII utilizing 0-(Diethylaminoethyl)-Sephadex and Sulfopropyl Sephadex Chromatography. Anal. Biochem. 136: 357-361, 1984.
4.
BAJAJ, S.P., RAPAPORT, S.I., PRODANOS, C. A simplified procedure for purification of human prothrombin, factor IX and factor X. Prep. Biochem. 11: 397-412, 1981.
5.
SANDERS, N.L., BAJAJ, S.P., ZIVELIN, A., RAPAPORT, S.I. Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component. Blood, 66: 204-212, 1985.
6.
RAO, L.V.M. and RAPAPORT, S.I. Affinity purification of human brain tissue factor utilizing factor VII bound to immobilized anti-factor VII. Anal. Biochem.165: 365-370, 1987.
7.
SCHREIBER, A.B., KENNEY, J., KOWALSKI, W.J., FRIESEL, R., MEHLMAN, T., MACIAG, T. Interaction of endothelial cell growth factor with heparin: characterization by receptor and antibody recognition. Proc. Natl. Acad. Sci. m. 82: 6138-6142, 1985.
8.
MOORE, K.L., ANDREOLI, S.P., ESMON, N.L., ESMON, C.T., BANG, N.U. Endotoxin enhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro. J. Clin. Invest. 79: 124-130, 1987.
9.
BEVILACQUA, M.P., POBER, J.S., WHEELER, M.E., COTRAN, R.S., and GIMBRONE, M.A., Jr. Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines. J. Clin. Invest. 76: 2003-2011, .1985.
All rights reserved.
BRIEF COMMUNICATION DECREASED INDUCIBILITY OF TISSUE FACTOR ACTIVITY ON HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS CULTURED WITH ENDOTHELIAL CELL GROWTH FACTOR AND HEPARIN
Fanny E. Almus, L. Vijaya Mohan Rao, and Samuel I. Rapaport* Departments of Medicine and Pathology, University of California, San Diego Medical Center (HBllK), San Diego, California 92103, U.S.A. (Received 12.11.1987; Accepted in original form 12.2.1988 by Editor J.H. Griffin)
INTRODUCTION Tissue factor (TF) is an apoprotein/phospholipid complex found constitutively on the surface membrane of some cells, such as glial cells or trophoblasts, and expressed on the surface membrane of other cells after stimulation. Unperturbed cultured vascular endothelial cells do not possess surface membrane TF activity but develop such activity to a variable degree after exposure to perturbing stimuli such as thrombin, phorbol, endotoxin or interleukin 1. Conditions of culture are known to affect many responses of endothelial cells (1). We report herein that growing primary cultures of human umbilical vein endothelial cells (HUVEC) with endothelial cell growth factor (ECGF) and heparin impairs the ability of monolayers of HUVEC to express surface membrane TF after exposure to thrombin or phorbol myristate acetate.
Key words:
tissue factor, human endothelium, growth factors, heparin
*TO whom correspondence should be addressed. 339
340
TISSUE FACTOR AND ECGF-HEPARIN
Vol. 50, No. 2
MATERIALS AM) METHODS Cell Culture: Human umbilical vein endothelial cells were cultured according to the method of Jaffe et al. (2) with slight modifications. Cells were suspended in RPM1 1640 (Whittaker Bioproducts, Walkersville, MD) supplemented with L-glutamine 200mM, 1 mL/lOOmL media, the usual antibiotics, and 20% v/v fetal calf serum (Irvine Scientific, Santa Ana, CA) an adjusted to pH 7.4. Cells were plated at a density of 2 x 10 !! cells/ml/dish in 35 mm Falcon 3001 (Becton Dickinson, CA) Petri dishes coated with fibronectin (Boehringer Mannheim, Indianapolis, IND), 40 ug/dish. After incubation for 24 hr at 37OC in humidified 5% CO2 atmosphere, the media was discarded, the cells were washed in phosphate buffered saline (PBS), pH 7.4 and new RPM1 media was added. The new media differed from the initial media in containing 14% v/v human serum (inactivated at 56'C for 30 min) instead of fetal calf serum. It was either not supplemented further or supplemented in one of the following ways: (1) with heparin (Organon, W. Orange, NJ), 90 ug/mL of media; (2) with ECGF (Meloy, Springfield, VA), 20 ug/mL of media; or (3) with these concentrations of both heparin and ECGF. Incubation was continued as described above and media were replaced every 2 days. Cells grown in media containing both ECGF and heparin usually reached confluence in 5 days; cells grown in the absence of both ECGF and heparin usually reached confluence in 7 days. Cells were characterized as endothelial cells by morphological criteria (uniform polygonal shape) and, on by immunofluorescent demonstration of von two occasions, Willebrand antigen. Purified Clotting Proteins. Human factors VII, IX, and X were purified as described elsewhere (3, 4). Tritiated sialyl factors IX and X were prepared by the general technique of Van Lenten and Ashwell with slight modifications as described ea ier from this laboraiory (5). Spec'fic radioactivity wa#: for ? H-factor IX, 3.2 x 10 CPM/mg; for 5 H-factor X, 2.0 x 10 CPM/mg. Factor VIIa: Purified factor VII at a concentration of loo-150 ug/mL was incubated with factor Xa at a ratio of 5O:l (wt/wt) in the presence of cephalin, 0.52 mM, and calcium, 5 mM, for 20 minutes at 37'C. The reaction was terminated by adding EDTA to a final concentration of 10 mM. Factor VIIa was stored at -8OOC in small aliquots. Reconstituted purified human TF and soat anti-TF u were prepared as described earlier (6). TF Induction: Only primary cultures were used in these studies. Confluent cell monolayers, (approximately 6 x lo5 viable cells per dish) were washed three times with PBS. For induction of TF with thrombin, human alpha thrombin (gift of Dr. J. W. Fenton, NY State Dept. of Health, Albany NY) was added in a final concentration of 2.5 U/mLto RPM1 containing 0.5 % bovine serum albumin, (Fraction V, ICN Immunobiologicals, Lisle, IL). For induction of TF with phorbol, Phorbol 12-myristate 13-acetate (Sigma, St. Louis MO) was added in a final concentration of 100 ng/mL to RPM1 containing 20% v/v human serum that had been heat inactivated at 56'C for 1 hr. One mL of either thrombincontaining or phorbol-containing medium was added to dishes
Vol. 50, No. 2
TISSUE FACTOR AND ECGF-HEPARIN
341
containing confluent monolayers that had been grown in media differing as described above in its added supplements. After incubation for 6 hrs at 37O C, the media were then decanted and the monolayers were washed 3 times with a buffer containing 0.01 M Hepes, 0.14 M NaCl, 0.004 M KCl, and 0.011 M glucose, pH 7.45 (buffer A). Then, 1 ml of buffer A containing 1 mg/mL bovine serum albumin and 5mM calcium (buffer B) was added and the TF activity of the monolayers was measured. Measurement of TF Activitv. Surface membrane TF activity was measured as the abilit of a monolayer to support activation peptide release from Y H-factor X or 3H-factor IX when one of these substrates was added with factor VIIa to the overlying buffer B. The concentrations of purified clott'ng factors in eaction mixtures were: factor VIIa, 0.5 ug/mL, 3 H-factor X or 5H-factor IX, 5 ug/mL. The activation peptide release assays were performed as described earlier (5). Multiple aliquots were sampled over 2 hours and the initial linear portion of progress curves of activation were used to calculate activation rates. One hundred per cent activation of factor IX or factor X, as determined by radioactivity profiles on reduced and nonreduced SDS gels, yielded approximately 35 to 37% trichloroacetic acid soluble counts. Two controls were performed with each experiment: a negative control with a monolayer not treated with thrombin or phorbol; and a positive control in which reconstituted purified human brain TF (0.5 ng/mL) was used instead of an HUVEC monolayer. This concentration of purified TF yielded rates of factor X and factor IX activation approximating the average rates obtained when treated monolayers of HUVEC not grown with ECGF and heparin were used as the source of TF. Procoagulant activity was verified as TF in two ways: first, showing that activation of the substrates required the by presence of F.VIIa; and second, by showing that adding a specific goat anti-human tissue factor IgG (6) abolished activation of the substrates. RESULTS m
DISCUSSION
Batches of HUVEC grown from different pools of cord cells differed in their ability to develop surface TF activity after treatment with thrombin or phorbol. Factor X activation at 1 hour varied from 11% to 32% with monolayers grown in the absence of ECGF and heparin from 12 different pools of cord cells. However, duplicate dishes of cells prepared in the same way from the same pool of cord cells gave closely similar rates of factor X Within primary cultures from the same cord cell activation. pool, we consistently found that monolayers grown in the absence of ECGF and heparin possessed greater surface TF activity after treatment with thrombin or phorbol than did monolayers grown with ECGF and heparin. The data from experiments, usually performed in duplicate, from primary cultures prepared from the 12 different pools of cord cells are summarized in Table I. The statistical significance of these data, as evaluated by a paired t test, is as follows: for phorbol treated-HUVEC and F. X as and F. X as substrate, p <0.05: for thrombin-treated-HUVEC
342
Vol. 50, No. 2
TISSUE FACTOR AND ECGF:HEPARIN
substrate, p < 0.001; for thrombin-treated HUVEC and F. IX as substrate, 0.05 < p
Phorbol Thrombin
Assay Substrate
+
Reaction rate nM/h
+ +
F.X 2.6 f 1.4, F.X 13.6 f 6.3, + + F.X 2.6 f 1.4, + F.X 16.7 2 6.4, + + F.IX 0.7 _+ 0.4, + F.IX 4.1 _+ 2.3, Group mean !:SD, n = number of primary cultures
n=4 n= 4 n=8 n=8 n= 4 n= 4
These data provide substantial evidence that HUVEC grown in RPM1 1640 in the presence of ECGF-heparin generate less measurable surface TF activity after thrombin or phorbol induction than do HUVEC grown in their absence. Similar results (data not shown) were obtained in several experiments in which the cells were cultured in Medium 199 (Cell Culture Facility, UC San Diego). Further experiments were carried out to investigate whether the impaired inducibility of TF expression in cells grown with both ECGF and heparin would also be observed in cells grown with ECGF alone or heparin alone. Rates of factor X activation calculated from duplicate dishes from monolayers cultured from four different cord pools and treated with thrombin to induce TF expression are summarized in Table II. F.X Activation
Table II (nM/hr) Observed with HUVEC Cultured Different Additives
Primary Culture ECGF/Heparin A B* C D
183:: 1.0 1.7
Additive ECGF Heparin 4.5 25.2 3;:: 4.2 6.2 9.4 7.7
with
Nothing 8.0 34.3 1:::
*The unusually high TF activity observed with HUVEC monolayers cultured from cord cell pool B illustrates strikingly the inherent and unpredictable variability of TF expression that we observed with primary cultures grown from different pools of cord cells.
Vol. 50, No. 2
TISSUE FACTOR AND ECGF-HEPARIN
343
One notes that growing cells in the presence of heparin alone had little effect upon the ability of the cells to express TF activity. Growing the cells with ECGF alone partially diminished inducibility of surface membrane TF activity; however, growing the cells with both ECGF and heparin was required for maximum suppression of TF activity after exposure of HUVEC to thrombin. These data suggest that ECGF is the material responsible for altering the ability of cultured endothelial cells to express surface TF activity after exposure to phorbol or thrombin, since heparin is known to interact structurally with ECGF to induce a conformational change in the polypeptide that stabilizes or increases its biological activity (7). In supplemental experiments, HUVEC monolayers grown without ECGF and heparin were treated with thrombin to induce expression of TF. Then ECGF 20 ug/mL, was add d to the overlying buffer B and, after 20 min, factor VIIa and 5H-F.X were added to initiate factor X activation. The added ECGF had no effect upon the rate of activation of F.X. In other experiments, to be reported elsewhere in detail, adding 1 U/mL of heparin to reaction mixtures overlying treated HUVEC monolayers was found to increase, rath r than decrease , rates of activation of both 3Hfactor IX and 3 H-factor X. Thus, it appears that neither ECGF nor heparin impedes the activity of TF once it is present on the surface membrane of HUVEC. The data support the hypothesis that HUVEC grown with ECGF and heparin have reduced surface TF activity after treatment withthrombin or phorbolbecause they synthesize less TF apoprotein. However, direct evidence for this hypothesis is still lacking. Since treatment with either thrombin or phorbol of HUVEC cultured with ECGF and heparin yielded diminished surface membrane TF activity, we suspect that decreased TF activity would also be found with other perturbing stimuli, e.g., interleukin 1 or endotoxin. Why culturing HUVEC with ECGF and heparin should lead to decreased responsiveness is unknown. Evidence is growing that noxious stimuli may induce an endothelial cell response to injury in which the surface membrane acquires TF activity, thrombomodulin surface membrane sites are down regulated (81, and the surface membrane acquires properties that cause adhesion of leukocytes (9). Whether culturing HUVEC with ECGF and heparin affects responses other than the generation of surface membrane TF activity remains to be determined. In any event, it would appear that although ECGF-heparin accelerates proliferation of HUVEC in culture, its use has a counterbalancing disadvantage when HUVEC are grown for studies of the TF pathway of coagulation on endothelium. ACKNOWLEDGEMENT We thank Dr. J. W. Fenton for the gift of alpha thrombin and Ms. Angela Wakeham for preparation of the manuscript. This research was supported by NIH grant HL27234. Dr. Fanny E. Almus was supported by NIH Hematology Training Grant HL07107-12.
344
TISSUE FACTOR AND ECGF-HEPARIN
Vol. 50, No. 2
REFERENCES 1.
BALCONI, G., DEJANA, E. Cultivation of endothelial cells: limitations and perspectives. Med. Biol. 64: 231-245, 1986.
2.
JAFFE, E.A., NACHMAN, R.L., BECKER, C.G. and MINICK, C.R. Culture of human endothelialcells derived from umbilical veins. J. Clin. Invest. 52: 2745-2756, 1973.
3.
RAO, L.V.M., BAJAJ, S.P. Purification of human factor VII utilizing 0-(Diethylaminoethyl)-Sephadex and Sulfopropyl Sephadex Chromatography. Anal. Biochem. 136: 357-361, 1984.
4.
BAJAJ, S.P., RAPAPORT, S.I., PRODANOS, C. A simplified procedure for purification of human prothrombin, factor IX and factor X. Prep. Biochem. 11: 397-412, 1981.
5.
SANDERS, N.L., BAJAJ, S.P., ZIVELIN, A., RAPAPORT, S.I. Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component. Blood, 66: 204-212, 1985.
6.
RAO, L.V.M. and RAPAPORT, S.I. Affinity purification of human brain tissue factor utilizing factor VII bound to immobilized anti-factor VII. Anal. Biochem.165: 365-370, 1987.
7.
SCHREIBER, A.B., KENNEY, J., KOWALSKI, W.J., FRIESEL, R., MEHLMAN, T., MACIAG, T. Interaction of endothelial cell growth factor with heparin: characterization by receptor and antibody recognition. Proc. Natl. Acad. Sci. m. 82: 6138-6142, 1985.
8.
MOORE, K.L., ANDREOLI, S.P., ESMON, N.L., ESMON, C.T., BANG, N.U. Endotoxin enhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro. J. Clin. Invest. 79: 124-130, 1987.
9.
BEVILACQUA, M.P., POBER, J.S., WHEELER, M.E., COTRAN, R.S., and GIMBRONE, M.A., Jr. Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines. J. Clin. Invest. 76: 2003-2011, .1985.