THROMBOSIS

RESEARCH

Volume 10, Pages 557-566.Pergamon Press,1977.PrintedinGt. Britain.

RADIOLABEL INCORPORATION ASSOCIATED WITH TISSUE FACTOR PRODUCTION IN CULTURED RUMAN FIBROBLASTS AND SMOOTH MUSCLE CELLS

F.A. Pitlick and M. Valentyn-Benz Departments of Internal Medicine and Molecular Biophysics and Biochemistry, Yale Medical School, 333 Cedar Street New Haven, CT. 06510 USA

21.12.1976; in revised form 3.2.1977. (Received Accepted by Editor H.L. Nossel)

ABSTRACT Incorporation of 14C leucine and 3N glucosamine into cellular components increases concomitant with production of tissue factor (thromboplastin) activity in human fibroblasts and smooth muscle cells after subculture. After an initial burst, coagulant activity and incorporation of leucine and glucosamine both decline (within 12 hours). In smooth muscle cells, a minor activity peak was associated with the early burst of synthetic activity. 48 hours after subculture of these cells, tissue factor had increased substantially with lesser but measurable increments of glucosamine and leucine incorporation. The radiolabel content and tissue factor activity of the material released from the cells by trypsin digestion, presumably representing the surface coat, rose and fell in a manner similar to the total cell culture. We conclude that changes of tissue factor levels in the cultured cell may be one aspect of more generalized synthetic events.

INTRODUCTION Tissue factor (thromboplastin) has been found in a variety of cultured human somatic cells (fibroblasts, smooth muscle, endothelial, WISH amnion, HeLa, (l-4).

The major portion of this activity is inaccessible to plasma

coagulation factors unless the cells have been disrupted by freezing or unless portions of the cell surface are digested with trypsin (1). In the latter instance, cell viability is maintained and less than.20% of several plasma membrane enzymes are released as most of the tissue factor is released. Thus, a major portion of the tissue factor activity of a cultured cell appears to be located in a protected state external to the plasma membrane and repre-

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sents one control point for hemostatic potential. The apparent coagulant activity of the cell is not only a function of the extent of disruption, but also depends upon the time elapsed since

sub-

culture; activity increases within several hours after transfer, then usually declines to a basal level within 24 to 36 hours, depending upon cell type (2,5).

This rise in potential activity is blocked by actinomycin and cyclo-

heximide (2,5), drugs which block synthesis of RNA and protein.

Such exper-

iments suggest that synthesis of some macromolecules are required for tissue factor expression and we shall report here that increased amounts of of radiolabelled leucine and glucosamine are found in cultured fibroblasts and smooth muscle cells and their surface coats after transfer into fresh medium under conditions in which tissue factor production is stimulated. Leucine and glucosamine were chosen as broad spectrum precursors of proteins, glycoproteins or proteoglycans which might be found in the cells or the surface coat.

We have compared the incorporation of radiolabelled

leucine and glucosamine to development of tissue factor activity in fibroblasts and smooth muscle cells at several intervals after transfer.

We have

also studied the partitioning of radiolabels and coagulant activity into the cell pellet and trypsin-released material for both cell types. MATERIALS Crude bacterial Collagenase (CLS, 125-200 U/mg) was obtained from Worthington Biochemical Corporation, Freehold, New Jersey and trypsin (2x crystallized), and N-2-hydroxyethylpiperaxine-N '-2-ethanesulfonic acid (HEPES) were from Sigma Chemical Company, St. Louis, Missouri.

D-glucosamine-6-3H (N)

hydrochloride (10.13 pCi/ mmole) and L-leucine- 14C(U) (308 mCi/mmole) were products of New England Nuclear, Boston, Ftissachusetts. Medium 199 was supplemented with penicillin (50U/ml), streptomycin (50 ng/ml), glutamine (2 mM) and fetal calf serum (heat inactivated at 60" for 30 minutes and adsorbed with CaP04 to remove coagulation factors VII, IX, X and thrombin (1); 10% fetal calf serum was used for fibroblasts, 15% for smooth muscle cells) which are all supplied by Gibco, Grant Island, New York.

Puck's Saline A (6) used

for washing and resuspending cells, and phosphate buffered saline (PBS) (7) were prepared in the laboratory from chemicals obtained from the usual sources and sterilized by filtration (0.45 urn) (Nalgene, Rochester, New York). Media and Saline A were buffered with HEPES (20 mM, pH 7.4). Plastic polystyrene cultureware was obtained from Corning Glass Works, Corning, New York (75 cm2 flasks) and Falcon Plastics, Oxnard California (60 mm diameter petri dishes). Glass bottles from Keystone, New Haven, Connecti-

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were used to maintain stock cultures of fibroblasts. METHODS Fibroblast cultures were initiated from collagenase digests (0.2% for

3 hours at 37") of human foreskin.

Smooth muscle cultures were obtained

from collagenase digests (0.2% in PBS for 30 minutes at 37") of traumatized umbilical veins (8,9).

Each cell type was subcultured by lifting with 0.2%

trypsin for 2 minutes (fibroblasts) or 30 minutes (smooth muscle cells), then diluted with medium 199 supplemented with fetal calf serum as described above and transferred to plastic flasks for labelling or petri dishes for experiments. Fibroblasts were studied in duplicate at transfers 7, 18 and 19; smooth muscle cells were studied in duplicate in their 6th subculture.

The

cells were prelabelled by growth for four days in appropriate medium containing 0.5 uCi/ml (0.05 pmol/ml) 3H glucosamine and 0.05 uCi/ml (0.2 pmol/ml) 14C leucine.

Since tissue factor activity increases several fold after

medium replenishment (see Results, 2,3), cells were subcultured into medium containing radiolabel and grown for four days to allow coagulant activity to return to basal level.

The cells were then lifted as above and transferred

to petri dishes containing fresh portions of the same medium and incubated at 37" in a moist atmosphere of 95% air, 5% CO2.

A portion of the cells

lifted for transfer was assayed for tissue factor activity and sampled for radiolabel content before and after centrifugation to separate cells from the surface coat.

At each interval, the cells were lifted from duplicate

washed monolayers by incubation with 0.002% trypsin in Saline A for one hour at 37".

A portion of the lifted cells was counted for cell number; another

was sampled for radioactivity and tissue factor; another was centrifuged (170 Xg, 5 min.), washed two times with Saline A, then resuspended in Saline A.

The cell-free supernatant will be referred to as the surface coat. 'IWO

100 ul portions of each fraction (lifted cells, supernatant, washed pellet and wash supematants)

were added to scintillant for counting.

Each major

fraction (lifted cells, washed pellet, supernatant) was also frozen and thawed three times before assay for tissue factor activity by previously described methods (1).

Each data point represents the average of two deter-

minations for coagulant activity and radioactivity.

Two monolayers were

sampled at each time point. The cells described here have been derived from human tissues generally used to culture fibroblasts (foreskin) and smooth muscle cells (traumatized umbilical vein) (8,9). ria.

The cell types can be distinguished by several crite-

Smooth muscle cells, which are slightly larger than the fibroblasts,

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attach poorly to glass but grow well on plastic although usually at a slower rate than fibroblasts. Fibroblastslift readily with trypsin digestion in comparison to smooth muscle cells. These cells have distinguishingcharacteristics by light microscopy as has already been noted by others (8,9,10). Finally, confluent cultures of fibroblastsgrow in whorl patterns,while the smooth muscle cells form hills and valleys. The kinetics of tissue factor productionare also different,as described previously (2) and reaffirmed here. RESULTS Fibroblasts: Lifted cells: Fibroblastswhich have been trypsin-liftedfor transfer into fresh medium (see methods) retain very little tissue factor activity. However, activity rises steadily after transfer,reaching a maximum at 10 hours, and clearly decliningby 12 hours (Fig lA). In the instance illustratedhere, the increase in activity was 30 fold. By 24 hours, the culture has apparently reached a lower steady state level. The amount of incorporatedradiolabel follows similar kinetics: leucine and glucosaminelevels increase about 5 fold over the amount in the cells at time of transfer,reaching the maximum 10 hours after transfer. Half the label is lost in the ensuing two hours, and the amount in cells has apparentlyreached a basal level. A gradual increase of radiolabel is found in the trypsin-releasedmaterial between 30 and 50 hours. During the course of this experimentand others with fibroblasts the cell number was essentiallyconstant until 12 hours; the number increased then and again by 50 hours. Cell Pellet: If cells in monolayers are incubatedover prolonged periods (30 minutes to one hour) with dilute trypsin, the cell is freed from its growth support and the apparent tissue factor activity is also increased. Furthermore, lifted cells have lost their ruthenium-red-staining surface coat, but retain viability (1). If the lifted cells are centrifuged,and the pellet and supernatantare consideredseparately,a slightly differentpicture emerges from that of the lifted cells (Fig 1B). After an initial decline, changes of radiolabei incorporationcoincide with coagulantactivity, reaching a peak at 10 hours. The rise is somewhatmore abrupt than in the whole culture and is approximatelytwo-fold for the radiolabelsand three to five fold for tissue factor. The absolute values are about one third that of the culture as a whole.

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Production of tissue factor activity, and incorporation of leucine and glucosamine by human fibroblasts after subculture. Human fibroblasts were cultured for four days in medium containing the indicated radiolabel, then subcultured into petri dishes in a fresh portion of the same medium. At the indicated intervals the cells were washed, lifted with dilute trypsin. The lifted cells (A), and the pellet (B) and supernatant (C) after low speed centrifugation were assayed for coagulant activity and radiolabel content. Cell-free Supernatant: An analysis of the material released by trypsin from these fibroblasts suggests that the burst If incorporation and coagulant activity found in the lifted cells is reflected in the cell surface coat released by trypsin (Fig 1C).

The data here do not differentiate between intracellular synthesis with

rapid transport to the surface coat or synthesis or assembly at the coat itself.

At the peak, coagulant activity in the coat is 15 times greater than

that found in the cells at the time of transfer; this activity accounts for 70% of the total in the culture.

After the cells had become attached to

their growth support (4 hours), the coagulant activity found in the trypsinreleased material increased five fold.

Badiolabel incorporation over this

period of time increases 2.5 to 3 fold, dropping abruptly at 12 hours. About 60% of the radiolabel is found in the trypsin-released material at the peak.

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FIG 2 Production of tissue factor activity and incorporationof leucine and glucosamineby human smooth muscle cells after subculture. Human smooth muscle cells were cultured and assayed in a manner similar to that described' in figure 1 for fibroblasts. Lifted cells (A) were also studied as the pellet (B) and supematant (C) after low speed centrifugation. Smooth Muscle Cells: Smooth muscle cells, like fibroblasts,exhibit changes of radiolabel incorporationwithin 11 hours after transfer (Fig 2A). At 5 hours, the glucosamine level had already increased 5 fold and was beginning to fall; leucine incorporationbehaved in a similar manner. Coagulant activity was three to four fold greater during this period of time before declining at 24 hours. If the stripped cells (Fig 2B) and trypsin-releasedmaterial (Fig 2C) are examined separately,it can be seen that these increaseshave occurred preferentiallyin the material released by trypsin. The amount found in the cell pellet is actually less than was associatedwith the cells at time of transfer implying transport of intracellularmaterial to the coat; the amount associatedwith the cell does gradually increase between five and eleven hours, but also declines by 24 hours, about 50% in each case. By 48 hours, there has been a large increase in the total amount of tissue factor activity

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in the whole cell, accompanied by a lesser increase of radiolabel incorporation.

This change in activity is observed both in the cell itself (four

fold) and the extracellular material (seven fold).

Changes in radiolabel

*incorporation are more pronounced in the cells (two fold) than in the trypsin-released material (less than 30%).

Examination of the radiolabel data

indicates that the ratio of leucine to glucosamine is increased at this time. At 72 hours, the tissue factor activity and the amount of radiolabel have both declined.

Furthermore, the cell number, which had remained constant

through 55 hours, had almost doubled but did not change thereafter. DISCUSSION In two strains of fibroblasts studied thus far, it is clear that generation of tissue factor activity coincides with production of protein and carbohydrate components of the cell surface coat.

The amount of radiolabel

is usually somewhat greater in the cell surface coat at each time point than in the stripped cell.

More than half the coagulant activity is found in the

coat following culture. The question arises as to disposition of these materials at the decline of incorporation and activity.

That query has not been pursued here, but

there are at least two possibilities:

incorporation into an extracellular

matrix not lifted by trypsin or shedding into the medium.

both types of

cells in culture produce collagen and proteoglycans which are found in the medium as well as the extracellular matrix (10-12).

The primary goal of the

experiments reported here, however, was to determine if there was coincidence of at least one metabolic event with development of tissue factor activity. In previous experiments with WISH amnion cells, a simultaneous incorporation of leucine with development of coagulant activity was not observed (5). The intervals between time points during the present experiment were shorter than those for the experiment with WISH cells in order to clearly establish a trend if it existed.

The incorporation observed in the experiments report-

ed here may reflect the fact that the cells themselves are larger.

Further-

more, the tissue factor specific activity in fibroblasts and smooth muscle cells is at least 10 fold greater than for WISH cells; this may be related to differences in incorporation observed here and for WISH cells. The absolute amount of radiolabel incorporated varies from one experiment to another, but the kinetics of incorporation and ratios are consistent for a given cell strain.

In comparing fibroblast lines cultured from two

different foreskins, the kinetics of incorporation are similar, but there are variations in the absolute amount of glucosamine incorporated and the

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amount relative to leucine. In the experimentpresented, the ratio of glucosamine to leucine was about half that found in another strain of fibroblasts. The increase in tissue factor activity of fibroblastsand smooth muscle cells appears to be associatedwith a general increase in cellular metabolism, reflected by an apparent increase of protein synthesis and incorporationof glucosamine. The rise in tissue factor is generally greater than the rise of radiolabel incorporation,indicatingthat tissue factor production is not a singular event, but may be a minor component of a general increased synthetic activity. The experimentswith smooth muscle cells are particularly illustrativein this regard, exhibitingtwo phases of synthesis: the early phase includesmarked shifts of glucosamineand leucine to the cell surface coat, with a slight increase in tissue factor activity; the later phase shows a gentle rise in general syntheticactivity, but a marked increase in tissue factor production as noted previously (2). The experimentscannot directly answer the question of whether tissue factor itself is synthesizedduring the observed increase in metabolic activity. The results do suggest that the disappearanceof activity may not be an unusual event in that the levels of glucosamineand leucine in the cell surface coat also rise and fall during cell culture. Subculture offers two stimuli to primary cells, namely the regeneration of the surface coat which has been removed by the trypsin digestion required to lift the cells, and the added stimulus of fresh medium. The techniques employed for these experimentsdo not separate these two components. It is possible that synthetic activity occurs in response to trypsin treatment and that tissue factor production is a result of medium changes. Tissue factor levels in WISH amnion cells in suspensionculture increase five to ten fold after transfer to fresh medium without prior trypsin treatfnent(5). Since this increase in activity in WISH amnion cells fails to occur in the presence of inhibitorsof protein synthesis,we assume that some change in metabolism also occurs with this particular type of subculturein which the surface coat is not removed. Surface changes have been described for a variety of synchronizedcells at the time of mitosis. Neutral sugars fall precipitouslyat mitosis and sialic acid shortly thereafterin KB cells (13). In HeLa cells, the H blood group antigen is expressed at mitosis (14). Chinese hamster ovary cells lose surface heparan sulfate just before mitosis (15). Thus, there are a number of molecules of types similar to that which might be found on the surface of primary cell types (glycoproteins,glycosaminoglycans)which are expressed

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at particular times or which may be present on the cell only at particular times, then shed into the medium (11,16,17). Thus, it need not be surprising to see a rise then an abrupt fall of materials and activities found in the cell, partfcularlythose associatedwith the surface coat. Furthermore,a variety of enzymatic activitiesfluctuatewith cell cycle in the BEK cell (18) The cells employed in the experimentsreported here were not deliberately synchronized. These data do not rule out the possibility that we are observing an event associatedwith certain portions of the cell cycle, but such has not been demonstrated. If tissue factor production is associatedwith the cell cycle, it would be an event occurring before cell division based on these and previous data (5,19). These observationssuggest that there is a burst of cellularmetabolic activity immediatelyafter trypsin treatmentand transfer to fresh medium and indicate that tissue factor productionmay be associatedwith some general changes in cellularmetabolism. ACKNOWLEDGEMENTS We thank Ma. Barbara Dreyer for providing the cells used in this erperiment. Supportedby NELBI BiomaterialsContract NOl-EV-42968.

REFERENCES

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MAYNARD, J.R., HECKMAN, C.A., PITLICK, F.A., AND NKKERSON, P. Aasociaion of tissue factor activity with the surface of cultured cells. J. Clin. Invest. 55, 814-824, 1975.

2. MAYNARD, J.R., DREYER, B.E., PITLICK, F.A., AND NEMERSON,Y. Tissue factor activity of endothelialand other cultured cells. Blood 66, 1046, 1975. 3. GREBN, D., RYAN, C., MANALDRUCCOLO,N., AND NADLER, H.L. ;Eict$rization of the coagulant activity of cultured husmn fibroblasts. -9 47-51, 1971. 4.

ZACEARSKI,L.R., HOYER, L.W., MCINTYRE, O.R. Immunologicidentification of tissue factor (thromboplastin)synthesizedby cultured fibroblasts. Blood 41, 671-678, 1973.

5. MArivARD,J.R., FINTEL, D.J., PITLICK, F.A., AND EEMERSON, Y, Tissue factor in cultured cells. Metabolic control. Lab. Invest. 35, 542-549, 1976. 6. PUCK, T.T., CIECIURA, S.J. AND FISHER, H.W. Clonal growth in vitro of humsn cells with fibroblasticmorphology. 3. Exp. Med. 106, 145-157, 1957.

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7. DULBECCO, R. AND VOGT, M. Plaque formation and isolationof pure lines with poliomyelitisviruses. J. Exp. Med. 99, 167-182, 1954. 8. JAPPE, E.A., NACBMAN, R.L., BECKER, C.G., AND MINICK C.R. Culture of human endothelialcells derived from umbilical veins. Identification by morphologicand immunologiccriteria. J. Clin. Invest. 52, 27472756, 1973. 9.

10.

GIMBRONE, M.A., JR., AND COTRAN, R.S. Buman vascular smooth muscle in culture. Growth and ultrastructure. Lab. Invest. 33, 16-27, 1975. LAYMAN, D.L., AND TITUS, J.L. Synthesisof type I collagen by human swath muscle cells -in vitro. Lab. Invest.33, 103-107, 1975.

11. WIGBT, T.N. AND ROSS, R. Proteoglycansin primate arteries. II. synthesis and secretion of glycosaminoglycansby arterial smooth muscle cells in culture. J. Cell Biol. 67, 675-686, 1975. 12. LAYMAN, D.L., McGOODWIN, E.G., AND MARTIN, G.R. The nature of the collagen synthesizedby cultured human fibroblasts.Proc. Nat. Acad. Sci. 68, 454-458, 1971. 13. GLICK, M.C., GERNER, E.W., AND WARREN, L. Changes in the carbohydrate content of the KB cell during the growth cycle. J. Cell. Physiol. 77, l-6, 1971. 14. KUBNS, L. AND BRAMSON, S. Variable behavior of blood group H on HeLs populations synchronizedwith thymidine. Nature 2, 938-939, 1968. 15. KRARMER, P.M. AND TOBERY, R.A. Cell-cycledependent desquamationof heparan sulfate from the cell surface. J. Cell Biol. 55, 713-717, 1972. 16. KRAEMER, P.M. Heparan sulfates of cultured cells. I. Membrane associated and cell-sap species in Chinese hamster cells. Biochemistry lo, 1437-1445,1971. 17. WARREN, L. AND GLICK, M.C. Membranes of animal cells. II. The metabolism and turnover of the surface membrane. J. Cell Biol. 21, 729-746, 1968. 18. GLICK, M.C. AND BUCK, C.A. Glycoproteinsfrom the surface of metaphase cells. Biochemistry12, 85-90, 1973. 19. MAYNARD, J.R., FINTEL, D.J., PITLICK, F.A. AND NEMERSON,Y. Tissue factor in cultured cells. Pharmacologiccontrol. Lab. Invest. 35, 550-557, 1976.