Alterations in the apparent tissue factor (thromboplastin) expression in HeLa cells by a cellular factor Xa inhibitor.

HeLa cells have undetectable tissue factor (thromboplastin) activity when measured by a one-stage coagulation assay. In contrast, these cells accelerated the factor VII-catalyzed cleavage of factor X. The two assays gave similar results after either heating the samples to 100 degrees C for 2 min or exposure to thrombin. Neither of these treatments altered the tissue factor activity of human foreskin fibroblasts, a cell type with high tissue factor activity. HeLa cells contain an inhibitor(s) directed against factor Xa but not thrombin. The inhibitor(s) was inactivated by exposure to thrombin or by heat treatment. Inhibition of factor Xa-catalyzed cleavage of a synthetic peptide was blocked by ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) so the inhibition was apparently dependent on divalent cations. Inhibition was not accelerated by heparin. The inhibitor(s) was not protein C or other serine proteases since it was not inactivated by diisopropylfluorophosphate. The factor Xa inhibitor(s) has been isolated from HeLa cells with an approximate 500-fold increase in specific activity. After SDS-polyacrylamide gel electrophoresis factor Xa-inhibitory activity was recovered from a region corresponding to the major Coomassie-staining band at 43 kDa and in lesser amounts from regions corresponding to 26 and 17 kDa. Cellular inhibitors of coagulation may partially explain the low apparent tissue factor observed in some in vitro cells and may serve a regulatory role in limiting the expression of tissue factor.

Induction of plasminogen activator by 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore. Suppression by inhibitors of fatty acid lipoxygenase.

HeLa cells incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and rat basophilic leukemia (RBL-1) cells incubated with calcium ionophore, showed increased levels of the protease plasminogen activator. These treatments have previously been shown to stimulate the cellular metabolism of arachidonic acid. The induction of plasminogen activator in both cell types was inhibited in a dose-dependent manner by 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid, two compounds known to inhibit arachidonate metabolism via lipoxygenases. In contrast, indomethacin, which selectively inhibits arachidonate metabolism via cyclooxygenase, was inactive. The levels of four enzyme markers in HeLa cells were unchanged by treatment with TPA plus the lipoxygenase inhibitors, indicating that the inhibitors did not exert their effects on plasminogen activator via general cell toxicity. HeLa cells preincubated with [3H]arachidonate and subsequently challenged with TPA produced small amounts of material with the chromatographic mobilities and resistance to indomethacin expected of hydroxylated fatty acids derived via lipoxygenase. RBL-1 cells have been shown previously to produce leukotrienes and other lipoxygenase metabolites when treated with calcium ionophore. Plasminogen activator in HeLa cells was stimulated by up to 2.5-fold by incubation with 0.5-2 micrograms/ml 5-hydroxyeicosatetraenoic acid. Our results suggest that the induction of plasminogen activator in HeLa and RBL-1 cells is not mediated by prostaglandins or thromboxanes, but may be mediated or modulated by arachidonate metabolites derived via a lipoxygenase pathway.

Purification of a platelet protein which stimulates fibrinolytic inhibition and tissue factor in human fibroblasts.

Platelets are able to stimulate an increase in two distinct activities, tissue factor (thromboplastin) and fibrinolytic inhibition, in human fibroblasts in vitro. A procedure has been developed which allows the purification of a platelet macromolecule which is able to stimulate both of these changes. Washed human platelets were homogenized, sonicated, and then centrifuged at 90,000 x g for 2 h. The resulting pellet was solubilized in 0.05 M sodium carbonate, pH 10.5, and chromatographed on Sephadex G-200, then on hydroxylapatite, resulting in a 135-fold purification and a 20% yield. When the purified material was further fractionated on sodium dodecyl sulfate-polyacrylamide gels, stimulatory activity for both tissue factor and fibrinolytic inhibition was found only in the 75,000-dalton region. The active material could be inactivated by mercaptoethanol with no change in its apparent molecular weight. It was readily inactivated by trypsin with the concomitant loss of the 75,000-dalton Coomassie-staining band. Assay of the purified material for carbohydrate was negative. After isoelectric focusing, the purified material had a major band at pH 5.8 which stimulated both tissue factor and fibrinolytic inhibition. Subcellular fractionation of platelet homogenates by sucrose density gradient centrifugation resulted in a 2-fold increase in stimulatory material in the granule/mitochondrial fraction. This platelet-derived protein may represent a physiologically important regulator for both cellular procoagulant and the net fibrinolytic activity of systemic cells.

Stimulation of fibrinolytic activity in human skin fibroblasts by prostaglandins E1, E2 and I2.

The effects of prostaglandins on the fibrinolytic activity of cultured human foreskin fibroblasts have been measured by a [125I]fibrin dish assay. Prostaglandin (PG) E1, added to fibroblasts in serum-containing medium, produced dose-dependent increases in the fibrinolytic activity of both cellular extracts and conditioned medium. PGE2 and PGI2, but not PGD2 or 6-keto-PGF1 alpha, also stimulated fibrinolytic activity. In each case, activity was due to the protease plasminogen activator because it was abolished by omitting plasminogen from the fibrinolytic assays. The effects of PGE1 were observed at 10 ng/ml and maximal stimulation occurred at 1 microgram/ml. Levels of both intra- and extracellular plasminogen activator increased, indicating that PGE1 stimulated the overall synthesis and release of the protease. The effects of PGE1 were slow in onset and persistent (greater than 48 hr) and were abolished by cycloheximide and actinomycin D. Cellular plasminogen activator was stimulated by 10 microM isoproterenol and 250 microM dibutyryl cyclic AMP; the effects of PGE1, isoproterenol and dibutyryl cyclic AMP were potentiated by the phosphodiesterase inhibitors 1-methyl-3-isobutylxanthine (100 microM) and dipyridamole (20 microM). The induction of plasminogen activator by PGE1 may therefore be initiated by stimulation of cellular adenylate cyclase. Increased fibrinolytic stimulation of cellular adenylate cyclase. Increased fibrinolytic activity could contribute to the prolonged beneficial effects which have been reported after the administration of PGE1 and PGI2 in the treatment of occlusive vascular disease.

Platelet effects on tissue factor and fibrinolytic inhibition of cultured human fibroblasts and vascular cells.

Platelets stimulate tissue factor, the initiator of the extrinsic coagulation pathway, and increase fibrinolytic inhibition in fibroblasts grown in vitro. Cellular tissue factor increases an average of 2.8-fold over the control levels after a 6-hr incubation with platelets, and no activity is present in the media. Fibrinolytic inhibition is stimulated in both the fibroblasts and their media in the presence of platelets and accumulates throughout a 24-hr incubation. Neither leukocytes nor erythrocytes stimulate these changes. Both tissue factor and fibrinolytic inhibition increases are dependent on platelet concentration and are blocked by inhibitors of RNA or protein synthesis. Control smooth muscle cells have higher tissue factor and fibrinolytic inhibition than fibroblasts, but their response to the presence of platelets is similar. Confluent monolayers of endothelial cells have very low levels of tissue factor that are not altered by the presence of platelets. However, the ability of endothelial cells to inhibit fibrinolysis is enhanced by the presence of platelets. The fraction that stimulates tissue factor and fibrinolytic inhibition is distinct from platelet-derived growth factor and from the fraction that enhances leukocyte tissue factor. It is associated with an insoluble, nonmitogenic fraction that is not inactivated by phospholipase C, or diisopropylfluorophosphate, nor is it chloroform:methanol extractable. Platelets are a physiologic modulator for both cellular tissue factor and the fibrinolytic system in vitro.

Induction of plasminogen activator and prostaglandin biosynthesis in HeLa cells by 12-O-tetradecanoylphorbol-13-acetate.

Recent reports suggest that many of the biological effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate are mediated via intracellular prostaglandin biosynthesis. We have investigated whether the induction of plasminogen activator by 12-O-tetradecanoylphorbol-13-acetate in cultured HeLa cells is similarly mediated. 12-O-Tetradecanoylphorbol-13-acetate (0.5 to 50 nM) increased intra- and extracellular plasminogen activators and stimulated E- and F-type prostaglandin production. Changes in prostaglandin biosynthesis preceded those in plasminogen activator by several hr. Indomethacin (0.5 microM) abolished prostaglandin production but had no effect on either the magnitude or the time course of induction of plasminogen activator. Similar results were obtained with human skin fibroblasts and MDCK cells. Prostaglandins E1, E2, F2 alpha, and I2 had no direct effect on plasminogen activator in HeLa cells or skin fibroblasts. We conclude that in these cells, phorbol ester independently induces plasminogen activator and prostaglandin biosynthesis.

Comparative pharmacologic effects on tissue factor activity in normal cells and an established cell line.

The tissue factor (thromboplastin) activity of cells grown in vitro is modulated by exogenous drugs. The activity of human foreskin fibroblasts and umbilical vein smooth muscle cells is enhanced by 10(-6) M hydrocortisone or 1 mM butyrate. Activity is suppressed in these cells by 10(-6) M colchicine whereas 10(-4) M chloroquine has little or no effect. Two established cell lines, WISH or HeLa cells, have elevated tissue factor activity in the presence of colchicine or chloroquine and suppressed activity with exogenous hydrocortisone. Their activity is also decreased by 10 mM butyrate whereas 1 mM butyrate does not alter activity. Colchicine and butyrate apparently act via a mechanism unrelated to their effect on microtubules since it is possible to dissociate activity changes from morphologic changes. Umbilical vein endothelial cell tissue factor activity responds uniquely to exogenous drugs. Hydrocortisone or 10(-5) M vinblastine (or colchicine) only minimally alters activity. Endothelial cells are not simply refractory toward all drugs, however, since chloroquine dramatically enhances activity whereas 1 mM butyrate suppresses it. The low specific activity of endothelial cells and their apparently unique drug response may be another measure of their function as an in vivo hemostatic barrier.

Tissue-factor coagulant activity of cultured human endothelial and smooth muscle cells and fibroblasts.

The tissue-factor (thromboplastic) activity of cultured human endothelial cells and fibroblasts is low at time of transfer into fresh medium but increases 3-10 fold. Endothelial cells reach peak activity (400 U/10(5) cells) 5-8 hr after subculture. Activity in fibroblast cultures peaks (3000-12,000 U/10(5) cells) 7-12 hr after subculture. After attaining maximum activity, endothelial and fibroblast tissue-factor content decreases in a time course similar to other cells studied in this laboratory, approaching basal levels by 24-50 hr after subculture. If medium over fibroblasts is changed every 12 hr, activity can be sustained at the peak level for an additional day but cannot be maintained at a high level indefinitely. The kinetics of expression of smooth muscle cell tissue factor are markedly different from other cell types. There is always a pronounced lag (30 hr or more) before the activity increases, and then, in most cases, there is no subsequent decline in activity even though the cells are not refed or restimulated. The activity of each of these cell types is cryptic but becomes available after freeze-thaw disruption of cells.

Tissue factor in cultured cells: pharmacologic effects.

Tissue factor activity in suspension cultures of WISH amnion cells is modulated by pharmacologic doses of agents which alter membrane structure and function. Lysosomal stabilizing steroids (hydrocortisone, dexamethasone, aldosterone, prednisolone, and estradiol) suppress the change in activity which follows subculture; lytic steroids (testosterone and progesterone) are ineffective. Chloroquine both increases the specific activity and extends the time before return to the basal level. Dimethyl sulfoxide and ouabain suppress the complete expression of activity but do not inhibit the subsequent decay. The effect of cytochalasin B is complex, the drug being either suppressive or slightly stimulatory depending on the time of addition. Cyclic nucleotides (AMP or GMP) or insulin do not regulate the expression of tissue factor in these cells. A dramatic increment and prolongation of activity occurs when colchicine or vinblastine is added to the cell suspension shortly after subculture; there is much less stimulation by griseofulvin. Lumicolchicine has no effect while deuterium oxide is inhibitory. From these experiments, we conclude that increased membrane fluidity or altered secretory processes resulting from microtubule disruption stabilize tissue factor in cultured cells. Since contradictory results were obtained with agents which stabilize lysosomes or inhibit transport, the role of these cellular functions in tissue factor production or decay is unclear.

Tissue factor in cultured cells: metabolic control.

Tissue factor content of WISH amnion cells in spinner culture increases 3- to 10-fold within 12 hours after subculture, then declines to a basal level within 30 to 50 hours. Maximal development of activity requires fresh serum and fresh medium. When added at the time of subculture, actinomycin D and cycloheximide completely inhibit development of coagulant activity; when added several hours after transfer, these inhibitors suppress the development but do not affect the disappearance of activity. Of the oxidative phosphorylation inhibitors tested, dinitrophenol had no effect whereas carbonyl cyanide m-chlorophenylhydrazone inhibited the activity increase but did not alter the decline. The kinetics of development and decay are similar over a pH range of 6.7 to 7.6 and with fetal calf serum concentration between 5 and 30 per cent. At pH 6.7 or in 30 per cent fetal calf serum, cell division did not occur. 3H-leucine and 35SO4= incorporation into the cell surface coat did not change appreciably during the burst of coagulant activity nor did the levels of naphthylamidase or alkaline phosphatase; 3H-thymidine incorporation reached a peak within 2 hours of the tissue factor maximum.

Association of tissue factor activity with the surface of cultured cells.

Tissue factor occurs in a dormant state on the surface of cultured normal human fibroblasts and WISH 1 amnion cells. The activity of undisturbed monolayers or cells lifted with brief trypsin treatment (0.125 per cent trypsin for 1 min) increases up to 60-fold upon prolonged digestion with dilute trypsin (0.0025 per cent trypsin for 30 min); activity appears subsequent to cell detachment. Up to 70 per cent of the total cellular tissue factor becomes active under these conditions and is released from the cells. The ruthenium red staining coat of the cells is lost during detachment, but cell viability (more than 90 per cent exclude trypan blue) and cell morphology do not change during the subsequent development of tissue factor activity. Furthermore, less than 10 percent of four intracellular enzymes and less than 20 per cent of two plasma membrane enzymes are released during this period of time. We therefore conclude that cells in culture do have tissue factor activity, that it exists in a latent form, and that total cell disruption is not necessary for this activity to initiate blood coagulation.