Latent tissue plasminogen activator produced by human endothelial cellsin culture: Evidence for an enzyme-inhibitor complex (sodium dodecyl sulfate activation/hydroxylamine)

Conditioned medium from cultures of human umbilical vein endothelial cells was analyzed for the presence of tissue plasminogen activator (tPA) and urokinase. Immunoprecipitation studies using metabolically labeled conditioned medium and anti-tPA IgG revealed a single band on autoradiographs corresponding to a Mr of 100,000. No bands were observed after immunoprecipitation with anti-urokinase IgG. The Mr 100,000 tPA was found to be inactive and did not bind to fibrin clots. However, exposure of this tPA to 1% NaDodSO4 resulted in the appearance of plasminogen activator activity with no apparent change in its Mr. Treatment with 10 mM diisopropylfluorophosphate prior to NaDodSO4 activation did not inhibit the NaDodSO4-induced appearance of plasminogen activator activity, indicating that the active site was not available for diisopropylfluorophosphate binding. The possibility that the properties of this Mr 100,000 tPA reflected a tPA-inhibitor complex was examined. Attempts to dissociate such a complex by denaturation, reduction, or extremes of temperature were not successful. However, after treatment of conditioned medium with 1 M hydroxylamine in the presence of 0.1% NaDodSO4, the Mr of the anti-tPA immunoprecipitable material declined by 40,000 to Mr 60,000, a Mr consistent with that of other human tPAs. Hydroxylamine has been shown previously to dissociate covalently coupled serine protease-inhibitor complexes. Furthermore, incubation of purified, human melanoma cell tPA with conditioned medium resulted in an increase iwits-Mr by.40,000 with a concomitant decline in tPA activity. The data suggest that the latent tPA present in the conditioned medium of endothelial cells is composed of a Mr 60,000 tPA associated with an inhibitor. The plasminogen activators are a class of serine proteases that convert plasminogen to the fibrinolytically active enzyme plasmin (1). Human plasma contains two plasminogen activators that are immunochemically distinct (2), tissue plasminogen activator (tPA) (3) and urokinase (4). tPA provides an extremely efficient pathway for vascular thrombolysis. Its high affinity for fibrin (2) in conjunction with a fibrin-dependent stimulation of tPA activity (5) ensures that activation of plasminogen by tPA is localized to the fibrin thrombus and reduces systemic plasminogen activation and the resulting degradation of fibrinogen and other plasma proteins (6). Activity neutralization studies using tPA-specific antibodies have demonstrated that tPA is responsible in large part for increases in plasma fibrinolytic activity after specific physiologic and pathologic stimuli (7). The source of plasma tPA has been presumed to be the vascular endothelium. Studies with cultured human endothelial cells have shown that these cells are associated with plasminogen activator activity (8), although relatively low levels were observed. Fibrinolytic inhibitors that also were found in human endothelial cell extract (8) were considered to be responsible for the depressed level of plasminogen activator activity. Although it has been established that plasminogen activators are present in cultures of human endothelial cells, the immunochemical or biochemical properties of these proteases have not been determined. This study examines the plasminogen activators secreted by primary cultures of human endothelial cells and demonstrates that a single form of plasminogen activator that is immunochemically, related to human melanoma cell tPA is released. No urokinase-like plasminogen activators were detected by activity measurements or immunoprecipitation with anti-urokinase IgG. The endothelial cell tPA (EC-tPA) was inactive and did not bind to fibrin clots. It is suggested that this latent. EC-tPA is present in the culture medium as a complex between a Mr 60,000 tPA and an inhibitor. MATERIALS AND METHODS Plasminogen was prepared from human plasma by affinity chromatography on lysine-Sepharose as described (9). Bovine fibrinogen fraction 1 (CalBiochem-Behring) was purified free of plasminogen by ethanol precipitation in the presence of lysine (10). Human a-thrombin was a gift from J. Fenton (Albany, NY). Human urokinase (World Health Organization first reference standard) and antiserum against human urokinase were obtained from G. Murano (National Institutes of Health). Human melanoma cell tPA and tPA antiserum was a gift from D. Collen (University of Leuven, Leuven, Belgium) (4). IgG fractions were prepared as described (11). Radioiodination of purified tPA was performed by the IODO-GEN method (12). Cell Culture, Conditioned Medium Preparation, and Metabolic Labeling. Endothelial cells were isolated from human umbilical cord veins as described (13) and cultured in 75-cm2 tissue culture flasks coated with calf skin gelatin (2 mg/ml; Eastman). Cells were grown to confluency in RPMI 1640 medium (GIBCO) containing 20% fetal bovine serum (Reheis) and 100 units of penicillin and 100 ,ug of streptomycin per ml. Primary cultures were used exclusively in these studies. I To prepare conditioned medium, confluent cultures were washed twice with RPMI 1640 and incubated in the absence of serum for 24 hr in 5 ml of RPMI 1640/Neuman and Tytell serumless medium (GIBCO), 1:1 (vol/vol). The medium was centrifuged at 600 x g to remove cell debris and made 0.01% with Tween 80 (Baker). Samples were frozen at -70°C until used. Abbreviations: EC-tPA, endothelial cell tissue plasminogen activator; tPA, tissue plasminogen activator; iPr2P-F, diisopropylfluorophosphate. 6804 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 80 (1983) 6805 Conditioned medium from metabolically labeled cells was prepared by washing confluent cultures three times with methionine-free RPMI 1640 and adding 2 ml of the same medium containing [3S]methionine at 100 ,uCi/ml (1,195 Ci/mmol, New England Nuclear; 1 Ci = 37 GBq) for 4 hr. Assays for Plasminogen Activator and Fibrinolytic Inhibitors. Plasminogen activator activity was assayed on "2I-labeled fibrin ('"I-fibrin)-coated multiwell tissue culture dishes (24 wells, 16 mm; CoStar) as described (14). Where indicated, EC-tPA was incubated with 1% NaDodSO4 for 30 min at 370C and then Triton X-100 was added to 5% prior to addition to the assay mixture. The presence of NaDodSO4 in the assay mixture did not affect either plasminogen generation nor fibrin degradation as demonstrated by parallel experiments with urokinase and melanoma cell tPA. When necessary, a urokinase dose-response assay was performed to permit the expression of EC-tPA activity in terms of units of urokinase activity. Inhibitor activity was measured in mixing experiments with 0.025 units of urokinase as described (8). NaDodSO4/Pol~yacrylamide Gel Electrophoresis, Fibrin Autography, and Autoradiofluorography. NaDodSO4/polyacrylamide gel electrophoresis was performed by the procedure of Laemmli (15) with resolving gels of 9% acrylamide and stacking gels of 4% acrylamide. Mr standards included plasminogen (90,000), human transferrin (76,000), bovine serum albumin (66,200), ovalbumin (45,000), and soybean trypsin inhibitor (21,500>. Gels to be analyzed by fibrin autography were soaked for 1.5 hr in 2.5% Triton X-100 and applied to the surface of a fibrin agar gel prepared as described (11) except that the final plasminogen concentration was increased to 38 jig/ml. For autoradiofluorography the acrylamide gel was stained with 0.1% Coomassie brilliant blue in 50% trichloroacetic acid, destained in 10% acetic acid, and treated with an autoradiography enhancer (EN3HANCE; New England Nuclear) according to the manufacturer's instructions. Gels were dried and placed on x-ray film (XR-5, X-Omat R film; Kodak) for 3-5 days at -70°C. Preparation of Partially Purified EC-tPA. Conditioned medium was applied to a lysine-Sepharose 4B column (1 X 25 cm; Pharmacia) equilibrated with 0.1 M sodium phosphate, pH 7.2/ 0.01% Tween 80 and eluted with 0.5 M ammonium thiocyanate. To locate plasminogen activator activity, 100-,ul aliquots from each fraction were incubated in the presence of 1% NaDodSO4 for 30 min at 37°C and analyzed by NaDodSO4/ polyacrylamide gel electrophoresis and fibrin autography. Fractions containing EC-tPA activity were pooled and dialyzed against sodium phosphate/Tween buffer. Hydroxylapatite powder (10 mg/ml; Bio-Rad) was added to the pooled fractions, and the slurry was mixed for 30 min at 4°C. After centrifugation at 2,000 x g, the pellet was washed two times with sodium phosphate/Tween buffer, and the enzyme was eluted with 0.2 M sodium phosphate, pH 7.2/0.01% Tween 80 at 1/10th the original volume. The final product is referred to as partially purified EC-tPA. Fibrin Binding and Diisopropylfluorophosphate (iPr2P-.F) Treatment. Partially purified EC-tPA (0. 1 unit/ml), melanoma cell-derived tPA (0.1 unit/ml), and urokinase (0.5 unit/ml) were added to plasminogen-free fibrinogen (final concentration, 0.53 mg/ml; CalBiochem-Behring). Thrombin (1 unit) was added, and the mixture was incubated for 10 min at room temperature. Clots were removed by centrifugation at 15,000 X g for 5 min and washed twice with 1 ml of sodium phosphate/Tween buffer. The clots were extracted with 1% NaDodSO4 for 30 min at 37TC (EC-tPA) or 5 M urea for 30 min at room temperature (melanoma cell tPA and urokinase). The appropriate clot supernatants were adjusted to contain 1% NaDodSO4 or 5 M urea, and all clots and supernatants were assayed for fibrinolytic activity on "2I-fibrin plates. A final concentration of 10 mM iPr2P-F (Sigma) was added to a 200-jul sample of partially purified EC-tPA (0.1 unit/ml in 0.2 M sodium phosphate, pH 7.2) previously treated with 1% NaDodSO4 and 5% Triton X-100 or to a duplicate untreated sample. The samples were, ineubated for 2 hr at 370C and dialyzed against sodium phosphate/Tween buffer for 16 hr. The sample that was not treated with.NaDodSOprior to iPr2P-F addition was then incubated with 1% NaDodSO4, and plasminogen activator activity in all samples was assayed by the 5Ifibrin plate method. Parallel experiments were performed with urokinase (0.1 unit/ml) to ensure that iPr2P-F could inactivate the serine protease in the presence of 1% NaDodSO4/5% Triton X-100 and that no residual iPr2P-F remained after dialysis. Immunoprecipitation. Immunoprecipitation of partially purified EC-tPA was performed as follows: serial 1:10 dilutions of an anti-tPA IgG fraction (5.4 mg/ml) were made in phosphatebuffered saline containing 0.01% Tween 80, 2% ovalbumin, and 5.4 mg of normal rabbit IgG per ml; 50 ,ul of each dilution was added to an equal volume of a partially purified EC-tPA (0.1 unit/ml), and the mixture was incubated overnight at 4°C. Immune complexes were incubated for 30 min at 4°C with 200 ,ul of protein A-Sepharose (0.1 mg/ml; Pharmacia) and removed by centrifugation at 15,000 X g. Supernatants were assayed for fibrinolytic activity by the "2I-fibrin plate assay. Immunoprecipitation studies with metabolically labeled conditioned medium were performed by 1:50 dilution of anti-tPA IgG or 1: 10 dilution of anti-urokinase IgG (1. 6 mg/ml) into 300 ,ul of conditioned medium containing 1% Nonidet P-40, 1% bovine serum albumin, 0.2 M sodium chloride, 0.3 mM phenylmethylsulfonyl fluoride (Calbiochem-Behring), 100 units of Trasylol (CalBiochem-Behring) per ml, and 50 mM Tris HCl (pH 8.0). Samples were incubated overnight at 40C, and the immune complexes were removed with 100 ul of protein ASepharose (0.1 mg/ml) as described above. The Sepharose beads were washed five times with 50 mM Tris HCl, pH 8.7/0.1% Nonidet P-40/1 M NaCI, two times with 50 mM Tris HCl, pH 6.8/1% Nonidet P40/0.3% NaDodSO4/0. 1 M NaCl, and once with water. The beads were suspended in 3% NaDodSO4 and boiled for 3 min, and the supernatants were analyzed by NaDodSO4/polyacrylamide gel electrophoresis. Hydroxylamine Treatment. Metabolically labeled conditioned medium (300 ,ul) was treated with a final concentration of 0.1% NaDodSO4 for 30 min at 37°C, and an equal volume of 2 M hydroxylamine (Sigma)/0.01 M sodium phosphate, pH 7.5, was added. The mixtures were incubated at 37°C for 1 hr and dialyzed at room temperature against 0.05 M Tris HCl (pH 8) prior to immunoprecipitation.