Tumor Cell Generation of Thrombin Via Functional Prothrombinase Assembly1

[CANCER RESEARCH 45, 5521-5525, November 1985] Tumor Cell Generation of Thrombin via Functional Prothrombinase Assembly1

Livingston VanDeWater, Paula B. Tracy,2 Denise Aronson, Kenneth G. Mann,2 and Harold F. Dvorak3

Departments of Pathology, Beth Israel Hospital and Harvard Medical School and the Charles A. Dana Research Institute, Beth Israel Hospital, Boston, Massachusetts 02215 (L. V. D. W., D. A., H. F. D.Â¡,and Hematology Research Section, Mayo Clinic, Rochester, Minnesota 55095 [P. B. T., K. G. M.Â¡

ABSTRACT These classical pathways are now recognized as a network of activation and feedback reactions that converge on a final com Prothrombinase affects the proteolytic conversion of pro- mon pathway of prothrombinase and thrombin generation. Pro- thrombin to thrombin and is the penultimate enzyme in the coagulants acting at steps proximal to this common pathway will common coagulation pathway. Prothrombinase is a complex in be ineffective in generating thrombin and depositing fibrin if which the proteinase, Factor Xa, a cofactor, Factor Va, and prothrombinase is not active. calcium are bound to a membrane surface to generate the active Prothrombinase complex formation is thus a critical step in enzyme. Guinea pig line 1 and line 10 tumor cells, grown as coagulation and one that has been largely neglected in studies primary cultures from ascites tumors or as cell lines in culture, of tumor-associated clotting. Prothrombinase is a complex of an provide a surface that interacts with coagulation Factor Va and active protease (Factor Xa) with a non-enzymatic cofactor (Fac Xa and with calcium ions to form this enzyme complex. Cultured tor Va) bound to an appropriate membrane surface in the pres human colorectal carcinoma cells (Colo 205) also participate in ence of calcium ions. Platelets (18-22), some inflammatory cells Prothrombinase complex assembly and function. Prothrom (23), endothelial cells (24), and certain phospholipid vesicles of binase generation was measured by following the kinetics of defined composition (25) all provide an appropriate surface for prothrombin conversion to thrombin. Thrombin generation was prothrombinase complex assembly; I.e., they afford a surface monitored continuously using the reversible thrombin inhibitor, that binds Factor Va and allows it to serve as a receptor for dansylarginine A/-(3-ethyl-1,5-pentanediyl)amide, which displays Factor Xa. In the absence of such a surface, the capacity of enhanced fluorescence upon binding to thrombin. Analyses of prothrombinase to cleave prothrombin to thrombin falls nearly kinetic data indicate that the apparent dissociation constants (1- three orders of magnitude (26); thus, provision of a suitable 4x10~10 mol/liter) and the number of Factor Va-Xa binding sites surface for prothrombinase activation, in addition to plasma per tumor cell are comparable to values reported for human and clotting factors, is required for fibrin formation under any circum bovine platelets, human lymphocytes, and monocytes. Guinea stance, including that occurring about tumors. pig lymphocytes were also active, while erythrocytes were inac We considered the possibility that tumor cells themselves tive, in the prothrombinase assay. Membrane vesicles, shed by might provide an appropriate surface for prothrombinase gener guinea pig and human tumor cells into conditioned medium, also ation and thus contribute directly to local fibrin deposition. We supported functional prothrombinase activity. Although earlier report here that several types of carcinoma cells, as well as studies indicated that tumor cells may initiate coagulation, this is membranes shed by such cells, provide an effective surface for the first demonstration that tumor cells are competent to bring activation of the prothrombinase complex. clotting to fruition by generating thrombin, a step essential to fibrin generation. These data suggest that tumor cells, in the MATERIALS AND METHODS presence of clotting initiators and appropriate coagulation fac tors, are sufficient to generate the fibrin deposited in solid tumors. Materials. Dulbecco's modified Eagle's medium, HEPES,4 calf serum, and fetal calf serum were obtained from Grand Island Biological Co. (Grand Island, NY), and fat-free bovine serum albumin was obtained from INTRODUCTION Miles (Elkhardt, IN). DAPA was prepared as described (25, 27). All other reagents were of analytical grade. Fibrin is a prominent component of the stroma of autochtho Coagulation proteins were purified from bovine plasma using published nous and transplanted tumors (1-5). Diverse functions such as procedures (28, 29). Factor V was activated by the addition of thrombin angiogenesis, desmoplasia, facilitation of fibroblast and endothe- (0.02 units/ml) for 3 min at 37Â°C (30). Factor X was activated using lial cell adhesion and motility, mast cell degranulation, and mod immobilized Factor X activator purified from Russell Viper Venom (29). ulation of the immune response have been attributed to fibrin Cell Culture. Guinea pig line 10 and line 1 bile duct carcinoma cells, and its catabolites (5-9). The fibrin deposited in tumors derives ascites variants, were passaged in the peritoneal cavities of inbred strain from plasma fibrinogen that has extravasated from local, hyper- 2 Sewall-Wright guinea pigs (11). The line 10 cells grow in culture as non-adherent cells. The ascites form of line 1 carcinoma cells and the permeable blood vessels. However, the mechanisms by which human colorectal carcinoma cell line (Colo 205) grow in culture as thrombin is generated in the extravascular space to convert adherent (tissue culture plastic) or non-adherent (bacteriological plastic) fibrinogen to fibrin are poorly understood. Several tumor cell cells. Cells (0.5-1 x 106 per ml) were maintained in Dulbecco's modified procoagulant activities have been described that act at various Eagle's medium supplemented with penicillin (50 Â¿Â¿g/ml),streptomycin steps in the intrinsic and extrinsic clotting pathways (10-17). (50 units/ml), glucose (4.5 mg/ml), and 5% calf serum (line 1 and line 10) 1This work was supported by USPHSgrants CA 28471 and HL 17430. or 10% fetal calf serum (Colo 205, HT1376) (31, 32). Cells were prepared 2Current address: Departments of Medicine (P. B. T.) and Biochemistry (K. G. for prothrombinase assay by washing two times in phosphate-buffered M.), Universityof Vermont, Burlington, VT 05405. saline and once in HEPES-glucose buffer (150 mw NaCI:11 Ã•TIMglu- 3To whom requests for reprints should be addressed, at Departmentof Pathol ogy, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. 4The abbreviations used are: HEPES, 4-(2-hydroxyethyl)-1-piperazineethane- Received 3/26/85; revised 8/9/85; accepted 8/12/85. sulfonic acid; DAPA,dansylarginineN-<3-ethyl-1,5-pentanediyl)amide.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1985 American Association for Cancer Research. TUMOR CELL PROTHROMBINASE ACTIVITY cose 10 mu HEPES, pH 7.2). from 0.01-5 nM. Changes in prothrombinase activity were inter Conditioned medium was obtained from guinea pig line 1 or 10 preted to reflect binding of Factor Va to a cellular site (21, 23, carcinoma cells or from human HT1376 by a modification of published procedures (10,11 ). Cells were washed and cultured in Eagle's Minimal 25). We found that the guinea pig line 1 and line 10 bile duct essential medium without phenol red (Flow Laboratories) and without serum at 2.5 x 10* cells/ml for 4 h at 37Â°C with 5% CO2. Cells (95% carcinomas and the human colorectal line, Colo 205, all provided viable by try pan blue exclusion) were removed by centrifugation at 160 efficient surfaces for prothrombinase generation. Using the anal x g for 10 min, and the medium was centrifuged at 10,000 x g for 20 ysis of kinetics of prothrombin activation to infer parameters of min. In some experiments, the resulting supernatant was centrifuged at Factor Va binding to the cell surface, we found that when Factor 100,000 x g for 90 min. Va concentrations were decreased below saturating levels, the Prothrombin Activation (Monitoring with DAPA. The formation of a reaction rate decreased according to a hyperbolic function (Chart functionally active prothrombinase complex on the surface of tumor cells 1) and represented a binding isotherm characteristic of a func was analyzed by assessing the influence of Factor Va and Xa on the tional Factor Va-cell interaction (21,23). Reaction velocities were rate of prothrombin activation to thrombin (21). This approach is based on the observation that deletion of the catalytic "membrane surface' assumed to be directly proportional to the number of occupied results in a 1000-fold decrease in reaction rate and, therefore, an Factor Va sites on the cells. The ratio of observed veloc- ineffective enzymatic complex (26). No thrombin is generated unless an ity:maximum velocity allowed calculation of the fraction of oc equivalent "membrane surface" is provided by tumor cells. Prothrom- cupied sites (21,23,25). A double reciprocal plot (Chart 1, inset) bmase activity was monitored continuously using DAPA, which emits depicts the reciprocal of free Factor Va cellular binding sites increased fluorescence when it binds to newly generated thrombin (21, plotted as a function of the ratio of nominal Factor Va concentra 23, 25, 27). Tumor cells (1 x 10* cells/ml) were preincubated for 2 min tion to the fraction of filled sites. The apparent dissociation at ambient temperature in a mixture containing prothrombin (1.39 ^ M) constant calculated from the slope is Ka Â»1.7 x 10~10mol/liter; and Ca2* (2 mw) and varying concentrations of Factor Va, in a final volume of 1 ml in HEPES-glucose buffer, pH 7.2. Addition of Factor Xa the number of functional cellular receptor sites calculated from the y-intercept is 45,000 sites per cell in this experiment. This (5 nM) initiated prothrombinase generation, and the time course of thrombin formation (OAPA fluorescence) was continuously recorded (26). calculation is based on the assumption that one Factor Va The reaction velocity was determined from the initial slope of the recorded molecule binds per receptor (see Ref. 21). data. The binding parameters derived from the various tumor cells studied are summarized in Table 1. The apparent Kds of tumor cells, 1-3 x 10~10mol/liter, were comparable to those observed RESULTS with platelets, monocytes, and lymphocytes (21,23). In addition, Kinetic and protein binding experiments with platelets and we found that some cell lines (line 10, Colo 205) had significantly monocytes have demonstrated that cell surface bound Factor more cellular binding sites for prothrombinase than did others Va functions as a receptor for Factor Xa, permitting assembly of (line 1 cells). Cells fresh from asertes (e.g., line 10, experiment 3; active prothrombinase at the cell surface (18-23). Deletion of Factor Va from the prothrombinase complex results in a 10,000- fold decrease in rate (26). In the present report using a kinetic assay, we have examined the possibility that tumor cells provide a suitable surface for prothrombinase generation. Three estab lished carcinoma cell lines (guinea pig lines 1 and 10; human Colo 205) were chosen for the study of cellular Factor Va binding. It was essential that tumor cells be in suspension for the kinetic assay and have intact surfaces unperturbed by chelating agents or proteases commonly used during harvest of attached cells from plastic. These cell lines met these criteria and, in addition, / 0.6 1.0 they are known to exhibit fibrin deposits when grown as solid â€¢/[FACTORVJ/HMxIO9 tumors in guinea pigs (lines 1 and 10) (2) or nude mice (all three cell lines).5 Prothrombinase activity was monitored continuously (25, 27) 1.0 2.0 5.0 using DAPA, which emits increased fluorescence when it binds [FACTOR Mxio9 to thrombin generated from prothrombin. Initial experiments were Chart 1. Piothrombmase activity of guinea pig line 1 tumor cells. Line 1 asertes carried out with each tumor cell type using concentrations of tumor cells, cultured as non-adherent cells for 1 day in Dulbecco's modified Eagle's Factor Va and Xa (5 nM) which were saturating with respect to medium with 5% calf serum, were washed in phosphate-buffered saline and HEPES-glucose buffer (8). Reaction mixtures in HEPES-glucose buffer and con rate of prothrombin activation as determined by preliminary taining bovine prothrombin (1.39 MM),DAPA (3 MM),Ca2* (2 CTIM),andcells (4x10* experiments. No prothrombinase activity was detectable in the per ml) were pre-incubated with varying amounts of bovine Factor Va for 2 min at absence of Factor Va, Xa, or tumor cells, indicating that thrombin ambient temperature. Assays were initiated by the addition of 5 nM bovine Factor generation was strictly dependent upon all these components. Xa. and initial velocities were obtained. Results are presented as mot prothrombin converted per min as a function of nominal Factor Va concentration. The double In subsequent experiments, we determined the influence of reciprocal plot obtained from the saturation curve is shown as an inset. The Factor Va on tumor cell-associated prothrombinase activity un reciprocal of the fraction of free Factor Va cell sites is plotted as a function of the der conditions in which saturating concentrations of Factor Xa nominal Factor Va concentration divided by the fraction of bound Factor Va cell sites. Bovine prothrombin (17), Factor V (6), and Factor X (17) were purified by (5 nM) were used, while concentrations of Factor Va were varied published procedures. Factor Xa was activated from Factor X (18) using Factor X activator, and Factor Va was activated from V by catalytic amounts of thrombin â€¢D.R. Senger. L. VanDeWater, and H. F. Dvorak, unpublished data. (6).

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Table 1 Binding parameters governing prothrombinase complex formation on tumor cells 0.7

(mol/liter)"2.8x10-'Â°0.7 CellGuinea 0.6 pigune 10Line!Human 0.5 x1Q-102.9 X10-102.4 03 x10-'Â°3.8 Z 0.4 x1Q-101.7 tu X1Q-102.0 xIO"101.1 0.3 (Colo 205)Experiment12312312K* x 10-'Â°n"179,000250,000205,00032,00083,00045,000380.000151,000 a Ka was derived from the inverse of slope of double reciprocal plot (e.g., Chart 0.2 1, inset). " n, number of functional binding sites per cell derived from the /-intercept (e.g., 0.1 Chart 1,/riser).

6 8 10 12 14 line 1, experiment 3) or in culture for as long as 14 days (e.g., 1IME..min line 1, experiment 2) expressed similar binding parameters. Chart 2. Prothrombinase activity of line 10 tumor cell conditioned medium. Therefore, the observed cell-associated capacity of viable tumor Conditioned medium was obtained from washed guinea pig line 10 cells grown in Dulbecco's modified Eagle's medium (1 x 106 cells/ml) without serum overnight at cells to participate in prothrombinase complex formation was 37Â°Cin 5% C02. Conditioned medium was centrifuged at 1000 x g for 10 min and stably expressed in tissue culture and was present on primary 10,000 x g for 10 min, and the supernatant was concentrated on an Amicon XM- 300 filter (Amicon Corp., Lexington, MA). Conditioned medium, derived from the explants as well as on cultured cells. equivalent of 1.4 x 107 cells, was incubated with prothrombin, DAPA, Ca2*, Factor It would have been desirable to compare the prothrombinase Va (5 MM),and Factor Xa (5 nu) as in Chart 1 and generated activity of approximately generating capacities of our tumor cells with those of normal 1 x 10* line 10 cells. The arrow marks the addition of Factor Xa. Results are shown cells from which tumors had been derived. Unforunately, no as increasing fluorescence intensity as a function of time. matched "normar cell pairs exist for the bile duct and colonie tumor epithelia we studied. However, we did test purified guinea and also procoagulant activity in the common coagulation path pig lymphocytes and erythrocytes (1 x 106/ml) at saturating way (10). It was therefore of interest to determine if shed vesicles, concentrations of Factor Va and Xa and found that spleen and present in serum-free conditioned medium, would also support lymph node lymphocytes exhibited rates of 0.6 and 0.4 x 10~7 functional prothrombinase activity. Vesicle preparations from line mol of thrombin per liter per min, respectively. These values are 10 and line 1 guinea pig cells and human HT1376 [bladder comparable to those reported for human peripheral blood lym carcinoma (31)] cell lines all promoted functional prothrombinase phocytes (23). Guinea pig, as human (23), erythrocytes were activity (Chart 2). With line 10 conditioned medium, the Factor totally inactive. Va concentration was varied to generate a binding isotherm It was important to document that the functional activity we analogous to that shown for intact line 10 cells in Chart 1. An observed reflected a direct association of prothrombinase with apparent dissociation constant of 1.6 x 10~10 mol/liter was the tumor cell surface. This was accomplished by monitoring observed, in the range observed of that for intact tumor cells prothrombinase activity in the DAPA assay before and after (Table 1). pelleting the cells. Assay mixtures were prepared as before Preliminary experiments were carried out to determine whether (Chart 1); however, a Factor Va concentration was chosen that phospholipid was critical to the vesicle-associated, prothrom- gave less than maximal reaction velocities. At the beginning of binase-stimulating activity. We found (results not shown) that the assay, the initial velocity was determined, and an aliquot was the prothrombinase-supporting activity of line 10 tumor cell con removed and centrifuged (39,000 x g, 10 min) to remove the ditioned medium could be extracted along with phospholipids in cells. No residual prothrombinase activity was found in the ethanol-ether and also was inhibited by an antiphosphatidyl supernatant. Moreover, the addition of saturating concentrations glycerol antibody. It thus appears that phospholipid may be of Factor Va to the supernatant did not stimulate thrombin essential to the expression of tumor vesicle prothrombinase generation. Direct evidence of Factor Va binding to line 10 tumor activity. cells was obtained with 125l-labeledbovine Factor Va (results not shown). The binding of Factor Va was dependent upon the number of cells and was reduced by an excess of unlabeled DISCUSSION Factor Va. Therefore, as in all other systems thus far tested (21- 23), the evidence suggests that the functional prothrombinase Our data demonstrate that viable tumor cells of guinea pig and we observed was physically bound to the tumor cell surface. human origin participate in prothrombinase complex formation. However, we also observed that centrifugation of reaction Earlier work had established that tumor cells could initiate clotting mixtures at lower forces (e.g., 10,000 x g) left significant (~40%) as tissue factor (11) or as a Factor X-activating protease (13, prothrombinase activity in the supernatant. This finding sug 14). However, for coagulation to proceed to thrombin and hence gested that some portion of the prothrombinase activity might fibrin generation, a functional prothrombinase activity such as be bound to a subcellular fraction. Earlier work had demonstrated that demonstrated here on tumor cells is essential. that viable tumor cells shed submicroscopic plasma membrane The kinetic assay used in our studies has been used previously vesicles into tumor cell conditioned medium in a time-dependent to measure a similar functional activity on the surface of platelets, fashion (10). These vesicles expressed tissue factor activity (11) monocytes, and lymphocytes (21-23). In accord with earlier

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1985 American Association for Cancer Research. TUMOR CELL PROTHROMBINASE ACTIVITY studies of several normal cells, Factors Va, Xa, and Ca2+ were An important related question concerns the relative capacities all required, in addition to tumor cells, for prothrombinase expres of normal and tumor cells to promote coagulation. In fact, this sion. In the absence of any one of these components, all detect comparison cannot be made properly because normal epithelial able prothrombinase activity was lost. Prothrombinase activity cells and fibroblasta, isolated directly from animals or carried in was saturable in that high concentrations (>5 nw) of either Factor tissue culture, are adherent cells and must be harvested with Va or Xa did not increase further the rate of thrombin generation. trypsin before they can be studied in suspension in the DAPA Lowering the Factor Va (Chart 1) or Xa concentration below assay. Trypsinized cells have modified surface properties and saturation levels resulted in reduced prothrombinase activity. We therefore cannot be studied meaningfully in this assay. To avoid conclude from these data that functional tumor cell binding sites this complication in the present experiments, we used tumor for Factor Va were saturated at plateau concentrations of Factor cells which were non-adherent in culture and which grew in Va and that at lower concentrations of Factor Va reaction velocity ascites form in animals. Lacking a matched normal cell for was directly proportional to the number of tumor sites binding comparison, we examined lymphocytes isolated freshly from Factor Va. The ratio of observed reaction velocity to the limit guinea pig blood; these cells also activated a functional pro velocity allowed calculation of the fraction of cellular sites bound thrombinase, in agreement with earlier reports of human lympho or free for Factor Va (Chart 1, inset). Thus, we conclude that the cytes and monocytes (23). That cells so diverse as tumor epithe Factor Va bound to the tumor cell surface served as receptor lium and normal lymphocytes express prothrombinase activity sites for Factor Xa. This conclusion had been substantiated in raises the possibility that all nucleated mammalian cells may earlier work directly demonstrating that Factor Va serves as a express this property. Factor Xa receptor on platelets (18-22) and monocytes (23). Other factors undoubtedly influence fibrin deposition in tumors; Tumor cells from either guinea pig tumor and from the human among these are products that are secreted by or are associated tumor line all participated efficiently in prothrombinase assembly with tumor cells. These include soluble factors (5, 34, 35) that and had binding sites for Factor Va of approximately equivalent render the local microvasculature leaky to plasma proteins and affinity (Ka = 1-4 x 10~10mol/liter). The capacity of both guinea also include initiators of coagulation, such as tissue factor (11, pig tumor cell lines to support prothrombinase activity remained 36), that may trigger extravascular clotting of fibrinogen. Thus, constant and equivalent whether cells were isolated directly from complex interactions between plasma proteins and tumor cell animal ascites or following culture in vitro for up to 2 weeks. products may promote the preferential deposition of fibrin ob That binding parameters can be properly inferred from kinetic served in tumors. Factor Va is itself subject to cleavage both by data has been established by correlation of extensive kinetic and Factor Xa and activated protein C (37), and thus the prothrom direct binding experiments involving platelets (21, 22) and by binase complex, essential to thrombin generation, may provide sedimentation experiments with monocytes (23). It is noteworthy one such regulatory mechanism within the tumor microenviron- that line 1 tumor cells had significantly fewer binding sites for ment. Factor Va than their line 10 counterparts (Table 1). This difference may relate, at least in part, to corresponding differences in cell REFERENCES surface area; line 1 cells are smaller cells that have approximately 1. O'Meara, R. A. Q., and Jackson, R. D. Cytological observations on carcinoma. one-fourth the packed cell volume and only about 40% of the Ir. J. Med. Sci., 6: 327-328,1958. surface area of line 10 cells. Alternatively, the number of sites 2. Dvorak, H. F., Dvorak, A. M., Manseau, E. J., Wiberg. L., and Churchill, W. H. per cell may be a characteristic of the cell type (33). Fibrin gel investment associated with line 1 and line 10 solid tumor growth, Prothrombinase activity similar to that expressed by viable angiogenesis, and fibroplasia in guinea pigs. 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Livingston VanDeWater, Paula B. Tracy, Denise Aronson, et al.

Cancer Res 1985;45:5521-5525.

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