Binding of Human Factors X and Xa to HepG2 and 582 Human

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 16, Issue of June 5, pp. 9105-9113.1990 Printed in U.S. A.

Binding of Human Factors X and Xa to HepG2 and 582 Human Tumor Cell Lines EVIDENCE THAT FACTOR Xa BINDS TO TUMOR CELLS INDEPENDENT OF FACTOR Va*

(Received for publication, November 20, 1989)

Toshiyuki Sakaij: and Walter Kisielg From the Blood S.ystems Research Foundation Laboratory, Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131

Previous studies demonstrated that several cultured human tumor cell lines potentiate the conversion of prothrombin to thrombin by factor Xa and calcium in the absence of exogenous factor Va. In the present study, the specific binding of radioiodinated preparations of human factor X and factor Xa to a human hepatocellular carcinoma cell line (HepG2) that constitutively synthesizes a factor V/Va molecule, and a human bladder carcinoma cell line (582) that does not synthesize factor VJVa, was examined. Radioiodinated factor Xa bound specifically to 582 and HepG2 cells, whereas no significant specific binding of “‘I-factor X to either cell was observed. The binding isotherm of ‘2eI-factor Xa to each tumor cell line exhibited a hyperbolic profile, and Scatchard analysis demonstrated a single class of binding site for factor Xa on each cell surface with Kd values of 1.66 2 0.39 and 1.64 +: 0.52 nM and 566,000 f 71,000 and 28,000 f 6,000 binding sites/cell for HepG2 and 582 cells, respectively. Thrombin formation by cell-bound factor Xa was hyperbolic and saturable at 5 nM factor Xa on each cell line. Hanes-Woolf plots of the prothrombin activation data indicated that half-maximal rates of thrombin formation occurred at factor Xa concentrations of 1.50 f 0.43 nM and 1.42 + 0.48 nM on HepG2 and 582 cells, respectively. Pretreatment of 582 cells with polyclonal anti-human factor V IgG had no measurable effect on either the binding of ‘2SI-factor Xa or prothrombin activation. However, pretreatment of HepG2 cells with anti-human factor V IgG inhibited prothrombin activation in a dose-dependent manner, but did not inhibit the binding of factor Xa to this cell. When both cell lines were preincubated with exogenous human factor Va, the binding of factor Xa to either HepG2 or 582 cells was marginally affected. These data indicate that HepG2 and 582 cells have cell surface factor Xa binding sites proximal to, but independent of, cell surface factor Va of either exogenous or endogenous origin.

The proteolytic conversion of prothrombin to thrombin is a key step in the coagulation cascade. Efficient proteolytic activation of prothrombin is catalyzed by the so-called “pro-

* This work was supported by Research Grant HL35246 from the National Institutes of Health and by a grant from Blood Systems, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a travel grant from the Ryoichi Naito Foundation for Medical Research.

5 To whom correspondence should be addressed.

thrombinase” complex that consists of the serine protease factor Xa, the nonenzymatic cofactor factor Va, calcium ions, and an appropriate phospholipid membrane that serves as a template for the proper assembly of this catalytic unit (1). It is widely accepted that the activated platelet provides the necessary phospholipid membrane for the assembly of the prothrombinase complex during hemostasis (2-5). In addition to the platelet, other cell surfaces, including monocytes (6), lymphocytes (6), neutrophils (7), macrophages (8), endothelial cells (9, lo), and tumor cells (ll), have been shown to provide the appropriate surface for binding of factor Va and factor Xa and the assembly of the prothrombinase complex. Follow- ing assembly on these cell surfaces, the prothrombinase complex converts prothrombin to thrombin at a rate roughly 300,000-fold greater than that observed for factor Xa alone (2, 12).

Factor Va plays a pivotal role in the assembly of the prothrombinase complex on the platelet membrane. Factor Va is a heterodimeric protein consisting of a heavy chain (Mr = 94,000) and a light chain (Mr = 74,000) held together by a single calcium ion (13). Factor Va is derived from procofactor factor V, a high molecular weight glycoprotein (Mr = 300,000), through thrombin proteolysis that results in the excision of a highly glycosylated peptide (M, = 150,000) from the middle portion of the factor V molecule (14-16). The lipophilic light chain of factor Va not only mediates its interaction with the phospholipid membrane (17), but also serves as a high affinity receptor for factor Xa (& = 10-l’ M) (17). Thus, factor Xa and factor Va form a 1:l stoichiometric complex on the platelet membrane, and factor Va is essential for factor Xa binding and the expression of its activity on this, as well as several other, cell surface (4, 17).

Recent studies investigating the interaction of factor Xa with cultured endothelial cells revealed that the association of factor Xa with this cell surface may be uniquely different from that observed on platelets and peripheral blood cells. In these studies, ‘251-factor Xa interacted with -39,000 binding sites on the endothelial cell surface in a specific, saturable, and reversible manner with a dissociation constant of 3.6 x lo-’ M (18). Cell-bound factor Xa activated prothrombin in a reaction that was blocked by anti-factor V IgG, suggesting the involvement of endothelial cell-derived factor V (18). Inter- estingly, the binding of radiolabeled factor Xa to these cells was not influenced by anti-factor V IgG indicating that, unlike platelets and peripheral blood cells, endothelial cell surface factor V did not serve as the factor Xa receptor. This observation has been confirmed recently by Rao et al. (19) who clearly demonstrated that the order of exposure of human umbilical vein endothelial cells to factor Xa and to factor Va had no effect on the prothrombin activation rate. In previous

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9106 Factor Xa-Tumor Cell Interaction

studies (20), we examined four human tumor cell lines (HepG2, Colo-205, 582, and Capan-2) with respect to their ability to support the factor Xa-mediated activation of prothrombin in the presence and absence of exogenous factor Va. We observed that each tumor cell line supported this reaction in the absence of factor Va, with the order of effectiveness being Colo-205 > HepG2 > 582 > Capan- (20). Anti-factor V IgG inhibited this reaction on HepG2 and Colo-205 cells, but was without effect on 582 and Capan- cells. Biosynthetic labeling and immunoprecipitation experiments revealed con- stitutive synthesis of factor V, or a molecule immunologically related to human factor V, by HepG2 and Colo-205 cells. In order to further characterize prothrombin activation by factor Xa on tumor cells, we have examined in detail the interaction of human factor Xa with a tumor cell line that constitutively synthesizes factor V (HepG2) and one that does not produce factor V (582). The results of these studies, reported herein, demonstrate that factor Xa binding sites on these cells do not involve factor V of either endogenous or exogenous origin, and provide additional evidence that, like endothelial cells, the assembly of a functional prothrombinase complex on these tumor cells is distinctly different from that observed on platelets and peripheral blood cells.

EXPERIMENTAL PROCEDURES

Materials-DEAE-Sepharose CL-GB, dextran sulfate, Sephadex G- 25, Sephadex G-150, protein A-Sepharose, and SDS’ low M, standard kit were obtained from Pharmacia LKB Biotechnology Inc. Ultrogel AcA-22 was purchased from IBF Biotechnics. Protein assay dye reagent concentrate, Coomassie Brilliant Blue R-250, acrylamide, bisacrylamide, TEMED, and Chelex 100 were products of Bio-Rad. Sodium dodecyl sulfate was obtained from British Drug House. Benz- amidine HCl was a product of Aldrich. H-D-Phe-Pip-Arg-D-nitroanilide (S-2238) was obtained from Helena Laboratories (Beaumont, TX). Iodo-Gen was purchased from Pierce Chemical Co. Sodium “‘1 was obtained from Du Pont-New England Nuclear. Bovine serum albumin (fatty acid-free), ovalbumin (Grade V), trypsin (1 x solution; tissue culture grade), penicillin-streptomycin (10’ units of penicillin and 10 mg of streptomycin/ml), polyethylene glycol 8000, and minimum essential medium-a were obtained from Sigma. Minimum essential medium (Eagle’s) and nonessential amino acids were purchased from Mediatech. Fetal bovine serum was obtained from HyClone Laboratories. Tissue culture flasks and 12-well plates were purchased from Corning. Seakem ME agarose was obtained from FMC. Monospecific rabbit antiserum against human factor V was obtained from Dakopatts, Copenhagen, Denmark. Rabbit anti-factor V IgG and pre-immune rabbit IgG were prepared by protein A- Sepiarose c&omatography. Rabbit anti-human-factor v IgG was also kindlv nrovided bv Dr. Biorn Dahlback. Universitv of Lund. Malmo. Swed-&. Human brain cephalin (mixed’phospholipids) was prepared according to Bell and Alton (21). All other reagents were of the highest purity commercially available.

Proteins-Human plasma factors X, Xa, VII, VIIa, and IX were purified to homogeneity essentially as described (22). Human prothrombin was prepared according to Kisiel and Hanahan (23). Human cu-thrombin was purified by SP-Sephadex C-50 chromatography essentially according to Lundblad et al. (24) following activation of human prothrombin by a factor Xa/calcium/phospholipid mixture (25). Factor V was purified from normal pooled plasma by a method involving barium citrate adsorption, 8-12% polyethylene glycolSOO0 precipitation, DEAE-Sepharose CL-6B column chromatography, and Ultrogel AcA-22 column chromatography as described by Suzuki et al. (16). Conversion of factor V to factor Va was carried out by incubation with the purified factor V activator from Russell’s viper venom at an enzyme:substrate weight of 1:lOO as described previously (26). Bovine antithrombin III and rabbit anti-bovine antithrombin III-IgG were prepared as described (27). Human antithrombin III was purified as described (28).

’ The abbreviations used are: SDS, sodium dodecvl sulfate: Hepes, 4-(2-hydroxyethyl)-l-piperazineethane sulfonic-acid; TEMED; N,N,N’,N’-tetramethylethylenediamine; S-2238, H-o-Phe-pipecolic acid-Arg-p-nitroanilide; BSA, bovine serum albumin.

Cell Culture-The human bladder carcinoma 582 cell line (ATCC:HTB-1) was obtained from American Type Culture Collec- tion, Rockville, MD. The human hepatocellular carcinoma HepG2 cell line was kindly provided by Dr. Paul Mann, University of New Mexico. Albuaueraue. NM. Cells were maintained at 37 “C in an atmosphere containing 6% CO, and 98% relative humidity. 582 cells were grown in T-75 flasks containing minimum essential medium (Eagle’s) supplement with 10% heat-inactivated fetal bovine serum, nonessential amino acids, and penicillin-streptomycin. Prior to sub- culturing, the cells were removed from T-75 flasks using 5 ml of trypsin solution, resuspended in 10 ml of fresh medium containing 10% fetal bovine serum, and replated either in new T-75 flasks or in 12-well tissue culture plates. Binding and activation studies were performed within 24 h after reaching confluency.

Radioiodination-Human factor X and factor Xa were each radiolabeled to a specific activity of 1.5-2.2 &i/pg using the method of Fraker and Speck (29). Iodo-Gen (Pierce Chemical Co.) was prepared according to the manufacturer’s instructions, and 10 pg was dried in a polypropylene tube. Protein (100 pg), 0.5 M sodium phosphate (pH 7.2) (15 ~1) and 0.5 mCi of NaY (5 ~1) were added to the tube and allowed to react at 4 “C for 4 min. Then, 100 ~1 each of 1% KI and 1% ovalbumin in phosphate-buffered saline (pH 7.2) was added to stop the reaction. Free iodine was separated from the radiolabeled protein by gel filtration in a g-ml colimn of Sephadex G-25 equili- brated with nhosnhate-buffered saline containing 1% BSA. Radiola- beled factor-X and Xa were each purified by aisorption to barium citrate followed by elution with Tris-buffered saline/30 mM EDTA/ 0.1% BSA (pH 7.5) as described (30). Greater than 95% of the radioactivity was adsorbed to barium citrate, and the purified radioiodinated proteins were 95-99% precipitable by 10% trichloroacetic acid. No measurable difference was found for unlabeled and radioiodinated preparations of factor Xa with respect to their abilities to react with a lo-fold molar excess of human antithrombin III in the presence of heparin, as judged by SDS-polyacrylamide gel electrophoresis and subsequent autoradiography or Coomassie Blue staining. The clotting activities of the radiolabeled factor X and Xa preparations were 0.11 unit/pg and 15 unitslpg, respectively, as assessed in a one-stage factor X clotting assay. The specific activities of radiolabeled factor X and Xa preparations were approximately 90% of that observed for the respective unlabeled preparation. The protein concentrations of Y-factor X and ‘251-factor Xa were determined by Laurel1 rocket immunoelectrophoresis (31) using rabbit anti-human factor X IgG (30).

Binding Assays-The methodology utilized for measuring the association of ‘251-factor X and ‘251-factor Xa to HepG2 or 582 cells was a modification (30) of that described by Stern et al. (32). Twelve-well plates were initially seeded with 1.3 x lo5 cells/cm* and the cells allowed to grow to confluence. At confluence there were 1.8-2.1 x lo5 cells/cm*. Confluent cultures were washed once with 1 ml of Hepes- saline buffer (10 mM Hepes (pH 7.45), containing 137 mM NaCl, 4 mM KCl, and 11 mM glucose) supplemented with 10 mM EDTA, and subsequently washed-three times-with 1 ml of Hepes-saline buffer. Duplicate wells were incubated with radioiodinated ligand in 1 ml of Hepes-saline buffer supplemented with 0.5% BSA (Sigma) and 5 mM Ca&, (Hepes-saline buffer+) for 2 h at 4 or 37 “C-with constant oscillation (50 rum) on an orbit shaker (Lab-Line). After this period, the plates were rapidly washed six times with cold Hepeslsaline buffer+. The amount of cell-associated radioiodinated ligand was determined after lysing the cells in 1 ml of 200 mM NaOH, 1% SDS, 10 mM EDTA and counting in a Packard model 578 y scintillation spectrometer. Bound radioactivity was expressed as an average value of radioactivity observed in duplicate wells. Specific cell-associated radioligand was determined by subtracting the radioligand associated with the cells in the presence of 50-fold molar excess of unlabeled ligand from the total cell-bound radioligand.

Analysis of Prothrombin Activation by Cell-bound Factor X or Xa- Prothrombin activation was evaluated in a two-stage assay using a thrombin-specific chromogenic substrate, S-2238. Varying concentrations of either factor X 0; Xa were incubated with c&-&ent mono- lavers of HenG2 or 582 cells in a 12-well elate for 2 h at 4 or 37 “C and washed $ix times. Fifty ~1 of prothroibin (2 GM), 25 ~1 of 0.1 M CaCl* (5 mM) and 425 ~1 of Tris-buffered saline/O.l% BSA were added to the cell monolayers and incubated at room temperature. At selected intervals, lo-p1 aliquots of the reaction mixture were removed and added to 890 gl of Tris-buffered saline/O.05 M imidazole (pH 8.3) and 100 ~1 of 1 mM S-2238. The absorbance at 405 nm was continu- ously recorded using a Beckman DU-65 spectrophotometer. Throm- bin concentration was interpolated from a standard curve of AA&


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Factor Xa-Tumor Cell Interaction

min versus thrombin concentration. Inhibition of Factor Xa Binding and Prothrombin Activation by

Polyclonal Anti-human Factor V ZgG-Confluent monolayers of each cell line were washed once with Hepes-saline buffer supplemented with 10 mM EDTA, and subsequently washed three times with Hepes- saline buffer. Duplicate wells were incubated with 1 ml of Hepes- saline buffer+ containing either pre-immune rabbit IgG or anti-human factor V IgG for 2 h at 37 “C. Following removal of the IgG solution, binding and prothrombin activation studies were performed as described above.

Effect of Exogenous Factor Vu on Factor Xa Binding and Prothrom- bin Actiuation-Washed confluent monolayers of the cells as described above were incubated with various concentrations of human factor Va in Hepes-saline buffer+ for 2 h at 4 “C. The plates were washed six times with cold Hepes-saline buffer+, and binding and prothrombin activation studies were performed as described above.

Electrophoretic Analysis of Cell-associated Radiolabeled Proteins- Confluent monolayers of the cells were washed once with Hepes- saline buffer supplemented with 10 mM EDTA, and subsequently washed three times with Hepes-saline buffer+. The wells were incubated with either 1 nM “‘I-factor X and Xa or ““I-factor Xa for 2 h at 4 and 37 “C. After this period, the cells were washed six times with cold Hepes-saline buffer+ and lysed with 20 mM Tris-HCl (pH 7.5), 1% SDS. Samples were subjected to electrophoresis in SDS-lo% polyacrylamide slab gels according to Laemmli (33), and developed by autoradiography.

RESULTS

The interaction of iz51-factors X and Xa with HepGZ cell monolayers that constitutively synthesize a factor V/Va molecule (20,33), and 582 cell monolayers which do not (20), was examined in radioligand binding experiments. In initial experiments, we examined the calcium dependence of ‘251-factor X and Xa specific binding to the HepG2 and 582 cell monolayers at 4 “C. In these experiments, tumor cells were incubated with either 5 nM iz51-factor X or 5 nM ‘251-factor Xa for 2 h at 4 “C in the presence of O-20 mM CaC12. As illustrated in Fig. 1, no significant specific binding of ‘251-factor X to either cell was observed as a function of calcium concentration (Fig. 1). In contrast, ‘251-factor Xa binding to each cell monolayer progressively increased from 0 to 5 mM calcium and declined between 5 and 20 mM calcium (Fig. 1). Interestingly, approximately 70-75% of maximal ‘251-factor Xa binding was observed in the absence of added calcium (0 mM) on both cell monolayers (Fig. 1). In addition, following a 2-h incubation at 4 “C in the presence of 5 mM calcium, approximately 30% of cell-bound ‘251-factor Xa was eluted from the cell surface by treatment with 10 mM EDTA (data not shown). To rule out the possibility of calcium contaminating the calcium-free Hepes-saline+ used in these experiments, we passed the Hepes-saline+ buffer over a Chelex 100 column and repeated the experiment using ‘251-factor Xa pretreated with 10 mM EDTA followed by dialysis against Hepes-saline buffer. Iden- tical results were obtained with the Chelex loo-treated buffer, which by atomic absorption spectrophotometric analysis (Per- kin-Elmer model 2380) revealed ~0.5 fig/ml calcium. Accord- ingly, it appears that significant binding of ‘251-factor Xa to both tumor cell monolayers occurs in the absence of calcium. For subsequent studies, however, 5 mM calcium was included in the incubation mixtures to effect maximal binding of lz51- factor Xa.

We next determined the time dependence, temperature dependence, and reversibility of ‘251-factor X and ‘251-factor Xa binding to each tumor cell monolayer (Fig. 2). Incubation of each monolayer with either 5 nM’251-factor X or Xa resulted in time-dependent total binding that reached an apparent steady-state after 2 h. Approximately lo-20-fold more factor Xa than factor X bound to each tumor cell line at both temperatures. The binding of ‘251-factor Xa to HepG2 or 582 at 37 “C was increased 35 and lo%, respectively, when com-

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FIG. 1. Calcium dependence of specific cell binding of factor X or factor Xa to HepG2 cells (A) or 582 cells (B) at 4 “C. lz51- Factor X (0) or ‘251-factor Xa (0) was incubated with the cells for 2 h at 4 “C in the presence of O-20 mM CaCb. Specific binding was determined by subtracting the radioactivity bound in the presence of a 50-fold molar excess of unlabeled offered ligand from the total radioactivity bound.

pared to binding at 4 “C. Addition of 50-fold molar excess of unlabeled factor Xa to HepG2 cells following a 2-h incubation with 5 nM ‘251-factor Xa demonstrated that radioligand binding was largely reversible at 4 “C. However, at 37 “C significant radioligand remained bound to the cell monolayer under these conditions. By comparison, there was little difference between 4 and 37 “C with respect to the reversibility of 1251- factor Xa bound to 582 cells. This suggested that HepG2 cells internalized ‘251-factor Xa at 37 “C to a greater extent in comparison to 582 cell-bound 1251-factor Xa. Significant reversibility of cell-bound factor X was not observed on either tumor cell monolayer (data not shown).

In order to determine the association constant and number of binding sites per cell for ‘Y-factors X and Xa, we assessed specific binding of each radioligand over a wide concentration range of offered radioligand. Specific binding was determined by subtracting the ‘251-factor X and Xa bound in the presence of 50-fold molar excess of respective unlabeled ligand from the total radioligand bound. As shown in Fig. 3, using concentrations of ‘251-factor X and Xa ranging from 0.5 to 10 nM, 1251-factor Xa binding to each tumor cell approached saturation at approximately 5 nM. Significant specific binding of ‘251-factor X to either tumor cell was not observed (Fig. 3). Specific binding of ‘251-factor Xa increased linearly at 4 “C on each tumor cell when radioligand concentrations of 50 pM to 1 nM were employed (data not shown). The binding isotherm of ‘251-factor Xa on each tumor cell monolayer exhibited a hyperbolic profile. Scatchard analysis indicated a single class


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Time (hr)

d m

1 2 3 4

Time (hr)

FIG. 2. Time course and reversibility of “‘I-factor X or Xa binding to HepG2 cells (A) or 582 cells (B) at 4 and 37 “C. Five nM ‘251-factor X (0, A) or 5 nM “‘I-factor Xa (0, A) were incubated with the cells at 4 “C (A, A) or 37 “C (0, 0). After incubation with ‘T-factor Xa for 2 h, a M-fold molar excess of unlabeled factor Xa was added to the cells (arrow). At selected intervals, the cells were washed, and the bound radioactivity counted.

of binding site for factor Xa with Kd values of 1.66 f 0.39 and 1.64 f 0.52 nM and 566,000 f 71,000 and 28,000 + 6,000 binding sites/cell to HepG2 and 582 cells, respectively (Fig. 4).

To demonstrate the specificity of radiolabeled factor X or Xa binding to each tumor cell, ‘251-factor X (5 nM) or 1251- factor Xa (5 nM) was incubated for 2 h at 4 “C! with each cell monolayer in the presence of 50-fold molar excess of either unlabeled factor X, factor Xa, human prothrombin, human factor VII, human factor VIIa, or human factor IX. Under these conditions, only unlabeled factor Xa specifically inhibited ‘251-factor Xa binding to both tumor cells by 65-90% (Tables I and II). Unlabeled factor X did not inhibit 1251- factor Xa binding to either tumor cell, and only weakly inhibited ‘251-factor X binding to each cell. Concerning the latter finding, it is perhaps important to emphasize that an accurate measurement of ‘251-factor X binding in the presence of the above competitors was technically difficult given the small amount of ‘251-factor X bound to each tumor cell. We then assessed the proteolytic activity of either cell-bound factor X on factor Xa toward prothrombin with respect to calcium dependence, time dependence, and concentration of the offered ligand. In the presence of O-20 mM CaC12, no prothrombin activation was observed on either tumor cell line offered 5 nM factor X (Fig. 5). In comparison, equilibration of either tumor cell monolayer with 5 nM unlabeled factor Xa in the presence of O-20 mM CaC12 revealed significant (50% of maximum) prothrombin activation on each tumor cell in

Factor X or Xa Added (nM)

0 5 10


FIG. 3. Specific binding of “‘I-factor X or Xa to HepG2 cells (A) or 582 cells (B) at 4 “C. Increasing concentrations of ‘251-factor X (0) or Xa (0) were incubated with the cells for 2 h at 4 “C. Specific binding was determined as indicated in Fig. 1 legend.

the absence of calcium (0 mM) consistent with the ‘Z51-factor Xa specific binding data. In addition, similar to the ‘251-factor Xa binding data, maximal prothrombin activation was observed at 5 mM calcium on each tumor cell and this rate declined slightly at calcium concentrations between 5 and 20 mM (Fig. 5). In the presence of 5 mM calcium and 5 nM factor Xa, maximal cell surface prothrombin activating activity was achieved at 4 “C in -2 h, in agreement with the time dependence for ‘251-factor Xa binding (data not shown). In subsequent saturation binding and activation studies, increasing concentrations of unlabeled factor X or factor Xa were added to each tumor cell and incubated at either 4 “C or 37 “C for 2 h in the presence of 5 mM calcium. After washing, the rate of prothrombin activation was determined in an amidolytic assay (Fig. 6). No thrombin formation by cell-associated factor X was observed on either tumor cell, suggesting that the factor X was not activated on the tumor cell surface. Throm- bin formation by cell-bound factor Xa on both tumor cells was hyperbolic with increasing concentrations of factor Xa added at 4 “C and 37 “C (Fig. 6). In addition, a linear rate of thrombin formation was observed at factor Xa concentration between 50 pM and 1 nM (data not shown). Thrombin formation approached saturation at 5 nM factor Xa on both tumor cells. The rate of thrombin formation on HepG2 cells was 5-fold higher than that observed on 582 cells at 4 “C. In addition, the rate of thrombin formation at 37 “C was lower than that observed at 4 “C on each tumor cell. Although not proven, it is highly likely that internalization of factor Xa by each cell occurred at 37 “C. Hanes-Woolf plots of the prothrombin activation data at 4 “C indicated that half-maximal rates of thrombin formation occurred at factor Xa concentra-


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Factor Xa-Tumor Cell Interaction 9109

500

Bound (fmol) 0

I I I

10 20 30

Bound (fmoll

FIG. 4. Scatchard plot of ‘251-factor Xa specific binding to HepG2 cells (A) and 582 cells (B) at 4 “C. Each point represents the mean of three separate experiments.

TABLE I Specificity of Factor X and Factor Xa binding to HepG2 cells

Competitof Y-Factor X Bound ‘“I-Factor Xa bound bound Bound

None Factor X Factor Xa Prothrombin Factor VII Factor VIIa Factor IX

fmol/well % fmol/well % 32 100 560 100 27 84 531 95 29 91 55 10 34 106 572 102 31 97 557 99 31 97 580 104 33 103 570 102

a Competing proteins were added at a 50-fold molar excess with 5 nM ‘*‘I-factor X or Xa on HepG2 cells and incubated for 2 h at 4 “C.

TABLE II Specificity of Factor X and Factor Xa binding to 582 cells

Competitof ‘*‘I-Factor X Y-Factor

bound Bound Xa Bound bound

fmol/well % fmol/well % None 6 100 40 100 Factor X 5 83 39 98 Factor Xa 6 100 13 33 Prothrombin 6 100 42 105 Factor VII 7 117 40 100 Factor VIIa 7 117 43 108 Factor IX 6 100 43 108

a Competing proteins were added at a 50-fold molar excess with 5 nM ““I-factor X or Xa on 582 cells and incubated for 2 h at 4 “C.

a 0 5 10 15 20


e

B 0 5 10 15 20


FIG. 5. Calcium dependence of prothrombin activation by cell-bound factor X (0) or factor Xa (0). Factor X or factor Xa (5 nM) was incubated with either HepG2 cells (A) or 582 cells (B) at 4 “C in the presence of varying concentrations of CaCh. The cells were then washed and prothrombin activation assessed in an amidolytic assay.

tions of 1.50 + 0.43 and 1.42 + 0.48 nM on HepG2 and 582 cells, respectively (Fig. 7).

To study the putative role of cell surface factor V with respect to factor Xa binding and prothrombin activation, we examined the binding of ‘251-factor Xa and thrombin formation by cell-bound factor Xa on each monolayer following preincubation of the cells with either rabbit anti-human factor V IgG or pre-immune rabbit IgG as a control. In these studies, cells were pretreated with various concentrations of IgG for 2 h at 37 “C, washed three times with Hepes-saline buffer+ to remove unbound IgG, and ‘261-factor Xa binding and prothrombin activation studies were examined at 4 “C. As shown in Fig. 8A, ‘251-factor Xa specific binding to HepG2 cells was unaffected by either anti-factor V IgG or pre-immune rabbit IgG, whereas prothrombin activation on these cells was inhibited in a dose-dependent manner by anti-factor V IgG. In

lz5 comparison, neither I- factor Xa binding to 582 cells nor prothrombin activation on 582 cells was blocked by anti- factor V IgG (Fig. 8B). Factor Xa did, however, appear to bind to a site on the HepG2 cell surface in the vicinity of factor V/Va, inasmuch as cell surface prothrombinase activity was blocked by anti-factor V IgG. This suggested that endogenous factor V/Va on the HepG2 cell surface did not serve as a receptor for factor Xa. We next examined the effect of exogenous factor Va on binding of Y-factor Xa and prothrombin activation on these tumor cells (Fig. 9). In these studies, tumor cells were pretreated with various concentrations of factor Va for 2 h at 4 “C, washed six times, and subsequently examined for ‘251-factor Xa binding and factor Xa-mediated prothrombin activation at 4 “C. Exogenous fac-


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Factor X or Xa Added (nMt

al ii i 0 5 10 K


FIG. 6. The rate of thrombin formation by factor X or factor Xa bound to HepC‘2 cells (A) and 582 cells (B) at 4 and 37 *C. Increasing concentrations of factor X (0, A) or factor Xa (0, A) were incubated for 2 h at 4 “C (0,O) or 37 “C (A, A) with the cells. After washing, prothrombin was added and the rate of thrombin formation was determined at selected intervals by an amidolytic assay described under “Experimental Procedures.”

tor Va had little effect on the binding of factor Xa to either HepG2 or 582 cell monolayers (Fig. 9). Prothrombin activation by HepG2 cell-bound factor Xa was increased -50% and this effect was achieved at 5 nM added factor Va. On the other hand, prothrombin activation by 582 cell-bound factor Xa was greatly augmented by pretreating the cell with increasing concentrations of exogenous factor Va. Furthermore, to elu- cidate the potential interaction of factor Xa and factor Va, we varied the order of adding factor Xa or factor Va to the 582 cells and subsequently assessed prothrombin activation by 582 cell-bound factor Xa. In these experiments, J82 cells were initially incubated with 5 nM factor Xa for 2 h at 4 “C, washed six times, and treated with various concentrations of exogenous factor Va for 2 h at 4 “C, washed six times, and finally examined for their ability to activate prothrombin. In this instance, the rate of thrombin formation was 2-fold higher than that observed when the cells were first pre-incubated with factor Va. This suggested that factor Xa binds to 582 cells independent of cell-associated factor Va. Thus, factor Va does not appear to be essential for functional binding of factor Xa to either HepG2 or 582 cells.

To study whether “‘I-factors X and Xa bound to the cells had experienced proteolytic processing at the cell surface, HepG2 and 582 cells were incubated with “51-factor X or Xa for 2 h at 4 and 37 “C. After washing, the cells were lysed and subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. As shown in Fig. 10, HepGP or 582 ceil-bound Y-factor Xa was identical to ‘251-factor Xa offered to the cells regardless of the incubation temperature. Due to the weak binding of ‘*?-factor X to each tumor cell, we were

2 1 5

Factor Xa Added (nM)

10 @

210 5 10

Factor Xa Added (nM)

FIG. 7. Hanes-Woolf plot of prothrombin activation by HepG2 (A) and 582 (B) cell-bound factor Xa at 4 “C. Each point represents the mean of three separate experiments.

unable to obtain sufficient recovered radioactivity to assess the structure of cell-bound factor X.

DISCUSSION

The data presented in this report indicate that factor Xa, but not its precursor factor X, interacts specifically with monolayers of human hepatocellular carcinoma (HepG2) and human bladder carcinoma (582) cell lines. These tumor cell lines were specifically chosen to study cell surface assembly of the prothrombinase complex on the basis of their ability to constitutively synthesize factor V. As HepGX cells synthesize a functional factor V molecule (20, 34), while 582 cells do not (20), we postulated that factor Xa interaction with the HepG2 cell surface may involve factor V. The results of our studies provide definitive evidence that this indeed is not the case. Factor Xa interacted with -566,000 binding sites per HepGP cell and -28,000 binding sites per 582 cell with essentially the same dissociation constant (1.66 uersus 1.64 nM). Factor Xa binding to each tumor cell was time-, temperature-, and calcium-dependent. The binding of factor Xa to these tumor cell lines was not inhibited by pre-treatment and/or co-incubation of the cells with monospecific antibodies against human factor V. Furthermore, while preincubation of each tumor cell with purified human factor Va potentiatedprothrom- bin activation on the tumor cell surface, this cell-associated factor Va had little, if any, affect on ‘251-factor Xa binding. Thus, it is highly unlikely that factor V/Va, whether derived from endogenous or exogenous sources, constitutes the cell surface receptor for factor Xa on these tumor cells. In this regard, these tumor cells appear to assemble a functional prothrombinase complex similar to that reported for bovine aortic endothelial cells (18), but distinct from that reported


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m / \ I c

IgG Concentration (mgiml)

z 03 2 I E

H

52 1.0 r

s .- 5 30 E b IL

20 0.5 .g

E 10

c

z 0

z 0 0.001 0.01 0.1 1 u

IgG Concentration (mg/ml)

FIG. 8. The effect of pre-treating HepG2 cells (A) and 582 cells (B) with anti-factor V IgG on ““I-factor Xa specific binding and prothrombin activation by cell-bound factor Xa. After the cells were pretreated with various concentrations of anti- factor V IgG (0, A) or pre-immune rabbit IgG (0, A), the binding of ‘““I-factor Xa (A, A) and the rate of thrombin formation (0, 0) was examined.

earlier for the platelet and a variety of peripheral blood cells (l-8).

Two aspects of the factor Xa interaction with these tumor cell lines merit further consideration and discussion. First, although the binding of factor Xa to each cell was largely reversible when examined at 4 “C, considerable radioligand remained bound to the cell after 2 h of incubation at 37 “C and treatment with a 50-fold molar excess of unlabeled factor Xa. Although not proven here, it is probable that a significant amount of the radioligand was internalized by endocytosis at this temperature. Other events, such as covalent complex formation between factor Xa and cell surface serpins derived either from the cell or the serum-containing media, conceiv- able may also contribute to the poor reversibility of factor Xa from the cell surface at 37 “C. In this regard, however, pretreatment of the tumor cell monolayers with rabbit anti- antithrombin III-IgG had no measurable affect on the binding of factor Xa to either of these cells (data not shown). In addition, SDS-polyacrylamide gel electrophoresis/autoradiography of cell-bound factor Xa revealed no evidence for either proteolytic degradation of factor Xa or complex formation with a protease inhibitor derived from either the cell or the media. Thus, although indirect, all of our experimental evidence points to endocytosis of factor Xa by these tumor cells at 37 “C.

The second issue relates to the rather substantial specific binding of factor Xa to each tumor cell observed in the absence of calcium ions. In our hands, 70-75% of maximal factor Xa- specific binding was observed using a calcium-free incubation

L---

0 05 10

Factor Va Added (nM)

L I I 0 5 10 15 20

Factor Va Added hM)

The effect of pretreating HepG2 Fro 9. cells (A) and 082 cells (B) with exogenous human factor Va on the specific binding of ““I-factor Xa and prothrombin activation by cell- bound factor Xa. After the cells were incubated with various concentrations of exogenous factor Va for 2 h at 4 “C, the binding of ““I- factor Xa (A) and the rate of thrombin formation (0) was examined. Alternatively after incubating the 582 cells with factor Xa for 2 h at 4 “C, the cells were washed, various concentrations of factor Va added, and the rate of thrombin formation (0) was determined.

- - y-30 - 111, m *20

12345678

FIG. 10. Autoradiography of ‘““I-factor X and Xa recovered from the cells. HepG2 cells and 582 cells were incubated with 1 nM ““I-factor X or Xa for 2 h at 4 “C or 37 “C. After the cells were washed and lysed with 20 mM Tris-HCl (pH 7.5), 1% SDS, the recovered “‘I-factor X or Xa was subjected to electrophoresis on SDS-lo% polyacrylamide slab gels and developed by autoradiography. Lanes I and 2, “‘I-factor X under nonreduced and reduced conditions, respectively; lanes 3 and 4, ‘“I-factor Xa under nonreduced and reduced conditions, respectively; lanes 5 and 6, HepG2 cell-bound IL’I- factor Xa recovered from the cells after incubation at 4 “C for 2 h under nonreduced and reduced conditions, respectively; lanes 7 and 8, 582 cell-bound ““I-factor Xa recovered from the cells after incubation at 4 “C for 2 h nonreduced and reduced conditions, respectively.

buffer. Furthermore, -30% of cell-bound ““I-factor Xa was eluted from the cells with 10 mM EDTA following incubation in the presence of 5 mM calcium. That cell-bound factor Xa in the absence of calcium is functionally active and unrelated to the radiolabeling process is supported by prothrombin activation studies. In these studies, using unlabeled factor Xa, prothrombin activation rates by cell bound factor Xa exhib-


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9112 Factor Xa-Tumor Cell Interaction

ited a dependence on calcium concentration similar to that observed for “‘I-factor Xa binding. Given that Scatchard plots, derived from factor Xa-specific binding in the presence of 5 mM calcium, each indicated a single class of binding sites on the tumor cell surface for factor Xa, it is intriguing as to precisely how calcium increases the number of factor Xa molecules bound to the cell over that observed in the absence of calcium. One could speculate that factor Xa binds calcium and, in the process, undergoes a conformational change that results in a factor Xa-calcium complex. This complex then interacts with a specific class of binding sites on the cell surface distinct in character and number from the cell surface binding sites that interact with factor Xa. The fact that approximately 30% of cell-bound iz51-factor Xa was eluted from the cell by rapid EDTA treatment would seemingly argue against this proposal. The latter observation would appear to be more consistent with lz51-factor Xa binding to a single class of binding sites on the cell surface and calcium, in some unknown manner, potentiating this interaction. Alterna- tively, it is not inconceivable that calcium may, in fact, alter the binding site for factor Xa on these cells rather than induce a conformational change in the ligand that facilitates its interaction with the cell surface. The possibility that factor Xa interacts with a nonproductive site in the absence of calcium and rapidly redistributes to a productive site following the addition of calcium and prothrombin would appear to be remote, as generation of thrombin was linear at several time points during the first 30 min of incubation. Thus, additional experiments are needed to further characterize the nature and significance of the factor Xa-tumor cell interaction observed in the absence of calcium.

Tumor cell-bound factor Xa activated prothrombin in the absence of added factor V. Half-maximal rates of prothrombin activation occurred at 1.50 and 1.42 nM factor Xa on HepG2 and 582 cells, respectively. Thus, binding of factor Xa to these cells (& = 1.65 nM) and the expression of cell-bound factor Xa proteolytic activity appear to be in reasonably good agreement, Inasmuch as 582 cells do not synthesize factor V (20), the rate of prothrombin activation by 582 cell-bound factor Xa was, as expected, unaffected by pretreatment of the cells with anti-factor V IgG. On the other hand, HepG2 cells constitutively synthesize factor V (20,33) that greatly poten- tiates the rate of prothrombin activation by cell-bound factor Xa, and pretreatment of these cells with anti-factor V IgG markedly inhibited thrombin formation by cell-bound factor Xa. Consistent with their ability to synthesize factor V, exogenous factor V offered to HepG2 cells produced only a modest increase in prothrombin activation rate in relation to that observed in comparable experiments with 582 cells. This suggested that, to a large degree, HepG2 cell surface binding sites for offered factor Va were occupied by endogenous factor V/Va synthesized by the HepG2 cell. Any remaining factor Va binding sites on the HepGP cell were completely occupied in the presence of 5 nM offered factor Va (Fig. 9). In comparison, 582 cells readily bound exogenous factor Va, and these cell surface binding sites appeared to saturate at -20 nM factor Va. Thus, despite its inability to synthesize factor V, 582 cells possess binding sites for factor V/Va that results in cell surface binding and functional expression of this cofactor.

Our present results, coupled with data reported earlier by our laboratory (30), as well as those from other laboratories (11, 35, 36), provide a plausible mechanism for the initiation and propagation of the extrinsic pathway of blood coagulation on tumor cells that express functional cell surface tissue factor. In this scheme, factor VII/VIIa binds to cell surface tissue factor and subsequently activates factor X by limited

proteolysis. Newly formed factor Xa, in turn, binds to a cell surface site adjacent to factor V/Va of either cellular or extracellular origin, and converts prothrombin to thrombin. Fibrin deposition in the pericellular space would rapidly ensue as a result of thrombin proteolysis of fibrinogen (37). Presum- ably, all the necessary components of this scheme, with the exception of tissue factor, are derived from the hyperperme- able tumor microvasculature (38). Whether this process is regulated by either serine proteinase inhibitors and/or components of the protein C anticoagulant pathway is unknown and will require several additional studies.

Acknowledgments-We would like to thank Dr. Bjorn Dahlback for providing us with a preparation of anti-human factor V IgG used in this study, and Dr. Masashi Noguchi for helpful discussions. We also wish to thank Nancy Basore for excellent technical assistance, and Margie Topper for typing the manuscript.

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T Sakai and W KisielEvidence that factor Xa binds to tumor cells independent of factor Va.

Binding of human factors X and Xa to HepG2 and J82 human tumor cell lines.

1990, 265:9105-9113.J. Biol. Chem.

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Binding of Human Factors X and Xa to HepG2 and 582 Human