mechanisms of binding of recombinant extrinsic …repi to two cell lines possessing surface tissue...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 2, Issue of January 15, pp. 876-882,1992 Printed in V. S. A.

Mechanisms of Binding of Recombinant Extrinsic Pathway Inhibitor (rEPI) to Cultured Cell Surfaces EVIDENCE THAT rEPI CAN BIND TO AND INHIBIT FACTOR VIIa-TISSUE FACTOR COMPLEXES IN THE ABSENCE OF FACTOR Xa*

(Received for publication, June 28, 1991)

Natalie S . CallanderSQ, L. Vijaya Mohan RaoSn, Ole NordfangJJ, Per Morten Sandset$, Bonnie Warn-CramerS, and Samuel I. RapaportS**$$ From the Departments of $Medicine and **Pathology, University of California, Sun Diego, California 92093 and ![Novo-Nordisk AIS, lDK2820 Gentofre Denmark

Extrinsic pathway inhibitor plays a key role in modulating tissue factor-dependent blood coagulation. We have studied binding of radioiodinated recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. rEPI in the absence of added reactants bound to a limited extent to three cell lines studied. Binding of rEPI to two cell lines possessing surface tissue factor, but not to a cell line lacking surface tissue factor, was markedly increased in the presence of both factor VIIa and factor Xa, and calcium ions. Moreover, some increased tissue factor-dependent binding was also demonstrated with factor VIIa alone. Binding isotherms of rEPI to factor VIIa-tissue factor obtained with an ovarian carcinoma cell line were hyperbolic. Scatchard plots indicated the following: a Kd value of 4.5 2 1.5 nM and 335,000 f 84,000 siteslcell when factor Xa was present; a Kd value of 11.9 f 3.5 nM and 236,000 f 68,000 sites/cell when factor Xa was absent. In functional studies, high concentrations of rEPI, e.g. 27-67.6 nM, were found to inhibit factor VIIa-tissue factor-catalyzed release of activation peptide from tri- tiated factor IX in the absence of factor Xa. Whereas factor Xa was thus shown not to be required for rEPI to inhibit factor VIIa-tissue factor catalytic activity, its presence markedly enhanced rEPI’s inhibitory function. Since the local concentration of extrinsic pathway inhibitor achieved at a site of tissue injury is unknown, the physiologic significance of the observation of extrinsic pathway inhibitor-induced inhibition of factor VIIa-tissue factor activity in the absence of factor Xa is not clear. However, factor Xa-independent inhibition could play a significant role when large doses of rEPI are administered in experimental studies of thrombosis.

* This work was supported by Grants HL-42813 (to L. V. M. R.) and HL-27234 (to S. I. R.) from the National Heart, Lung, and Blood Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 Supported by Training Grant CA 09290 from the National Insti- tutes of Health during this research.

T Recipient of a research career development award from the National Heart, Lung, and Blood Institute.

$3 To whom reprint requests should be addressed Dept. of Medi- cine (8423), UCSD Medical Center, 225 Dickinson St., San Diego, CA 92103. Tel.: 619-543-3552.

Extrinsic pathway inhibitor (EPI)’.’ (l), also known as lipoprotein-associated coagulation inhibitor (2), is a protease inhibitor containing three tandem Kunitz-type domains (3) that plays a key role in modulating TF-dependent blood coagulation (4-6). Generation of factor Xa has been thought necessary before EPI can inhibit factor VIIa-TF enzymatic activity (1, 7, 8). Inhibition presumably results from a two- step reaction leading to the formation of a quaternary EPI- factor Xa-factor VIIa-TF complex (2, 9). In the first step factor Xa binds to the second Kunitz domain of EPI (10) with resultant neutralization of factor Xa. In the second step this EPI-factor Xa complex is thought to bind to a factor VIIA- TF complex through an interaction between the first Kunitz domain and factor VIIa-TF (10). Recently a genetically en- gineered hybrid molecule consisting of the y-carboxylated light chain of factor Xa and the first Kunitz domain of EPI has been reported to be a more potent inhibitor of factor VIIa-TF than the combination of an intact EPI molecule and factor Xa (11).

Functional studies have supported the hypothesis that factor Xa is required for EPI to bind to factor VIIa-TF (l), but direct binding studies of EPI to cell surfaces possessing TF activity have not as yet been reported. The availability of rEPI has made it possible to conduct such studies, which are described here. They reveal that factor Xa enhances but is not an absolute requirement for the binding of EPI to factor VIIa-TF complexes on cell surfaces with subsequent neutralization of factor VIIa-TF catalytic activity.

EXPERIMENTAL PROCEDURES

Reagents-Sodium [1251]iodide and sodium b~ro[~H]hydride were purchased from Amersham Corp. Iodogen was from Pierce Chemical Co. Heparin (bovine lung) was from either Calbiochem or Abbott Laboratories. Chromozym X was from Boehringer Mannheim. Dul- becco’s modified Eagle’s medium was obtained from JRH Biosciences, and RPMI-1640 was from Whittaker. Ex-Cell 320 serum-free medium was from Hazelton Biosciences. All chemicals for analytical SDS- PAGE were obtained from Bio-Rad. All other chemicals, reagent grade or better, were from Fisher or Sigma.

The abbreviations used are: EPI, extrinsic pathway inhibitor; rEPI, recombinant extrinsic pathway inhibitor; TF, tissue factor; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; Hepes, 4-(2-hydroxyethyl)-l-pi- perazineethanesulfonic acid.

The Scientific and Standardization Committee of the Interna- tional Society on Thrombosis and Haemostasis has recently recom- mended that the name tissue factor pathway inhibitor (TFPI) be used for the inhibitor described herein in lieu of its current two names: extrinsic pathway inhibitor (EPI) and lipoprotein-associated coagulation inhibitor (LACI).

876

Binding of rEPI to Cultured Cell Surfaces a77 Cell Lines-A human ovarian carcinoma cell line OC-2008, which

constitutively expressed abundant TF, and a second ovarian carcinoma cell line, OC-735, with no measurable TF, were obtained from Dr. Stephen Howell, University of California, San Diego Medical Center. Human lung fibroblasts WI-38 were obtained from American Type Culture Collection. Carcinoma cells were grown in T-75 flasks in RPMI-1640 medium supplemented with 5% fetal calf serum, 2 mM glutamine, and penicillin/streptomycin in a humidified incubator with 5% C02 at 37 "C. Prior to subculturing the cells were removed from T-75 flasks using 5-7 ml of trypsin solution, resuspended in 10 ml of fresh medium containing fetal calf serum, and replated either in new T-75 flasks or culture plates. Fibroblasts were grown using the same procedure in Dulbecco's modified Eagle's medium supplemented with 1 g/liter glucose, 10% fetal calf serum, 1% glutamine, and penicillin/streptomycin. For some studies, cells were grown under identical conditions except that the serum supplemented RPMI or Eagle's medium was replaced with Ex-Cell 320 serum-free medium. Binding studies were performed when cells had reached confluency, approximately 72 h after seeding onto 12-well polystyrene culture plates (Corning Glass Works). At confluency, the cell viability and number were calculated using the trypan blue exclusion method. The viability of both carcinoma cell lines and fibroblasts was more than 98%. Cell numbers were as follows: OC-2008, 1.4 k 0.15 X lo6 cells/ well; WI-38 fibroblasts, 2.5 rt 0.25 X lo6 cells/well.

Coagulant Proteins-Recombinant human EPI was expressed in baby hamster kidney cells and purified as described in detail by Pedersen et a!. (12). Its protein concentration was determined spec- trophotometrically using an extinction coefficient of 5.3 at 280 nm (13). Molecular mass was estimated from SDS-PAGE to be 37 kDa. The specific activity of the preparation was 15,000 units/mg, when activity was measured in a two-stage chromogenic assay. In the first stage of this assay, a dilution of rEPI was incubated for 30 min with a saturating concentration of factor VIIa, a limiting concentration of TF, a low concentration of factor Xa, and calcium ions. In the second stage, a high concentration of factor X was added to the reaction mixture as a substrate for residual factor VIIa-TF catalytic activity, and the factor Xa activity generated was measured with an amidolytic substrate. The assay was performed using human reagents essentially as described in detail earlier (6). The activity present in 1 ml of a pooled normal human plasma standard was defined as 1 unit of EPI activity.

Human clotting factors IX and X were prepared as described previously (14). Factor Xa was prepared by activating purified factor X with insolubilized Russell's viper venom (14). Recombinant factor VIIa was purchased from Novo-Nordisk. Annexin V (15) was the gift of Dr. Jonathan F. Tait, University of Washington, Seattle. Purified human brain T F apoprotein was prepared as previously described (16), and the apoprotein was incorporated into phospholipid vesicles, 40% phosphatidylserine and 60% phosphatidylcholine, as described earlier (17) according to the method of Mimms et al. (18).

Antibodies-Antibodies to human factor X were raised in a rabbit, and the IgG fraction was purified as described previously (16). The IgG was adsorbed with barium sulfate (100 mg/ml) for 20 min at room temperature to remove any possible traces of contaminating rabbit factor X or factor Xa. The supernatant after centrifugation at 12,000 X g for 20 min was dialyzed overnight a t 4 "C against 10 mM Hepes, 0.15 M NaCI, pH 7.5. This anti-factor X IgG at a concentration of 0.2 mg/ml neutralized over 99% of the factor X clotting activity of normal plasma.

Rabbit anti-human TF antibodies were prepared as previously described (19). Antibodies to human recombinant factor VIIa were raised in a rabbit, and the IgG fraction was purified as described previously (16).

Radiolabeling-Recombinant EPI was radiolabeled using the Io- dogen method (20). '261-rEPI had a specific radioactivity varying from 3 to 6 X lo6 cpm/pg protein and retained 83% of its original activity in the two-stage chromogenic EPI activity assay. Sialyl [3H]factor IX was prepared by the general technique of Van Lenten and Ashwell (21). Specific radioactivity of [3H]factor IX was 2-4 X l@ cpm/mg. The labeled preparations retained about 90% of the biological activity as measured in a one-stage factor IX clotting assay.

Binding Assays-Confluent monolayers were washed with 1 ml of a buffer containing 10 mM Hepes, 0.15 M NaCl, 4 mM KCl, and 11 mM glucose, pH 7.45 (Buffer A) supplemented with 5 mM EDTA, and then washed 3 more times with 1 ml of Buffer A not containing EDTA. Duplicate wells were incubated at room temperature with lZ6I- rEPI (2.7 nM), factor Xa (10 nM), and factor VIIa (10 nM) in a total volume of 0.5 ml of the above buffer supplemented with 5 mM CaClz

and 1 mg/ml BSA (Buffer B). In individual experiments incubation mixtures were modified as described under "Results" and in the figure legends. After 2 h, incubation mixtures were removed, and the cells were washed 6 times in ice-cold Buffer B as quickly as possible. Surface-bound counts were eluted by incubating the monolayers for 5 min with Buffer A supplemented with 5 mM EDTA and 1 mg/ml BSA. This eluate was collected and counted in a y counter. The cells were then lysed with 0.5 ml of 0.2 M NaOH, 1% SDS, and 10 mM EDTA, and the lysate was collected and counted. The partitioning of counts between the EDTA eluate and the cell lysate was similar when experiments were performed at 4 "C and room temperature. There- fore, the counts in the cell lysate were thought to represent surface- bound rEPI not elutable with EDTA, and the the sum of the counts from the EDTA-eluate and from the cell lysate was defined as the bound rEPI.

Measurement of the Ability of rEPI to Inhibit Factor VIIa-TF- catalyzed Activatwn of PHJFactor ZX-Activation peptide release from [3H]factor IX (22) was used to monitor the effect of rEPI upon factor VIIa-TF-catalyzed activation of factor IX. The sources of T F were monolayers of OC-2008 cells or a suspension of purified, reconstituted TF. OC-2008 cells were incubated with factor VIIa, 10 nM, and various dilutions of rEPI with or without factor Xa, 10 nM, at room temperature in a final volume of 0.5 ml of Buffer B. After 2 h, monolayers were washed 6 times in ice-cold Buffer B, and [3H]factor IX, 88 nM, in a volume of 0.5 ml Buffer B was added. Serial aliquots were removed over the next hour for determination of trichloroacetic acid-soluble 3H-labeled activation peptide as described in detail earlier (23). Reaction mixtures made with T F in suspension contained: purified reconstituted TF, 0.11 nM (5 ng/ml); factor VIIa, 5 nM; and various dilutions of rEPI with or without factor X, 17.5 nM, in Buffer B. After a 30-min incubation, [3H]factor IX, 88 nM, was added. Samples were removed for measurement of 3H-labeled activation peptide release at time zero and after 2 h. When reaction mixtures did not contain factor X, the [3Hjfactor IX was incubated with anti- factor X IgG, 0.5 mg/ml, for 30 min before being added as substrate.

Electrophoresis-SDS-PAGE was performed according to the method of Laemmli (24) on 12% SDS-polyacrylamide slab gels.

Data Processing and Statistical Methods-All values reported for each group of experiments are means k S.D. Statistical comparisons of the groups were first performed with a one-way analysis of variance followed by a Mann-Whitney two-sample test or a two-tailed unpaired Student's t test where applicable.

RESULTS

The presence of TF on cell surfaces was assessed by the ability of monolayers to support factor VIIa-catalyzed activation of factor X. Monolayers were incubated with 10 nM factor VIIa and 175 nM factor X in buffer B, and serial aliquots were tested for factor Xa activity in a chromogenic assay. Mean rates of factor X activation (n = 4) were as follows: for OC-2008 cells, 1.8 nM/min; for WI-38 fibroblasts, 0.6 nM/ min; for OC-735 cells, none.

Calcium, Time, and Temperature Dependence of rEPI Bind- ing to OC-2008 CeZZs-In early experiments from this labora- tory (1) binding of EPI to factor VIIa-TF complexes formed with a crude TF suspension appeared to require the presence of Xa. Therefore, the calcium, time, and temperature depend- ences of binding of '251-rEPI to OC-2008 cells were determined using incubation mixtures containing factors VIIa and Xa. When the calcium ion concentration of incubation mixtures was increased from 0 to 10 mM, binding was found to reach a plateau at 5 mM CaC12. The time dependence of binding was investigated at 4 "C and room temperature (24 "C). Maximum binding, which was similar at both temperatures, required about 180 min of incubation at 4 "C as contrasted with about 90 min at room temperature (Fig. 1). Similar values for time dependence of binding were obtained in additional experiments with incubation mixtures containing 5 mM CaClz and rEPI alone or with only factor VIIa or factor Xa as an additional reactant. Based on these results, further binding experiments were carried out with a 2-h incubation period at room temperature in reaction mixtures containing 5 mM

878 Binding of rEPI to Cultured Cell Surfaces

TIME (nin)

FIG. 1. Effect of time and temperature upon the binding of rEPI to OC-2008 cells in the presence of factors VIIa and Xa. Monolayers were incubated with lZ5I-rEPI, 2.7 nM, factor VIIa, 10 nM, and factor Xa, 10 nM, in buffer B at either 4 "C (0) or room temperature (0) for increasing time periods. Cells were then washed, and bound rEPI was measured as described under "Experimental Procedures."

TABLE I Comparison of rEPI bound among cell lines tested

Monolayers were incubated with '251-rEPI in Buffer B in the presence of other indicated reactants at room temperature. At the end of 2 h cells were washed, and bound rEPI was determined. Final concentration of reactants: '251-rEPI, 2.7 nM; factor VIIa, 10 nM; factor Xa, 10 nM; anti-TF IgG, 100 pg/ml; annexin V, 20 pn/rnl.

Reaction mixture

EPI, VIIa, Xa EPI, VIIa, Xa,

annexin V EPI, VIIa, Xa,

anti-TF EPI, Xa EPI, VIIa EPI alone

'z61-rEPI bound"

0C-2008b

522 f 164 526 f 93

155 f 44

152 f 26 206 f 38 99 f 11

fmollwell 460f 57 2 5 7 f 9 5 7 f 17

205 f 41 46 f 13

89 +- 9 123 f 24 52 -t 13

83 k 5 124 f 24 48 f 7 196 -t 35 87% 10 2 9 f 5 39 +- 2 4 6 f 9 2 9 f 8

Values represent mean f S.D. of at least three experiments.

Cell line without T F activity. * Cell line with T F activity.

CaCL (buffer B) to assure that the binding data represented equilibrium measurements.

lZ5I-rEPI Binding to Monolayers in the Absence of Factors VIIa and Xa-12sI-rEPI, 2.7 nM in buffer B, bound to a limited extent to monolayers of all three cell lines (Table I, line 6). Binding was unaffected by prior incubation of OC-2008 cells with antibodies to TF, factor VIIa, or factor X and by prior incubation of the cells with annexin V, a material binding with high affinity to anionic phospholipids (Fig. 2). This "base-line" binding of rEPI to OC-2008 cells (mean, 99 fmol/ well) was also not calcium ion-dependent, since it was also unaffected by substituting buffer A/BSA for buffer B (Fig. 2). Because the supply of rEPI was limited, binding isotherms of rEPI to OC-2008 cells in the absence of factors VIIa and Xa were not determined. However, on two occasions adding a 50- fold molar excess of unlabeled rEPI reduced the binding of Y- rEPI by about 40%.

lZ5I-rEPI Binding in the Presence of Factors VZZa and Xa- '2sI-rEPI binding to OC-2008 cells was enhanced by adding either factor Xa or factor VIIa to incubation mixtures. How- ever, maximum binding, a &fold excess over base-line binding, required the presence of both factors VIIa and Xa. It exceeded the sum of the binding obtained with the individual factors alone (Table 1). On two occasions adding a 50-fold molar excess of unlabeled rEPI to an incubation mixture of lZ5I-

FIG. 2. Binding of rEPI to OC-2008 cells in the absence of factors VIIa and Xa. All incubation mixtures contained 2.7 nM 12'1-

rEPI in buffer B. Control mixtures contained no additional materials. Experimental mixtures were incubated for 30 min before the addition of 1251-rEPI with one of the following: anti-TF IgG, 100 pg/ml; annexin V, 20 pg/ml; anti-factor VIIa IgG, 100 pg/ml; anti-factor X IgG, 100 pg/ml. In additional experiments monolayers were incubated with 2.7 nM Iz5I-rEPI in a buffer not containing calcium ions. At the end of 2 h cells were washed and bound rEPI was determined.

A B C D FIG. 3. Effect of anti-TF IgG on binding of rEPI to OC-

2008 cells in the presence or absence of factors VIIa and Xa. The cell monolayers were incubated for 30 min with either buffer B (solid bars) or with 100 pg/ml anti-TF IgG in buffer B (open bars) before incubation for 2 h with '251-rEPI, 2.7 nM, and other reactants. Other reactants were: A , factor VIIa, 10 nM and factor Xa, 10 nM; B, factor Xa alone, 10 nM; C, factor VIIa alone, 10 nM; D, none.

rEPI, factor Xa, and factor VIIa reduced the binding by 79 and 81%.

The majority of the binding obtained when both factor Xa and factor VIIa were present was TF-dependent. Adding anti- TF IgG to OC-2008 cells (Fig. 3) reduced mean binding from 522 f 164 to 155 f 44 fmol/well ( p = 0.006). The latter was equivalent to the mean value obtained with incubation mixtures containing only rEPI and factor Xa (Table I). This fits the observation that preliminary incubation of OC-2008 cells with anti-TF IgG failed to reduce binding of rEPI to OC-2008 cells when incubation mixtures contained only rEPI and factor Xa (Fig. 3). Mean values were: without anti-TF IgG, 152 f 26 fmol/well, n = 5; with anti-TF IgG, 138 f 14 fmol/ well, n = 4.

In additional experiments annexin V, 20 pg/ml, was added to incubation mixtures to determine whether the binding of annexin V to anionic phospholipid on the surface of OC-2008 cells would alter binding of rEPI. As shown in Table I when annexin V was added to incubation mixtures containing both factor VIIa and factor Xa, binding was not affected. When annexin V was added to incubation mixtures containing only factor Xa mean rEPI binding was reduced, but the decrease was not significant (without annexin V, 152 +- 26 fmol/well, n = 5; with annexin V, 124 f 12 fmol/well, n = 4, p = 0.11). In previous work this annexin V preparation was found to bind with high affinity to OC-2008 cell surfaces (25).

Similar results were obtained when monolayers of fetal lung

Binding of rEPI to Cultured Cell Surfaces 879

fibroblasts, a second cell line constitutively expressing surface membrane TF activity, were incubated with factor VIIa, factor Xa, or both (Table I). In contrast, an overall low level of binding was noted regardless of added reactants when binding assays were carried out with monolayers of OC-735 cells, an ovarian carcinoma cell line lacking measurable surface membrane TF activity. '251-rEPI binding was slightly higher in the presence of both factor Xa and factor VIIa (57 f 17 fmol/ well, n = 3) than when the cells were incubated with rEPI alone (29 +- 8 fmol/well, n = 3). The increased binding was not abolished by prior incubation with TF antibodies (52 f 13 fmol/well, n = 3) and could be attributed to the presence of factor Xa. Pooled data from the four experimental mixtures containing factor Xa (Table I, column 4, lines 1-4) differed significantly (p = 0.001) from pooled data from the two experimental mixtures lacking factor Xa (Table I, column 4, lines 5 and 6).

"'I-rEPI Binding in the Presence of Factor VIIa Alone- As mentioned above, mean binding of lZ5I-rEPI to OC-2008 monolayers in the presence of factor VIIa alone (206 f 30 fmol/well, n = 5) significantly exceeded (p = 0.003) mean base-line binding of '"1-rEPI (99 f 11 fmol/well, n = 9). In contrast to the increased binding observed with factor Xa alone, incubating OC-2008 cells with anti-TF IgG reduced the binding to base line (Fig. 3). Similar data were obtained with monolayers of fetal lung fibroblasts. Excess binding of rEPI in the presence of factor VIIa alone was not found with OC- 735 cells, which do not express measurable TF activity (Table I).

This unexpected finding suggested that factor Xa was not an absolute requirement for the binding of EPI to a factor VIIa-TF complex. Therefore, although no factor Xa could be detected in our incubation mixtures in a chromogenic assay sensitive to 5 ng/ml (0.088 nM) of factor Xa, additional experiments were performed to rule out any possibility of contaminating factor Xa. Adding antibodies to human factor X, 100 pg/ml, to incubation mixtures did not decrease binding which ruled out the possibility of contamination with human factor Xa due to synthesis of factor X by OC-2008 cells. Moreover, excess binding in the presence of factor VIIa alone was observed when monolayers of OC-2008 cells were grown in a serum-free medium (Table I), which ruled out the possibility of contamination with bovine factor Xa from media containing fetal calf serum.

Binding Constants for rEPI to Factor VIIa-TF Complexes on OC-2008 Cells-Monolayers were incubated with increasing concentrations of lZ5I-rEPI from 0.27 to 27 nM and, at each concentration, with a &fold molar excess of factor Xa to rEPI. From the published K; for the binding of factor Xa to EPI ( 5 ) , this should have yielded essentially full binding of rEPI to factor Xa. All incubation mixtures contained factor VIIa at 10 nM, a concentration saturating TF sites on the monolayer^.^ Under these conditions total Iz5I-rEPI binding represented the sum of EPI-factor Xa binding to factor VIIa- T F complexes plus EPI-factor Xa binding to the cell surface independent of factor VIIA-TF. The latter was measured by repeating binding experiments in the presence of factor Xa but in the absence of factor VIIa. Specific EPI-factor Xa binding to factor VIIa-TF complexes was calculated by subtracting EPI-factor Xa binding in the absence of factor VIIa from total binding.

Binding isotherms from a mean of three experiments are shown in Fig. 4A, and a Scatchard plot of specific EPI-factor Xa binding to factor VIIa-TF (see above) is shown in Fig. 4B. Scatchard analysis showed a single class of binding sites with

,'ID. T. Le, S. I. Rapaport, and L. V. M. Rao, unpublished data.

1261- rEPI (nH) Bound (fmol) FIG. 4. rEPI binding to OC-2008 cell surface TF-VIIa sites

in the presence of factor Xa. A , monolayers of OC-2008 cells were incubated for 2 h with increasing concentrations of Iz5I-rEPI from 0.27 to 27 nM and with a 5-fold molar excess of factor Xa to rEPI at each concentration in the presence or absence of 10 nM factor VIIa. Binding in the presence of factor VIIa (0) represented total binding, i.e. EPI-factor Xa binding to factor VIIa-TF plus EPI-factor Xa binding to the cell surface independent of factor VIIa-TF. Binding in the absence of factor VIIa (A) represented only the latter. The difference (e) represented specific binding of EPI-factor Xa to factor VIIa-TF. B, Scatchard plot of '251-rEPI-specific binding to cell surface factor VIIa-TF complexes in the presence of factor Xa. Each point is the mean of three experiments.

t 4 i - Y L

V

3 Ig6I- rEPI (nM) Round (fmol)

FIG. 5. rEPI binding to OC-2008 cell surface TF-factor VIIa in the absence of factor Xa. A, monolayers of OC-2008 cells were incubated with increasing concentrations of 1261-rEPI for 2 h in the presence (0) or absence (A) of factor VIIa, 10 nM. Factor VIIa- TF-specific binding (e) was determined by subtracting the radioactivity bound in the absence of factor VIIa from the radioactivity bound in the presence of factor VIIa. B, Scatchard plot of IZ5I-rEPI- specific binding to the cell surface factor VIIa-TF complexes in the absence of factor Xa. Each point is the mean of three experiments.

a mean Kd (n = 3) of 4.5 f 1.5 nM and 335,000 f 84,000 binding sites/cell.

Binding isotherms were also determined for '251-rEPI binding to factor VIIa-TF in the absence of factor Xa (Fig. 5A) . Monolayers were incubated with 0.27-27 nM concentrations of lZ5I-rEPI and 10 nM factor VIIa. Under these conditions total lZ5I-rEPI binding represented factor Xa-independent binding of rEPI to factor VIIa-TF complexes plus cell surface binding of rEPI independent of both factor Xa and factor VIIa-TF. The latter was measured by repeating binding experiments in the absence of both factor Xa and factor VIIa. The difference between the binding isotherms represented factor Xa-independent specific binding of rEPI to factor VIIa- TF. Scatchard analysis gave a mean Kd of 11.9 f 3.5 nM and 236,000 f 68,000 binding sites/cell (Fig. 5B).

The Effect of Heparin Upon rEPI Binding to OC-2008 Cells-Since the intravenous injection of a bolus of heparin increases human plasma EPI levels (26, 27), presumably because of the release of EPI bound to glycosaminoglycans on vascular endothelium, we investigated the effect of heparin upon '"I-rEPI binding to monolayers of OC-2008 cells. Fig. 6 shows mean data from four experiments in which concentrations of heparin from 0.0001 to 1.0 unit/ml were incubated for 30 min with monolayers before the addition of "'I-rEP1. Heparin in a concentration of 0.01 unit/ml or higher substan- tially diminished the base-line binding of rEPI to OC-2008


E O L--"-l X 0 .ooo1 .001 .Ol .l 1

HEPARIN CONCENTRATION W m l )

FIG. 6. Effect of heparin upon rEPI binding to OC-2008 cells. Monolayers of OC-2008 cells were incubated for 30 min with buffer B containing increasing concentrations of heparin before the addition of either "'I-rEPI, 2.7 nM, alone (0) or '*'I-rEPI, 2.7 nM, factor VIIa, 10 nM and factor Xa, 10 nM (0). At the end of 2 h of incubation at room temperature the cells were washed, and the bound rEPI was determined. Binding is expressed as percent of binding of rEPI in the absence of heparin.

-97 - 66 -43

-31

-22

-14

FIG. 7. Autoradiography after reduced SDS-PAGE of '''I- rEPI offered to OC-2008 cells in the presence of factors VIIa and Xa. Lune I , rEPI starting material; lane 2, rEPI in supernatant after 2 h of incubation; lane 3, rEPI eluted from the cells with EDTA.

cells. However, when incubation mixtures also contained both factor Xa and factor VIIa, i.e. when binding primarily resulted from the binding of EPI-factor Xa complexes to factor VIIa- TF complexes, heparin had no significant effect upon the total binding of l2'1-rEPI to OC-2008 cells ( p > 0.8).

SDS-PAGE Analysis of Cell-bound rEPI-In order to determine whether rEPI underwent proteolytic cleavage during a 2-h incubation of 2.7 nM '2sI-rEPI with monolayers of OC- 2008 in the presence of factors VIIa and Xa, samples of the starting l2'1-rEPI, of the supernatant after 2 h of incubation, and of EDTA-eluate that had been concentrated by ultrafiltration were subjected to 12% SDS-PAGE under reducing conditions. Autoradiography of the gels revealed that the rEPI in all samples migrated as a single band at the same apparent molecular mass of 37 kDa (Fig. 7). After elution with EDTA the monolayer was lysed, and the lysate was concentrated by ultrafiltration to attempt a similar analysis of the non-EDTA elutable rEPI. Cell debris in the concentrate clogged the lane and prevented the entrance of 1251-rEPI into the gel.

The Ability of rEPI to Inhibit Factor VIIa-TF Activity in the Absence of Factor Xa-The evidence that rEPI could bind to factor VIIa-TF on cell surfaces in the absence of factor Xa prompted us to determine whether high concentrations of rEPI in the absence of factor Xa could inhibit the ability of factor VIIa-TF to catalyze the activation of factor IX. This was studied first in experiments using monolayers of OC-2008 cells as the TF source. Monolayers were incubated a t room temperature with factor VIIa, 10 nM, and increasing concentrations of rEPI, from 2.7 to 67.5 nM, in buffer B. After 2 h the cells were washed 6 times with ice-cold buffer B. Then buffer B containing ['Hlfactor IX, 88 nM, was added to the monolayers, and the time course of factor IX activation was determined by activation peptide release. At an approximate

normal plasma concentration of 2.7 nM (100 ng/ml), only a small, questionable inhibition of factor IX activation was observed. However, as the rEPI concentration was increased a progressive, unequivocal inhibition was demonstrated. At 5 times the plasma concentration (13.5 nM) the activation rate of factor IX was decreased by 30%, and a t 25 times the plasma concentration the activation rate was decreased by about 75% (Fig. 8). It is important to emphasize that no factor Xa could be detected in these experiments when the chromogenic substrate Chromozym X was added to monolayers of the OC- 2008 incubated with factor VIIa and factor IX in the absence of rEPI.

In additional experiments the inhibitory capability of rEPI in the absence of factor Xa was tested in reaction mixtures containing reconstituted, purified TF, factor VIIa, increasing concentrations of rEPI, anti-factor X IgG, and ['Hlfactor IX and calcium ions. The same progressive inhibition of factor IX activation was observed as the concentration of rEPI was increased above plasma concentration (Fig. 10).

In contrast to the inhibition of factor VIIa-TF catalytic activity observed in the absence of factor Xa, a manyfold lower concentration of rEPI in the presence of factor Xa was sufficient to inhibit factor VIIa-TF catalytic activity formed on OC-2008 cells (Fig. 9) or with suspensions of TF (Fig. 10).

DISCUSSION

The present data establish that maximum binding of EPI to monolayers of two cell lines possessing surface membrane TF activity requires the presence of factor VIIa, factor Xa, and calcium ions. This observation fits with the current belief (4, 5) that under physiologic conditions EPI inhibits factor VIIa-TF enzymatic activity as the result of the formation of an inactive quaternary EPI-factor Xa-factor VIIa-TF complex.

A limited binding of rEPI to monolayers (Table I) was also observed in the absence of factors VIIa and Xa. This binding, which was independent of both TF and calcium ions, was reduced in the presence of a 0.1 unit/ml concentration of heparin. An intravenous injection of a bolus of heparin in normal human subjects has been shown to increase substan- tially plasma EPI levels (26, 27), presumably because of the release of EPI bound to glycosaminoglycans on the luminal

EPI Conc. (nM1 I

c o 10

a ;ad r(

.-I :5 z:.;16

1.8 h-42

x O

a m X I - 0 4

m m o 0

0 10 20 30 40 50 60 Time (min)

FIG. 8. The ability of high concentrations of rEPI to inhibit cell surface factor VIIa-TF-catalyzed activation of factor IX in the absence of factor Xa. Monolayers of OC-2008 cells were incubated at room temperature with factor VIIa, 10 nM, and an increasing concentration of unlabeled rEPI, from 0 to 67.5 nM in 0.5 ml of buffer B. After 2 h the cells were washed 6 times with ice-cold buffer B. Then, 0.5 ml of buffer B containing ["Hlfactor IX, 88 nM, was added to the monolayers, and the time course of activation was monitored by activation peptide release. The inset is a secondary plot of the rate of ['Hlfactor IX activation versus rEPI concentration.

Binding of rEPI to Cultured Cell Surfaces 88 1

2

n "0 10 20 30 40 50 60 70

Time ( m i d FIG. 9. rEPI inhibition of cell surface factor VIIa-TF-cata-

lyzed activation of factor IX in the presence of factor Xa. Experimental conditions are as described in Fig. 8 except that the incubation mixture contained factor Xa, 10 nM, and rEPI concentrations between 0 to 2.7 nM. Inset is as described in the legend of Fig. 8.

0 .OOl .Ol . l 1 10 loo

EPI Conc. (nM) FIG. 10. Progressive inhibition of factor VIIa-TF catalytic

activity by increasing concentrations of rEPI in reaction mixtures containing reconstituted, purified TF. Reaction mixtures contained purified reconstituted TF, 0.11 nM, factor VIIa, 5 nM, and increasing concentrations of rEPI in buffer B. Reaction mixtures denoted by the symbol (0) also contained factor X, 17.5 nM. After 30 min of incubation at room temperature [3H]factor IX, 88 nM, was added. In reaction mixtures not containing factor X (O), the [3H] factor IX was incubated for 30 min with anti-factor X IgG, 0.5 mg/ ml, before being used as substrate. The values plotted are trichloroacetic acid-soluble counts in a subsample removed 2 h after addition of the [3H]factor IX.

surface of vascular endothelium. Adding factor Xa alone to incubation mixtures containing calcium ions resulted in an increase in cell surface binding of rEPI. Gemmell and col- leagues (28) have reported that factor Xa can bind to TF embedded in acidic phospholipid in a continuous flow capil- lary reactor. They have suggested that such binding can lead to the subsequent formation of a factor Xa-EPI-TF complex in the absence of factor VIIa. Our finding of increased rEPI binding to cell monolayers in the presence of factor Xa cannot be attributed to such a mechanism. Increased binding of rEPI in the presence of factor Xa was not abolished by prior incubation of monolayers with anti-TF antibodies and could also be demonstrated with monolayers of a cell line lacking measurable TF activity (Table I). Therefore, we believe that the increase in rEPI binding to monolayers in the presence of factor Xa observed in our experiments reflects the binding of a EPI-factor Xa complex to factor Xa binding sites on cell surfaces other than TF. At this time the nature of these binding sites is unknown. Although at a 2.7 nM '"I-rEPI and 10 nM factor Xa concentration, annexin V did not appear to impair the binding of EPI-factor Xa to OC-2008 cells, further studies with annexin V at different ratios of rEPI and factor Xa and at higher concentrations of both are needed to confirm

this hint of independence of the binding site from anionic phospholipid.

Adding factor VIIa alone to reaction mixtures containing calcium ions also increased rEPI binding to monolayers. In contrast to increased rEPI binding in the presence of factor Xa alone, increased binding in the presence of factor VIIa alone was TF-dependent. It was abolished by prior incubation of OC-2008 cells and WI-38 cells with anti-TF antibodies, and it was not observed with OC-735 cells lacking TF activity (Table I). Moreover, Scatchard analysis revealed that the mean number of binding sites to OC-2008 for rEPI in the presence of factor VIIa alone, 236,000 f 68,00O/cell, did not differ significantly ( p = 0.19) from the mean number of rEPI binding sites in the presence of both factor VIIa and factor Xa, 335,000 1 84,00O/cell. Thus, the evidence is convincing that rEPI can bind to factor VIIa-TF complexes on monolayers both in the presence and in the absence of factor Xa (Figs. 4 and 5). The presence of factor Xa increases the affinity of binding. The mean K d value of rEPI binding to factor VIIa- TF complexes on monolayers of OC-2008 cells was about 12 nM in the absence of factor Xa and 4.5 nM in its presence ( p = 0.03). For this Scatchard analysis the data obtained both in the presence and absence of factor Xa (Figs. 4 and 5) have been accepted as showing that EPI binds to factor VIIa-TF complexes on OC-2008 monolayers with a single affinity. However, in view of the large standard deviation of the binding data in the presence of factor Xa (Table I), such Scatchard plots might fail to reveal the presence of a limited number of high affinity factor VIIa-TF binding sites for EPI- factor Xa complexes.

In summary, it appears that rEPI can bind to cell monolayers by at least three mechanisms. The first requires the expression of cell surface TF and is factor VIIa-TF-dependent. Factor Xa enhances the binding but is not an absolute requirement. Adding heparin to reaction mixtures containing factors VIIa and Xa does not reduce binding by this mechanism (Fig. 6). A second mechanism involves factor Xa-EPI complexes that appear to bind to a cell surface site that is not TF. Finally, a limited amount of rEPI can bind to cell surfaces in the absence of other reactants. Since this binding is reduced by heparin (Fig. 6), it may reflect the binding of rEPI to glycosaminoglycans on cell surfaces. Thus, in studies of EPI binding to different cell lines in the presence and absence of factor VIIa, factor Xa, or both (Table I), at least three variables will determine the partition of binding: the number of TF sites, the number and type of factor Xa binding sites, and the concentration and types of glycosaminoglycans on the cell surface.

The evidence for binding of '*'I-rEPI to factor VIIa-TF in the absence of factor Xa made it important to test further the earlier hypothesis that the presence of factor Xa was an absolute requirement for EPI to inhibit factor VIIa-TF catalytic activity (4). Pedersen et al. (12) recently reported that factor Xa is not required for EPI to inhibit the amidolytic activity of factor VIIa in the presence of relipidated, purified TF and calcium. Nevertheless, these investigators, using a factor IXa coagulant assay to measure activation of factor IX, were unable to demonstrate inhibition of factor VIIa-TF- catalyzed activation of factor IX in the absence of factor Xa. Using an activation peptide release assay, we have now demonstrated that EPI can inhibit factor VIIa-TF activation of factor IX in the absence of factor Xa. This was found both with cell surface TF (Fig. 8) and with purified relipidated TF in suspension (Fig. 10).

However, it is important to emphasize that only a questionable, at best minimal, inhibition could be demonstrated in the


absence of factor Xa when reaction mixtures contained a 2.7 nM rEPI concentration (Fig. 8), which is equivalent to the EPI concentration in normal human plasma (29). Th' 1s con- trasts with the complete inhibition of factor VIIa-TF-catalyzed activation of factor IX induced by this concentration of rEPI in the presence of factor Xa (Fig. 9). Nevertheless, a substantial inhibition of activation of factor IX by EPI in the absence of factor Xa could be demonstrated when the concentration of rEPI in reaction mixtures was increased to approximately 10 times normal plasma EPI concentration.

The functional data on rEPI-induced inhibition of factor VIIa-TF activity on OC-2008 cells in the absence of factor Xa is concordant with the binding data, i.e. with a Kd of 12 nM. In contrast, the functional data and binding data obtained in the presence of factor Xa are discordant. Whereas the binding data yielded a Kd of 4.5 nM for the binding of rEPI to OC-2008 cells in the presence of factor Xa, substantial inhibition of factor VIIa-TF-catalyzed activation of factor IX was observed at an rEPI concentration of 0.27 nM (Fig. 9). Several possible causes for this discordance, including the earlier mentioned possibility of a small number of high affinity factor VIIa-TF binding sites for an EPI-factor Xa complex, are currently being evaluated.

It is possible that the local concentration of EPI at a site of tissue injury is much higher than plasma concentration as a result of the release of EPI from activated, aggregated platelets (30) and the release of EPI from an endothelial cell pool (26). If so, the present observation that a concentration of EPI many times that of plasma concentration can inhibit factor VIIa-TF activity in the absence of factor Xa could have physiologic implications. Moreover, the present data make it clear that EPI-induced inhibition of factor VIIa-TF independent of the generation of factor Xa should be taken into consideration when evaluating the results of experimental studies of prevention of thrombosis in which very large, pharmacologic doses of rEPI have been administered (31,32). The very high plasma concentrations achieved under such conditions could possibly prevent thrombosis triggered by a tissue factor-dependent mechanism without the need for first generating factor Xa.

Acknowledgment-We would like to thank An D. Hoang for tech- nical assistance.

REFERENCES 1. Rao, L. V. M., and Rapaport, S. I. (1987) Blood 69,645-651 2. Broze, G. J., Jr., Warren, L. A., Novotny, W. F., Higuchi, D. A.,

Girard, T. J., and Miletich, J. P. (1988) Blood 71,335-343 3. Wun, T-C., Kretzner, K. K., Girard, T. J., Miletich, J. P., and

Broze, G. J., Jr. (1988) J. Biol. Chem. 263,6001-6004 4. Rapaport, S. I. (1989) Blood 73,359-365

5. Broze, G. J., Jr., Girard, T. J., and Novotny, W. F. (1990) Biochemistry 29,7539-7546

6. Sandset, P. M., Warn-Cramer, B. J., Rao, L. V. M., Maki, S. L., and Rapaport, S. I. (1991) Proc. Natl. Acud. Sci. U. S. A. 88,

7. Sanders, N. L., Bajaj, S. P., Zivelin, A., and Rapaport, S. 1. (1985)

8. Broze, G. J., and Miletich, J. P. (1987) Blood 69,150-155 9. Warn-Cramer, B. J., Rao, L. V. M., Maki, S. L., and Rapaport,

S. I. (1988) Thromb. Huemostusis 60,453-456 10. Girard, T. J., Warren, L. A., Novotny, W. F., Likert, K. M.,

Brown, S. G., Miletich, J. P., and Broze, G. J., Jr. (1989) Nature

11. Girard, T. J., MacPhail, L. A., Likert, K. M., Novotny, W. F., Miletich, J. P., and Broze, G. J., Jr. (1990) Science 248,1421- 1424

12. Pedersen, A. H., Nordfang, O., Norris, F., Wilberg, F. C., Chris- tensen, P. M., Moeller, K. B., Meidahl-Pedersen, J., Beck, T. C., Norris, K., Hedner, U., and Kisiel, W. (1990) J. Biol. Chem.

13. Novotny, W. F., Girard, T. J., Miletich, J. P., and Broze, G. J.,

14. Bajaj, S. P., Rapaport, S. I., and Prodanos, C. (1981) Prep.

15. Funakoshi, T., Heimark, L. R., Hendrickson, L. E., McMullen,

16. Rao, L. V. M. (1988) Thromb. Res. 51,373-384 17. Rao, L. V. M., and Rapaport, S. I. (1988) Proc. Natl. Acud. Sci.

18. Mimms, L. T., Zampighi, G., Nozaki, Y., Tanford, C., and Rey-

19. Fleck, R. A., Rao, L. V. M., Rapaport, S. I., and Varki, N. (1990)

20. Fraker, P. J., and Speck, J. C. (1978) Biochem. Biophys. Res.

21. Van Lenten, L., and Ashwell, G. (1971) J. Biol. Chem. 246,1889-

22. Zur, M., and Nemerson, Y. (1980) J . Biol. Chem. 225,5703-5707 23. Almus, F. E., Rao, L. V. M., and Rapaport, S. I. (1989) Thromb.

24. Laemmli, U. K. (1970) Nature 227,680-685 25. Rao, L. V. M., Tait, J. F., and Hoang, A. D. (1990) Blood 76,

26. Sandset, P. M., Abildgaard, U., and Larsen, M. L. (1988) Thromb.

27. Novotny, W. F., Brown, S. G., Miletich, J. P., Rader, D. J., and

28. Gemmell, C. H., Broze, G. J., Jr., Turitto, V. T., and Nemerson,

29. Broze, G. J., Jr., and Miletich, J. P. (1987) Proc. Natl. Acud. Sci.

30. Novotny, W. F., Girard, T. J., Miletich, J. P., and Broze, G. J., Jr. (1988) Blood 71,2020-2025

31. Day, K. C., Hoffman, L. C., Palmier, M. O., Kretzner, K. K.,

T. G., and Wun, T.-C. (1990) Blood 76,1538-1545 Huang, M. D., Pyla, E. Y., Spokas, E., Broze, G. J., Jr., Warren,

32. Haskel, E. J., Torr, S. R., Day, K. C., Palmier, M., Wun, T.-C., Sobel, B. E., and Abendschein, D. R. (1990) Circulation 82, (suppl.) 604a

708-712

Blood 66,204-212

338,518-520

265,16786-16793

Jr. (1989) J. Biol. Chem. 264, 18832-18837

Biochem. 11,397-412

B. A., and Fujikawa, K. (1987) Biochemistry 26,5572-5578

U. S. A. 86,6687-6691

nolds, J. A. (1981) Biochemistry 20,833-840

Thromb. Res. 57,765-782

Commun. 80,849-857

1894

Huemostusis 62,1067-1073

(suppl.) 1730a

Res. 50,803-813

Broze, G. J., Jr. (1991) Blood 78,387-393

Y. (1990) Blood 76, 2266-2271

U. S. A. 84,1886-1890

mechanisms of binding of recombinant extrinsic …repi to two cell lines possessing surface tissue...

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