Binding of factors IX and IXa to cultured vascular endothelial cells (hemostasis/thrombosis/clot localization/intrinsic system)

Proc. Natl. Acad. Sci. USA Vol. 8, pp. 4119-4123, July 1983 Medical Sciences Binding of factors IX and IXa to cultured vascular endothelial cells (hemostasis/thrombosis/clot localization/intrinsic system) DAVID M. STERN, MICHAEL DRILLINGS, HYMIE L. NOSSEL, ANNE HURLET-JENSEN, KALLIOPE S. LAGAMMA, AND JOHN OWEN Department of Medicine, Columbia University College of Physicians and Surgeons, 63 West 168 Street, New York, New York 132 Communicated by Earl W. Davie, March 21, 1983 ABSTRACT Factor IX and its activated form IXa have been found to bind to confluent cultured bovine aortic and human umbilical vein endothelial cells. Binding of bovine factors IX and IXa to the bovine endothelial cells was saturable and specific and reached a plateau in 75 min at 4C and 3 min at 37C. Binding was half-maximal at a total factor IX or IXa concentration of 2.3 ±.2 nm. At 4TC, a maximum of 42 fmol of tritiated factor IX or IXa bound to 16 cells (an average of 2, molecules per cell). The binding of tritiated factor LX or ]Xa was inhibited by excess unlabeled factor IX or IXa but not by factor X, prothrombin, or thrombin. Competition studies indicated that factors IX and IXa interacted with the same site. Binding was reversible, with 5% of the specifically bound factor IX or IXa eluted in 4 min by a 4-fold excess of unlabeled protein. Specific binding required Ca2+ with half-maximal binding at 1.2 mm CaC12. Factor ]Xa bound to the cells was tested for procoagulant activity in a clotting assay with factor ]X-deficient plasma, cephalin, and CaC12. Cell-bound factor IXa was at least 3-fold more active than was factor IXa in solution. The retention of procoagulant activity by cell surfacebound factor IXa provides a mechanism for the localization of clotpromoting activity. Localized blood coagulation is important in both hemostasis and thrombosis. When vessel walls are transected, localized formation of thrombin may be promoted on the surface of platelets adherent to subendothelial tissues. Endothelial cells inhibit hemostasis and thrombosis by secretion of prostacyclin (1) and plasminogen activator (2, 3) and by the action of a specific cellmembrane component termed "thrombomodulin," which binds thrombin and alters its substrate specificity (4-6). Potential clotpromoting activities of endothelial cells have received little attention. We hypothesized that the initial reactions of thrombosis occur on the endothelial cell surface and asked how the clot-promoting activity is localized. One possible mechanism would be through the binding of factor IX to endothelial cells, if bound factor IXa (the activated form of factor IX) were active in promoting activation of later zymogens which promote thrombin formation. This paper reports reversible, calcium-dependent binding of both factor IX, the zymogen, and the serine protease, factor IXa, to cultured endothelial cells. Bound factor IXa retains its coagulant activity and appears to be more active bound than in solution. MATERIALS AND METHODS Preparation and Radiolabeling of Coagulation Factors. Human factor IX was isolated from human plasma by the method of W. Kisiel (7). Tritiated bovine factor IX (bovine [3H]IX) used in initial studies was a gift of Y. Nemerson (Mt. Sinai School of Medicine), and bovine factor IX used in later studies was a The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact. 4119 gift of C. Esmon (Oklahoma Medical Research Foundation). Tritiation of factor IX (8, 9) yielded a tracer with minimal aggregation that behaved reproducibly and was readily activated. The specific activities of human and bovine [3H]IX were 2.3 X 16 and 3.24 x 16 cpm/,ug, respectively. Protein concentration was determined by the method of Lowry et al. (1) and by using Al*m at 28 nm of 14.9 for bovine factor IX, 14.3 for bovine factor IXa (11), and 13.2 for human factor IXa (12). Factor IX was activated with human factor XIa from I. Ohkubo and K. Kurachi (University of Washington). The factor XIa was bound to cyanogen bromide-activated Sepharose (13) and incubated at 37C with factor IX and 5 mm CaCl2. The reaction was stopped by separating the factor XIa by centrifugation. Factor IXa formation was monitored by its procoagulant activity and the release of the 5% trichloroacetic acid-soluble activation peptide (9). Cleavage of factor IX reached a plateau when 43-52% of the total initial radioactivity remained in solution with 5% CC13COOH. Purified proteins were analyzed by NaDodSO4/ polyacrylamide gel electrophoresis (14). The reduced and unreduced forms of bovine and human factor IX were homogeneous on 1% NaDodSO4/polyacrylamide gel electrophoresis (see electrophoresis of reduced bovine [3H]IX in Fig. 1A). The major peak migrated identically with the unlabeled protein, and a variable earlier peak containing from.5% to 2% of the total counts was thought to represent aggregated factor IX produced during radiolabeling. NaDodSO4/polyacrylamide gel electrophoresis of bovine [3H]IXa without reduction shows two major peaks and two minor peaks that comprise <2% of the total radioactivity (Fig. 1B). The minor peaks represent aggregated protein (higher molecular weight) and residual unactivated factor IX. The earlier major peak migrates identically with unlabeled factor IXa, and the later peak is presumably the activation peptide. Factor IXa used in these experiments consists of two disulfide-linked polypeptide chains from which the activation peptide has been cleaved [factor IXao (11)]. Human factor X and prothrombin were prepared by a modification of the method of Amphlett et al. (7). Bovine factor X and prothrombin were gifts of W. Kisiel (University of Washington). Prothrombin used in initial studies was a gift of K. Mann (Mayo Clinic). Human fibrinogen (grade L) was from Kabi, and platelet factor 4 was a gift of K. Kaplan (Columbia University). Bovine serum albumin (fraction V) was from Sigma, and human IgG was from Calbiochem. Cell Culture. Bovine aortic endothelial cells (BAEC) were isolated from calf aorta (gift of the Great American Veal Co., Newark, NJ) as described by Schwartz (15) and were grown in Dulbecco's modified Eagle's medium with penicillin (5 units/ ml), streptomycin (5 jig/ml), and 2% calf serum (Sterile Systems). Cells were separated for subculture with.25% trypsin/ Abbreviations: [3H]IX and [3H]IXa, tritiated factors IX and IXa (activated form); BAEC, bovine aortic endothelial cells.

412 Medical Sciences: Stern et al. Proc. Natl. Acad. Sci. USA 8 (1983) 4 cm 3 o x 2 $1 4 3 2 1 B 21 I I I 6 1 2 3 4 5 6 1 2 3 4 5 6 Gel slice number FIG. 1. NaDodSO4/polyacrylamide (1%) gel electrophoresis of bovine [3H]IX (A) and [3H]IXa (B). (A) Bovine [3HIIX in solution (i) and after elution from BAEC (x). The latter sample was obtained by binding bovine [3HIIX (1.7 nm) to BAEC at 4C for 2 hr and washing away unbound tracer as described, except that the last three washes with buffer B contained no bovine serum albumin. The bound factor IX then was eluted with 1 mm EDTA. All samples were reduced (5% 2-mercaptoethanol). Gels were run at room temperature for 3 hr at 8 ma per gel with.1 M Tris-phosphoric acid, ph 7./.1% NaDodSO4. Gel slices (1 mm) were incubated overnight at 37 in 5% Protosol in Econofluor (New England Nuclear), and radioactivity was assayed. (B) Bovine [3H]IXa in solution (a) and after elution from BAEC (x). The latter sample was obtained by the identical method described for cell-bound bovine [3H]TX. Samples were not reduced..5% EDTA (GIBCO). At confluence there were 1.2-1.5 X 15 cells per cm2. The cells were characterized by a cobblestone appearance in monolayer (15) and by the presence of von Willebrand antigen with indirect immunofluorescence (16). Human umbilical vein endothelial cells were grown as described by Jaffe et al (17). Binding Studies. BAEC at passages 2 through 5 were plated at 3 X 14 cells per cm2 in 1 x 35 mm plastic culture dishes (Costar) and allowed to grow to confluence. Binding assays were performed 24-36 hr after confluence. Just prior to an experiment, dishes were washed five times (1 ml per wash) with buffer A (.1 M Hepes/. 137 M NaCI/.4 M KCI/.11 M glucose, ph 7.45). Then 1 ml of incubation buffer [buffer A containing 2 mg of bovine serum albumin per ml (Sigma) and 2 mm CaCl2] was added. After cells were equilibrated at the temperature of the experiment for 2 min, tracer and other components of the reaction mixture were added, and the dishes were incubated on an orbital shaker (6 oscillations per min). The supernatant was then aspirated, and dishes were washed six times at 4C with buffer B (1 ml per wash consisting of.5 M Tris-HCI,.14 M NaCI, 2 mg of bovine serum albumin per ml with or without 2 mm CaC12 (ph 7.45). This took -45 sec per dish, and the radioactivity in the last wash was at the background level. After being washed, the cells were dissolved in.5 ml of.2 M NaOH/1% NaDodSO4/.1 M EDTA for 15 min at room temperature, and the radioactivity was assayed. Coagulation Assays. The procoagulant activity of cell-bound factor IXa was assessed by measuring the clotting time of factor IX-deficient plasma added to monolayers of BAEC to which factor IXa was bound. Different concentrations of human [3H]- IXa were incubated with BAEC for 1 hr at room temperature, and then the dishes were washed free of unbound tracer. Then 5 p1 of 5 mm CaCl2, 5 ul of a phospholipid preparation (Thrombofax, Ortho Diagnostics), 5 A1l of incubation buffer, and 1 A.l of factor IX-deficient plasma (G. King) were added at room temperature, and the time at which the first definite fibrin strands formed was noted. Controls consisted of monolayers of BAEC treated as above except that the human [3H]- IXa was added with the factor IX-deficient plasma. Where indicated, factor IX-deficient plasma was replaced by factor VIIIdeficient plasma (G. King). RESULTS Characteristics of Factor IX and IXa Binding to BAEC. Time course of binding. Binding of bovine [3H]IX or [3H]IXa (1.7 nm) to BAEC reached an apparent maximum after 6-8 min at 4C (Fig. 2A). At 37C, binding reached a maximum between 3 and 45 min and was about 25-45% less. The rate and extent of binding of the zymogen and activated enzyme were similar at both temperatures. At 4C the eluted cell-bound [3H]IX was unaltered on NaDodSO4/polyacrylamide gel electrophoresis (Fig. 1A). The small peak of radioactivity preceding the major peak could represent binding of aggregated bovine [3H]IX or a covalent complex of [3H]IX with a cell-associated protein that was dissociated from the cells by EDTA. In binding experiments at 37C, no change in coagulant activity was measured up to 5 min. The solubility of the tracer in 5% CC13COOH increased slowly (1 fmol/hr per 16 cells) during the incubation at 37C, suggesting degradation of factor IX by proteases derived from the cells or the calf serum. When bovine factor IX is activated in vitro, the activation peptide is noncovalently bound to the enzyme (18). In contrast, when factor IXa binds to BAEC, the activation peptide is not associated with the cell-bound ligand eluted with 1 mm EDTA. NaDodSO4/polyacrylamide gel electrophoresis of eluted cellbound bovine [3H]IXa shows one major peak of radioactivity that migrated similarly to unlabeled factor IXa, a small earlier peak as discussed above, and minimal radioactivity in the activation peptide region (Fig. 1B). By using radiolabeled factor IX before or after incubation with cells at 4C, >95% of the radioactivity was precipitated by CC13COOH; after full activation, =5% of the radioactivity was precipitated by 5% CC13COOH. Cell-bound bovine [ H]IXa eluted from the cell surface with 1 mm EDTA was 95% precipitable in 5% CCI3COOH, presumably due to absence of the CCI3COOHsoluble activation peptide. Because the activation peptide contained 4-5% of the radioactivity, the specific radioactivity of bovine [3H]IXa bound to the cell was decreased by 4-5% on a molar basis in comparison with [3H]IX. Specific binding was not seen when either tracer was incubated in dishes without cells. Reversibility of binding. Bovine [3H]IX was eluted from the cell surface either by removing calcium or by adding excess unlabeled ligand. EDTA (1 mm) removed specifically bound bovine [3H]IX and a variable but small amount of nonspecifically bound tracer within 2 min (Fig. 2B). Specifically bound tracer was eluted by washing the cells five times with buffer B without added calcium. The elution of bound bovine [3H]IX by unlabeled factor IX does not appear to be a first-order process (Fig. 2B). This may reflect the presence of negative cooperativity,

. 1 1 a 12 6 1 8 6 4 Medical Sciences: Stem et al. A Proc. Natl. Acad. Sci. USA 8 (1983) 4121 x x- X x man or bovine factor X (1.8,tM), human or bovine prothrombin *.-. (7.4 /.M), human thrombin (12 /M), fibrinogen (8.8 NM), platelet factor 4 (1.4 x 1.tM), immunoglobulin G (.16 mm), and bo- /5/ vine serum albumin (.29 mm). /x/ Competition betweenfactors IX and IXa. The results of competition studies suggest that factors IX and IXa bind to the same sites with similar affinities (Fig. 3B). Calcium dependence of binding. Specific binding of bovine [3H]IX to BAEC increased with increasing concentration of CaC12 from.4 to 8 mm in the incubation buffer and then reached a plateau. Half-maximal binding occurred at 1.2 mm CaC12. Endothelial cell binding of humanfactor IX. Similar specific binding of human factor IX to BAEC was observed, but non-, I I ^ ^ I, ^ Is specific binding was greater and amounted to =4% of the total 2 4 6 8 1 12 14 counts bound. Human [3H]IX also bound to confluent human umbilical vein endothelial cells (passage one) at 4TC. The binding was calcium-dependent and 6%% inhibitable in the presence of a 1-fold excess of unlabeled material in the incubation mixture. The rate and amount of human factor IX binding to human endothelial cells were in the same range as in the experiments with BAEC. Procoagulant Activity of Human [3H]IXa Bound to BAEC. The procoagulant activity of human [3H]IXa bound to BAEC * was assessed in dishes containing cells previously incubated with human [3H]IXa and washed free of unbound ligand. After incubation with human [3H]IXa, monolayers washed with buffer 2 B without calcium still contained factor IXa thought to represent nonspecific binding, which amounted to about 17% of total binding. This nonspecifically bound factor IXa had little pro- I I I I I I coagulant activity: with up to 5 fmol per dish, the clotting time 5 1 15 2 25 3 of factor IX-deficient plasma was >35 sec. Monolayers washed Time, min with buffer B containing calcium showed significant coagulant activity (Fig. 4), indicating that factor IXa bound to calcium-derime course of factor IX (x) and IXa (a) specific binding pendent binding sites retained coagulant activity. Fig. 4 shows FIG. 2. (A)'] to BAEC at 4C Confluent dishes of BAEC (1.2 x 1n cells per dish) the amount of human [3H]IXa bound to the calcium-dependent were incubated with bovine [3H]IX or [3H]1X8 (1.7 nm) alone or tracer along with unlaibeled factor IX or IXa (7 nm), respectively. Nonspe- binding sites (the difference between the amount of factor IXa cific binding (nc)t shown) in the factor IX and IXa dishes was 17% and bound after washing with calcium-containing buffer B and the 2%, respective] ly, of total binding. Specific binding is the difference of amount bound when the wash buffer did not contain calcium) total minus nonespecific binding. (B) Elution of bovine [3H]IX from BAEC plotted versus clotting time. The coagulant activity of factor IXa by unlabeled faxctor IX or EDTA. BAEC (1.3 x 1' cells per dish) and added immediately prior to the clotting assay, without allowing bovine [3H]IX (' 2.2 nm) were incubated at 4TC. To one series of dishes, time for significant cellular binding, is approximately 1/3 the unlabeled facto: r IX (1 um) was added at the beginning of the experi- set of dishes, the same amount of unlabeled factor coagulant activity of cell-bound factor IXa. In 5 mm at 22C, ment (). To a seecond IX was added a: fter 4 hr (.). To a third set of dishes, EDTA at a final <5% of the total factor IXa that will bind to BAEC in 6 min concentration o: f 1 mm was added at 4 hr (x). Each observation was is cell-associated (data not shown). Thus, the coagulant activity made in duplica tedishes, and the experimentwasrepeated three times. of cell-bound factor IXa is due to ligand bound to the specific, calcium-dependent binding sites. In four experiments similar cell-surface tr*acer aggregation, or other mechanisms. to that shown in Fig. 4, there was a consistent increase in the Saturabilitty of binding. Semilogarithmic plots of bovine [3H]- coagulant activity of the human [3H]IXa bound specifically to IX or [3H]IXa bound to BAEC at 4TC versus total added tracer the cells over that in solution. If no human [3H]IXa was added showed a clas;s of saturable binding sites that were half-maxi- to the cells, the clotting time was >6 sec. When factor IX- at 2. 1 nm factor IX and 2.5 nm factor IXa (Fig. deficient plasma was replaced with factor VIII-deficient plasma, mally occupied 3A). If each cuell bound the tracer equivalently, there would be the clotting time was increased from 168.3 to 388.4 sec in the ecules per cell. These binding data may not rep- presence of 6.9 fmol of cell-bound human [3H]IXa. -2, mob resent equilibrium conditions because of perturbation by six washes. Because the rate of dissociation of bovine [3H]IX from BAEC is slow (about 1% per 6 min) at 4C in the absence of unlabeled factor IX and because the washing procedure was complete within 45 sec, the perturbation is probably minor. The binding data demonstrate that class of binding sites observed after washing; ligand bound to other classes of binding sites may have been washed away. Specificity of binding. Specificity of binding for factor IX was demonstrated in competition studies. The binding of bovine [3H]IX or [3H]IXa to BAEC was inhibited by unlabeled bovine factor IX or IXa (Fig. 3B). The following proteins did not affect the binding of bovine [3H]IX or [3H]IXa to BAEC at 4C: hu- DISCUSSION The coagulation mechanism can be viewed as grouping coagulant proteins into activator complexes associated with surfaces. Surface binding serves to localize and modulate coagulant reactions. The contact factors interact with surfaces with negative charges such as glass, celite, human skin, and collagen-containing extracts (19-21); factors VII and VIa interact with cell surface-bound tissue factor on stimulated monocytes (22); and factors Xa and Va bound to the platelet surface greatly accelerate prothrombin activation compared with reagents in solution (23, 24). A surface that supports the participation of

4122 Medical Sciences: Stern et al. Proc. Natl. Acad. Sci. USA 8 (1983) 5 4 > 1 3 a 2 PQ A ~ 9 r 7 5 3 1 Oa. a & a * *a.1.2.5 1 2 5 1 2 5 6 [3H]IX or [3H]IX. added, nm.6 1 2 5 1 2 5 Unlabeled factor IX or IX., nm FIG. 3. (A) Saturability of [3HIIX and [3H]IXa binding to BAEC; semilogarithmic graph in which specifically bound [3H]IX () or [3H]IX. () is plotted against the concentration of added tracer. Binding was studied by using confluent BAEC (1.4 x 16 cells per dish) after a 2-hr incubation at 4C. Each observation was made in duplicate dishes, and the experiment was repeated four times. Error bars denote the SEM. (B) Inhibition of [3H]IX or [3H]IX, binding by unlabeled factor IX or IXM. BAEC (1.4 x 1' cells per dish) were incubated for 2 hr at 4C either with bovine [3H]IX (2.2 nm) alone or with increasing concentrations of unlabeled bovine factor IX (e) or factor IXa (o). Another set of dishes was incubated with bovine [3H]IXM (2.2 nm) alone or with increasing concentrations of unlabeled bovine factor IX (A) or factor IX, (A). Specific binding is the percentage of maximal binding that occurs in the absence of unlabeled ligand. The mean of duplicates is plotted, and the experiment was repeated twice. factor IX,, in coagulation has not been reported to our knowledge. The data presented in this paper demonstrate that factors IX and IXa bind to monolayers of cultured vascular endothelial cells. [Similar results have been obtained ley R. Heimark and S. Schwartz (personal communication).] Autoradiograms of bound radiolabeled factor IX (data not shown) demonstrated uniform distribution of the ligand over the cells that were >95% viable by trypan blue exclusion. Binding is slower (Fig. 2A) than is thrombin binding to platelets (25). However, if factor IX in plasma (concentration, -6 nm) binds to vascular endothelium in vivo as purified factor IX binds to cultured cells, then the constant contact of the plasma with the vessel wall should lead to an equilibrium between circulating and bound factor IX. Factor IX also binds to human foreskin fibroblasts and lymphoid B cells (Wil-2) but not to red cells, platelets, or melanoma Colorado 38. The binding demonstrated is specific for the factor IX molecule. The failure of prothrombin and factor X to inhibit factor IX binding indicates that binding is not solely a -y-carboxyglutamic acid-dependent phenomenon. One consequence of the finding that factors IX and IXa bind to the same site is that small amounts of factor IX,, produced in solution would not bind to the vessel wall in the presence of the large excess of plasma Q 6) on bla 25 2 15 1 8 1 2.5 5 1 2 [3HIIX,, fmol/dish FIG. 4. Coagulant activity of cell-bound factor IX,. Confluent BAEC (P5; 1.2 x 16 cells per dish) were incubated with various amounts of human [3H1IX. from.4-2.5 nm or with incubation buffer alone for 1 hr at room temperature. Monolayers preincubated with the [3HIIXa were then washed with buffer B with or without calcium, and the factor IXa coagulant assay was carried out in the dish. The amount of factor IXa bound to the calcium-dependent sites versus clotting time is plotted (.). In dishes not preincubated with factor IXa, after washing with calciumfree buffer B, the [3H]IXa was added just prior to doing the coagulant assay. In this case, the amount of added factor IX. is plotted versus clotting time (x). Clotting times shown represent the mean of duplicates. The experiment was repeated four times. zymogen. If activated on the cell surface, however, bound factor IX,, would remain on the vessel wall for a finite time. The mechanism whereby factor IXM bound to the cells is more active than factor IXa in solution (Fig. 4) remains to be determined. The factor IXa is presumed to remain associated with the cells based on the rate of dissociation shown in Fig. 2B, but its actual location during coagulation must be determined directly. The procoagulant effect of cell-bound factor IXa remains dependent on factor VIII, indicating that the observed procoagulant activity on BAEC is not due to induction of tissue factor, factor Xa, or thrombin associated with the cells. These observations immediately prompt questions concerning mechanisms of factor IX activation and inhibition and how perturbation of the cell influences binding and function. The observation that factor IXa bound to the cell surface retains its coagulant activity provides a mechanism for the localization of a potent clot-promoting activity to the surface of the vessel wall which may be important in thrombosis and hemostasis. We acknowledge Dr. Walter Kisiel's invaluable assistance in providing reagents and advice. Both Drs. Kurachi and Ohkubo kindly provided vital reagents. We thank Drs. Karen L. Kaplan and Tom Detwiler for comments on the manuscript and Dr. Gabriel C. Godman for autoradiography. The work was supported by research grants from the National Institutes of Health (HL-15486 and HL-216). D.M. S. is a research fellow supported by Training Grant HL-7461 and A.H.-J. was supported by the same grant. 1. Weksler, B. B., Marcus, A. J. & Jaffe, E. A. (1977) Proc. NatL Acad. Sci. USA 74, 3922-3926. 2. Astrup, T. (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 1-57. 3. Loskutoff, D. J. & Edgington, T. S. (1977) Proc. Natl. Acad. Sci. USA 74, 393-397. 4. Esmon, N. L., Owen, W. G. & Esmon, C. T. (1982)J. Biol. Chem. 257, 859-864. 5. Owen, W. G. & Esmon, C. T. (1981) J. BioL Chem. 256, 5532-5535. 6. Esmon, C. T., Esmon, N. L. & Harris, K. W. (1982)J. Biol. Chem. 257, 7944-7947. 7. Amphlett, G. 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