Cultured Bovine Aortic Endothelial Cells Promote Activated Protein C-Protein S-mediated Inactivation of Factor Va

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1 THE JOURNAL OP BIOLOGICAL CHEMISTRY by The American Society of Biological Chemists, Inc. Vol. 261, No. 2, Issue of January 15,p ,1986 rznted cn U.S.A. Cultured Bovine Aortic Endothelial Cells Promote Activated Protein C-Protein S-mediated Inactivation of Factor Va (Received for publication, April 29, 1985) David M. Stern$, Peter P. Nawroth, Kevin Harris, and Charles T. Esmong From the Columbia Uniuersity, Department of Medicine, New York, New York and the Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma Previous studies have demonstrated that protein S is required for optimal activated protein C-mediated inactivation of Factor V, on the surface of either the platelet or phospholipid vesicles. In this report we demonstrate assembly of the activated protein C-protein S complex on the surface of cultured bovine aortic endothelial cells. Endothelial cell surface acceleration of Factor V. inactivation by activated protein C required the presence of protein s. Kinetic studies indicated that the rate of Factor V. inactivation was halfmaximal at a protein s concentration of 0.2 nm and an activated protein C concentration of 0.05 nm. Binding of laai-activated protein C to endothelial cell monolayers was absolutely dependent on the presence of protein S. At saturating levels of protein S, activated protein C binding was saturable with & = 0.04 nm. In contrast, specific, time-dependent, and saturable binding of lzaiprotein S to endothelium occurred in the absence of activated protein C. Addition of activated protein C increased the affinity of protein S from K d = 11 nm to 0.2 nm, but did not change the number of molecules bound per cell at saturation (85,000 molecules/cell). These studies suggest that activated protein C increases the affinity of protein S for pre-existing sites on the endothelial cell surface. The close correlation between the parameters of protein S-activated protein C binding to endothelium and Factor V, inactivation supports the concept that it is bound protein S and activated protein C that are the active species. For- mation of functional activated protein C-protein S complexes thus occurs effectively on the endothelial cell surface and represents a new addition to the list of vessel wall anticoagulant properties. Protein C and protein S are two regulatory plasma coagulation proteins (reviewed in Refs. 1 and 2). After activation, protein C is a potent anticoagulant enzyme capable of inactivating factors V. and VIIIa, thereby blocking activation of the coagulation system (3-6). Activated protein C (APC ) * This work was supported by a Young Investigator Award from the Oklahoma Affiliate of the American Heart Association and National Institutes of Health Grants HL-34625, HL (D. M. S.), and HL (C. T. E.). 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. $ Completed this work during the tenure of a Clinician Scientist Award from the American Heart Association with funds contributed in part by the New York and Oklahoma Affiliates. 5 Established Investigator of the American Heart Association. The abbreviations used are: APC, activated protein C; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; MES, 4-morpholineethanesulfonic acid. 713 requires the presence of negatively charged phospholipid surfaces (3,4) and protein S (7) for optimal anticoagulant activity. Protein S has been shown to function as a nonenzymatic cofactor for the binding of APC to phospholipid surfaces by forming a complex with the enzyme (8,9). This complex has enhanced affinity for membrane surfaces (8,9). A recent study has demonstrated assembly of the APC-protein S complex on the platelet surface with subsequent acceleration of Factor V. inactivation (10). As the cells forming the luminal vascular surface, endothelial cells are strategically located to play an important role in the regulation of coagulation. Anticoagulant properties of endothelium include the presence of anticoagulant heparinlike molecules (11-13) and thrombomodulin (14) on the cell surface. Elaboration of tissue plasminogen activator (15) and prostacyclin (16) also contributes to the antithrombotic nature of endothelium. In addition, endothelium has recently been shown to participate in a series of procoagulant reactions (17-21) including Factor 1X.-VII1,-mediated Factor X activation (17-19) and Factor X.-V,-mediated prothrombin activation (18, 21). This suggests that an effective vessel wall anticoagulant mechanism would involve inactivation of the cofactors, Factors V. and VIII,, which play an integral role in the activation of coagulation. These considerations prompted us to examine the interaction of APC and protein S with endothelium. The results indicate that cultured endothelial cells can provide a surface capable of assembling the protein S-APC complex, thereby promoting Factor V. inactivation. EXPERIMENTAL PROCEDURES Protein Purification and Radiolabeling-All purified coagulation factors were of bovine origin. Purification of protein S was carried out as described (7). Protein S (0.9 mg/ml) in 2 mm Tris (ph 7.5), 0.1 M NaCl was inactivated (9) by treatment with 5% purified bovine a-thrombin (22) (w/w) for 4 h at 37 C. The ph of the reaction mixture was then adjusted to 6.0 using MES, and thrombin was removed by chromatography on sulfopropyl-sephadex (bed volume, 5 ml) (Pharmacia). Reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)(23) demonstrated complete conver- sion of protein S to its thrombin-cleaved form (9). Thrombin-cleaved protein S had no anticoagulant activity in the protein S functional assay described below. Radiolabeling of protein S was accomplished by the lactoperoxidase method (24), using Enzymobeads according to the manufacturer s instructions. The reaction was carried out at room temperature for 15 min by incubating Enzymobeads (Bio-Rad) (50 pl), protein S (40 pl; 29 pg), Na *I (2 mci), and glucose (2 pl). Free iodine was separated from protein S by gel filtration using a column (1 X 20 cm) of Sephadex G-25. The specific radioactivity of Iprotein S was 9,000-12,000 cpm/ng (corresponding to approximately 0.1 mol of Z51/mol of protein S) over five radioiodinations. Radioiodinated protein S co-migrated with unlabeled material on SDS-PAGE. Protein S anticoagulant activity (see below) was not affected by the radiolabeling procedure. Protein S anticoagulant activity was assayed (25) using oxalated bovine plasma previously adsorbed with barium sulfate (50 mg/ml)

2 714 Activated Protein C-Protein S and Endothelium and supplemented with prothrombin (26) (150 pg/ml). This plasma (0.1 ml) was mixed with purified Factor X. (27) (the concentration of Factor X. was selected to give a clotting time of 30 s), activated protein C (2 pg/ml; 10 pl), cephalin (0.1 ml; Ortho Pharmaceutical), and varying concentrations of protein S and CaC12 (25 mm; 0.1 ml) at 37 "C. Protein C was purified as described previously (4) and was activated by incubation with 5% thrombin (w/w) for 3 h at 37 "C in 1 mm Tris (ph 7.4), 0.1 M NaCI. The reaction mixture was adjusted to ph 6.5 with MES and chromatographed on QAE-Sephadex (0.6 X 5 cm) equilibrated with MES, 0.1 M NaCl. Activated protein C was eluted using a M linear salt gradient (5 ml/reservoir). Radiolabeling of protein C was accomplished by the method of Bolton and Hunter (28) usingn-succinimidyl-3-(4-hydroxy-5-['~i]iodophenylpropionate (Amersham Corp.) with a specific activity of 1950 Ci/mmol. After evaporation to dryness, Bolton-Hunter reagent (2 mci) was incubated with protein C (50 pg) in borate buffer (0.1 M, ph 8.8) for 20 min at 4 "C. The reaction mixture was chromatographed on a Sephadex G-25 column (0.9 x 20 cm) (1 mm Tris (ph 7.5), 0.1 M NaCl, 0.2% gelatin) to remove excess reagent. The radiolabeled preparation was activated as described for unlabeled protein C. The pass-through fractions containing APC from the sulfopropyl-sephadex column were pooled and applied to a rn-aminobenzamidine Affi- Gel column (bed volume, 2 ml) prepared by the method of Guinto (29). After washing the column with 10 bed volumes of 0.02 M Tris/ HCl (ph 7.5),0.1 M NaCl, the column was eluted with the same buffer containing 1 M benzamidine hydrochloride. The peak fractions were pooled (2 ml), dialyzed at 4 "C uersus three changes of 0.02 M Tris (ph 7.5), 0.1 M NaCl (ll/dialysis), and stored at 4 "C. SDS- PAGE demonstrated that this material co-migrated with unlabeled APC and had a specific radioactivity of X lo4 cpm/ng (corresponding to approximately 0.25 mol of '251/mol of APC). The radiolabeling procedure had no effect on APC anticoagulant activity measured in a clotting assay as described (25). Factor V, was purified as described (301, and the preparations used in this study were recombined from isolated subunits in the presence of 10 mm CaC12 overnight at 4 "C. Factors IX (260 units/mg) and X (100 units/mg) and prothrombin (13 units/mg) were purified as described (26, 31-32). Factor IX was activated by incubation with Factor XI, bound to CH-Sepharose as described (18), and Factor X was activated by incubation with the coagulant protein from Russell's viper venom (33) coupled to CH-Sepharose as described (18). Burro anti-bovine Factor V IgG (34) was generously provided by Drs. Tracy and Mann (University of Vermont, Burlington, VT). Nonimmune burro IgG was prepared by standard methods (35). Protein concentrations were determined colorimetrically (36). SDS-PAGE was carried out as described (24), and slab gels were dried (Bio-Rad slab gel drier, model 224) and processed for autoradiography using Kodak X-Omat (XAR 5) film. A Cronex intensifying screen (DuPont) was used and exposure times were 24 h. Standard proteins including myosin heavy chain (Mr 200,000), phosphorylase b (M, 97,400), bovine serum albumin-(m, 68,000), ovalbumin (Mr 43,000), and a-chymotrypsin (M, 25,700) (Bethesda Research Laboratories) were run simultaneously. Cell Culture-Bovine aortic endothelial cells were grown from aortae of newborn calves as described (37) and were used from passages 2 to 9. Experiments were carried out within 24 h after the cells achieved confluence using either or 9.6-cm2 wells. At confluence there were X lo5 cells/cm2. Endothelial cells with sprouting morphology, vacuolations, or from older passages did not promote APC-protein S-mediated Factor V. inactivation. Factor V, Znuctiuation Assay-The medium from 0.32-cm2 wells with confluent endothelial cells was aspirated, and the cells were washed twice with Hanks' balanced salt solution containing dextran sulfate allowing 15 min of incubation between washes. Then incubation buffer (10 mm HEPES (ph 7.45), 137 mm NaC1, 4 mm KC1, 11 mm glucose, 3 mm CaC12, and 3 mg/ml bovine serum albumin) (85 pl) was added along with Factor V. (80 nm) (5 pl) and the indicated amounts of APC and/or protein S, each in a volume of 5 pl. Where indicated, thrombin-treated protein S was also added to reaction mixtures. The reaction mixture was incubated at room temperature with constant gentle mixing, and one 25-p1 aliquot was removed from each well. Samples were removed at 10, 30, 40, 60, 90, and 120 s of incubation. Samples were immediately assayed in a one-stage clotting assay (10, 38) by adding 25 p1 of diluted sample to 25 pl of incubation buffer, 50 pl of rabbit brain thromboplastin (Dade), and 50 pl of CaClz (25 mm). Finally, 50 rl of Factor V-deficient human plasma was added and the clotting time determined. Factor V-deficient plasma was prepared by the method of Bloom et al. (39). Standard curves were constructed using purified Factor V., and all clotting times were done in duplicate or triplicate. The rate of Factor V. inactivation was determined from the slope of the linear initial portion of a plot of Factor V. activity uersus incubation time and generally included the lo-, 30-, 40-, and 60-s points. Binding Studies-Binding studies were carried out after washing endothelial cells twice with calcium/magnesium-free Hanks' balanced salt solution containing dextran sulfate. Incubation buffer containing radiolabeled proteins alone or in the presence of other proteins, as indicated, was then added. APC binding studies were carried out in a final volume of 1 mlof 9.6-cm2wells. Protein S binding studies employed a final volume of0.1ml in 0.32-cm2 wells. When antibovine Factor V IgG or nonimmune IgG was used, endothelial cells were preincubated with the antibody preparation in incubation buffer for 45 min at 23 "C and washed twice with ice-cold incubation buffer, and then coagulation proteins were added. Binding assays were carried out at 15 "C for the indicated times and were terminated by three rapid washes (0.1 ml/wash) over 3 s with incubation buffer (4 "C). Cells were solubilized with 0.2 N NaOH, 1% SDS, and 10 mm EDTA. No binding was observed in wells without cells. Data from binding experiments were fit to the equilibrium binding equation: B=- nk 1+KA (40) assuming a one-site model, where B is the amount bound, n is the number of sites/cell, A is the free concentration of radioligand, and K is the association constant. A nonlinear least squares program (SAS Institute, Cary, NC) was used to obtain the best fit curve, to solve for n and K, and to determine the S.E. A plot of residuals uersus free radioligand for the binding data shown in Figs. 5A and 7B indicated that no systematic error was involved in fitting the binding to the model used (data not shown). Dissociation studies were carried out using the method of infinite dilution (Fig. 3B) as described by Lollar et al. (41): after endothelial cell monolayers were incubated with '%I-protein S and washed free of unbound protein, fresh incubation buffer was added (0.1 ml). At the indicated times, incubat.ion buffer was aspirated, and the wells were washed twice (0.2 ml/wash) with the same buffer and solubilized as described above. Dissociation of cell-bound lz5i-protein S was also studied by adding excess unlabeled protein S to the incubation mixture (Fig. 3B). RESULTS When Factor V, and APC are incubated with cultured bovine aortic endothelial cells, rapid Factor V, inactivation is dependent on the presence of protein S (Fig. 1). Since these concentrations of APC (1 nm) and protein s (2 nm) do not, Seconds FIG. 1. Factor V. inactivation by APC-protein S on endothelia1 cell monolayers. Endothelial cell monolayers were incubated with Factor V. (70 nm) and APC (1 nm) in the presence of protein S (2 nm) (0) or thrombin-cleaved protein S (2 nm) (A) or in the absence of protein S (X). Thrombin-treated protein S (50 nm) was added to reaction mixtures along with protein S (2 nm), APC (1 pmol/ml), and Factor V. (70 nm), where indicated (0). Aliquots were withdrawn for Factor V. assay at the indicated times and assayed as described under "Experimental Procedures." The mean of duplicates is shown.

3 Activated C-Protein under comparable conditions, result in effective Factor V, (70 nm) inactivation in a test tube without cells, the results in Fig. 1 indicate that the endothelial cell surface promotes APCprotein S-mediated Factor V. inactivation. Addition of thrombin-treated protein S in place of native protein S did not promote APC-mediated Factor V, inactivation. Thus, cleaved protein S, which has been reported to have no anticoagulant activity (9), does not substitute for protein S in this system. To better characterize the involvement of endothelial cells in this reaction, the dependence of the rate of Factor V, inactivation on the concentration of both APC and protein S was examined. Endothelial cell-dependent enhancement of Factor V, inactivation was saturable with respect to APC (Fig. 2A) with half-maximal rates at an APC concentration of 0.05 f 0.01 nm. The enzyme system was also saturable with respect to protein S (Fig. 2B) with half-maximal Factor V, inactivation rates occurring at a protein S concentration of 0.20 k 0.04 nm. This is considerably below the plasma concentration of protein S (approximately 100 nm). These data suggest that a limited number of cellular binding sites might mediate the interaction of protein S and APC with endothelium. The binding of '251-protein S to endothelial cell monolayers occurred in a time-dependent manner (Fig. 3A). The second-order rate constant for association, calculated from the data in Fig. 3A, was approximately lo6 min" M-'. The concentration of binding sites used in this calculation was taken from the data in Fig. 5A (see below). Even at the lowest concentrations of lz5i-protein S employed, binding reached an apparent maximum by 45 min (Fig. 3A). Dissociation studies indicated that the interaction of lz5i-protein S and endothelial cells was reversible with a first-order dissociation constant of 8.6 x min-' (Fig. 3B). A similar dissociation rate was observed whether elution of cell-bound protein S was studied by the method of infinite dilution or in the presence of excess unlabeled protein S. Since this dissociation rate is quite slow, requiring about 4 h for 90% of the specifically bound lz5i-protein S to dissociate, binding studies were carried out at 15 "C to prevent internalization of surfacebound protein s. Under these conditions, addition of dextran sulfate (10 mg/ml) effected rapid elution of cell-bound 'Ti- A B "- I I /rate I /[APCl [APC] (nm) -.L (SI (nm) FIG. 2. Factor V. inactivation on endothelial cells: effect of protein S and APC. A, endothelial cell monolayers were incubated with Factor V. (80 nm), protein S (10 nm), and the indicated concentrations of APC. The initial rate of Factor V. inactivation is plotted uersus the concentration of APC. Inset, calculated using nonlinear regression analysis applied to the Michaelis-Menten equation. B, endothelial cell monolayers were incubated with Factor V. (80 nm), APC (20 nm), and the indicated concentrations of protein S. The initial rate of Factor V. inactivation, determined as described under "Experimental Procedures," is plotted uersus the concentration of protein S. Inset, calculated using nonlinear regression analysis applied to the Michaelis-Menten equation. 1.0 Endothelium S and A 100 h, B Time (min) FIG. 3. Time course and reversibility of ""I-protein S binding to endothelial cells. A, time course. Endothelial cell monolayers were incubated for the indicated times with lz5i-protein S (0, 30 nm; X, 10 nm; 0, 2 nm; A, 2 nm plus APC at 2 nm) (total binding). A duplicate set of wells was incubated with the same concentrations of '?-protein S in the presence of a 220-fold molar excess of unlabeled protein S (nonspecific binding). Specific binding, the difference be- tween total and nonspecific binding, is plotted uersus incubation time. The mean of duplicates is shown and nonspecific binding was 16-21% of the total binding. B, reversibility: endothelial cell monolayers were incubated with '261-protein S (20 nm) for 25 min and then washed three times as described under "Experimental Procedures." At this point (time 0), a 100-fold molar excess of unlabeled protein S (1.9 p ~ (01, ) fresh incubation buffer (0.1 ml) (O), or incubation buffer containing dextran sulfate (10 mg/ml) (X) was added. The wells were then incubated at 15 "C for the indicated times, washed, and solubilized. Nonspecific binding was determined from a set of wells in which '251-protein S (20 nm) and unlabeled protein S (4 p ~ were ) added simultaneously. Maximal specific binding (time 0) was 3.2 fmol/well. The mean and S.E. of the per cent maximal specific binding are plotted uersus time. protein S (Fig. 3B, X) even at later times, indicating that protein S was present on the cell surface. Dextran sulfate did not result in endothelial cell detachment or loss of viability. SDS-PAGE of the pool of dextran sulfate-elutable lz5i-protein S indicated that it co-migrated with the initial tracer prior to incubation with endothelial cells (Fig. 4). lz51-protein S binding to endothelial monolayers was facilitated by calcium, being maximal at 2-3 mm. Employing these conditions for equilibrium binding, the association of lz5i-protein S with endothelial cell monolayers was observed to be saturable in the absence of other coagu- lation factors (Fig. 5A, 0). Binding was half-maximal at 11 f 1 nm, and at saturation there were 8.5 f 1.2 X lo4 molecules bound per cell. Although not required for protein S binding, activated protein C enhanced the affinity of protein S for the endothelial cell-binding sites (Fig. 5A, x). Consistent with this enhanced protein S-endothelial cell interaction observed in the presence of APC, the time course of lz5i-protein S- endothelial cell binding was accelerated approximately 10- fold (Fig. 3A, A) when APC was added. Optimal lz5i-protein S binding occurred at or above an activated protein C concentration of 0.5 nm (Fig. 5B). At saturating levels of activated protein C, the affinity of '251-protein S binding increased from 11 f 1 to 0.2 f 0.03 nm, although the number of sites was unchanged. Scatchard analysis (Fig. 5B, insets 1 and 2) clearly demonstrated this change in the affinity of protein S binding in the presence of APC. Binding of '251-protein S to endothelial cells was not subject to competitive inhibition by other vitamin K-dependent coagulation proteins including Factors IX, IX, and X and pro- thrombin (Fig. 6). Unlabeled protein s, however, was an effective inhibitor. Pretreatment of endothelial cells with antibody to bovine Factor V ( pg/ml) had no effect on '251-protein S binding, suggesting that endothelial cell Factor V (42) may not be involved in the binding site. Furthermore, addition of Factor V. ( nm) had no effect on the

4 716 C-Protein Actiuated Protein FIG. 4. Reduced SDS-PAGE of '*V-protein S before and after endothelial cell binding. Endothelial cells were incuhated with "'I-protein S (80 nm)for 15 min, washed, and then incuhated with incuhation huffer containing dextran sulfate (10 mg/ml) for 5 rnin. Samples were processed for reduced SDS-PAGE and autoradiography as descrihed under "Experimental Procedures." Lane A, '"1-protein S eluted from the endothelial cell surface with dextran sulfate: lane R, "'I-protein S prior to incuhation with endothelial cells. Endothelium S and S, ""'I-APC hound in a high affinity fashion to themonolayers with Kd = 0.04 k nm and 180 k 15 molecules hound per cell a t saturation (Fig. 7 R ). Scatchard analysis (45) (Fig. 'ib, inset) of the binding data was consistent with a single class of binding sites. It is apparent from the binding data that the observed ratio of cell-boundprotein S (Fig. 5 A ) exceedsbound activated protein C (Fig. 7 R ) by approximately 400-fold. If one expects a stoichiometry of 1:l for the protein S-activated protein C complex, as suggested by the resultsof experiment9 employing synthetic phospholipids (8),then the number of activated proteinc-bindingsitesshould be equal to the number of protein S-binding sites. The results of the cellular binding studies in Figs. 5 and 7 would then be difficult to explain. If the problem was technical and related entirely to radiolabeling, then one might expect the total number of activated protein-binding sites to equal the number of protein S sites. This led us to carry out pilot studies employing radiolabeled protein S and activated proteinc prepared by different methods. Radiolabeled protein S prepared by tritiation of the sialic acidresidues (44) is uniformly modified and had a similar affinity and number of cellular binding sites compared with 1251-proteinS (Fig. 5). Activated protein C was also radiolabeled by additional methods. Studies with radioiodinated activated proteinc prepared hy the lactoperoxidase method (23) indicated a n approximate Kd of 0.05 nm and 120 sites/cell. Furtherstudieswithtritiatedactivatedprotein C ( cpm) (10) demonstratedless than 40 cpm bound per 10" cells (corresponding to less than 4,000 sites/cell). This is far less than anticipatedif the numberof activated protein C-binding sites equaled the numberof protein S sites (85,000/cell). The latter would correspond to approximately 800 cpm of 'Hactivated protein C bound per 10' cells. Thus, the bindingof activated protein C labeled by three different methodsinvolving three different sites on the molecule consistently yields low numbers of cellular sites, suggesting that there are many more protein S- than activated protein C-binding sites. DISCUSSION The physiologic significance of theproteinc-protein S system in vivo is implied by the thrombotic diathesisobserved in kindreds withdeficiencies of either protein (25, 46-48). Since formation of functional activated protein C-protein S complex requires assembly on membrane surfaces, this indibinding of T - p r o t e i n S or "'1-APC (see below) when the cates the potentially central role that cellular surfaces can experiments were not carried out as described for Figs. 5 and play as modulators of this anticoagulant mechanism. The 7. Thus, in contrast to the substrate-dependent enhancementresultsreported heresuggest that the endothelial cell can providea surface for assembly of the activated protein Cof enzyme binding observed in the tissue factor pathway(43) protein S complex. The relatively high affinity of activated and formation of the intrinsic Factor X activation complex protein C for the endothelial cell surface suggests that comontheendothelial cell surface(19),thesubstratedidnot plex formation of activated protein C with protein S should enhance enzyme or cofactor binding in this system. occur onthe vessel wall in response toconcentrations of To further characterize the nature of these endothelial cell activatedprotein Cformed in uiuo as predicted from the sites, APC-endothelialcell interaction was examined. Bindingclinical studies of Bauer et al. (49). Particularly in the microof l2'1-apc to endothelial cell monolayer was absolutely de- circulation, where a high surface to volume ratio exists, the pendent on the presence of protein S (Fig. 7 A ). In the absence vessel wall protein S-activated C system should provide an of protein S, no specific binding was observed. In the presence effective clearancepathway for circulatingfactor V, and, of protein S, "'1-APC binding occurred, being optimal at a presumably, by analogy, Factor VIII.. protein S concentration greater than1.0 nm. The low amount Compared with previous data from studies on the platelet of specifically bound '"I-APC precluded detailed definitionof surface (10) and synthetic phospholipids ( 8 ),activated protein APC-endothelial cell binding parameters even in the 9.6-cm2 C appearsto haveaconsiderablyhigher affinity for the wells. However, "'I-APC binding was observed to be reversi- endothelial cell surface in the presence of protein S (11 nm ble following the addition of a 100-fold molar excess of unla- for activated protein C and 14 nm for the activated protein beled APC. Furthermore, even at the lowest APC concentra- C-protein S complex on the platelet surface and phospholipid, tions employed in these studies, ""I-APC binding reached an respectively, uersus 0.05 nm on the endothelial cell surface). S complex apparent. maximumby 45 min. At saturating levels of protein This indicates that the activated protein C-protein

5 Activated Protein C-Protein S and Endothelium I 0 IO 40 76' 'E-" (Y II) c I c 0 Fro. 1*sl-Protoin S CnM) APC CnM) m FIG. 5. The binding of 12'I-protein S to bovine aortic endothelial cells: the effect of APC. A, endothelial cell monolayers were incubated for 45 min with the indicated concentrations of '=I-protein S alone (total binding) or '"I-protein S and a 200-fold molar excess of unlabeled protein S (nonspecific binding) in the presence (X) OE absence (0) of APC (2 nm). The binding assay was carried out as described under "Experimental Procedures. Specific binding (total minus nonspecific binding) is plotted versus free '"I-protein S. Data were analyzed by the nonlinear least squares program, and the curve (-1 indicates the best fit line. Nonspecific binding accounted for 16-21% of the total binding. Insets, Scatchard analysis of the same data: inset 1, '=I-protein S binding in the presence of APC; inset 2, '=I-protein S binding in the absence of APC. B/F, bound/free; B, bound. B, endothelial cells were incubated with '"I-protein S (0.2 nm) in the presence of the indicated concentrations of APC. The mean and S.E. of specific '%I-protein S binding are plotted versus added APC. 0.3 I IO Unlabelled Protein (nm) FIG. 6. Competitive "'I-protein S-endothelial cell binding studies. Endothelial cell monolayers were incubated for 40 min with '"I-protein S (8.8 nm) and the indicated concentrations of the following unlabeled proteins: protein S (.), Factor IX (A), Factor IX. (A), Factor X (0), Factor V. (B), and prothrombin (0). Maximal specific binding, the difference between the binding observed in wells incubated with '=I-protein S alone and '"I-protein S in the presence of a 250-fold excess of unlabeled protein S, was 2.6 fmol/well. Per cent maximal specific binding, the mean of duplicates, is plotted versus the concentration of unlabeled protein added. assembles effectively on endothelium and suggests that the cellular binding site(s) differ from those observed in the previous studies (8). Whereas a stoichiometry of 1:l for activated protein C-protein S complex was observed on phospholipids (8), the present studies (Figs. 5 and 7) suggest that there are an excess of protein S-binding sites on endothelium. Based on the arguments presented under "Results," it is unlikely that the high ratio of protein S to activated protein C sites is due to a tracer artifact. Thus, the binding of one activated protein C molecule influences the binding of multiple protein S molecules. Since the total number of sites does not change, it is likely that this represents activated protein C-dependent enhancement of protein S binding to the preexisting site FIG. 7. The binding of 12'I-activated protein C to bovine aortic endothelial cells: the effect of protein S. A, endothelial cells were incubated for 45 min with the indicated concentrations of protein S and '=I-APC alone (0.1 nm) (total binding) or '=I-APC in the presence of a 200-fold molar excess of unlabeled APC (nonspecific binding). The mean and S.E. of specific '=I-APC binding (total minus nonspecific binding) are plotted versus added protein S concentration. The protocol for binding studies is described under "Experimental Procedures." Nonspecific binding accounted for 22-26% of the total binding. B, endothelial cells were incubated for 45 min with protein S (2 nm) and the indicated concentrations of '=I-APC alone or '"I- APC in the presence of a 200-fold molar excess of unlabeled APC as described in A above. An incubation time of 45 min was sufficient to allow '"I-APC binding to reach an apparent maximum at the lowest concentrations used. Specific binding is plotted versus the amount of free '"I-APC. Data were analyzed by the nonlinear least squares program, and the curve (-) indicates the best fit line. Inset, Scatchard analysis of the same data. B/F, bound/free; B, bound. The effective assembly of activated protein C and protein S on the endothelial cell surface with subsequent expression of activated protein C anticoagulant indicates that this mechanism may be an important component of the antithrombotic nature of the vessel wall.

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