Activated Protein C Stimulates the Fibrinolytic Activity of Cultured Endothelial Cells and Decreases Antiactivator Activity

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Activated Protein C Stimulates the Fibrinolytic Activity of Cultured Endothelial Cells and Decreases Antiactivator Activity
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  Proc. Nati. Acad. Sci. USA Vol. 82, pp. 1121-1125, February 1985 Cell Biology Activated protein C stimulatesthe fibrinolyticactivity ofcultured endothelial cells and decreases antiactivator activity (plasminogen activators/plasminogen activator inhibitor/cell-mediated effect/cell-independent effect/diisopropyl fluorophosphate) YOICHISAKATA, SCOTT CURRIDEN, DAN LAWRENCE, JOHN H.GRIFFIN, AND DAVID J. LOSKUTOFF Department of Immunology, Scripps Clinic and ResearchFoundation, La Jolla, CA 92037 Communicated byK. M. Brinkhaus, October 9, 1984 ABSTRACT The effects of bovine activated protein C (APC) on the fibrinolytic activity ofcultured bovine aortic en- dothelial cells were investigated. Confluent monolayerswere incubatedwith purified APC under variousconditions and changes in total fibrinolytic activity and in the level of plasmin- ogen activator and plasminogen activator inhibitor antiactiva- tor) were monitored. The additionof APC to the cells in the absence of other bloodor plasmacomponents ledto a rapid, dose-dependent increase of fibrinolytic activity both in the me- dia and in cellularextracts. For example, 3.4 jtg of APC per ml resulted in a 15-fold increase of fibrinolytic activity in the medium within 1 hour. The enhanced fibrinolytic activity re- flected increases in both theurokinase-related and tissue-type plasminogen activators producedby these cells. Interestingly, treatment of -cells with APC also caused a rapid, dose-depen- dentdecrease in antiactivator activity. Diisopropyl fluoro- phosphate-inactivated APC did not decrease antiactivator or increase plasminogen activator. Although a small but signifi- cant direct  i.e., cell-independent) effect of APC onboth fibrinolytic activity and antiactivator activity could be demon- strated, the major portion of these changes appeared to be cell- mediated. These observations indicatethat thefibrinolytic po- tential ofcultured endothelial cells is increased by APC and thatthe enzyme active site is essential for this change. More- over, the results suggest that one of the primary mechanisms for this stimulation of endothelial cell fibrinolytic activity in- volves an APC-mediated decrease in antiactivator. Protein C (PC) may function as a physiologic regulator of thrombosis since its inherited deficiency is associated with thromboembolic disease  1-3). In addition to its well-docu- mented anticoagulant activity  4-6), activated PC (APC) also exerts a potent profibrinolytic activity in vivo. For example, infusion of bovine APC into dogs results in increased circu- lating plasminogen activator  PA), presumably reflectingtherelease of this molecule from the endothelium  7, 8 . APC may not interactdirectly with endothelium but rather withanother blood component of unknown nature, which, in turn, elicits the release of PA  8 . We have been studying both PC  9) and the fibrinolytic system of cultured bovine aortic endothelial cells (BAEs; ref. 10). These cells produce multiple forms of PA  11, 12), including both the urokinase-like (u-PA) and tissue-type  t- PA) molecules  13). They also produce an antiactivator that can inhibit the activities of these PAs  14, 15). In this report, we show that purified bovine APC can increase the fibrino- lytic activity of BAEs in the absence ofblood cells or plasma proteins. The increase of fibrinolyticactivity is associatedwith an APC-dependent decrease in the antiactivator. MATERIALS AND METHODS Reagents. Materials werepurchased from the following sources: plasticware from Coming; mediafrom Flow Labo- ratories; calf serum, trypsin, penicillin, and streptomycin from GIBCO; diisopropyl fluorophosphate  iPr2P-F),hiru- din grade IV), bovine serum albumin,TritonX-100, and chloramine-T from Sigma; Tween 80 from Baker; Bio-Rex 70 from Bio-Rad; LPG-agarosefrom Miles; blue-Sepharose CL-6B and DEAE-Sephadex A-50 from Pharmacia; S-2366  pyro-Glu-Pro-Arg-paranitroanilide) a gift fromKabi (Moln- dal, Sweden). All other chemicals were analytical grade. Proteins. Bovine PC was purified from bovine plasma by using barium citrate adsorption, ammonium sulfate precipi- tation, and DEAE-Sephadex  16) and blue-Sepharose CL-6B chromatography. Blue-Sepharose CL-6B removed pro- thrombin from the DEAE-Sephadex fractions containing PC. The purified PC appeared to be at least 95 homoge- neous when analyzed on 9 polyacrylamide gels in the pres- ence of NaDodSO4 andwas free of detectablefactorsVII, IX, and   and of prothrombin activity. Bovine APC was prepared by activation of PC with a- thrombin at an enzyme-to-substrate weight ratio of 1:5. Acti- vation was monitored by the appearanceof amidolytic activ- ity toward the chromogenic substrateS-2366. In this assay, 10 ,ul of APC  0.5-1 mg)wasadded to 490 ,ul of 0.05   Tris -HCl containing 0.15   NaCl (pH 8.3), 0. 1 bovine se- rum albumin, and 0.15   S-2366.Activity wasmeasured at 37°C as the initial AA405/min using a Cary 210 spectropho- tometer. Thrombin was removed from APC by chromatogra- phy on a column of Bio-Rex 70. Although no thrombin activ-ity was detected in the APC as measured in fibrinogen clot- tingassays, hirudin  0.5 unit/ml final concentration) was added to insure that the preparation was devoid of all throm- bin activity. iPr2P-F-inactivated APC was prepared by incu- bating APC with 5   iPr2P-F for 2 hr at 22°C, followed by dialysis. The iPr2P-F-treated sample exhibited no detect le amidolytic activity. Plasminogen and fibrinogen were puri- fied as described  17, 18). 1251-labeled fibrinogen was pre- pared by using the chloramine-T method  11, 19). Human urokinase (World Health Organization 1st International Ref- erence Preparation) was purchasedfrom the National Insti-tute for Biological Standards and Control (London, U.K.). Cell Culture. BAEs were isolated from the aortae of fresh- ly slaughtered cowsand cultured as indicated  20). The cells used for these studies hadbeen passaged 5-20 times andwere positiveforfactor VIII-related antigen. All cultures were grown to confluency in 60-mm dishes. Confluent cul- tures were fed every day for 4 days and again 1 hr before use, with modified Eagle s medium  ME medium) containing 10 calf serum. Abbreviations: PC, protein C; APC, activated PC; PA, plasminogen activator; u-PA, urokinase-type PA; t-PA, tissue-type PA; iPr2P-F, diisopropylfluorophosphate; BAE, bovine aorticendothelial cell. 1121 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 thisfact.  Proc. NatL. Acad. Sci. USA 82  1985 Conditioned Media (CM) and Cellular Extracts. Confluent cultures werewashed three timeswith phosphate-buffered saline  Pi/NaCI 0.01 M sodiumphosphate/0.14 M NaCl, pH 7.2 to remove the serumand then incubated at 370C for various times in serum-free M medium containing various amounts of APC or iPr2P-F-inactivated APC and hirudin. The CM was collected,centrifuged at 600 xg for 10 min at 220C to remove detached cells and cellular debris, and stored in 0.01 vol/vol Tween 80 at -80 C. To prepare cellular extracts forstudies of cell-associated PA, cultures were washed three times with cold  40C Pi/NaCl and then ex- tracted at 40C with500 pul of Triton X-100 [0.5 vol/vol in P1/NaCl]. The culture dish waswashed again with 500 gl of P,/NaCl,and the extract and wash werepooledand stored at -800C. FibrinolyticActivity. PA activity was assayedon 1251-la- beled fibrin-coated multiwell tissue culture dishes  24 wells, 16 mm; Costar, Cambridge, MA as described  11 . The stan- dard cell-free assay contained in 1 ml:4 ,ug of human plas- minogen, 0.1 gelatin, 0.25 Triton X-100, 0.1 M Tris-HCl, pH 8.1, and a source of PA. Fibrinolyticactivity was not observed in the absence of plasminogenand was expressed in relative urokinase international units. Polyacrylamide Gel Electrophoresis. NaDodSO4/polyacryl- amide slab gel electrophoresis was carried out by using the method of Laemmli  21 , employing 10-cm resolvinggels containing 9 acrylamide and 2-cm stacking gels of 4 ac- rylamide. Afterelectrophoresis,portions of the gel contain- ing molecularweight standards were removed and stained with 0.25 Coomassie blue, and the remaining portion of thegel wasprocessed foreither fibrin autography orreverse fi- brin autography. Fibrin Autographyand Reverse Fibrin Autography. To pre- pare fibrin agar films, a 2 solution of agarose wasmixed with prewarmed  45°C P1/NaCl containing plasminogen and thrombin. Fibrinogen  10 mg/ml) in P,/NaCl  37°C was add- ed, and the solution was mixed and poured onto a glass slide. Final concentrations were 1 agarose, 25 ,g of plasminogen per ml, 0.18 unit of thrombin per ml, and 2 mg of fibrinogen per ml. After electrophoresis, the NaDodSO4/polyacryla-mide gels to beassayed by fibrin autographywere soaked in 2.5 Triton X-100 for 1.5 hr at room temperature, patted dry with a paper towel, and applied to the surface of a fibrin-agar indicator film  13 . Fibrin-agar indicatorfilms to beused for reverse fibrin autography were prepared as above but also containedurokinase  0.025 unit/ml final concentration . The interaction of this urokinasewith plasminogencaused the in- dicatorfilm to lyse. The opaque lysis-resistant zones that develop in the otherwise clear indicator film result from the presence of inhibitors in the NaDodSO4/polyacrylamide gel 14, 22 . Miscellaneous. Protein wasdetermined by the method of Bradford with bovinealbumin as a standard  23 . 1251I was measured by using a Micromedic (Horsham, PA) y spec- trometer. RESULTS Effect of APC on the Fibrinolytic Activity of BAEs. Washed monolayers wereexposed to APC in serum-free M medi- um, and at various times thereafter an aliquot of the medium wasremoved and testedfor fibrinolytic activity. The addi-tion of APC led to a rapid, dose-dependent increase of thefibrinolytic activity of  M  Fig. 1 . This increase was appar- ent within the first 15 min and continued for the next6 hr. iPr2P-F-inactivated APC did not increase the fibrinolytic ac-tivity of CM. In these experiments, hirudin was added rou-tinely to thereaction mixtures to neutralizetraces of throm- bin present in the APC preparation. Controls showed thatthe APC-dependent increase of fibrinolytic activity was the 32 Incubation Time  h FIG. 1. Accumulation of fibrinolyticactivity in mediafrom APC-treated and untreated BAEs. Increasing amounts of active or inactive APC wereadded to BAE monolayers asfollows: 0  0 , 0.68  A , and 3.4  u ,ug of APC perml; 3.4 ,ug of iPr2P-F-inactivated APC  X per ml. At varioustimes an aliquot  100pJ of the media wasremoved and tested forfibrinolytic activity. In separate experi- ments, aliquots 100 ,ui of the media collected from untreated BAEs at theindicated times were removed andadded to tubescontaining0.68  A or 3.4  in ,ug of APC per ml. The fibrinolyticactivity ofthese mixtures was measured after 1 hrof incubation  370C . same in the presence or absence of hirudin and that APC itself had no effect on the activity of purified urokinase, t- PA, or plasminogen  data not shown). No change in the mor- phology of BAEs incubated in the presence of 3.4 ,ug of APC per ml was observed over a24-hr period. Experiments were performed to determinewhether the APC-mediated increaseof fibrinolytic activityactually re- quired cells  i.e., cell-dependent effect or resulted from   directeffect of APC on some component(s) in  M  i.e., cell- independent effect .  M was collected at varioustimes from cells not previously exposed to APC, and floating cells and cellular debris were removed by centrifugation. Each sample then was incubated in vitro for 1 hr at 370C in th presence or absence of APC and retested for fibrinolytic activity. A small but significant anddose-dependent increase of fibrino- lytic activity was observed  Fig. 1 . For example, the fibrin- olytic activity of 6-hr  M increased by~2-foldupon incu- bation with 0.68 pug of APC per mlandby 4- to5-fold upon incubation in the presence of 3.4 ,ug of APC per ml.In con- trast, when added to cells for 6 hr, these concentrationsof APC resulted in 8- and 17-fold increases,respectively. In the invitro studies, no further change in fibrinolyticactivity was observed after increasing either the amount of APC em- ployed or the incubation time.In fact, brief in vitro incuba- tion  e.g., 15 mmn of  M with APC resulted in maximal stim-ulation of fibrinolyticactivity  data not shown). Incontrast, when APC wasadded to the medium in the presence of cells, the fibrinolytic activity continued to rise for atleast 6 hr  Fig. 1 . When confluent BAEs were fed with fresh M medium containing 10 calf serum, incubated for 96 hr without re- feeding, and thenchallengedwith APC, increases in fibrino- lytic activity similarto those seen in Fig. 1 were observed  data not shown).Thus, although the feeding regimen of BAEs may markedly influencethe overall fibrinolytic activi- 1122 Cell Biology: Sakata et aL  Proc. Natl. Acad. Sci. USA 82  1985 1123 Table 1. Effect of APC on cell-associated fibrinolytic activityFibrinolytic activity, IUx 1iO Sample 0 hr 1 hr 3 hr Control 2.52.5 3.0 APC 0.68 ,g/ml 9.0 13.0 3.4,ug/ml 25.0 35.03.4  5g3ml iPr2P-F 2.5 3.0 Monolayers of BAEs were exposed to various amounts of APC forthe indicated times under culture conditions, washed exten- sively, and extracted with Triton X-100. The fibrinolytic activity of an aliquot of each extract containing 50 ,ug of protein was measuredand expressed as international units  IU of urokinase. ty of these cells 11 , it did not alter their response to APC. The addition of APC butnot iPr2P-F-inactivated APC to BAEs increased cell-associated fibrinolyticactivity in a dose-dependent manner  Table 1 similarto its effect on extracellular fibrinolytic activity. Effect of APC on the PAs Producedby BAEs.Experimentswere conducted to determinewhether the APC-induced in- crease of fibrinolyticactivityresulted from changes in differ- ent forms of PA.  M was collected from cells exposed to APC for 6 hr, subjected to NaDodSO4/polyacrylamide gel electrophoresis, andanalyzed by fibrin autography  Fig. 2 .  M from confluent BAEs contained multiple molecular forms of PA, seen as distinctlytic zonesof Mr 52,000, 74,000, and 100,000  Fig. 2, lane 1 . A weaker activity was observedthroughout the regionof the gel between the two higher molecularweight forms. APC caused a dose-depen-dent increase in the activity of all PA forms  Fig. 2, lanes 2 and 3 . The PA profiledid not change when iPr2P-F-inacti- vated APC wasemployed  Fig. 2, lane 4 , in agreement with thefunctional assay data  Fig. 1 . NoPA activity was detect- ed in purified APC itself  Fig. 2, lane 5 . Effect of APC on Antiactivator. BAEs produce both PAs and an antiactivator  13-15 . Thus, agents that alter the fibrin- olytic activity of these cells may do so by changing PA, antiactivator, or both  24 . The effect of APC on the antiacti- vator activity of BAE  M was studied by reverse fibrin au-   00> i 74 52 4 1 23 4 5 FIG. 2. Analysis of PA forms released by BAEs exposed to APC. Aliquots of  M were fractionated by NaDodSO4/poly- acrylamide gel electrophoresis andanalyzed by fibrin autography. The indicatorgel was allowed to develop for 6 hr and then photo- graphed. Gel lanes contained 100  tl of  M from BAEs that were exposed for 6 hr to no APC  lane 1 0.68 jug of APC per ml  lane 2 , 3.4 /ig of APC per ml  lane 3 , and 3.4 ,ug of iPr2P-F-inactivated APC per ml  lane 4 . Lane 5 shows the profile when 100  ul of purified APC at 3.4 ,ug/ml was analyzed. Molecular weights are shown as Mr X lo-,.   2 3 FIG. 3. Analysisof antiactivator released from BAEs exposed to APC. Confluent BAEs were incubated in serum-free medium in the absence of APC  lane 1 or in the presence of either APC  lane 2 or iPr2P-F-inactivated APC  lane 3 at 3.4 ,4g/ml. Six hours later, the  M was removed and 100 u.l of it was analyzed by NaDodSO4/ polyacrylamide gel electrophoresis and reverse fibrin autography. tography  Fig. 3 , a semi-quantitative technique in which th size of th lysis-resistant zone in the indicator film reflects the amount of inhibitor applied to the NaDodSO4 gel  22 .  M collected from untreated cultures gave rise to a singlelysis-resistant zone of Mr 50,000  Fig. 3, lane 1 , reflectingthe presence of the antiactivator  22 . Considerably less an tiactivatoractivity was detected in  M prepared from APC treated cells  Fig. 3, lane 2 . This decrease in antiactivator was not apparent when iPr2P-F-inactivated APC was em ployed  Fig. 3, lane 3 , consistent with thelack of effect of inactive APC on total fibrinolyticactivity  Fig. 1 . The APC mediateddecrease in theantiactivator activity of  M was dose-dependent  Fig. 4A .   dose-dependent decrease in cell-associated antiactivator activity also was obvious  Fig. 4B , but to a lesser degree than that observed for CM Ex- periments similar to thosedescribed in Figs. 1 and 2 were performed to determine whether the decrease in antiactiva- tor resulted from a direct, cell-independent effect of APC on antiactivator or was cell-mediated  Fig. 4C . The pattern in Fig. 4C shows a smallbut significant effect of APC in th absence of cells  compare Fig. 4C, lanes 1 and 2 . However, this cell-independent effect was relatively minor when com pared to the decrease in antiactivator observed when APC was added to cells  compare Fig. 4C, lanes 2 and 3 . A   1 2 3 1 23C 1 23 FIG. 4. Effect of APC on antiactivator. Confluent BAEs wereincubated in serum-free ME medium in the presenceof increasing amountsof APC. After 6 hr, 100 /A of the resulting  M  A or 50  ag of the cell extracts  B was analyzed forantiactivator activity by reverse fibrin autography. The concentrations of APC employed were 0  lane 1 0.68  lane 2 , and 3.4  lane 3 ,ug/ml. In separate experiments  C , an aliquot 100  ul of  M collected from untreated BAEs after 3 hr wasincubated for an additional 1 hr in a test tube in the absence  lane 1 or presence  lane 2 of APC  3.4 jug/ml and similarly analyzed. Lane 3 shows  M collected from cells exposed to APC  3.4 tkg/ml for 3 hr. Cell Biology: Sakata et aL  Proc. NatL. Acad. Sci. USA 82  1985 DISCUSSION Bovine APC increasesthe fibrinolyticactivity of blood when it is administered intravenously to dogs  7,8 , possibly through the generation of a secondary messenger molecule that stimulates PA release from endothelium  8 . Our results indicate that  PC alsostimulates the fibrinolyticactivity of isolated endothelial cells. However, in apparent contrastto the in vivo results  8 , the effect on cultured cells occurs in the absence of plasma or blood cells  Fig. 1 suggesting that the increase in this system is initiated by  PC itself andnot by a second molecule. The  PC effect is dependent on the enzyme active site  Figs. 1 and 2 , and both cell-associated and secreted fibrinolyticactivity are increased. Interesting- ly, the APC-mediated stimulation of fibrinolytic activity is achieved both through the direct action of  PC on a compo- nent s in CM and as a consequence of a cellular response to the presence of APC. The direct, cell-independent effect is rapid, dose-dependent, and reproducible. However, its mag- nitude and duration are relatively small compared to the in- creases observed in the presence of cells  Fig. 1 . Thus, the APC-dependent stimulation ofthe fibrinolytic system of cul- tured BAEs appears to be mediated, in large part, by the cells themselves. Whether the responsiveness of the cells to APC is influenced by their growth state  10,11 and passage number remains to be determined. The exact mechanisms by which APC increases the fibrin- olyticactivity of BAEs remain to be elucidated. They are likely to be complex. For example, because BAEs produce both fibrinolytic activators  11-13 and an antiactivator that inhibits them  14,15 , the increaseof fibrinolyticactivity may bedue to increased PA, decreased antiactivator, or both. APC does cause a significant increase in the activity of all PA formsrevealed by fibrin autography gels  Fig. 2 . However, it also decreases antiactivator activity  Figs. 3 and 4 anddoes so in a way that is consistent with, and may account for, its effect on total fibrinolytic activity. For ex- ample, the APC-mediated decrease in antiactivator in both CM  Fig. 4A and cells  Fig. 4B requires theactive site of the enzyme  Fig. 3 and occurs in a dose-dependent manner. Moreover, there is both a cell-independent and cell-mediated decrease in antiactivator in response to APC  Fig. 4C . The cell-independent effect of  PC on antiactivator, like its di- rect effect on fibrinolytic activity, is small compared to the cell-mediated changes in these activities. These consider- ations support the hypothesis that one of the major mecha- nisms by which  PC increases the fibriholytic activity of BAEs is through these effects   antiactivator. Itis notclear whether APC also stimulatesthe rate of PA synthesis and release or whether the APC-inediated increase of overall fi- brinolyticactivity  Fig. 1 and of PA activity  Fig. 2 can be accounted for entirely by its effect on antiactivator. APC it- self is a serine proteinase  6,7 , and its active site is required both to stimilate fibrinolyticactivity  Figs. 1 and 2 and to decrease antiactivator  Fig. 3 . However, it is not clear from these experimentswhether the direct, cell-independentchange s is in fact a proteolytic event  e.g., cleavage and inactivationof the antiactivator . APC does not appear to alter directly the apparent molecularweight or fibrinolytic activity of purified t-PA,urokinase, or plasminogen  data not shown , suggesting that if theproteolytic activity of APC is important fdr the direct effect, it is probably not important at the level of thesecomponents. The nature of the cleavage event that initiates the cell-mediated changes also remains to be elucidated. Obviously, many further studies are required to distinguish among these and other possibilities and to es- tablish the exact mechanismbywhich APC enhances endo- thelialcell fibrinolyticactivity. It shouldbe noted that Colucci et al.  25 wereunable to detect increased fibrinolytic activity in blood after infusion of human APC into spider monkeys. This failure to demon- strate a profibrinolytic property of human APC may reflect inherentdifferences in the fibrinolytic system of the spider monkey as compared tothat of the dog  7, 8 and the cul- tured bovine endothelial cell. Alternatively,the inability to measure a fibrinolytic response in the spider monkey may indicatethat high circulating levels of antiactivator preventdetection of free t-PA in the euglobulin fractions prepared by Colucci et al.  25 . A similar complicationhas been suggest- ed for the failure to detect t-PA in the culture medium of human umbilical vein endothelial cells  24, 26-28 . In conclusion,these results show that APC directly in- creases the fibrinolyticactivity of endothelial cells and fur- ther support the hypothesis that APC generated in the vascu- lature by thrombin bound to thrombomodulin  29 may pre- vent thrombosis by increasing fibrinolytic activity as well as by inactivating factors Va andVIIIa  6, 7 . Since deficien- cies in PC  1-3 , plasminogen  30 , and fibrinolyticactivity  31 are associated with thrombotic disease, the APC-depen- dent fibrinolytic activity of endothelial cells may provide a useful modelsystem for studying the regulation of physiolog- ic thrombolysis. Bariuniprecipitateofbovine plasma was kindly provided by Drs. Gary Nelsestuan, Michael Nesheim, and Ken Mann.The technical assistance of Mary K. Brophy in thepurification of bovine PC is appreciated. The excellent secretarial assistance of Gerry Josephs and Cheryl McLean is gratefully acknowledged. Thisresearch was supported inpart by the National Institutes ofHealthGrants HLBI- 22289, HLBI-16411, RR00833, andHLBI-24891and by Eli Lilly Re- searchLaboratories. i. Griffin, J. H., Evait, B., Zimmerman, T. S., Kleiss, A. J.   Wideman, C. 1981 J. Clin. Invest. 68, 1370-1373. 2. Broekmans, A. W., Veltkamp, J.J.   Bertina, R.  1983 N. Engl. J. Med. 309, 340-344. 3. 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