Proc. Natl. Acad. Sci. USAVol. 89, pp. 2317-2320, March 1992Cell Biology

Coagulation factors X, Xa, and protein S as potent mitogens ofcultured aortic smooth muscle cells

(Xa inhibitors/atherosclerosis)

GREGORY P. GASIC*t, CARMEN P. ARENASt, TATIANA B. GASICO, AND GABRIEL J. GASICt*The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037; and tLaboratory for Experimental Oncology, Department of Medicine, ThePennsylvania Hospital, 800 Spruce Street, Philadelphia, PA 19107

Communicated by Robert W. Holley, December 10, 1991 (received for review October 2, 1991)

ABSTRACT Smooth muscle cells (SMCs) in the rat carotidartery leave the quiescent state and proliferate after ballooncatheter injury. The precise signals responsible for this SMCmitogenesis need to be elucidated. Although platelet-derivedgrowth factor (PDGF), a potent SMC mitogen, is released fromactivated platelets, damaged endothelium, and macrophages, itcannot be solely responsible for this proliferation. In search ofother SMC growth factors, we have examined several proteinsof the coagulation cascade. At nanomolar concentrations,factors X, Xa, and protein S promote cultured rat aortic SMCmitosis. In contrast, factor IX is only weakly mitogenic,whereas factor VII and protein C fail to stimulate SMCdivision. Protein S, the most mitogenic of these coagulationcascade factors, stimulates DNA synthesis in cultured SMCswith a time course similar to that ofPDGF-AA and without thedelay observed for transforming growth factor .3. Antistasinand tick anticoagulant peptide, two specific factor Xa inhibi-tors, inhibit SMC mitogenesis due to Xa and protein S.Coagulation factors that possess mitogenic activity may con-tribute to intimal SMC proliferation after vascular injury as aresult of angioplasty or vascular compromise during athero-genesis.

Our knowledge of intimal smooth muscle cell (SMC) prolif-eration in atherosclerosis derives from studies with animalmodels in which hyperlipidemic, fat-fed, or balloon-injuredanimals were used (1-4). SMC replication is well documentedin these models, an extensive list of cell types has beenpostulated to provide the mitogenic signal, and based onthese studies several theories of atherogenesis in humanshave been advanced (5-10). During the early stages ofatherogenesis, macrophages that adhere and infiltrate vas-cular endothelium are good candidates for such growth factorproduction as platelet-derived growth factor (PDGF) andinterleukin 1 (11-14). Platelets, however, probably do notinitiate SMC proliferation but contribute to lesion growththrough the release of PDGF, epidermal growth factor(EGF), and other mitogens (15-18). Based on in situ hybrid-ization and immunohistochemical studies of endothelial cellsand SMCs underlying regions of intimal thickening, thesecells can produce PDGF, fibroblast growth factor (FGF), andother molecules capable of stimulating SMCs (19-24). Theprimary cause of arterial intimal SMC hyperplasia duringatherogenesis and the regulation of this growth factor pro-duction in vivo, however, are unknown.

Vascular endothelial alterations may be inexorably linkedwith SMC proliferation via the failure of endothelial cellbarrier function due to denudation or to a reduction ofhomotypic endothelial cell adhesion and an increase in ad-hesivity for macrophages and platelets (24, 25). When theendothelium is damaged, cell components of the vascular

wall are exposed to circulating plasma proteins or productsresulting from activation of the coagulation cascade. Al-though PDGF released from activated platelets has for sometime now been proposed to play a leading role as a SMCmitogen, studies with PDGF neutralizing antibodies can onlyattribute 20o of the mitogenic activity in rat serum to PDGF(26). Also, Bowen-Pope et al. (27) found that rat serum,which contains 16 times less PDGF than mouse serum, isequipotent as a SMC mitogen. Collectively, these experi-ments strongly suggest that other mitogens are present in ratsera if not in the plasma itself.

In search of other SMC mitogens present in plasma, wediscovered that some proteins of the coagulation cascade,containing EGF-like domains (28-31), stimulate cultured rataortic SMCs to divide. They are mitogenic at nanomolarconcentrations, which are well within the range of physio-logical circulating levels of these proteins. Except for proteinS, they are all proteases in their activated forms. In addition,we investigated several agents that can inhibit this coagula-tion factor-mediated SMC mitogenesis. Intimal SMC hyper-plasia is the primary cause of vascular reocclusion andrestenosis after angioplasty (32-36). If these coagulationfactors serve as SMC mitogens in vivo, they could contributeto the high rate arterial restenosis following angioplasty(32-36) and even to atherogenesis.

MATERIALS AND METHODSGrowth and Coagulation Factors. Recombinant PDGF-AA

produced in yeast was obtained as a gift from Chiron.Transforming growth factor ,8 (TGF-,/) was obtained fromCollaborative Research. Highly purified human a-thrombinwas a gift of J. W. Fenton II (New York State Department ofHealth, Albany). Bovine factor VII (BfVII), BflX, BfX,human protein S (HPS), and bovine protein C (BPC), purifiedfrom plasma and chromatographically pure as ascertained bySDS/PAGE, were obtained from Enzyme Research Labs(South Bend, IN). Purified human factor X (HfX) was a giftof W. Kisiel (Department of Biochemistry and Pathology,University ofNew Mexico). BfXa, prepared from plasma anddetermined to be free of other proteases and contaminatingproteins by reverse-phase HPLC, was obtained from Boeh-ringer Mannheim. Although the human and bovine proteinswere used in place of their less readily available rat counter-parts, the primary amino acid sequences are highly conservedamong these three species.

Antibody and Other Proteins. Purified human C4b-bindingprotein was a gift from Charles T. Esmon (Howard Hughes

Abbreviations: SMC, smooth muscle cell; PDGF, platelet-derivedgrowth factor; EGF, epidermal growth factor; FGF, fibroblastgrowth factor; TGF-#B, transforming growth factor(; BfX, bovinefactor X; HfX, human factor X; HPS, human protein S; BPC, bovineprotein C; mAb, monoclonal antibody; ATS, antistasin; TAP, tickanticoagulant peptide; BSA, bovine serum albumin.tTo whom reprint requests should be addressed.

2317

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Medical Institute, Oklahoma Medical Research Foundation,Oklahoma City). Monoclonal antibodies (mAbs) to highlypurified HPS (mAb2l, mAb4l, and mAb54) were a gift fromBjorn Dahlback (University of Lund, Malmo General Hos-pital, Malmo, Sweden). Affinity-purified rabbit antibody toHfX was a gift of W. Kisiel. Purified recombinant antistasin(ATS) and tick anticoagulant peptide (TAP), two specificfactor Xa inhibitors, were generously provided by C. Dun-widdie, G. P. Vlasuk, and Paul A. Friedman ofMerck Sharpe& Dohme.SMC Cultures. Primary SMC cultures were prepared from

the thoracic and abdominal aorta of 2- to 3-month-old Wistarrats essentially by the procedure of Reilly (37). After as muchfat and adherent adventitia as possible was removed withscissors and forceps, the tissue was finely minced, enzymat-ically dissociated by successive 370C incubations with Hanks'buffered salt solution containing collagenase (0.5 mg/ml for 15min; Sigma), elastase (0.125 mg/ml for 30 min; Sigma), andcollagenase (60 min). The dissociated cells had a spindle-shaped morphology. They were grown in 10%1 fetal bovineserum/Dulbecco's modified Eagle's medium (DMEM) withantibiotics. At confluency, they exhibit extensive overlapping.Using a mAb to a-actin (Sigma), 70-90%o (preparation depen-dent) of these confluent cells stained positive for this SMCmarker. After 10 days in culture, the cells were dissociatedwith trypsin-EDTA, split 1:10, and subcultured in the mediumdescribed above. At confluency, the cells were transferred to0.1% bovine serum albumin (BSA)/DMEM for 24 hr beforethey were used for assay. All SMCs used in these studies werefrom passages 1-4. A simian virus 40 transformed SMC line(37) was provided by C. F. Reilly (Merck Sharpe & Dohme).

Experimental Protocol. Aortic SMC cultures were washedtwice with DMEM and incubated for 24 hr with 0.1% BSA(RIA grade; Sigma)/DMEM containing the buffer (control)used to dissolve the coagulation factors or 0.1 nM PDGF-AAor one of the following coagulation cascade factors at 10 nM:human a-thrombin (a-thrombin), BfVII, BfIX, BfX, HfX,BfXa, HPS, or BPC. At the end of the incubation, the cul-tures were washed twice with DMEM and pulsed for 2 hr in[3H]thymidine (6.7 mCi/mmol; 1 pCi/ml; 1 Ci = 37 GBq;DuPont/New England Nuclear) in 0.1% BSA/DMEM. Thecells were washed twice with ice-cold phosphate-bufferedsaline (PBS), treated twice with 5% trichloroacetic acid for 5

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min, dissolved with 0.25 M NaOH (0.4 ml) incubated at 370Cfor 60 min, and mixed with 5 ml of Ecolume (ICN); theirradioactivity was determined in a scintillation counter. As-says for each experiment were in triplicate.

Statistics. Error bars represent the SEM (n = 6 or moreexperiments).

RESULTS AND DISCUSSIONCultured rat aortic SMCs (passages 1-4) were treated withone of the following coagulation factors: factor X (5-50 nM),Xa (1-50 nM), IX (10-50 nM), VII (10-50 nM), protein C(20-60 nM), a-thrombin (1-50 nM), or protein S (10-50 nM).These proteins share several conserved domains; amongthem are variable numbers of EGF-like repeats (28, 29). Fig.la shows that 24-hr treatment of primary cultures of SMCswith factor X (BfX and HfX) BfXa, and HPS effectivelystimulates DNA synthesis. Since thrombin has previouslybeen shown to stimulate SMC DNA synthesis (39), it isshown here for comparison. Protein S, with the largest effect,stimulated DNA synthesis as much as 4-fold. Preincubationof 10 nM HPS for 15 min with 100 nM purified mAb (mAb4l)to epitopes of the third and fourth EGF-like domains ofHPS(38) abolished its mitogenic activity (Fig. lb). mAbs toepitopes in the y-carboxyglutamic acid domain (mAb2l) andin the first and second EGF-like domains (mAb54) had slightor no effects on mitogenic activity. Cell counts done between24 and 48 hr of SMC incubation with the mitogens confirmedthat [3H]thymidine incorporation was a fair index of celldivision (Table 1). In contrast, similar concentrations offactor Xa or protein S added to calf skin fibroblasts, 3T3fibroblasts, orto a simian virus 40 transformed SMC line failedto produce a significant increase in mitosis or in [3H]thymidineincorporation (data not shown). These results rule out con-taminating fibroblasts as contributors to the observed mito-genic response. Also, they eliminate the possibility that afibroblast mitogen (e.g., basic FGF) is a significant contami-nant of these coagulation factor preparations.To determine whether these coagulation factors act di-

rectly as SMC mitogens or through the autocrine induction ofPDGF-AA as observed for interleukin 1 (40) and TGF-p (41),a time course experiment for [3H]thymidine incorporationwas carried out. Whereas [3H]thymidine incorporation for

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FIG. 1. (a) SMC mitogenic activity of coagulation factors. SMC cultures were washed twice with DMEM and incubated for 24 hr with 0.1%BSA (RIA grade)/DMEM containing the buffer (control) used to dissolve the coagulation factors or one of the following coagulation cascadefactors at 10 nM: human a-thrombin, BfVII, BfIX, BfX, HfX, BfXa, HPS, or BPC. The results obtained with HfX and BfX were very similarand therefore they are represented by one bar in the graph. (b) Effect of mAbs against HPS on SMC mitogenic activity mediated by proteinS. SMC cultures were washed twice with DMEM and incubated for 16 hr with 0.1% BSA (RIA grade)/DMEM containing the buffer (control),100 nM each of three mAbs (mAb2l, directed against epitopes of the y-carboxyglutamic acid domain of protein S; mAb4l, directed against theepitopes of the third and fourth EGF-like domains; and mAb54, directed against epitopes of the first and second EGF-like domains; see ref.38), 10 nM HPS, or 10 nM HPS plus 100 nM mAb (preincubated at room temperature for 15 min). At the end of the incubations, the cultures(2-3 x 105 cells per well) were washed twice with DMEM and pulsed for 2 hr with [3H]thymidine (1 ,uCi/ml) in 0.1% BSA/DMEM. The cellswere washed twice with cold PBS, treated twice with 5% trichloroacetic acid for 5 min, dissolved with 0.25 M NaOH (0.4 ml), incubated at 37°Cfor 60 min, and mixed with 5 ml of Ecolume (ICN); their radioactivity was determined in a scintillation counter. Assays for each experimentwere in triplicate. The value obtained for the control samples was subtracted from the experimental values. Error bars represent the SEM (n= 6 or more experiments).

2318 Cell Biology: Gasic et al.

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Proc. Natl. Acad. Sci. USA 89 (1992) 2319

Table 1. Comparison of [3H]thymidine incorporation and cellproliferation in rat aortic SMC cultures

cpmTreatment incorporated SMCs

Buffer 4,710 ± 272 3.1 x 105BfXa (10 nM) 7,470 ± 368 4.5 x 105BfXa (50 nM) 14,709 ± 1439 5.7 x 105HPS (10 nM) 30,074 ± 2837 6.5 x 10-

[3H]Thymidine incorporation with cell proliferation in aortic SMCcultures treated for 36 hr with or without one of two differentcoagulation factors. At the end of the incubation, half of the groupswere pulsed with [3H]thymidine for 2 hr while their identically treatedcounterparts were dissociated with trypsin and the cells (SMCs onthe basis of a reddish-brown staining pattern with Masson's tri-chrome) were counted. Although there was not a direct correspon-dence between the percentage increase in thymidine incorporationand cell number, the increase in DNA synthesis correlated well withan increase in cell number.

protein S (10 nM), factor Xa (10 nM), and PDGF-AA (10ng/ml) stimulated SMCs reached a maximum at =18 hr ofincubation, TGF-f3 (0.1 ng/ml) stimulated SMCs were onlystarting to show some incorporation at this time point (Fig. 2).This outcome suggests that protein S and factor Xa either actdirectly on SMCs without the induction of transcription of agrowth factor gene (e.g., PDGF), which delays the onset ofthe mitogenic response, or they cause a release of a growthfactor (e.g., basic FGF) from a cell-surface pool (42).Because in human plasma, 40% of protein S occurs in free

form and 60%o occurs as a C4b-binding protein complex (43),we assayed the mitogenic activity of protein S, which con-tained a significant fraction of protein S C4b-binding proteincomplex, and found no apparent difference in mitogenicactivity between the free and complex-containing forms (43).Factor IX is only weakly mitogenic, whereas factor VII andprotein C were inactive in the SMC assay. SMC divisionstimulated by these coagulation factors is obtained withconcentrations below human plasma levels (29) for factor X(178 nM), factor IX (89 nM), and protein S (278 nM). In vivo,compromise of the endothelial cell barrier could permitsufficient amounts of these proteins to reach SMCs to stim-ulate them to divide.From one SMC primary culture preparation to another, we

observed some degree of variation in the mitogenic responseobtained with a given concentration of coagulation factor.The percentage of cells that immunostained positive fora-actin, a resting state SMC marker, also varied (70-90%)with each preparation. Despite some quantitative differences

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FIG. 3. Inhibition of factor Xa-mediated SMC mitogenic activityby specific factor Xa inhibitors. Details of rat SMC cultures aredescribed in Fig. 1. At confluency, cells were washed twice withDMEM and incubated at 37°C for 24 hr in 0.1% BSA/DMEMcontaining 10 nM BfXa (Boehringer Mannheim) alone or in combi-nation with recombinant ATS or TAP. Whether ATS or TAP wasadded after factor Xa or was preincubated with factor Xa and addedto the SMC cultures, the results were the same. At the end of the24-hr incubation, [3H]thymidine incorporation into DNA was mon-itored and SMC mitogenic activity was accessed as described in Fig.1. Vertical line at the top of each bar represents the SEM (n = 6 ormore experiments); where no bar is present, <3 experiments wereperformed.

observed forDNA synthesis for disparate SMC preparations,a consistent mitogenic result is obtained for several of thecoagulation factors.To rule out that a contaminant in the coagulation factor

preparations was the SMC mitogen, affinity-purified rabbitantibody to HfX was used to deplete factor X preparationsbefore assay with SMCs. No mitogenic activity remained.Since the coagulation factors used in this study are purifiedfrom bovine or human plasma, a minor contaminant that ismitogenic for SMCs but not fibroblasts cannot be completelyexcluded. In the case of protein S, however, this possibilityappears to be less likely since a mAb (mAb4l) to epitopes ofthe third and fourth EGF-like domain of HPS is able toneutralize the mitogenic effect.

Also, two specific factor Xa inhibitors, ATS (44) and TAP(45), were tested to determine whether they could inhibit thecoagulation factor-mediated SMC mitogenic response. Atnanomolar concentrations, which abolish factor Xa-mediatedcoagulation, recombinant ATS and TAP inhibit the SMCmitogenic activity of BfXa (Fig. 3). By themselves, ATS andTAP have no effect on SMC cell division. Surprisingly, ATSand TAP abolish the SMC mitogenic activity of purifiedprotein S but do not affect the factor X-mediated activity(Fig. 4). These results suggest that factorXa and factor X mayproduce their mitogenic effects on SMCs by different mech-anisms. SMCs may possess a factor Xa receptor analogous to

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FIG. 2. Time course of [3H]thymidine incorporation in SMCcells. SMC cultures as described in Fig. 1 were treated for 14, 16, 18,24, and 36 hr with buffer (control), 10 nM HPS (e), 10 nM BfXa (A),10 ng of PDGF-AA (ut) per ml, and 0.1 ng of TGF-,8 (o) per ml. Atthe end of the incubations, the cells were washed and pulsed for 2 hrwith [3H]thymidine as described in Fig. 1. The control value wassubtracted from each experimental value; thus, the [3H]thymidineincorporation represented is relative to the control value.

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FIG. 4. Effect of specific factor Xa inhibitors on HPS andBfX-induced SMC mitogenesis. Average mitogenic activity andSEM (n = 6 or more experiments) ofSMC incubated with 10 nM HPSor BfX in the absence and presence of specific factor Xa inhibitors(ATS and TAP). See Fig. 1 for details of methods.

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the endothelial cell thrombin receptor, which exhibits aproteolytic mechanism of receptor activation (46). Factor X,however, could interact with another receptor or couldactivate the same receptor simply through binding.

Protein S, which is not itself a protease or a precursor buta cofactor for protein C, may activate another SMC receptorthrough an interaction that is blocked by ATS or TAP.Despite considerable sequence similarities between thesecoagulation factors (28), not all of them are mitogenic, whichsuggests that the presence of an EGF-like domain alone doesnot confer activity. By virtue of the fact that protein S andfactor Xa stimulate DNA synthesis with a time course thatmore closely parallels that ofPDGF-AA than TGF-,f, a directmitogenic mechanism is favored. The SMC receptor(s) in-volved and mechanisms for mitogenic signal transductionremain to be determined.

If the endothelial cell barrier in vivo is altered, the coag-ulation factor-mediated SMC mitogenesis that we observedin vitro could contribute significantly to intimal SMC prolif-eration. Fibrinopeptide B, a product of clotting cascadeactivation, appears to be a chemotactic agent for macro-phages (47). Their transvascular migration during the earlyphases of atherosclerotic lesion formation increases the per-meability ofthe endothelium to the clotting factors. Exposureof underlying SMCs to protein S may be sufficient to stim-ulate cell division and lead to hyperplasia. In addition, factorXa, which promotes mitogen release from endothelial cells(48), may alter the junctional properties of these cells.Angioplasty treatment for vascular occlusion denudes arter-ies of endothelium and exposes SMCs to plasma proteins.Within 6 months of this treatment, 40% of patients haverestenosis, which is attributed primarily to intimal SMChyperplasia (32-36).

Experiments to determine whether these coagulation fac-tors contribute to the process remain to be done. Confirma-tion that coagulation factors (e.g., protein S) contribute tointimal SMC proliferation may pave the way for therapeuticuse of inhibitors such as ATS or TAP as a means of prevent-ing restenosis after balloon angioplasty.

We thank Drs. Russell F. Doolittle, Adrienne Bennett, David Schu-bert, and Pablo Valenzuela for invaluable discussions and critical readingofthe manuscript; Drs. B. Dahlback, C. Dunwiddie, G. P. Vlasuk, PaulA. Friedman, C. F. Reilly, C. T. Esmon, and W. Kisiel for providingrecombinant factor Xa inhibitors, transformed SMCs, C4b-binding pro-tein, purified HfX, factorX antibodies, and mAbs to protein S. This workwas supported by grants from the National Institutes of Health and theAmerican Cancer Society.

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