Proc. Nati. Acad. Sci. USAVol. 81, pp. 3424-3428, June 1984Cell Biology

Plasma binding proteins for platelet-derived growth factor thatinhibit its binding to cell-surface receptors

(carrier protein/wound healing/atherosclerosis/a2-macroglobulin)

ELAINE W. RAINES*, DANIEL F. BOWEN-POPE*, AND RUSSELL ROSS*tDepartments of *Pathology and Biochemistry, University of Washington, Seattle, WA 98195

Communicated by Arno G. Motulsky, February 17, 1984

ABSTRACT Evidence is presented that the binding ofplatelet-derived growth factor (PDGF) to plasma constituentsinhibits the binding of PDGF to its cell-surface mitogen recep-tor. Approximately equivalent amounts of PDGF-binding ac-tivity were found in plasma from a number of different speciesknown by radioreceptor assay to contain PDGF homologues intheir clotted blood. Activation of the coagulation cascade didnot significantly alter the PDGF-binding activity of the plasmacomponents. Three molecular weight classes of plasma frac-tions that inhibit PDGF binding to its cell-surface receptorwere defined by gel filtration: -40,000, 150,000, and>500,000 Specific binding of 125I-labeled PDGF to the highestmolecular weight plasma fraction could also be demonstratedby gel filtration. The binding ofPDGF to these plasma compo-nents was reversible under conditions of low pH or with guani-dine'HCl, and active PDGF could be recovered from the high-er molecular weight fractions. Immunologic and functional ev-idence is presented that the highest molecular weight plasmafraction may be a2-macroglobulin. A model is proposed inwhich the activity of PDGF released in vivo may be regulatedby association with these plasma binding components and byhigh-affinity binding to cell-surface PDGF receptors.

Platelet-derived growth factor (PDGF), a protein releasedfrom the platelet a granule during coagulation, is a majormitogen for connective tissue cells in culture (1-3). It hasalso been shown to be chemotactic for smooth muscle cellsand fibroblasts (4-7). Because of the specificity of PDGF forconnective tissue cells (8, 9) and the focal release of PDGFfrom platelets at sites of injury, it has been postulated thatPDGF may play a physiological role in wound healing andtissue repair (10, 11) and a pathological role in the formationof the lesions of atherosclerosis characterized by smoothmuscle cell proliferation (12-14).Since PDGF is an extremely potent mitogen (ED50,

=10-11 M) (9) that is not normally present in the plasma (15-17), modulation of its action on release is of interest. Thisreport presents evidence for the presence of PDGF-bindingproteins in plasma that inhibit the binding of PDGF to itscell-surface receptor and consequently alter the effective-ness of PDGF at the point of initial interaction between thehormone and the cell. General properties of the plasmaPDGF-binding activity and its phylogenetic distributionwere examined to evaluate its possible role in regulating theaction of PDGF.

MATERIALS AND METHODSProcedures and Materials. Highly purified PDGF and 1251_

labeled PDGF (125I-PDGF) were prepared as described (9,18-20). PDGF-Sepharose or bovine serum albumin-Sepha-rose were prepared by incubating 60 pgg ofPDGF in the pres-

ence of 6.7 mg of bovine serum albumin (Pentex crystallized,Sigma), or 6.7 mg of bovine serum albumin alone, with 6-aminohexanoic acid-activated-Sepharose 4B (10-14 gmol ofactivated spacer groups per ml of gel; Sigma). Incubations ofPDGF-Sepharose and bovine serum albumin-Sepharosewith plasma components were performed for 3 hr at roomtemperature and eluted with 1.0 M acetic acid. Human a2-macroglobulin (a2-M) and rabbit antiserum to human a2-Mwere obtained from Calbiochem-Behring. Anti-a2-M IgGwas prepared by chromatography on Protein-A Sepharose(Sigma). Binding and other procedures are described in thefigure legends and tables.

RESULTSPlasma Inhibits the Binding of PDGF to Its Cell-Surface Re-

ceptor. The effect of plasma on the specific high-affinitybinding ofPDGF to its cell-surface receptor was analyzed bydetermining the competitive activity of unlabeled PDGF inthe presence and absence of plasma. As demonstrated inFig. 1, 60% plasma inhibited the binding of unlabeled PDGFto the human fibroblasts by =50%. Sixty percent plasmalacking added PDGF (the point on the curve in Fig. 1 repre-sented by 0 PDGF) had no effect on the subsequent bindingof 125I-PDGF to cells. Since the test samples (unlabeledPDGF, unlabeled PDGF in 60% plasma, or 60% plasmaalone) are incubated with the cells and then removed beforethe addition of the 1251-PDGF, the lack of an effect on 1251_PDGF binding with 60% plasma alone suggests that the inhi-bition by plasma is not mediated by direct stable interactionwith the receptor, as would be the case, for example, if theplasma contained PDGF. Rather, the difference between theknown concentration of PDGF added to the plasma and theapparent PDGF concentration presumably represents PDGFbound to plasma proteins and, therefore, unavailable forbinding to the PDGF cell-surface receptor. For example, 0.5ng of PDGF per ml added in the presence of 60% plasmaproduces only as much inhibition of subsequent 125I-PDGFbinding as does 0.23 ng of PDGF added in the absence ofplasma. The remaining 0.27 ng/ml is proposed to be boundto plasma constituents.This can also be demonstrated by direct incubation of

plasma and 125I-PDGF with cultured cells at 40C. When plas-ma lacking PDGF (as shown in Fig. 1) is incubated simulta-neously with 125I-PDGF and cultured cells, the binding of125I-PDGF to the cultured cells is decreased (data notshown). Plasma also significantly inhibits binding of PDGFto its receptor at 370C (data not shown). Since cellular bind-ing at 370C is determined under nonequilibrium conditions,only cellular binding studies at 40C are reported here.

Presence of PDGF-Binding Activity in Plasma from VariousSpecies. Using the radioreceptor assay for PDGF, plasma, orplasma-derived serum (PDS; prepared from plasma by recal-

Abbreviations: PDGF, platelet-derived growth factor; a2-Ma2-mac-roglobulin; PDS, plasma-derived serum; IGF, insulin-like growthfactor; 1251-PDGF, 125I-labeled PDGF.

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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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FIG. 1. Inhibition of PDGF binding to its cell-surface receptor.125I-PDGF binding to human fibroblast preincubated in the presenceof increasing concentrations of unlabeled PDGF in binding medium(e) or in 60% human plasma (0) was determined as described (9, 20).Test samples (1.0 ml) were incubated with 2.4-cm2 cultures of hu-man fibroblasts (3.6 x 104 cells per well) at 40C on an oscillatingtable; after 3 hr, test samples were removed and the cultures werewashed once, followed by incubation with 125I-PDGF at 0.16 ng/ml(25,000 cpm/ng) for 1 hr at 40C. 1251-PDGF bound is expressed asthe percentage of the "251-PDGF bound in the absence of any PDGFor test substance during the 3-hr preincubation with the cells (817 +12 cpm per well). The data are plotted without correction for non-specific binding, which is normally 10%-12% of total binding (datanot shown). Binding in the absence of added PDGF was the same forno addition (817 ± 12 cpm per well) and 60% plasma alone (808 ± 4cpm per well). The mean ± range of duplicate determinations is plot-ted. Human plasma was prepared by drawing blood from normalhumans into precooled syringes containing acid citrate dextrose(NIH formula A) to give a final concentration of 10% (vol/vol). Thecitrate-treated blood was immediately centrifuged at 39,000 x g for30 min at 40C, and the cell-free plasma was removed.

cification and centrifugation to remove the fibrin clot) from anumber of different species was examined for inhibition ofPDGF binding to its cell-surface receptor (Table 1). Clottedwhole blood serum from all of the species listed in Table 1 isknown to contain a PDGF homologue that competes withhuman PDGF for receptor binding (16). The plasma of all ofthe species examined contained approximately equivalentamounts of PDGF-binding activity. In addition, no signifi-cant difference was detected in the PDGF-binding activitybetween human plasma and PDS that contained products re-sulting from the activation of the coagulation cascade.

Fractionation of Plasma PDGF-Binding Components by GelFiltration: Evidence for Specificity. The plasma PDGF-bind-ing activity was further characterized by fractionation of hu-man PDS on Bio-Gel A-0.5 m (nominal exclusion, Mr500,000) in 0.01 M Tris.HCl/0.15 M NaCl, pH 7.4 (Tris-buff-ered saline). Fig. 2 shows the A280 elution profile and theinhibition of PDGF-binding to its cell-surface receptor forpooled fractions. None of the fractions, when tested alonefor competitive activity, had detectable levels ofPDGF (datanot shown). The approximate molecular weights of the bind-ing fractions as determined by a logarithmic plot of the mo-lecular weights of the indicated calibration standards were>500,000, 150,000, and 40,000.

Fig. 3 demonstrates that this PDGF-binding activity can

Table 1. Plasma and PDS from various species inhibit PDGFbinding to its cell-surface receptor

Cell-bound PDGF in 50% plasma,Species % of control

PlasmaChicken 44 + 9 (n = 2)Goat 47 + 9 (n = 3)Human 46 + 3 (n = 4)Rabbit 51 + 14 (n = 2)Rat 33 ± 14 (n = 3)

PDSBovine 48 + 13 (n = 3)Fetal bovine 57 + 1 (n = 2)Human 42 ± 14 (n = 12)Macaca nemestrina 34 + 4 (n = 2)

Plasma or PDS from various species was tested at 2-4 doses ofunlabeled PDGF (0.05, 0.1, 0.2, or 0.3 ng/ml) as described in Fig. 1.For each condition, inhibition of binding of unlabeled PDGF wasevaluated by determining the apparent PDGF bound from the stan-dard curve of unlabeled PDGF incubated in binding medium and isexpressed as the percent of the original PDGF concentration addedto the test samples. Results are tabulated as the mean ± SEM of thenumber of determinations indicated in parentheses. In the absenceof unlabeled PDGF, 50% plasma or PDS had no effect on 251I-PDGFbinding (see Fig. 1). Plasma was prepared as described in Fig. 1 andPDS was prepared from the citrate-treated cell-free plasma by theaddition of calcium (final concentration, 14 Amol/ml), incubationfor 2 hr at 370C, and centrifugation at 39,000 x g for 30 min to re-move the fibrin clot. Adult bovine and fetal bovine PDS were ob-tined from Sterile Systems.

8.0

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I

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._

._

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FIG. 2. Fractionation of human PDS on Bio-Gel A-0.5 m anddetermination of fractions that inhibit PDGF binding to cells. Hu-man PDS (6 ml) prepared as described in Table 1 was chromato-graphed on a Bio-Gel A-0.5 m column (2.5 x 95 cm) equilibrated in0.01 M Tris'HCl/0.09 M NaCl, pH 7.4. Flow rate was 15 ml/hr; 8.2-ml fractions were collected, and for each fraction, absorbance at 280nM (v) was determined. The inhibition of PDGF binding to cells(bars) by column fractions has been determined from the differencebetween the amount of PDGF bound to the cells in the presence andabsence of the test fraction. Briefly, a known concentration of unla-beled PDGF (0.3 ng/ml) was added to each fraction, and the effec-tive concentration of PDGF in the presence of the test fraction wascalculated from a standard curve using pure PDGF alone (see Fig.1). The difference between the effective concentration and the actu-al concentration (0.3 ng/ml) represents the inhibition ofPDGF cellu-lar binding resulting from the presence of the test fraction, and it isplotted as a percentage of the actual concentration of unlabeledPDGF (0.3 ng/ml) added to the test fraction. All fractions from 20 to60 were examined (600 A.l per well) in pools of 2 fractions (concen-trated 4-fold with a PM-10 membrane). Only pools demonstrating>10o inhibition are plotted. Blue dextran (void volume, VO) aldol-ase (161,000), ovalbumin (43,000), chymotrypsinogen (25,700), and3H20 (total column volume, Vt) were used to calibrate the column.

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0

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0*10a)

90(k8070605040302010

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Protein concentration, g/ml

FIG. 3. Specificity of plasma inhibition of PDGF binding to itscell-surface receptor. The ability of increasing concentrations of testsubstances to decrease PDGF binding to human fibroblasts was de-termined as described in Fig. 1. Test substances were PDS (0); frac-tion I (fractions 23-26 in Fig. 2) ofPDS chromatographed on Bio-GelA-0.5 m (A); Bio-Gel A-0.5 m (fractions 23-26 in Fig. 2) of PDS thatwere incubated with and adsorbed material eluted from PDGF-Sepharose (0) or from bovine serum albumin-Sepharose (i); bovinefetuin (Sigma, type II) (A); and bovine serum albumin (Miles, Pentexcrystallized) (A). For each condition, inhibition of binding of unla-beled PDGF (0.25 ng) to fibroblasts was evaluated as described inTable 1. The standard errors of triplicate determinations of 125-

PDGF binding were normally 2%-5% of the mean.

be purified up to 60,000-fold over its representation in un-fractionated plasma by gel filtration followed by affinitychromatography on PDGF covalently bound to Sepharose.In Fig. 3, the PDGF-binding capacity of different fractionshas been evaluated by determining their ability to decreasethe binding of known concentrations of PDGF to test mono-layers, as described for Fig. 1. The ordinate indicates thepercent of added PDGF still able to interact with cell-surfacePDGF receptors. In the presence of increasing concentra-tions of plasma, decreasing amounts of PDGF bound to thecells (half-maximal inhibition at -3 x 10-2 g/ml). The high-est molecular weight fraction after gel filtration of plasma onBio-Gel A-0.5 m (Fig. 2) was >43-fold more potent (half-maximal inhibition at -7 x 10-4 g/ml) than unfractionatedplasma in preventing PDGF binding to its cell-surface recep-tor. This fraction was further purified 1400-fold by affinitychromatography on PDGF covalently bound to Sepharose(half-maximal inhibition at -5 x 10-7 g/ml). The affinity-purified PDGF-binding fraction used for Fig. 3 was elutedfrom PDGF-Sepharose in 1.0 M acetic acid, immediatelyneutralized, and tested for PDGF-binding activity. Subse-quent preparations that have not been neutralized and testedimmediately have shown as low as 1/20th the specific activi-ty (data not shown). This decrease may reflect instability ofthe purified protein (see Discussion). Since all fractions areevaluated by inhibition of binding of unlabeled PDGF to fi-broblasts (Fig. 1), any leakage of PDGF from the Sepharosewould have been detected when the fraction was assayedwithout unlabeled PDGF and none was found (data notshown). The specificity of the affinity purification is demon-strated by the fact that the same gel filtration fraction, ad-sorbed to and eluted from bovine serum albumin covalentlybound to Sepharose, had no effect on PDGF binding to itscell-surface receptor.

Fig. 3 also shows that this inhibitory effect is specific toplasma and not a general property of concentrated proteinsolutions. Bovine serum albumin and fetuin, at protein con-centrations equivalent to the total protein concentration ofplasma, had no effect on the PDGF binding to cultured cells,Fetuin, as a glycoprotein with an isoelectric point of 3.5 (21),was chosen as an anionic protein that might bind highly cat-ionic PDGF (p1, 9.8).

Specific Association of '2SI-PDGF with Higher MolecularWeight Plasma Fractions. Fig. 4 demonstrates the gel filtra-tion profile obtained when I-PDGF was incubated withplasma and then chromatographed on Bio-Gel A-0.5 m inTris-buffered saline (pH 7.4). Approximately 20% of the 125I-PDGF was found associated with fractions 20-27 (void vol-ume of column, Mr > 500,000). In the presence of 10-6 Munlabeled PDGF, 80% of the I251-PDGF previously associat-ed with this higher molecular weight fraction was shifted to abroad peak of lower molecular weight. The broad peak of5I-PDGF between fractions 35 and 50 (approximate Mr,

150,000-30,000) increased in the presence of 1 ,uM unlabeledPDGF and was also found when 125I-PDGF was chromato-graphed without plasma (data not shown). This broad peakof 1251-PDGF and the shift from the expected elution positionof unbound 125I-PDGF (fractions 45-55) may represent mul-timers of PDGF.

Dissociation of the Binding Protein(s)-PDGF Complex. Toexamine the nature of the binding protein(s)-PDGF complexand to verify its presence under physiological conditions, hu-man whole blood serum was fractionated on a Bio-Gel A-0.5m column equilibrated in Tris-buffered saline. Four fractionswith molecular weights greater than native PDGF were re-chromatographed under dissociating conditions on Bio-GelA-0.5 m equilibrated in 4 M guanidine'HCl in Tris-bufferedsaline and fractions 30-45 (the elution position of monomerPDGF) were pooled, dialyzed, and assayed by radioreceptorassay for the presence of dissociated PDGF. The results areshown in Fig. 5. Dissociated and biologically active PDGFwith an apparent Mr of 30,000 was detected in fractions I, II,and IV after gel filtration in guanidine.The distribution of the recovered PDGF from the gel filtra-

tion of whole blood serum was 15% in fraction I, 8% in frac-tion II, 6% in fraction IV, and 71% as monomer PDGF.Since overall recovery from the column was only 21%, it isnot possible to use these results to estimate the absolute dis-tribution of PDGF in whole blood serum. However, this ex-periment does demonstrate that active PDGF with an appar-ent Mr of 30,000 can be recovered from the same higher mo-lecular weight fractions of human whole blood serum shownin Fig. 2 to contain PDGF-binding activity.

Vo 161,000 25,700 Vt

0.6

E0.50.24-*""0 0.3L0 seet*

0.1 ,

10 20 30 40 50 60Fraction

c0

1200 -<1000

800 C;600 4400

200 eta

FIG. 4. Specific binding of 125I-PDGF to a high molecular weightplasma fraction. Human PDS (50 ILI) was incubated for 6 hr at 370Cin 400 1.l of 0.01 M Tris.HCl/0.15 M NaCi, pH 7.4/1 mM MgSO4/1mM ZnSO4/1 mM CaCl2 containing 0.8 mg of bovine serum albumin(a) or 15 ,g of unlabeled PDGF and 0.8 mg of bovine serum albumin(o); 50 ILI of 125I-PDGF was added to each fraction to give a finalconcentration of 2.5 ng of 125I-PDGF per ml and incubated an addi-tional 15 min at 370C followed by gel filtration on a Bio-Gel A-0.5 mcolumn (2.0 x 28 cm) equilibrated in the same buffer at 20'C. Thecolumn flow rate was 12 ml/hr and 1-ml fractions were collecteddirectly into tubes for counting in a Beckman Gamma Counter andfor determination of absorbance at 280 nm (-). Thyroglobulin(void volume, V0), aldolase (161,000), bovine serum albumin(67,000), chymotrypsinogen (25,700), and 3H20 (total column vol-ume, Vt) were used to calibrate the column.

0

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I L , f al 5,4f, ,tl,, l20 40 20 40 20 40 20 40

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FIG. 5. Dissociation of PDGF from higher molecular weightfractions of whole blood serum by chromatography in 4 M guani-dine HCl. Human whole blood serum was prepared by incubationof freshly drawn blood to clot at 370C for 6 hr and removal of the clotby centrifugation. (A) Whole blood serum (5 ml) was chromato-graphed on a Bio-Gel A-0.5 m column (2.5 x 95 cm) equilibrated in0.01 M Tris HCl/0.15 M NaCl, pH 7.4. (B-E) Fractions I, II, III,

and IV, respectively, were pooled, lyophilized, solubilized in 4 Mguanidine HCl in Tris-buffered saline, and 2.5-ml whole blood se-rum equivalents were rechromatographed on Bio-Gel A-0.5 m (1.5 x90 cm) in guanidine HCl. Fractions 31-45 (the elution position offree PDGF) from each of the guanidine columns were pooled, dia-lyzed against 1.0 M acetic acid in the presence of 0.25% bovine se-rum albumin (Miles, Pentex recrystallized), 1yophilized, and as-sayed at 3 doses for free PDGF (bars) using the radioreceptor assayas described (9, 19). Fraction III, when rechromatographed, showedno detectable PDGF. The PDGF content of fractions 44-46 and 47-56 (the peak of free PDGF) from the Tris-buffered saline column(bars in A) were also determined by radioreceptor assay after lyophi-lization and chromatography on the same guanidine column used forfractions I, II, III, and IV. A280 nm values are shown by solid lines.Molecular weight standards used to calibrate the columns are indi-cated by arrows as follows: a, blue dextran (void volume); b, aldol-ase (Mr, 161,000); c, bovine serum albumin (Mr, 67,000); d, ovalbu-min (Mr, 43,000); e, chymotrypsinogen (Mr, 25,700); and f, 3H20(total column volume).

Immunologic and Functional Evidence Suggesting that a2-M Is Responsible for Some of the PDGF-Binding Activity inPlasma. The fact that a2-M is the major plasma protein of Mr>500,000 and that a2-M has been implicated in the binding ofother growth factors (22, 23) led us to investigate whether a2-M is capable of inhibiting PDGF binding to its cell-surfacereceptor. Table 2 demonstrates that commercially availablepurified human a2-M inhibits the binding of PDGF with half-maximal inhibition at 5.5 x i0-5 g/ml. Antiserum to a2-Mpartially removed the PDGF-binding activity from plasma.

DISCUSSIONSpecificity of Plasma Inhibition ofPDGF Binding to Its Cell-

Surface Receptor. This report presents evidence for an asso-ciation of PDGF with specific PDGF-binding proteins inplasma that inhibit binding of PDGF to its cell-surface recep-tor. These binding proteins do not act by blocking or perma-nently altering the properties of the PDGF receptor (Figs. 1and 3). Specific inhibitory fractions can be isolated fromplasma by adsorption to, and elution from, PDGF covalentlybound to Sepharose (Fig. 3). Half-maximal inhibition by af-finity-purified fractions was in the range of 5 x 1o-7 g/ml to1 x 10-5 g/ml. Two other plasma proteins, fetuin and albu-min, had no effect on PDGF binding at total protein concen-

Table 2. The effect of a2-M on the binding of PDGF to its cell-surface receptor

Cell bound PDGF,Sample Concentration % of control

a2-M 5 pg/ml 8250 ug/ml 56100 tg/ml 40

PDS 40% (vol/vol) 5660% (vol/vol) 3880% (vol/vol) 28

PDS and anti-a2-M 60% (vol/vol) 61

PDGF (0.25 ng/ml) in binding medium or in the presence of in-creasing concentrations of PDS or a2-M were assayed by the PDGFradioreceptor assay on human fibroblasts as illustrated in Fig. 1. Foreach condition, inhibition of binding of the unlabeled PDGF wasexpressed as the percent of the original PDGF concentration addedto the test samples, as described in Table 1. The standard errors oftriplicate determinations of 125I-PDGF binding were normally 2%-5% of the mean.

trations comparable to protein concentrations of plasma (1-4x 10-2 g/ml) (Fig. 3).Three fractions, obtained by gel filtration of plasma, of Mr

=40,000, -150,000, and >500,000 were found to inhibit thebinding of PDGF to its receptor. A candidate for the largestmolecular weight fraction is a2-M, which has a molecularweight of about 800,000 and is present in plasma at a concen-tration of -2 mg/ml (24). a2-M has been studied most exten-sively as a protease inhibitor (24), but it has also been report-ed to bind nerve growth factor (22) and human growth hor-mone (23). When purified a2-M was assayed for its ability toinhibit PDGF binding, it was found to be active with half-maximal inhibition at -5 x 10-5 g/ml (Table 2)-i.e., to bemuch less potent than the PDGF-binding protein purifiedfrom plasma by affinity chromatography. It is therefore pos-sible that the PDGF-binding activity of commercially avail-able a2-M is accounted for by a contaminant. The ability ofthe antibody to a2-M to remove 50% of the PDGF-bindingactivity from PDS might similarly be due to a contaminant inthe immunizing preparation of a2-M. However, it is knownthat the protease inhibiting activity of a2-M is extremely la-bile (25), and the fact that the activity of the affinity-purifiedfractions of plasma have varied 20-fold in their specific activ-ity suggests that the PDGF-binding activity may also be la-bile. This could also explain why the a2-M preparations test-ed do not have as much PDGF-binding activity as freshlyprepared plasma. One could also speculate that only a subsetof a2-M is capable of inhibiting PDGF cellular binding.Monoclonal antibodies to a2-M and further characterizationof purified PDGF-binding fractions relative to a2-M and itssubunits will be necessary to clarify the relationship betweena2-M and plasma fractions inhibiting PDGF cellular binding.PDGF Binding to Plasma Components. The above experi-

ments demonstrate the presence in plasma of a component(s)able to inhibit PDGF binding to its cell-surface receptor. Todemonstrate that this inhibition results from the associationof PDGF with plasma proteins, we incubated plasma with125I-PDGF and analyzed the mixture by gel filtration. 1251_PDGF behaved as though present in a complex of Mr>500,000 and in a broad peak with a Mr range of 150,000 to30,000 (Fig. 3). However, only the association with highestmolecular weight fraction (>500,000) was inhibited when10-6 M unlabeled PDGF was preincubated with the plasmaprior to the addition of 125I-PDGF. The lack of demonstrablecompetable binding in the Mr range 40,000 to 150,000 sug-gests that these plasma fractions have a low affinity forPDGF. Huang et al. (17) recently reported the association of125I-PDGF with a plasma component with an estimated Mr of280,000, which appears, except for molecular weight esti-

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mates, to be comparable to the void volume fraction(>500,000) reported here.The association of PDGF with other blood components

was also demonstrated by gel filtration analysis of wholeblood serum (Fig. 5). By analyzing each fraction for the pres-ence of bound PDGF (determined by radioreceptor assay af-ter dissociation and gel filtration), the distribution of PDGFbetween different serum components could be determined.Part of the PDGF released from platelets during the prepara-tion of whole blood serum was found in the fractions previ-ously shown to contain PDGF-neutralizing and 125I-PDGFbinding activities.

Binding Proteins in Plasma as Modulators ofPDGF Activityin Vivo. One example of binding proteins in plasma that re-versibly inhibit the efficacy of mitogenic agents is the bind-ing proteins for the somatomedins/insulin-like growth fac-tors (IGFs) (26, 27). These binding proteins have been shownto increase the circulating half-life of injected somatomedinactivity from a few minutes to several hours (28) and to in-hibit the biological activity of IGF in vitro (26, 27). It hasbeen suggested that the plasma may serve as an inactivestorage pool for the somatomedin/IGFs (29), and quantita-tive alterations in the levels of different binding proteinsmight be one mechanism through which IGF activity is regu-lated in vivo. In vivo clearance studies with 1251I-PDGFshowed a t1/2 for clearance from the blood of -2 min (15).These studies would argue against a role for the binding pro-teins and plasma as a storage pool for PDGF.What does appear clear is that at low concentrations of

PDGF and 100% plasma-i.e., under the conditions existingin vivo at sites peripheral to vascular injury and platelet re-lease-80%-85% of the PDGF would be bound to, and in-hibited by, the plasma components (Fig. 3, at 40C; and un-published observations demonstrating comparable inhibitionat 370C). The fact that we found approximately equivalentamounts of PDGF-binding activity in the plasma and PDS ofall species examined (Table 1) lends support to the hypothe-sis (see below) that PDGF-binding proteins may play a rolein regulating the action of PDGF. PDGF binds with high af-finity (10-1 M) (9) to a cell-surface receptor (8, 30, 31) onresponsive connective tissue cells. We therefore proposethat PDGF-binding proteins in plasma and high-affinity cel-lular binding may jointly regulate the action of PDGF. Theinteraction of PDGF with responsive cells near the site ofPDGF release from platelets would be favored over bindingto the plasma components because of its very high bindingaffinity. Any PDGF not quickly bound to responsive cellswould bind to specific binding proteins in plasma, whichwould prevent the binding of PDGF to cells at sites distal tothe point of focal release. The plasma flow at the site of re-lease would serve as a fresh source of binding proteins andfurther dilute any released PDGF. Based on this model, onewould also speculate that alterations in either the number ofcellular binding sites or plasma binding proteins could affectthe fate of PDGF and, consequently, PDGF-stimulatedgrowth.

Note Added in Proof. Recently, Huang et al. (32) reported the bind-

ing of "'I5-labeled PDGF to a2-macroglobulin.

We wish to acknowledge the excellent technical assistance of Bil-lie Fortune, Janet Hansom, and Li-Chuan Huang; Karen Tittle forher assistance with cell cultures and plasma collections; Arnie Hest-ness for drafting the figures; and Mary Hillman for tireless typing.The research was supported by National Institutes of Health GrantHL 18645 and by a grant from R. J. Reynolds.

1. Ross, R., Glomset, J., Kariya, B. & Harker, L. (1974) Proc.Natl. Acad. Sci. USA 71, 1207-1210.

2. Kohler, N. & Lipton, A. (1974) Exp. Cell Res. 87, 297-301.3. Westermark, B. & Wasteson, A. (1976) Exp. Cell Res. 98, 170-

174.4. Grotendorst, G. R., Chang, T., Seppa, H. E. J., Kleinman,

H. K. & Martin, G. R. (1982) J. Cell. Physiol. 113, 261-266.5. Bernstein, L. R., Antoniades, H. & Zetter, B. R. (1982) J. Cell

Sci. 56, 71-82.6. Seppa, H., Grotendorst, G., Seppa, S., Schiffmann, E. & Mar-

tin, G. R. (1982) J. Cell Biol. 92, 584-588.7. Senior, R. M., Griffin, G. L., Huang, J. S., Walz, D. A. &

Deuel, T. F. (1983) J. Cell Biol. 96, 382-385.8. Heldin, C.-H., Westermark, B. & Wasteson, A. (1981) Proc.

Natl. Acad. Sci. USA 78, 3664-3668.9. Bowen-Pope, D. F. & Ross, R. (1982) J. Biol. Chem. 257,

5161-5171.10. Ross, R. & Vogel, A. (1978) Cell 14, 203-210.11. Scher, D., Shepard, R. C., Antoniades, H. N. & Stiles, C. D.

(1979) Biochim. Biophys. Acta 560, 217-241.12. Ross, R. & Glomset, J. A. (1976) N. Engl. J. Med. 295, 369-

377.13. Ross, R. & Glomset, J. A. (1976) N. Engl. J. Med. 295, 420-

425.14. Ross, R. (1981) Arteriosclerosis 1, 293-311.15. Bowen-Pope, D. F., Malpass, T. F., Foster, D. M. & Ross, R.

(1984) Blood, in press.16. Singh, J. P., Chaikin, M. A. & Stiles, C. D. (1982) J. Cell Biol.

95, 667-671.17. Huang, J. S., Huang, S. S. & Deuel, T. F. (1983) J. Cell Biol.

97, 383-388.18. Raines, E. W. & Ross, R. (1982) J. Biol. Chem. 257, 5154-

5160.19. Raines, E. W. & Ross, R. (1984) Methods Enzymol., in press.20. Bowen-Pope, D. & Ross, R. (1984) Methods Enzymol., in

press.21. Pedersen, K. 0. (1947) J. Phys. Colloid Chem. 51, 164-171.22. Ronne, H., Anundi, H., Rask, L. & Peterson, P. A. (1979) Bio-

chem. Biophys. Res. Commun. 87, 330-336.23. Adham, N. F., Chakmakjian, Mehl, J. W. & Bethune, J. E.

(1969) Arch. Biochem. Biophys. 132, 175-183.24. Barrett, A. J. & Starkey, P. M. (1973) Biochem. J. 133, 709-

724.25. Nagasawa, S., Han, B. H., Sugihara, H. & Suzuki, T. (1970) J.

Biochem. 67, 821-832.26. Zapf, J., Schoenle, E., Jagars, G., Sand, I., Grunwald, J. &

Froesch, E. R. (1979) J. Clin. Invest. 63, 1077-1084.27. Knauer, D. J. & Smith, G. L. (1980) Proc. Natl. Acad. Sci.

USA 77, 7252-7256.28. Cohen, K. L. & Nissley, P. S. (1976) Acta Endocrinol. 83,

243-258.29. Zapf, J., Froesch, E. R. & Humbel, R. E. (1981) Curr. Top.

Cell. Regul. 19, 257-309.30. Huang, J. S., Huang, S. S., Kennedy, B. & Deuel, T. F.

(1982) J. Biol. Chem. 257, 8130-8136.31. Williams, L. T., Tremble, P. & Antoniades, H. N. (1982)

Proc. NatI. Acad. Sci. USA 79, 5867-5870.32. Huang, J. S., Huang, S. S. & Deuel, T. F. (1984) Proc. Natl.

Acad. Sci. USA 81, 342-346.

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