J. Cell Sri. 62, 117-127 (1983) \ \ 7Printed in Great Britain © The Company of Biologists Limited 1983

FIBRINOGEN AND FIBRONECTIN AS SUBSTRATES

FOR EPIDERMAL CELL MIGRATION DURING

WOUND CLOSURE

DONALD J. DONALDSON* AND JAMES T. MAHANDepartment of Anatomy, University of Tennessee, Center for the Health Sciences, 875Monroe Avenue, Memphis, Tennessee 38163, U.SA.

SUMMARY

Pieces of glass coverslip coated with human fibronectin or human fibrinogen were implantedunder one margin of a skin wound on adult newt (Notophthalmus viridescens) hind limbs. Incontrast to uncoated glass or glass coated with newt serum, bovine serum or bovine serum albumin,glass treated with either fibronectin or fibrinogen supported considerable epidermal cell migration.When optimal amounts of each protein were used, the amountof migration on fibrinogen-coatedglass did not differ from the amount on fibronectin-coated glass or from the amount on the woundbed. Migration on a fibronectin substrate could be blocked by treating the substrate with an anti-serum against fibronectin just prior to implantation. Similarly, migration on a fibrinogen substratecould be blocked by exposing it to an antiserum against fibrinogen. While we have yet to determineif fibrinogen and fibronectin are interacting directly with the cell surface, our observations suggestthat these two proteins may play an important role in wound closure by providing a suitable substratefor epithelial cell migration.

INTRODUCTION

Skin wounds on adult newt hind limbs are rapidly covered by a wound epitheliumeven if the limbs are amputated and placed in saline (Donaldson & Dunlap, 1981).We are currently investigating the influence of the extracellular matrix on migrationof epidermal cells in this system. Previously we found that nucleopore filters coatedwith collagen types I, II or IV would all support considerably more migration thanuncoated filters (Donaldson, Smith & Kang, 1982). While this clearly established thecapacity of collagen-coated substrates to support migration, epidermal cells may notencounter naked collagen molecules in a fresh wound. Immunohistochemical studiesof wound healing (Fujikawa et al. 1981; Grinnell, Billirigham & Burgess, 1981;Repesh, Fitzgerald & Furcht, 1982) suggest that epithelial cells are more likely toencounter fibrin(ogen) and/or the adhesive glycoprotein, fibronectin. (See recentreviews by Mosher & Furcht (1981) and Akiyama, Yamada & Hayashi (1981) forthe structure and functions of fibronectin.) Therefore, in the present study wehave examined the ability of fibronectin and fibrinogen to support epidermal cellmobility.

•Author for correspondence.

118 D.jf. Donaldson andj. T. Mahan

MATERIALS AND METHODS

GeneralAdult male newts {Notophthalmus viridescens) obtained from Connecticut Valley Biological were

stored at 4°C in 1/10 strength operating solution (Rose & Rose, 1965) containing 0-14ml/1 ofWardley's Aquatonic (a commercial product commonly used for disease prevention in tropical fish).At least a week prior to use, the animals were moved to room temperature where they were kept inthe same solution and exposed to a natural photo-period.

Wounding, glass implantation and measurement of migrationRectangular wounds with their long axis extending proximo-distally were made by removing a

piece of skin from the dorsal surface of each hind limb between the knee and ankle. Wounded limbswere amputated through the thigh and explanted into 5 ml of Holtfreter solution (HS) containing0-05 g/1 of streptomycin in 35 mm X 10 mm plastic dishes. The clotted blood was then cleaned fromeach wound, the limbs were transferred to 5 ml of fresh HS and a 1 mm X 2 mm piece of coverslipglass was then inserted under the anterior wound margin (Fig. 1). Eight hours later, limbs were fixedovernight in 10 % formalin, and then the migrating cells were stained by immersing the entire limbbriefly in 0-1 % crystal violet. After the glass was dissected free from the limb, it was placed undera compound microscope equipped with a Leitz drawing tube. The magnified image of the glass andthe wound epithelium was drawn onto a sheet of paper, and the area occupied by a standardizedwidth of wound epithelium (distance migrated) was determined using a polar planimeter. Thesearbitrary migration units were used to compare the effectiveness of various substrates. Experimentswere designed so that one hind limb of an animal served as the control, and the other as theexperimental. This allowed us to analyse the results by a paired <-test, thereby reducing the impactof animal variability. A difference was judged to be significant if the P value was less than 0 0 1 .

Coating of glass implantsGlass coverslips were immersed overnight in a 2% Contrad 70 solution (Scientific Products).

ft TTWound

bedGlass implant

T

1 fm ' * • - ' • »

Wound epithelium Intact skin

Fig. 1. A diagram of a typical wound and its glass implant. The arrows show the directionin which migrating epidermal cells move to close the wound. The glass is shown as opaquerather than transparent.

Epidermal cell migration 119

The next day they were rinsed in several changes of distilled water, placed in 1 % HC1 for 5 min,rinsed again in distilled water (four changes), dipped briefly in 95 % alcohol and wiped dry with aKimwipe. All handling of the cleaned glass was by forceps or rubber-gloved hands. After thecoverelips were cut into 1 mm X 2 mm pieces, 10 /il of test material was pipetted onto each piece andthey were air-dried at 24 °C. The drop of test material completely enveloped the implant so that someof the applied material dried onto the floor of the container holding the implants. Prior to implanta-tion the coated glass was washed in HS for 1 h (two changes).

Test materialsLyophilized human fibrinogen purchased from Kabi was dissolved in accord with their instruc-

tions and frozen in 1 ml samples. On the day of use it was thawed and diluted to the desiredconcentration in either phosphate-buffered saline (PBS) or Tris buffer. When samples wereanalysed by gel electrophoresis under reducing conditions, the only appreciable staining byCoomasie Blue occurred in three closely spaced bands corresponding to the a, /Sand ysubunits offibrinogen in the 50-70 000 molecular weight range. The maximum amount of contaminatingfibronectin as estimated by a solid-phase immuno-assay using monoclonal antibodies against humanfibronectin was 015/ig/lOO/ig fibrinogen. This assay was kindly performed by Dr David Hasty,University of Tennessee Health Sciences Center. While fibrinogen and not fibrin was dried onto theglass, it is possible that thrombin in the wound environment may have converted some or all of thefibrinogen to fibrin after the implants were made (Unkeless et al. 1973). Thus when we speak offibrinogen, it is with this qualification.

Fig. 2. Immunodiffusion plate showing the specificity of the anti-fibrinogen and anti-fibronectin used in this study.

Anti-fibronectin (A-Fn) shows a broad precipitin line against human fibronectin(Fn, 900^<g/ml), which forms an identity chevron with a sharp line against humanplasma (HP). Anti-fibrinogen (A-Fgn) shows a sharp line against human fibrinogen(Fgn, 900^g/ml), which forms an identity chevron with a line formed against humanplasma. The two chevrons are not identical and the non-immune rabbit serum (RS) didnot react with fibronectin, fibrinogen or human plasma.

120 D. J. Donaldson andjf. T. Mahan

Human plasma fibronectin purified by the method of Vuento & Vaheri (1979) was generouslysupplied by Dr Waits A. Simpson, Jr, Veterans Administration Medical Center, Memphis. Puritywas verified by gel electrophoresis, which under reducing conditions showed the only appreciablecomponent to be a doublet of approximately 220 000 molecular weight. The fibronectin was storedat 4CC in Tris buffer until needed. Then it was diluted to the desired concentration with either Trisbuffer or PBS.

Newt blood obtained by cardiac puncture was collected in a capillary tube and allowed to clot.The clot was centrifuged and the serum was diluted with either distilled water or PBS.

Antibodies

Goat antiserum to human fibrinogen was purchased from Sigma, while rabbit antiserum tohuman plasma fibronectin was a generous gift from Dr Magnus Hook, University of Alabama inBirmingham. The specificity of these antisera is shown in Fig. 2. When antisera were tested for theirability to block migration, fibrinogen or fibronectin-coated implants were washed twice in HS andthen 10/il of undiluted antiserum was applied to each implant. The antisera were allowed to reactwith the substrates for 30 min in a moist chamber. Immediately following a second 30 min incuba-tion with a fresh 10/il sample of antiserum, each implant was washed twice in two changes of HSand tested as a migration substrate.

RESULTS

Preliminary experiments showed that glass implants treated with 1 mg/ml offibrinogen were far superior to untreated glass as a migration substrate. We thenconducted a dose-response study in which one limb of each animal received an implanttreated with 1 mg/ml of fibrinogen while the contralateral limb received an implanttreated with a smaller amount of fibrinogen. Fig. 3 shows that implants treated with100^g/ml were as effective as those receiving 1 mg/ml. A concentration of 10jUg/ml

Table 1. Comparative migration on fibrinogen and fibronectin-treated glass

Group

In TrisFibrinogen (100/ig/ml)Fibronectin (100 /ig/ml)

Fibrinogen (100/ig/ml)Fibronectin (1-5/ig/ml)

In PBSFibrinogen (100/ig/ml)Fibronectin (100/ig/ml)

Fibrinogen (100/ig/ml)Fibronectin (1-5/ig/ml)

Number oflimbs

OO

OO

OO

O

O

88

85

Distancemigrated*

364 ± 34f422 ±30

357 ±3578 ±9

462 ±8493 ±23

295 ±4085 ±19

% Difference

14NSJ

457 P = 0-0002(paired / test

NSJ

347 P = 0-003(unpaired f-test)

Experimental design was as described in the legend to Fig. 3, except that one limb of each animalreceived an implant coated with fibrinogen while the contralateral limb received one treated withfibronectin.

•As defined in Materials and Methods.•(• Standard error.JNS, not significant.

Epidermal cell migration

Fibrinoqen

121

Fibronectin

S CO § o o

s -o ,-

sProtein concn (/<g/ml)

Fig. 3. Dose-response data showing the effect on migration of decreasing amounts offibrinogen or fibronectin on the substrate. Fibrinogen in PBS or fibronectin in Tris bufferwas air-dried onto small pieces of coverslip-glass, which were then inserted under one edgeof a skin wound on limbs explanted into a small dish of Holtfreter solution. Eight hourslater, when epidermal cells had moved onto the implant, the area occupied by woundepithelium (distance migrated) was determined from a standard portion of each implant.This was done using a planimeter on drawings of the stained wound epithelium, made withthe aid of a Leitz drawing tube mounted on a compound microscope. For each concentra-tion of fibrinogen to be tested, 6-8 limbs (the fibrinogen controls) were implanted withglass treated with 1 mg/ml of human fibrinogen, while the contralateral limbs (the experi-mentals) received implants treated with the test concentration. Each pair of bars comparesthe mean distance covered by wound epithelium in the fibrinogen controls with the meanfor the companion experimentals. The fibronectin bars were derived similarly, except thatthe fibronectin controls received 0-5 mg/ml of protein. NS, no significant differencebetween experimental and control limbs; S* (P = 0-001), S " (P = 0-0001), S# #*(P<0-0001).

however, allowed significantly less migration than the higher concentrations. Theamount of migration on implants treated with 1 |Ug/ml was similar to that on untreatedglass.

Implants treated with human fibronectin also supported more migration than un-coated implants. Fig. 3 shows that fibronectin was as effective at 10/ig/ml as it wasat 500 fig/m\. Its effectiveness was greatly diminished at 1 ^ig/ml. A comparison ofthe right and left halves of Fig. 3 suggested that the maximum amount of migrationpossible on fibronectin might be less than on fibrinogen. However, since there was

CEL62

122 D. jf. Donaldson andj. T. Mohan

considerable variation in the maximum from experiment to experiment, and since thefibrinogen was diluted in PBS and the fibronectin was diluted in Tris buffer, wedecided to conduct direct comparison experiments using 100^g/ml of each proteindiluted in the same buffer. Table 1 shows that in either Tris or PBS there was nosignificant difference in the amounts of migration that fibronectin and fibrinogenwould support.

Since fibrinogen preparations commonly contain a small amount of fibronectin, wewere concerned that migration on fibrinogen might actually be due to thiscontaminant. We therefore directly compared the amount of migration on 100 /ig/mlof fibrinogen with the amount produced by 1-5 /ig/ml of fibronectin (an amount 10times more than our fibrinogen preparation contained). The results (Table 1) showthat, regardless of the buffer, there was considerably more migration on fibrinogenthan on fibronectin. Thus, the migration on fibrinogen is not due to contaminationby fibronectin. This conclusion is verified by the specific effects of antibody treatmentof the substrates (Fig. 4). When one group of fibrinogen substrates was treated withanti-fibrinogen and another group was treated with anti-fibronectin just prior toimplantation, migration was significantly less on the implants exposed to anti-fibrinogen. Similarly, when fibronectin was the substrate, this same antibody treat-ment resulted in significantly less migration on the anti-fibronectin-treated implants.This shows that in each case the migration-promoting effect of the substrate was notdue to some contaminant in the coating material.

400"

2 300-m

it'ECD£ 200

n100-

Fibrinogen substrate

A

i.

-Fn

IIt

::

!:: A-Fgn

I•1

i:

ft

Fibronectin substrate

A-Fgn

A-Fr

ft

Group

Fig. 4. Effect of antiserum treatment on migration over fibrinogen or fibronectin sub-strates. Substrates coated with 100/ig/ml of either fibrinogen or fibronectin were treatedwith antisera against fibrinogen (A-Fgn) or against fibronectin (A-Fn) and were thentested for their ability to support epidermal cell migration. P< 0'005 for fibrinogen sub-strate experiment, P= 0-0001 for fibronectin substrate experiment. Distance migrated isin arbitrary units (see Materials and Methods).

Epidermal cell migration 123

Intact skin

Fig. 5. Drawing tube tracings of three representative pairs of limbs from an experimentin which one hind limb of each animal received an untreated glass implant (AI , BI , Ci)while the contralateral limb (A2, vz, Cz) received an implant treated with 100/ig/ml ofhuman fibrinogen.

We also learned that the maximum amount of migration produced by these twoproteins is very similar to that occurringon the natural wound bed. This is shown clearlyin Fig. 5, where the leading edge of the wound epithelium on fibrinogen-treated glass

124 D.jf. Donaldson andj. T. Mohan

Table 2. Migration on serum or albumin-treated glass

Group Number of limbs Distance migrated*

Newt serum (10%)Untreated

Newt serum (1%)Untreated

Newt serum (0-2%)Untreated

Foetal bovine serum (10%)Untreated

Bovine serum albumin (1 mg/ml)Untreated

oo o

o00

O

O

88

OO

00

00

OO

90 ± 35f127 ±44

74 ±32121 ±55

86 ± 16103 ±25

38 ±1348 ±17

116 ±24166 ± 29

NSJ

NS

NS

NS

NS

Experimental design was as described in the legend to Fig. 3, except that one limb of each animalreceived an implant coated with serum or albumin while the contralateral limb received an uncoatedimplant.

•As defined in Materials and Methods.f Standard error.\ NS, not significant.

implants is generally adjacent to the leading edge on the wound bed. The fact that theleading edge on the control implants (untreated glass) was left far behind the leadingedge on the wound bed shows that the advancing front on the fibrinogen implants hadnot simply been pulled along by the adjacent wound bed epithelium.

In contrast to the ability of fibrinogen and fibronectin-coated glass to supportmigration, glass coated with newt serum (0-2%, 1% and 10%) or foetal bovineserum (10%) was no better than uncoated glass (Table 2). This might at first seemsurprising, in view of the fact that serum contains fibronectin. However, the work ofUniyal & Brash (1982) shows that the composition of protein layers adsorbed toforeign surfaces from plasma (and presumably serum) cannot be predicted fromadsorption patterns of single proteins from solution. Therefore, serum proteins suchas albumin, which cannot support migration (Table 2), may attach to the glass andprevent a migration-supporting amount of fibronectin from binding. Alternatively,proteins such as albumin may secondarily cover adsorbed fibronectin. Certainly in oursystem, when bovine serum albumin (BSA) is dried down onto a fibronectin-coatedsubstrate, migration is blocked (data not shown). Even after an hour in HS (twochanges) serum and albumin-treated glass had a Coomassie-Blue-stainable film onthem, showing that the failure of epidermal cells to migrate on those substrates wasnot because the implants had not bound any protein.

DISCUSSION

Our results show that the rate of epidermal cell migration on fibrinogen-coated glassand fibronectin-coated glass implants is generally comparable to the rate on the wound

Epidermal cell migration 125

bed. Since an earlier study (Donaldson et al. 1982) showed that collagen types I, IIand IV also supported migration, it might appear that epidermal migration onprotein-coated substrates is a relatively non-specific phenomenon. Migration on BSAand serum-coated glass, however, was poor. This and the fact that migration wasblocked when fibrinogen or fibronectin substrates were coated with immunoglobulinsshow that there are proteins on which newt epidermal cells cannot migrate. To thisextent we can say that migration on collagen, fibronectin and fibrinogen-coated im-plants is a specific response.

For a time it appeared that epithelial cells and fibroblasts might utilize differentadhesive molecules in their interaction with extracellular matrix components. Murrayet al. (1979) showed that, while fibroblast attachment to collagen types I and IV wasenhanced by fibronectin, this glycoprotein produced little or no increase in attach-ment of guinea pig epidermal cells to these collagens. Similarly, Terranova, Rohrbach& Martin (1980) found that the fibronectin in serum did not enhance the attachmentof PAM212 cells (an epidermal cell line) to plastic, or to type I or type IV collagen.They did show, however, that the preference of these cells for type IV in adhesionassays was mediated by a high molecular weight glycoprotein called laminin. Lamininhad no effect on attachment of Chinese hamster ovary (CHO) cells (presumably afibroblastic line) to either type I or type IV collagen.

It is now known that fibronectin can enhance the attachment of several types ofepithelial cells to certain substrates. For example, intestinal epithelial cells adhere ingreater numbers to bacteriological plastic or type I collagen if the substrates arepretreated with fibronectin (Burrill, Bernardini, Kleinman & Kretchmer, 1981). Rathepatocytes will not attach to bacteriological plastic but will readily adhere if the dishis coated with fibronectin (Johansson, Kjellen, Hook & Timpl, 1981). In fact,Gilchrest, Calhoun & Maciag (1982) have recently reported that human keratinocytesattach much better to fibronectin-coated plastic dishes than to uncoated dishes.

While adhesion assays and mobility studies do not measure exactly the same events,cells clearly cannot migrate on a substrate unless they can adhere to it. Thus ourfinding that newt epidermal cells can migrate on fibronectin substrates is compatiblewith the observation that human keratinocytes can bind to fibronectin. The fact thatmouse epidermal cells migrating in vitro require serum but do not seem to needfibronectin, suggests, however, that the availability of fibronectin in vivo is not alimiting factor in the ability of some types of epidermal cells to close wounds (Feder-green & Stenn, 1980).

In adhesion studies using chick presumptive myoblasts as the responder cell, themyoblasts adhered very well to fibrinogen-coated substrates and very poorly to thosecoated with gamma globulin or BSA (Ratner, Horbett, Hoffman & Hauschka, 1975).CHO cells cultured on fibrinogen-coated plastic have a different morphology and aremore adherent to the substrate than those cultured on uncoated plastic (Nozawa,1977). Similarly, platelets adhere in greater numbers to glass if fibrinogen is in themedium (Zucker & Vroman, 1969). Grinnell, Feld & Minter (1980) have shown thatbaby hamster kidney (BHK) cells will not attach to fibrinogen-coated plastic unlessthe fibrinogen substrate is pretreated with fibronectin. It is not necessary for the

126 D. Jf. Donaldson andjf. T. Mohan

fibronectin to be covalently bound to increase adhesion on fibrinogen, but when it isthe fibronectin is even more effective. Antibody blocking studies showed that BHKcells exposed to fibronectin-treated fibrinogen were adhering to the bound fibronec-tin. Since serum was present in the other three fibrinogen adhesion studies describedabove, those cells and cell products may also have been adhering to fibronectin. Wecould have a similar situation in our experiments. The wound environment or eventhe epidermal cells themselves may modify the fibrinogen coating on our implants.Because of this possibility we cannot say that newt epidermal cells have receptors forfibrinogen; nor can we say they have receptors for fibronectin even thoughfibronectin-coated implants supported migration. It is clear, however, that if thesesubstrates are in fact being altered, this is likely to be part of a sequence of events thatnormally occurs in wound healing. Certainly, this hypothetical sequence does notoccur if the implants are coated with albumin, serum or immunoglobulins, since theydo not support migration. We are currently testing a variety of proteins for their abilityto support migration in order to understand better the significance of the epidermalresponse to collagen, fibronectin and fibrinogen. We are also studying these positivesubstrates at various times after implantation to learn if they are being modified bythe wound environment before cells cross them.

This research was supported by grant AM-27940 from the National Institute of Arthritis,Diabetes, Digestive and Kidney Diseases, National Institutes of Health.

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{Received 11 February 1983-Accepted 18 February 1983)