[CANCER RESEARCH 41, 4629-4636, November 1981]0008-5472/81 /0041 -OOOOS02.00

Effect of Plasminogen Activator (Urokinase), Plasmin, and Thrombin on

Glycoprotein and Collagenous Components of Basement Membrane

L. A. Liotta,1 R. H. Goldfarb, R. Brundage, G. P. Slegai, V. Terranova, and S. Garbisa

Laboratories of Pathophysiology [L. A. L., R. B., G. P. S.], and Immunodiagnosis [R. H. G.], National Cancer Institute, and National Institute of Dental Research,[V. P. T.], W/H, Bethesda, Maryland 20205, and Institute of Histology [S. G.], University of Padova, Italy

ABSTRACT

Tumor cells traverse basement membranes (BM) during thestages of the metastatic process. Penetration of the BM mayinvolve proteolysis by enzymes directly or indirectly associatedwith tumor cells. This study evaluated the role of the serineproteases urokinase (plasminogen activator), plasmin, and another regulatory protease, a-thrombin, in the degradation of

the BM. Homogeneously pure enzyme preparations were incubated with isolated components of BM and with whole humanamnion BM. The BM components consisted of acid-extracted

type IV collagen, pepsin fragments of collagen type IV, laminin,and fibronectin. Collagen type V (aAaB) associated with theperi-BM zone was also studied. The purity of the enzymes was

verified by gel electrophoresis and inhibitor studies. Digestionof the BM components was performed at 25°using matched

activity for the different enzymes. Urokinase failed to significantly degrade fibronectin or any of the other BM components.Under the same 25°(native) conditions, plasmin and thrombin

cleaved fibronectin and laminin into multiple specific fragmentsbut did not produce a major cleavage of acid-extracted type IV

collagen, pepsinized type IV collagen, or aAaB (type V) collagen. a-Thrombin selectively degraded only the m.w. 400,000

chain of laminin, whereas plasmin degraded both of the lamininchains. Digestion of laminin by the serine proteases was timeand concentration dependent, as verified by a new degradationassay using [14C]laminin. A variety of normal and neoplastic

cells were tested for the presence of laminin-degrading pro

teases. Macrophages, polymorphonuclear leukocytes, andmetastatic tumor cells contained a significant laminin-degrading activity. The activity was enhanced by the addition ofplasminogen. Type V collagen was cleaved by thrombin andplasmin at 35° but not at temperatures below 33°. Following

treatment of whole-amnion BM with any of these enzymes,

electron microscopy demonstrated preservation of the laminadensa. Immunohistology studies indicated that laminin, but nottype IV collagen, was removed from the whole BM by plasmintreatment. The results suggest that these BM components arepoor substrates for plasminogen activators and that plasminalone is not sufficient to completely degrade the whole BM.Plasmin, generated through the action of plasminogen activator, may play a significant role in the degradation of noncollag-enous components of the BM.

INTRODUCTION

The extracellular BM2 matrix is a stable structure with a slow

1 To whom requests for reprints should be addressed.2 The abbreviations used are: BM, basement membrane; PA, plasminogen

activator; UK, urokinase.Received December 17,1980; accepted August 11,1981.

turnover rate in normal adult tissues (3, 13, 33, 48). However,under certain developmental and pathological states, e.g.,mammary gland involution and tumor invasion, histologicalstudies have demonstrated local rapid dissolution of the BM(2, 27, 47, 49). Multiple enzymes may be responsible for suchdissolution. Separate enzymes may degrade the collagenousand noncollagenous constituents of the BM.

Type IV collagen is a major structural protein present in allBMs (13). This collagen (pro IV) can be acid extracted fromtissues rich in BM and consists of 2 chains with helical andnonhelical domains (3, 13, 16, 28, 42, 44). Type IV collagencan also be extracted by pepsinization of tissues rich in BM,yielding a family of components with a range of molecularweights from 140,000 to 20,000 (14). Laminin is a glycoproteindiscovered by Timpl ef al. (43), which is present exclusively inall BM and is thought to mediate epithelial cell attachment (41 ).Laminin exists as M.W. 400,000 and M.W. 200,000 components on polyacrylamide gel electrophoresis. Fibronectin, aM.W. 220,000 serum glycoprotein (11, 30) and cell attachmentfactor, is associated with some BM such as those found invessels. Type V collagen (aAaB) or (aAaBaC) is a newly discovered collagen which can be extracted from tissues rich inBM (3, 4, 23, 38). The exact distribution of this collagen isunclear at this time, but it may be located at the stroma-BM

interface.BM type IV collagen is resistant to previously described

classic mammalian collagenases but can be specificallycleaved by a neutral metal protease isolated from invasivetumor cells (16, 21). Type IV collagen is also susceptible toleukocyte elastase (24). Type V collagen is resistant to classicmammalian collagenase (16) and leukocyte elastase but canbe degraded by tumor cell and macrophage metal proteases(19). Outside of these previous studies, there is very limitedinformation concerning the specific types of enzymes whichcan degrade the collagenous as well as the noncollagenousportions of the BM zone under physiological conditions.

Serine proteases, e.g., PA, plasmin, and thrombin, havebeen shown to be associated with cell migration, cell adhesion,malignant transformation, and tissue remodeling (reviewed inRefs. 5 and 35 to 37). Since these phenomena may also beassociated with BM changes, we studied the effects of purifiedforms of these enzymes on isolated components of BM and onwhole BM. The BM-associated components studied were acid-

extracted type IV collagen (3, 13, 19, 21, 28, 42, 44), pepsinfragments of type IV collagen (14), the glycoprotein laminin(41, 43), collagen type V (aAaB) (4, 23, 38), and fibronectin(30). The ability of these native components to act as a substrate for the purified enzymes was studied by incubation withthe enzymes and identification of any degradation products bygel electrophoresis. Jones and DeClerk (12) showed plasmin-ogen-dependent tumor cell degradation of extracellular matrix

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L. A. Liotta et al.

glycoproteins. We therefore studied the effect of these serineproteases on laminin, a specific BM glycoprotein. For this partof the study, a new assay for ['"CJIaminin degradation was

used.Human amnion membrane was used as a source of whole

BM. This tissue has previously been shown to be composed ofepithelium attached to a typical BM by hemidesmosomes andoverlying an avascular stroma (20). At least, some lines ofhighly invasive tumor cells can invade the full thickness of theamnion in vitro (20). Following a brief exposure to ammoniumhydroxide or deoxycholate, the amnion epithelium could beremoved, leaving the BM intact as judged by electron microscopy and ¡mmunohistology (20). A solution of each purifiedserine protease was placed on the surface of the intact denuded amnion BM. Following incubation, the structure of thetreated BM was studied by transmission electron microscopyand immunohistology using antibodies to type IV collagen andlaminin.

MATERIALS AND METHODS

Substrates. Type IV BM collagen was obtained in pepsinized andunpepsinized native form. The unpepsinized type IV collagen was aproduct of EHS sarcoma tissue cultures and was purified as describedpreviously (21, 44). The unpepsinized type IV collagen consists of 2chains and contains nonhelical extension peptides (42, 44). Pepsinizedlens capsule type IV collagen was a generous gift of Dr. Edward J.Miller and contained C,, C, and D chains as reported previously (14).

Fibronectin, purified from human plasma (30), was the generous giftof Dr. David L. Amrani and Michael W. Mosesson, State University ofNew York Downstate Medical Center, Brooklyn, N. Y. After reductionof disulfide bonds, this material migrates as a dimer in the 220,000molecular weight range on polyacrylamide gel electrophoresis.

Laminin, a BM-specific glycoprotein (43), was purified (41) from

EHS sarcoma tissue harvested from C57BL/6 mice. Polyacrylamidegel electrophoresis of this preparation reveals m.w. 200,000 and m.w.400,000 components, which require reduction of disulfide bonds toenter the gel.

Native type V (aAaB) collagen, prepared from pepsinized humanplacenta tissue as described previously (4, 38), was a gift from Dr. P.Fietzek, Rutgers Medical School, New Brunswick, N. J. This materialconsists of 2 chains migrating slightly slower than the «-chains of type

I collagen on gel electrophoresis.Preparation of Antibodies. Rabbit antisera to unpepsinized EHS

type IV collagen were prepared as described previously (20, 41). Theantisera were passed over a 5-ml CNBr-activated Sepharose 4B affinity

column containing bound laminin (5 mg) to remove any contaminatinganti-laminin antibodies. Antibody specificity was verified by immuno-

precipitation of both chains of labeled EHS type IV collagen andcyanogen bromide cleavage products of these chains. The purifiedantibodies failed to immunoprecipitate laminin; fibronectin; or types I,II, III, or V collagen. Indirect immunofluorescence that used theseantibodies showed uniform fluorescent staining of all BM. The stainingwas specifically blocked by exogenous competition with type IV collagen. Antibodies to fibronectin were the generous gift of Dr. D. L. Amraniand Dr. M. W. Mosesson (30). Purified anti-laminin antisera were

prepared as described previously (41). These antibodies precipitatedboth of the ['"CJIaminin chains as judged by gel electrophoresis fol

lowed by fluorography. Indirect immunofluorescence was performedon frozen sections of amnion BM as described previously (20).

Digestion of Substrates. The purified plasmin, PA, and thrombinwere incubated with the substrates, and digestion was assessed bypolyacrylamide gel electrophoresis of the reaction mixture. The collagen samples were dissolved in 0.5 M acetic acid, followed by neutralization and dilution in buffer (50 mM Tris-HCI:0.2 M NaCI:5 mM CaCI2,

pH 7.6). A solution of the enzymes in Dulbecco's phosphate-buffered

saline (Grand Island Biological Co., Grand Island, N. Y.) was added tothe solution of substrate with an estimated enzyme:substrate ratio ofless than 1:100. Digestion was conducted at 25° for 15 hr with a

reaction volume of 400 jul. Digestion was stopped by the addition ofprotease inhibitors specific for the protease used followed by 9.6 ml ofice-cold absolute ethanol. The ethanol precipitate was collected by

centrifugation at 27,000 x g and was directly dissolved in gel electrophoresis sample buffer. Polyacrylamide gel (5%) electrophoresis wasperformed according to the method of Laemmli (15), except that 0.5M urea was included to improve the resolution of the bands.

Preparation of Whole BM. Whole intact surfaces of human amnionBM were prepared as described previously (20). Human placentaswere obtained from normal deliveries and were kept on ice until use.Within 4 hr after delivery, the amnion membrane was dissected awayfrom the chorion and washed in Dulbecco's modified Earle's media

containing Fungizone (1 ng/ml) and 1% gentamicin. Histologically, theamnion consists of a uniform epithelium resting on a uniform BMoverlying an avascular stroma. The epithelial cell layer of the amnionwas denuded from the BM by treatment with 0.1 M ammonium hydroxide for 30 min at 25°(20). Following this treatment, the BM remained

attached to the stroma. The BM was verified to be continuous andintact by: (a) impermeability to carbon particles and labeled serumproteins; (t>) uniform immunohistological staining with antibodies totype IV collagen and laminin; and (ci ultrastructural appearance bytransmission electron microscopy (20). The denuded amnion BM,clamped in individual 1-cm diameter culture holders, was washedextensively with serum-free media. A 0.4-ml solution of each purified

enzyme was then layered on the exposed BM surface. Purified bacterialcollagenase (Form III; Advanced Biofactures), 50 units with 10 mMphenylmethysulfonyl fluorine, was used as a positive control. Thestroma side of the amnion was exposed to serum-free media. Incubationproceeded at 25°for 15 hr. The membranes were then fixed with 2.5%

glutaraldehyde and processed for transmission electron microscopy.Collateral experiments were performed as described previously (20) toverify that the specific amnion membrane used for the enzyme studiescould be invaded by tumor cells in vitro.

Neutral Serine Proteases. Pure human plasmin (10.16 CU/ml) andpure human plasminogen (9.7 CU/ml) were kind gifts of Dr. GenesioMurano, Bureau of Biologies (Food and Drug Administration), Be-

thesda, Md. Pure human PA (UK, 165,000 units/mg) was also agenerous gift of Dr. Murano (32). Purified (97.3%) human a-thrombin

was a generous gift of Dr. John Fenton, II, New York State Departmentof Health (6). Trypsin and chymotrypsin were purchased from Worth-

ington Enzymes (Freehold, N. J.).Fibrinolysis Assay. Neutral serine proteases were assayed on iodi-

nated fibrin, a well-characterized substrate for proteolytic activity (40,45). 125l-labeled fibrin substrates were prepared as previously reported

(9). lodination was performed by the lactoperoxidase method, as described previously (10), except that human plasminogen-depleted fi-brinogen was used rather than bovine plasminogen-depleted fibrino-

gen. The human fibrinogen, purified by the method of Blomback andBlomback and depleted of plasminogen by the method of Mosesson(29), was a generous gift of Dr. John S. Finlayson, Bureau of Biologies(Food and Drug Administration), Bethesda, Md. (31). All fibrinolyticdeterminations used a standard UK, which allows for the calculation ofPA activity in Ploug units. The UK reference standard was purchasedfrom Leo Pharmaceutical Products, Denmark.

Laminin Degradation Assay. Labeled laminin was prepared fromorgan cultures of the EHS sarcoma using methods described previously(41, 43). [MC]Laminin was extracted from the tissue extracts with 0.5

M NaCI. The labeled laminin comigrated with purified laminin (41)prepared separately. Immunoprecipitation of the labeled laminin wasobtained with purified anti-laminin antibodies. Assays for laminin deg

radation used 2% trichloroacetic acid:0.05% fannie acid for precipitation of undigested substrate. The precipitation method was identical tothat described for type IV collagen (21). Routinely, 0.1 ng of ['"C]-

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Effect of Enzymes on BM Components

laminin containing 4000 cpm was used in 200 /il of 0.05 M Tris:0.2 MNaCI 5 mM CaCI2 solution buffer, pH 7.4. The enzyme solution wasadded in a volume of 200 ¡i\

Cell Culture Media Extracts. A variety of cell types was examinedfor laminin-degrading activity. Human Ewing s sarcoma cells were

supplied by Dr. T. Triche, National Cancer Institute. These cells aretumorigenic in nude athymic mice. Human fibroblasts, mouse PMTsarcoma cells, human leukocytes, and mouse-activated peritoneal

macrophages were isolated and cultured as described previously (17,

22).Electrophoretic Analysis of Proteases and Assay Substrate. PA,

plasmin, a-thrombin, and fibrinogen were examined by sodium dodecyl

sulfate:polyacrylamide gel electrophoresis according to the method ofLaemmli (15), as described previously (10). The electrophoresis used4% stacking and 10% resolving gels.

Protease Inhibitors. Leupeptin and chymostatin were generous giftsof Dr. Walter Troll, Department of Environmental Medicine, New YorkUniversity Medical Center, New York, N. Y. Aprotinin (trasylol) andhirudin were obtained from Sigma Chemical Co. (St. Louis, Mo.).

RESULTS

Verification of Enzyme Purity. The purity of PA, plasmino-gen, and a-thrombin was ascertained by electrophoretic mo

bility, enzymatic assay, and selective inhibition studies. Electrophoresis was also used to determine the purity of plasmin-ogen-free fibrinogen used as the substrate for protease assay.

The human PA used in this study (UK) exists in 2 forms withmolecular weights of approximately 53,000 and 34,000. Thisis in close agreement with published reports (32). a-Thrombin

used in this study has a molecular weight of approximately38,000, which is similar to reported values (6). The plasmino-

gen used in this study has a molecular weight of approximately88,000 to 90,000, which is in agreement with values in theliterature (36). Plasmin, the activation product of plasminogen,was derived from the plasminogen exposed to UK and showedmolecular weights of 76,000, 48,000, and 26,000, which is inagreement with published reports (39).

Fibrinolysis assays with or without inhibitors were an independent check on the purity of the proteases used in this study.The fibrinolytic activity of UK was completely dependent uponthe inclusion of plasminogen in the assay mixture and thereforeproves that it is indeed a PA. In contrast, trypsin, thrombin,chymotrypsin, and plasmin were fibrinolytic in the absence ofplasminogen. The specificity and enzymatic activity of eachprotease were determined by protease assay in the presenceor absence of specific inhibitors: leupeptin (tryptic specificity);chymostatin (chymotryptic specificity); hirudin (specificity forthrombin); and aprotinin (specificity for plasmin). Followingpreincubation with chymostatin or hirudin, the activity of UKwas inhibited by less than 10%, whereas preincubation withleupeptin inhibited plasminogen-dependent enzymatic activity

for more than 95%. Plasmin activity (99%) was inhibited byaprotinin but only slightly (2.1%) by hirudin. Thrombin wasinactivated by hirudin by 90% but not by aprotinin. The specificity of the inhibitors was checked by examination of theireffect on other proteases. Hirudin could not appreciably (2 to9%) inhibit trypsin, UK, or plasmin; leupeptin inhibited trypsinbut not chymotrypsin; and chymostatin inhibited chymotrypsinbut not trypsin. The results demonstrate that the proteasesjudged to be homogeneously pure by sodium dodecyl sulfate:polyacrylamide gel electrophoresis were also displaying theproper inhibition-susceptibility spectrum.

Effect of Enzymes on Isolated Components of BM. Theeffects of purified UK (PA), plasmin, plasminogen, and a-throm

bin were examined on native fibronectin (Fig. 1). UK (PA) failedto significantly degrade fibronectin, even at a concentration100-fold greater than physiological levels. The activity of theUK was biochemically verified before and after the incubationwith fibronectin. Plasmin cleaved fibronectin, producing specific cleavage products. The plasmin-produced fibronectin

cleavage products were specific and reproducible when thedigestions were partially or completely reduced prior to electrophoresis. Plasminogen alone showed no ability to degradefibronectin. Thrombin significantly cleaved fibronectin into specific fragments. Digestions were performed at identical conditions (28°and pH 7.6 for 24 hr) for all enzymes (Fig. 1).

Laminin was incubated with highly purified plasmin and a-

thrombin. Plasmin degradation of laminin produced specificcleavage products in which both the 400,000 and 200,000chains of the molecule were significantly cleaved (Fig. 2).Thrombin under the same incubation conditions degraded onlythe m.w. 400,000 chain of laminin but failed to show significanteffect on the m.w. 200,000 chain (Fig. 3). UK (PA) failed todegrade acid-extracted type IV collagen (Fig. 4) and pepsinizedtype IV collagen (not shown). Plasmin at 25°(enzyme:substrate

ratio, 1:100) failed to degrade type IV collagen. At high concentrations, plasmin produced a small reduction in the lower ofthe 2 type IV collagen chains, consistent with nonspecificremoval of a part of the nonhelical telopeptide region (Fig. 4).

Type V collagen, when incubated at 25° with a-thrombin,

was not significantly degraded (Fig. 5). With incubation as highas 33°and prolonged digestion times, type V was not cleavedby a-thrombin. At 35°,a-thrombin did produce specific cleav

age fragments of the type V collagen (Fig. 5). Plasmin alsoshowed a temperature-dependent cleavage of type V collagen

(1:50, enzyme:substrate ratio). Plasmin produced cleavageproducts at 37°but failed to produce any significant cleavageat temperatures less than 35°even when digestion times were

prolonged (not shown).The assay for laminin degradation using ['"Cllaminin pre

pared from cultured EHS cells showed appropriate saturationkinetics for trypsin digestion of laminin (Charts 1 and 2). Alinear relationship was found between the amount of laminin

1.0 2.0 3.0

HOURSChart 1. Laminin degradation assay. Time course of degradation by trypsin.

[14C]Laminin (50 jig) was incubated with 20 /ig of trypsin at pH 7.4, 37°.Following

incubation, the undigested substrate was precipitated with trichloroacetic acid:tannic acid. The radioactivity of the digested substrate (which remained insolution) was counted. Ninety % of the substrate was degraded by 1 hr. Bars,S.D.

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ENZYME CONCENTRATION

Chart 2. Laminiti degradation assay. Enzyme concentration dependence.[14C]Laminin (50 fig) was incubated with plasmin, trypsin, or UK at the indicatedconcentrations. Incubation was carried out at 37" for 2 hr. A linear relationship

was observed between enzyme concentrations and amount of substrate degraded. UK alone failed to degrade laminin. Trypsin degraded laminin at a fasterrate than did plasmin. Enzyme concentration is expressed in fig/assay. Bars.S.D.

degraded and the concentration of plasmin or trypsin used inthe assay. To study the biological relevance of laminin degradation, extracts from a variety of different cells were tested forlaminin-degrading enzyme activity. Leukocytes, macrophages,

and metastatic mouse sarcoma cells all exhibited significantdegradative activity. For the tumor cells, a major fraction of theactivity was plasminogen dependent. The plasminogen-inde-pendent laminin-degrading tumor cell activity was abolished by

metal protease inhibitors. Thrombin degraded 50% of the totallabeled substrate at 37°, consistent with the finding by gel

electrophoresis that only the M.W. 400,000 chain of lamininwas degraded by this protease. Degradation of laminin byplasmin was inhibited by aprotinin (Chart 3).

Since plasmin degraded 2 noncollagenous components associated with the BM zone and had a small effect on acid-extracted type IV collagen, we studied the effect of this enzymeon whole isolated amnion epithelial BM. Fibrinolytic activity ofthe plasmin was verified before and after incubation with thewhole BM. Histological study indicated that the amnion iscomposed of a uniform epithelium with surface microvilli overlying a typical BM zone (20). Following treatment of the denuded BM with plasmin for 15 hr at 28°,the BM ultrastructure

is not greatly altered (Fig. 6). The amnion BM exhibits uniformbinding of antibodies to both type IV collagen and laminin (20).The antigenicity is preserved after epithelial denudation (Fig.7). Even though the BM lamina densa is not completely destroyed by the plasmin treatment, it does lose its immunoreac-tivity for anti-laminin antibodies (Fig. 7D) and anti-fibronectinantibodies (data not shown). Immunoreactivity for anti-type IV

collagen antibodies is preserved after plasmin treatment (Fig.7C).

DISCUSSION

Current information suggests an enzymatic mechanism fortumor cell penetration of BM. During the hematogenous metastatic process (7), tumor cells must traverse the continuousvascular endothelial BM (2, 16, 18, 20, 22, 47, 49). Thisextracellular matrix comprises the major structural stability ofthe capillary and venule (33) and does not contain pores large

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Chart 3. Various cell lines and purified enzymes were assayed for laminin-degrading activity. The cells were grown in serum-free media and dilutions of themedia were performed, as described previously (22), so as to represent theactivity per 2 x 105 viable cells. The value shown is the mean ±range with amaximum of 2000 cpm. The Ewing's sarcoma cells exhibit invasive growth innude athymic mice. The assays were performed at pH 7.4. 37°for 2 hr. PLG,

plasminogen. Bars. S.D.

enough to permit passage of carbon particles even in regionsof endothelial retraction (25). Electron microscopy of tumorcell extravasation has demonstrated local dissolution of the BM(49). When tumor cells are placed on whole amnion BM invitro, local dissolution of the BM is followed by penetration ofthe cells into the stroma (20). Our previous studies haveindicated that at least some lines of metastatic tumor cellscontain metal protease activity which can degrade BM type IVcollagen (16, 18, 21, 22). These assays (16, 21, 22) for typeIV collagenolytic activity were done in the presence of a plasmininhibitor (soybean trypsin inhibitor); hence, the role of thisenzyme in degrading the type IV collagen was unknown. Thepresent study was therefore undertaken to clarify the role ofplasmin and associated PA and a-thrombin in degrading var

ious components of BM.The data indicate that none of these serine proteases alone

can completely degrade all the components of the BM. Plasminreadily degrades the glycoprotein components laminin andfibronectin at an enzyme:substrate ratio of 1:100. The newlaminin degradation assay introduced here shows that a varietyof cells, both tumor cells and normal inflammatory cells, secretelaminin-degrading proteases. The addition of plasminogen tothe serum-free conditioned media enhanced the laminin-degrading activity significantly. Such a response supports a plas-min-mediated degradation of laminin. However, since degradation also occurs in the absence of plasminogen, there are

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Effect of Enzymes on BM Components

other neutral proteases, different from plasmin, involved in thedegradation of laminin as well. The collagenous componentstype IV collagen or type V collagen are much less sensitivethan the glycoprotein components to degradation by plasminat the same enzymeisubstrate ratio. Nevertheless, very high(50- to 100-fold greater than were those used for laminin

degradation) concentrations of plasmin did produce a reduction in the staining intensity of the type IV collagen on gelelectrophoresis. Nonphysiological excessive concentrations ofplasmin may degrade the collagenous components of BM. It isprobable that enzymes other than plasmin degrade the collagenous components of the BM under normal physiological conditions (16, 21, 24). Since plasmin does not destroy whole BM(Fig. 7) under conditions where the laminin antigenicity isremoved, this may indicate that the structural stability of theBM is not determined by laminin or fibronectin. Type IV collagenmay be the main structural component.

PA has been proposed to have a direct biological role independent of plasminogen (35). The concurrent loss of cellsurface fibronectin and the enhanced production of PA in vitrohave led to the speculation that this enzyme may be able todegrade fibronectin (1). The present data conclusively demonstrate that fibronectin is not a direct substrate for PA (Fig.1). Perhaps the in vitro effect of PA on cells relates to an effecton the cell membrane-associated fibronectin-binding site. It is

also possible that PA may have a direct effect on anothercomponent of BM not studied here.

The production of PA (45) and the generation of plasmin byproteolytic activation of plasminogen (36) have been correlatedwith various aspects of the malignant state including: anchorage-independent growth; the induction of cell division; cell

migration, morphology, and adhesion; and cytoskeletal andcell surface alterations (reviewed in Refs. 10, 11, and 35). Theexpression of PA activity has also been correlated with thetumorigenicity of viral transformants in nude mice; with thetemperature-sensitive expression of the Rous sarcoma virus

src (sarcomagenesis) gene product; with the tumorigenic potential of malignant melanoma cells; and with tumor promotertreatment of normal and tumor virus-transformed cell cultures

(reviewed in Refs. 5, 9, and 35).PA exists in normal as well as malignant cells (5) and has

both multiple molecular weight forms and multiple immunolog-ical specificities. Normal cells that display PA activity areusually involved in some aspect of tissue remodeling or inva-siveness (37). The prototype human PA, UK, is produced inthe kidney and is present in urine. UK is a PA that exists in 2predominant molecular weight forms: molecular weights ofapproximately 54,000 to 55,000 and 31,000 to 35,000 (32,50). The majority of human PA produced by tumor cells isbiochemically and immunologically similar to or identical to UK(reviewed in Refs. 1, 5, and 26), whereas others have uniquemolecular weights and are immunologically distinct (reviewedin Refs. 46 and 51 ). It is therefore possible that other forms ofPA distinct from the type studied here might have differentsubstrate specificities.

In agreement with the study of Furie and Rifkin (8), thrombinwas shown to degrade fibronectin (Fig. 1). This enzyme alsodegraded the M.W. 400,000 chain but not the M.W. 200,000KD chain of laminin (Fig. 5). The specific thrombin cleavage ofthis laminin chain will be useful for structural, immunological,and biological studies of this molecule. Thrombin also de-

Fig. 7. Immunofluorescent localization of anti type IV collagen and antl-lamininantibodies in denuded amnion BM. Effects of plasmin treatment. Control denudedamnion indirect immunofluorescence with anti-IV antibodies (A) and anti-lamininantibodies IB). Plasmin-treated BM (conditions given in text), indirect immunofluorescence with anti-IV (C). and anti-laminin antibodies (D). control, in the absenceof antibody (E). The plasmin removed the laminin but not the type IV collagenimmunoreactivity. Identical results were obtained upon plasmin treatment ofhuman capillary basement membranes in frozen sections (data not shown). Tumortype IV collagenase (21) removed the type IV collagen immunoreactivity.

graded type V collagen at 35°but not at temperatures below33°(Fig. 3). Since pepsin extracted type V collagen may be at

least partially denatured at this temperature (38), the physiological significance of thrombin degradation of type V collagenis an open question. The thrombin-derived cleavage products

of type V collagen should be useful for structural studies.These data indicate that plasmin alone cannot completely

degrade the BM. This enzyme may nevertheless aid in theenzymatic destruction of BM through its activation of latenttumor cell metal proteases (19, 21, 34), which degrade collagen, glycoprotein or glycosaminoglycan components of theBM. Plasmin may play a role in removing the glycoproteincomponent of the BM and exposing the type IV collagen for

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degradation by different enzymes; plasminogen activator mayplay an important role in the regulation of plasmin action in thisregard.

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4634 CANCER RESEARCH VOL. 41

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A B C

Effect of Enzymes on BM Components

G H l440K

220K*»l

440K

220K

160K

130K

100K

60K

1

400K^|NátMé

200K-Õ-

400K

200K 180K140K

A B C DA B

Fig. 1. Effect of purified UK (PA), plasmin, plasminogen, and o-thrombin on fibronectin. Digestion was performed at 28°, pH 7.6, for 24 hr. Sodium dodecyl

sulfate: 5% polyacrylamide gel electrophoresis. A, fibronectin substrate (50 jig) alone; ß,substrate plus PA (0.6 /ig); C, substrate plus PA (6.6 /ig); D, substrate plusplasmin (0.6 /ig); F, substrate plus plasminogen (0.6 /ig); F, substrate plus o-thrombin (0.6 fig). All samples were reduced with 10 mM dithiothreitol. Digestion withplasmin was repeated, and gel electrophoresis was performed under 2 different reducing conditions. G, substrate alone plus 10 mM dithiothreitol; H, substrate plusplasmin (6.6 /ig) and 20 mm dithiothreitol; /, substrate alone plus 20 mM dithiothreitol.

Fig. 2. Effect of highly purified plasmin on laminin. 5% gel, all samples reduced (10 mM dithiothreitol). A, laminin (30 /ig) alone; 8. laminin (30 /ig) plus plasmin(0.3 /ig) at 25°for 1 hr; Both chains are cleaved. C, laminin (30 /ig) plus plasmin (0.3 /ig) at 25°for 5 hr; 0. laminin (30 /ig) plus plasmin (0.3 /ig) at 25°for 25 hr.

Fig. 3. Effect of highly purified a-thrombin on laminin. A, laminin (50 /ig) alone; M.W. 200,000 and M.W. 400,000 chains are present in 1:1.5 proportion. B,laminin (50 /ig) plus thrombin (0.5 /ig) at 25°for 30 min; C. laminin (50 /ig) plus thrombin (0.5 /ig) at 25°for 2 hr; D, laminin (50 /ig) plus thrombin (0.5 /ig) at 25°for

25 hr. The M.W. 400.000 K chain is selectively digested. 5% gel, all samples reduced with fresh 20 mM dithiothreitol.

NOVEMBER 1981 4635

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W *

ABCD E F G ^

Fig. 4. Effect of serine proteases on acid-extracted type IV collagen. A, type IV collagen (50 fig) alone; 8, type IV collagen (50 fig) plus UK (6.6 fig) at 28°,for 24hr; C, type IV collagen (50 fig) + a-thrombin (6.6 fig) at 28°for 24 hr; D, type IV collagen (50 fig) plus a-thrombin (0.6 fig) at 35°for 3 hr. No degradation is observed.E. type IV collagen (50 fig) plus plasmin (0.6 /ig) at 28°for 24 hr; F, type IV collagen (50 fig) plus plasmin (6.6 fig) at 28°for 24 hr; G, type IV collagen (50 fig) plusplasmin (25 fig) at 28°for 24 hr. Arrows, denote a, and a2 chains of type IV collagen.

f p

sr «M

B

t..-¿

B/M

~

•¿�

BST

B/W

BFig. 5. Temperature-dependent cleavage of type V collagen by a-thrombin.

Arrowed letters A, B, C, aA, aB, oC chains, respectively. A, type V collagen (50fig) plus a-thrombin (0.5 fig) at 25°for 20 hr; B, type V collagen (50 fig) plus a-thrombin (0.5 jig) at 30° for 20 hr; C, type V collagen (50 fig) plus a-thrombin(0.5 fig) at 33.5° for 20 hr; 0, type V collagen (50 fig) plus a-thrombin (0.5 fig) at35°for 3 hr. Specific cleavage was produced only at high temperatures. Plasmin(6.6 jig) produced cleavage products at 37° but not at low temperatures. UK

failed to degrade type V collagen at any temperature.

ST ».¿6

Fig. 6. Effect of purified plasmin (33 fig) on whole human amnion BM studiedby transmission electron microscopy. Digestion conditions are given in text. A.untreated amnion prior to epithelial denudation; the epithelial cells (EP) withcharacteristic nuclei (fV) are shown attached to the BM overlying a connectivetissue stroma (S7"). Approximately x 82,000. 6, control denuded amnion with

continuous amorphous BM surface. Approximately x 120.000. C, denudedamnion BM treated with plasmin for 24 hr. The BM lamina densa is retained[purified bacterial collagenase is known to remove the lamina densa within 20min (20)]. Approximately x 120,000.

4636

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1981;41:4629-4636. Cancer Res   L. A. Liotta, R. H. Goldfarb, R. Brundage, et al.   Basement MembraneThrombin on Glycoprotein and Collagenous Components of Effect of Plasminogen Activator (Urokinase), Plasmin, and

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