Latent and Active Plasminogen Activator in Corneal liberation - IOVS

Latent and Active Plasminogen Activator in Corneal liberation Hsin-Min Wong, Michael Berman, and Mary Low Previous studies of alkali burns have provided evidence for an important role of the plasminogen activator (PA)/plasmin system in corneal ulceration. Current studies have utilized a sensitive, plasminogen-dependent fluorescent assay to demonstrate that PA is present mostly in a latent (trypsinor plasmin-activatable) form (proactivator) in cultures of rabbit corneal epithelial cells or normal corneas. Cultures of ulcerating corneas demonstrate only active PA early in organ culture, whereas, latent PA levels increase later in culture. Thus, ulceration is correlated with the apparent conversion of latent to active PA. Moreover, profiles of proactivator and latent collagenase and of active PA and active collagenase in vitro, respectively, are similar, suggesting that activator and collagenase are under coordinate control. Cultures of normal epithelial cells and nonulcerating corneas contain PA molecular weight species of 72,000 and 46,000 MW, and ulcer corneas, species of 72,000, 46,000, and 35,000 MW. Double-diffusion analysis indicates that rabbit epithelial cells, fibroblasts, and ulcer corneas produce urokinase (UK)-like PA; and human cornea extracts and tears also contain PA immunoreactive with anti-UK antibodies. The existence of PA in a latent form identifies another level of regulation in the cascades that lead to stromal ulceration. Invest Ophthalmol Vis Sci 26:511— 524, 1985

regulatory properties of PA species, in order to understand their role in corneal ulceration.5

Previous work has indicated that the plasminogen activator (PA)/plasmin system initiates fibrinolytic activity at the corneal surface to effect resorption of fibrin and fibronectin after an alkaline injury.1 It has been suggested that regulation of this system goes awry after an alkali burn, such that increased levels of PA cause a persistent epithelial defect to develop, and the cornea is trapped in a phase of proteolytic debridement that eventually leads to stromal ulceration. 12 Other studies have indicated that the PA/ plasmin system can stimulate the enhanced secretion of latent collagenase from fibroblasts in organ cultures of ulcerating corneas after alkali burns 3 and in fibroblast cultures4; and that the latent collagenase can be activated by plasmin,3 leading to increased degradation of corneal stromal collagen. Thus, although various other biochemical systems no doubt also contribute to enzymatic degradation of the corneal stroma, previous work has indicated that the PA/plasmin system has important roles in such degradation. The current work was undertaken to begin to study the

Materials and Methods The plasmin substrate D-valine-leucine-lysine-4 methoxy-/3-naphthylamide (D-val-leu-lys-4M/3NA) was purchased from Enzyme Systems Products, Livermore, California. Human plasminogen was purified by affinity chromatography on lysylagarose6 from plasma provided by the American Red Cross, Boston, Massachusetts. Bovine fibrinogen containing plasminogen; and bovine fibrinogen, plasminogen-free; bovine thrombin; and human urokinase (UK, a mixture of 46,000 MW and 35,000 MW species) were obtained from Calbiochem-Behring, La Jolla, California. Dulbecco's Modified Eagle's Medium (DMEM) 4,500 mg/1 dextrose; nonessential amino acids; Eagle's Minimal Essential Medium (EMEM); fetal bovine serum (FBS), heated for 30 min at 56°C; streptomycin sulfate; penicillin-G; and pancreatin (0.25%) were obtained from Gibco, Grand Island, New York. Amphotericin B (Fungizone) was obtained from Squibb, New York, New York. N^N^N'-tetramethylethylenediamine, acrylamide, bis-acrylamide, and /3-mercaptoethanol were obtained from Eastman, Rochester, New York. Clostridial collagenase (type I) was purchased from Worthington-Millipore, Freehold, New Jersey. Silicone grease was obtained from Dow Corning, Midland, Michigan, and glass coverslips

From the Department of Ophthalmology, Laboratory for Ophthalmic Research, Emory University, Atlanta, Georgia. Supported by NIH Grant No. EY03879 and by a Departmental Grant from Research to Prevent Blindness, Inc. Submitted for publication: March 6, 1984. Reprint requests: Hsin-Min Wang, Ph.D., Emory University, Department of Ophthalmology, Eye Research Laboratories, Atlanta, GA 30322.

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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1985

were obtained from Corning Glass Works, Medfield, Massachusetts. P-nitrophenyl, p'-guanidinobenzoate (NPGB) for active site titration of trypsin and plasmin7 was obtained from Research Organics, Inc., Cleveland, Ohio. Amounts of trypsin, plasmin, or plasminogen are reported in active site equivalents. Thirty-three thousand and 55,000 MW species of human UK were obtained through the courtesy of Dr. Paul Kelly of Collaborative Research, Inc., Waltham, Massachusetts. Antisera to 33,000 MW UK made in rabbits was purchased from Collaborative Research, Inc., and to that species of UK, made in goats, was obtained through the courtesy of Dr. Kenzo Tanaka, Kyushu University, Japan. Molecular weight markers were obtained from Sigma Chemical Co., St. Louis, Missouri: myosin subunit (205,000); /3-galactosidase subunit (116,000); phosphorylase B subunit (97,400); plasma albumin (66,000); egg albumin (45,000); and carbonic anhydrase (29,000). Alkali Burns Animals (3-5 lb albino male rabbits) whose corneas were to be alkali burned were anesthetized systemically by sodium pentobarbital (approximately 20-25 mg/ kg) and topically by 0.5% proparacaine hydrochloride to ensure that they would not be caused pain by the alkali burns. Animals, once adequately anesthetized, were alkali-burned unilaterally by the application for 2 min of an 8-mm filter disc saturated with 4 N NaOH. After the burn was made, the cornea was irrigated copiously with 0.15 M NaCl. Antibiotic ointment (erythromycin) was instilled in the lower fornix immediately postburn and daily to prevent corneal infection. Animals treated in this way showed no signs of ocular pain either during the administration of the topical burn or during the postoperative period. Animals were killed (euthanized) by the intravenous injection of sodium pentobarbital. The methods of anesthesia used and care were approved by the University Veterinarian, pursuant to the requirements of the Animal Welfare Act, Amended public law 94-279, April 1976, and those of the Guide for the Care and Use of Laboratory Animals, 1978, National Research Council. This investigation adheres to the ARVO Resolution on the Use of Animals in Research. Burns of the severity used typically produce ringshaped stromal ulcers between 5 and 10 days postburn in 100% of animals so treated.1 In this model, the cornea is resurfaced completely by epithelium by 3 5 days postburn. Later, however, a persistent epithelial defect develops, followed by stromal ulceration. Alkaliburned corneas for culture were taken at the same

Vol. 26

time for a given experiment. That is, in one experiment all corneas were taken at 10 days postburn. In the second experiment all corneas were taken at 11 days postburn (see legend for Fig. 6). At those times the severity of ulceration varied between animals and even between eyes of the same animal. Thus, even though burned under standard conditions, the extent of ulceration varied from superficial to deep stromal ulcer (see below). Correlated with the severity of ulceration, alkali-burned ulcerating corneas demonstrate histologically, as reported previously by Kenyon et al,8 many polymorphonuclear leucocytes (PMNs) as well as some monocyte-macrophages and fibroblasts in the corneal stroma. Typically, PMNs are the predominant, if not the only, cell type detectable in the actual stromal ulcer region. Organ Cultures Routinely, for purposes of organ culture, 24 corneas (normal or ulcerating) each were divided into four equal sectors, one of which was distributed to each of four culture plates. All sectors per plate were minced to give the equivalent of six minced corneas (24 quarters) per culture plate and the same approximate ratio of corneal tissue per milliliter medium per experiment. Where ulcerating corneas were involved, attempts were made to distribute equal amounts of the macroscopically identifiable translucent ulcer region of each cornea to each plate. Since, as mentioned above, different corneas had different amounts of stromal ulceration when cultured, comparison is made in this work between "ulcerating" and "nonulcerating" corneas. No attempt is made here to determine ratios of latent and active plasminogen activator as they relate to stages of ulceration, ie, superficial, moderate, deep. Epithelium was left on the corneal tissue. Previous work9 had shown that such a distribution of ulcerating corneal tissue results in the same profiles of collagenase over time in the different replica plates. For a given experiment, one culture plate of a set of four replica plates was used for plasminogen activator assays. The other plates were used for other purposes (eg, prostaglandin assay). Thus, the plasminogen activator results are derived from two plates, one from each of two different culture experiments (done at different times), for both normal and ulcerating corneas. The culture medium was a mixture of DMEM and Tyrode's solution (1:1, v:v) (standard medium) containing penicillin (100 units/ml); streptomycin (100 mcg/ml); and amphotericin B (2.5 ixg/m\). Cultures were gassed continuously at 37°C under 5% CO 2 /95% O 2 in a moist chamber and were changed daily for 7 days.

No. 4

513

PLASMINOGEN ACTIVATOR IN CORNEAL ULCERATION/Wong er ol.

Epithelial Cell Cultures

Plasminogen proactivator |-«Activating protease ; Trypsin or Plasmin Plasminogen activator

Epithelial layers from normal (nonburned) or ulcerating corneas, respectively, with minimal superficial stroma, were separated mechanically from the rest of the rabbit corneas and were minced and distributed evenly on the bottoms of 35-mm plastic Petri dishes (Falcon, Oxnard, CA). Epithelial layers were obtained at different times and from different corneas than those used for organ culture. Glass coverslips then were pressed over the explants and secured by silicone grease. The explants then were incubated with clostridial collagenase (1 mg/ml) in DMEM at 37 °C for 20 min to loosen the epithelium from the subjacent stroma. Preliminary experiments had shown that epithelium grows out from the superficial stroma after treating the explants with the clostridial collagenase. After the completion of incubation, the medium was changed to DMEM, supplemented with nonessential amino acids, with D-valine substituted for L-valine (to select for epithelial cell outgrowth10) and with 10% FBS that had been dialyzed for 48 hr at 4°C against three changes of 0.15 M NaCl. Culture medium was changed every 2 days. After 4-5 days, when epithelial cells had migrated out onto the dish surface, the initial corneal explants were removed, and epithelial cells were allowed to spread over the entire dish. Confluent monolayer cultures of epithelial cells were detached by treatment with pancreatin (0.25%) at 37°C for 3 min; and the cells obtained were seeded to other dishes and allowed to grow to confluence. At confluence the cells were distinctly epithelial in appearance and easily distinguished from cultures of stromal fibroblasts. Assay of Plasminogen Activator (coupled assay) Prior to obtaining harvests of epithelial cultures for PA assays, the culture medium (serum containing) of the confluent cells was removed, and cultures were washed four times with PBS (pH 7.5) containing 1 X 10"4 M CaCl 2 . Calcium was added to the PBS to prevent the detachment of the cells in serum-free medium from the culture plates. All other reagents contained CaCl 2 . The washed cells then were incubated with DMEM (without phenol red which fluoresces) and antibiotics, at standard levels, for 2 days. In some experiments DMEM was supplemented with 0.2% lactalbumin hydrolysate. Harvests (1 ml) of primary cultures and secondary cell subcultures or of organ cultures were used. Harvests of both cell and organ cultures were dialyzed for 20-24 hr at 4°C against 0.025 M Tris-HCl, 0.005 M CaCl 2 , and 0.1 M NaCl, pH 7.5 (25°C), and

(Fluorophor) >4M0NA

Plasminogen — Plasmin—•

D-val-leu-lys-j- 4MpNA 2.4-r 2.22.01.81.61.41.21.00.80.60.40.20

0.1

0.2

0.3

0.4

0.5

0.6

UK (CTA UNITS)

Fig. 1. Coupled, plasminogen-dependent fluorescent plasminogen activator (PA) assay using UK (Calbiochem-Behring; 46,000 plus 35,000 MW species). The rate of fluorescence generation (in relative units) is proportional to UK, over the range 0.1-0.5 CTA units (see text for details).

concentrated tenfold by freeze-drying and redissolution in '/10th volume distilled, deionized water. PA concentrations in harvests were determined in a coupled, plasminogen-dependent fluorescence assay, in which PA generates plasmin from plasminogen in the first reaction. In the second, coupled reaction, plasmin, in turn, hydrolyzes off the fluorophor 4M/3NA from the substrate D-val-leu-lys-4M/?NA. The coupled assay combines the specificity of plasminogen used as initial substrate with the amplification of a two-stage assay and the increased sensitivity of fluorescence." Fluorescence was measured at room temperature (22°C) in a Turner Model No. 111 Fluorometer. Controls consisting of fluorescent substrate alone in buffer, with harvest or with plasminogen alone, were included in each determination. The linear rate of increase of fluorescence intensity (in relative units) was determined by the calculation of the slope value of net fluorescence (ie, after subtracting relevant reagent control values) over elapsed time. Using urokinase as a standard, a linear relationship between the rate of fluorescence units generated per minute and CTA units of UK [(1 CTA unit = 46.2 X 10"3 emotes methanol/hr/37°C from N-acetyl-Llysine methyl ester (12) = 5 relative fluorescent units/ min/22°C) (standard curve)] was established (Fig. 1). UK was used as a standard because it is available commercially to other laboratories in known international biologic units (CTA unit = unit defined by

514


100-r 90100% 80%

0 %

604 so-

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3020%

20-

2.2 -n

0 10 20 30 40 50 60 70 80 90 100

2.0-

TIME(min)

1.8-

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of the coupled assay, except that, because trypsin itself is a plasminogen activator 1213 and also has some direct activity on the plasmin substrate, additional controls of trypsin plus D-val-leu-lys-4M/?NA, trypsin plus plasminogen plus D-val-leu-lys-4M/3NA, and harvest plus trypsin plus D-val-leu-lys-4M/3NA also were included in each determination in order to obtain net harvest PA activity. By comparing the net activity in fluorescence units per minute after trypsin treatment of the harvest with that of harvest without such treatment, the levels of proactivator could be determined.

1.61.4-

Collagenase Assay

1.21.00.8-

5g

0.60.4-

0.2-j 0 20

40

60

80

100

% HARVEST CONCENTRATION

Fig. 2. Characteristics of the fluorescent PA assay using corneal harvests, a, Dilutions of harvest from organ culture of ulcerating corneas produce progress curves linear with both time and relative concentrations (see text for details), b, A linear relationship is observed when rates of fluorescence formation (fluorescence units/ min) are plotted against serial dilutions of harvest.

the Committee on Thrombolytic Agents). Thus comparison of PA results in other systems to those reported here for corneal plasminogen activator can be made by reference to the equivalent units of UK under defined conditions. Serial dilutions of a harvest from ulcerating corneas gave a linear dose-response of fluorescence values, within the range of the UK standard curve, when assayed under comparable conditions (Fig. 2). By comparing the rate of formation of fluorescence intensity by plasminogen activator in the harvests with the UK standard curve, the activity per unit volume,of harvest of active PA could be determined. Results are expressed in equivalent CTA U/ml. To measure the levels of latent, activatable PA (ie, plasminogen proactivator) in culture harvest, 10 /A of dialyzed, concentrated harvest first were incubated with 1 ixl of trypsin (12 ng) at 37°C for 20 min (to activate proactivator) before the addition of plasminogen. (Preliminary experiments with trypsin levels of 0-40 ng/10 fx\ harvest showed that 12 ng trypsin/10 ix\ harvest gave maximal activation of 10 /x\ of concentrated normal harvest. Higher levels of trypsin caused a decrease in total measurable activatible PA, possibly due to degradation of activator). Thereafter, the procedure of determination was identical to that

Active and latent collagenase assays were performed on harvests of organ culture as previously described.1415 Cultures of epithelial cells were not assayed for type I collagenase (active against type I collagen, the major collagen of the corneal stroma), since our previous unpublished work had detected no collagenase in such cultures. Other workers16 also have been unable to detect type I collagenase in harvests of rabbit corneal epithelial cells. Gel Electrophoresis To determine the molecular weight (MW) species of PA, dialyzed, concentrated harvests were electrophoresed in the presence of sodium dodecylsulfate (SDS-PAGE).17 Molecular weight species of PA were identified by lysis zones on fibrin-plasminogen-agarose plates, and MW were estimated by comparison to the electrophoretic mobilities of MW markers run under the same conditions18 (Fig. 3). PA detector plates were made by clotting a mixture of 5.7 ml of fetal bovine serum, as the source of plasminogen12 (neutralized to pH 7.5 after having been acid-treated to pH 2.0 and heat treated at 56°C for 30 min to inactivate antiproteases), 3.9 ml of bovine fibrinogen (1%) with plasminogen, and 9.6 ml of agarose (2.5%) prewarmed to 45 °C with 0.2 NIH U/ml (final concentration) of bovine thrombin. A control plate of plasminogen-free fibrin/agarose, formed the other half of the "sandwich" enclosing the acrylamide gel. The enclosed gel was sealed in a plastic wrap and incubated in a moist chamber at 37°C. The developing lysis zones on the fibrin-plasminogen-agarose plate were photographed after various incubation times, under dark-field illumination, to document PA activities. To characterize the nature of plasminogen cleavage by PA (ie, plasmin generation), plasminogen was incubated with concentrated culture harvests in the presence of bovine pancreatic trypsin inhibitor (BPTI; also known as Trasylol or Aprotinin; Sigma) to

No.

4

515


inhibit plasmin but not PA, and cleavage products were analyzed by SDS-PAGE, as described previously. 319

FIBRIN/AGAROSE (PLASMINOGEN-FREE) -

SDS-PAGE FIBRIN/AGAROSE (PLASMINOGEN)

Double-Diffusion (Ouchterlony Assay) The cross-reactions of PA from rabbit corneal organ and cell cultures with goat antibodies against low MW (33,000 MW) human UK and of PA in human tears (collected in capillary tubes from the external canthus) and corneal extracts against both rabbit and goat anti-UK antibodies were performed according to the method of Ouchterlony.20

a

PLASMINOGEN • FIBRIN " LYSIS

FIBRIN

Molecular Weight vs Rf(SDS-Page) 1 X 101 9 X 10' I X 10' _ X 10' 6 X 10' _ bX 10'

^ ^ ^ -

72 000 MW PA

_

• ^ r- 46.000 MW PA

4 X 10'

^ ^ V ^ t t . O O O MW PA

Adsorption of PA to Fibrin To examine the affinity of PA for fibrin, 90 /x\ of a 10-times-concentrated harvest from organ culture of ulcerating rabbit cornea was added to an Eppendorf tube containing 100 ^1 of plasminogen-free fibrinogen solution (6 mg/ml) in 6 mM veronal-HCl buffer, pH 7.3. The mixture was clotted by the addition of 10 ix\ of thrombin (40 NIH U/ml in 0.15 M NaCl) at 37°C for 10 min. The clot so formed was centrifuged at 50,000 X g at 4°C for 15 min. The supernatant was removed, and the packed clot was washed four times with veronal buffer. Then 100 /A of 2 M potassium thiocyanate in 0.1 M Tris-HCl, pH 7.5, was used to desorb the PA from the packed clot. The PA activities in the initial supernatant, washes, and thiocyanate extract were detected by spotting on fibrin-plasminogen-agarose films clotted on microscope slides or by a combination of SDS-PAGE and fibrin-plasminogen-agarose plate. A control film not containing FBS (ie, no plasminogen) also was used to determine if fibrin degradation was plasminogen dependent (Fig. 4).

Results By using the coupled, plasminogen-dependent fluorescent assay, a linear relationship was established between the rates of fluorescence generation (0-2.4 relative fluorescence units/min) and UK (0.1-0.5 CTA units) (Fig. 1). Dilutions of harvest from organ culture of ulcerating corneas gave a linear doseresponse relationship over the same range of fluorescence (Fig. 2). In two separate experiments, the organ culture harvests (N = 2) of pooled ulcerating rabbit corneas contained mostly active PA, with maximal activity at day 2 or 3, while proactivator (latent PA) was not detectable in harvests from days 1 and 2 but gradually increased from day 2 to maximal levels at days 4-5 (Fig. 6). In contrast, in two experiments PA

3 X 10'

-

1XID1

-

b IX

IB"

I

1

1

1

0.2

0.4

0.6

0.8

I

1.0

Rf Fig. 3. Molecular weight determination of PA by SDS-PAGE and plasminogen-dependent lysis on fibrin/agarose detector gels. After electrophoresis on SDS-PAGE, PA species were localized by relating lytic zones on the detector gel (a) to the mobilities of calibrating MW protein markers (b) (see text for details).

in organ culture harvests (N = 2) of pooled normal rabbit corneas, although variable in level between experiments, was mostly in proactivator form, with maximal levels early in culture. Some active PA was also present (Fig. 7). The total PA, ie, active plus latent, was higher per cornea in harvests of ulcerating corneas than in those of normal corneas; and a shift of the maximal total activity occurred from day 2 of the normal cultures to days 4-5 of the ulcerating cultures. Examination of the profiles of proactivator and active PA, in comparison with the profiles of latent collagenase and active collagenase in the same culture of ulcerating corneas, suggests that active PA and active collagenase have similar rates of appearance and disappearance in culture, as do plasminogen proactivator (proPA) and latent collagenase (Fig. 8). The harvests of epithelial cells from normal rabbit corneas (Fig. 9) also contained both proactivator and active forms of PA. Primary cultures of ulcer epithelium also contained both pro PA and PA. Because of the possible presence of PMNs and monocyte-macrophages in such cultures and their contribution to the PA detected, data from ulcer epithelial cultures are not reported here as due specifically to the ulcer epithelium. The ratio of proactivator to active PA produced in cell culture varied from culture to culture.

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CULTURE HARVEST

+

THROMBIN

+ FIBRINOCEN MIX, CLOT AT 37°C

CENTRIFUGE

SUPERNATANT

5 1/2HRS

I

-FILM +• F I L M

•

W A S H C L O T X 4

@

I C CENTRIFUGE

r

•

5 1/2 HRS

FOURTH WASH

+ FILM

t

(OR)

NON-EXTRACTED

* THIOCYANATE EXTRACTION

R.

4-THIOCYANATE

5 1/2 HRS

Y

[(^CENTRIFUGE

12 HRS

f

}

THIOCYANATE EXTRACT

EXTRACTED CLOT t X I K A C IhD CLOT WASH CLOTx4@ £ , CENTRIFUGE

21 HRS + FILM

-FILM

v..y FOURTH WASH 50 HRS + FILM

FILM

Fig. 4. Evidence for adsorption of corneal PA to fibrin. Ulcer culture harvest was mixed with fibrinogen, which then was clotted by the addition of thrombin. After centrifugation of the clot, assay of the supernatant demonstrated plasminogen-dependent lysis on fibrin films. Extensive washing of the clot resulted in no further demonstration of activator in the supernatant, as indicated by failure to detect lysis on plasminogen-containing films. Streaking of the clot (nonextracted) on plasminogen-containing (+) films demonstrated progressive lysis of the films. Thiocyanate extraction of the clot (thiocyanate extraction) resulted in the reappearance of activator in the extract, which lysed + films (plasminogen containing) but not - films (plasminogen free). Subsequent extensive washing of the extracted clot resulted in no further appearance of activator in the-wash. The extracted and washed clot showed only low levels of residual activator (see text for details).

517

PLASMINOGEN ACTIVATOR, IN CORNEAL ULCEIWION/Wcmg er al.

No. 4

Activation of normal epithelial pro PA could be carried out by plasmin as well as by trypsin (Fig. 10). The plasmin generated from plasminogen by PA from normal or ulcerating corneas in organ culture and from epithelial cells yielded heavy (H) and light (L) chains, which were similar or identical in mass to those produced by UK, based on electrophoresis under reducing conditions (Fig. 11). The heavy chains had an apparent MW of approximately 67,000 daltons, and the light chains had an apparent MW of approximately 27,000 daltons. Multiple H chains are thought to reflect derivation from both Glu-plasminogen and Lys-plasminogen in the plasminogen substrate preparation.12 In the current studies, fibroblast cultures were found to contain very low levels of both latent and active plasminogen activator activity. Because trypsin, itself a plasminogen activator, had been used to pass the fibroblasts, however, and because the plasmin cleavage pattern observed upon activation with fibroblast harvests (H chains of approximately 60,000 MW as well as traces of 67,000 MW;" and L chains of approximately 40,000 M W as well as traces

MWX10 3 72

1

•

O

3

'i

3

4

5

6

7

DAYS IN CULTLtHE

Fig. 6. Plasminogen activator (PA) and plasminogen proactivator (proPA) in harvests of ulcerating rabbit corneas. In two separate experiments, plasminogen activator was present as active PA I

I with maximal activity at day 2 or 3, while proactivator

I

, latent PA I was very low to nondetectable in harvests

from days I and 2 but gradually increased from day 2 to maximal levels at days 4-5. I days postburn;

, ulcer tissue taken for culture 11 I ulcer tissue, taken for culture 10 days

postburn.

Fig. 5. Molecular weight determination of PA by SDS-PAGE after desorption from fibrin by thiocyanate (see Fig. 4). SDS-PAGE and localization of PA on plasminogen-containing detector plates shows that of the three MW species present in the original adsorption mixture {pathway 3) (72,000; 46,000 and 35,000 MW); only the 46,000 MW species (arrow) was detected in the thiocyanate extract of the fibrin clot.

of 27,000 MW) is not compatible with the site of plasminogen cleavage by other known activators, but is similar to the cleavage caused by trypsin alone, it cannot be decided yet with certainty on the basis of activity measurements or plasmin cleavage fragments that corneal fibroblasts themselves secrete PA. Fibroblast harvests do form a precipitin band with antiUK antibodies, however. The molecular weights of PA species from the harvests of corneal organ and cell cultures were determined by the locations of lysis zones on fibrinplasm inogen-agarose plates, in comparison with the mobilities of MW markers, after separation by SDSPAGE (Fig. 3). Multiple MW species, that is two

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INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / April 1985

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100-n

30-n

90-

80proPA 70-

st

100,000). No lysis zones developed on the plasminogen free-fibrin aga-

PA

20^ ^ > - ^

DAYS IN CULTURE

Fig. 8. Latent and active forms of plasminogen activator and collagenase in cultures of ulcerating rabbit corneas. Active PA (— A —) and active collagenase (— • —) levels rose early in culture; and latent PA (- - • - -) and latent collagenase ( - - • - - ) levels rose later in culture, suggesting coordinate control between active and latent forms of PA and collagenase.

20

30

40

_ — ProPA

50

ProPA

60

TIME (min)

Fig. 10. Activation of latent PA from normal rabbit corneal epithelial cells by trypsin and plasmin. Activation by trypsin (1 ng/ ml) or plasmin (2 Mg/mO w a s done as described in the text. Activation by trypsin (- - • - -) or plasmin ( - - • - - ) revealed comparable levels of proactivator (proPA).

lit

PLASMINOGEN ACTIVATOR IN CORNEAl ULCERATION/Wong er QI.

No. 4

MWX 103 205

1

116 97.4 ? H

66

45

Fig. 11. Plasminogen cleavage fragments generated by plasminogen activators. Harvests from normal (3) or ulcer corneas (4) and from normal epithelial cells (5) produced heavy {H) chains of approximately 67,000 daltons and light (L) chains of approximately 27,000 daltons, comparable to those produced by UK (2). Plasminogen control (1). Molecular weight markers (6) (see text for details).

rose control plates with any of the harvests, indicating that the observed lysis zones are due, indeed, to PA. Some PA from ulcerating rabbit corneas could be recovered from fibrin by desorption with thiocyanate (Fig. 4), although most of the PA had not been adsorbed to the fibrin clot, under the conditions used. The desorbed PA had a MW of 46,000 (Fig. 5), as determined by SDS-PAGE and fibrin plate lysis. It would seem that the 35,000 MW species, which was not detected in the extract from the clot (Fig. 5), had not been bound to fibrin. The total level of 72,000 MW PA in the incubation was too low to determine if it had been bound to fibrin. Concentrated harvests from rabbit ulcer corneas and from normal epithelial cells and fibroblasts formed distinct precipitin bands in double-diffusion assay (Ouchterlony), with goat IgG antibodies directed against the 33,000 MW UK heavy chain (Fig. 14c). Antiserum raised in rabbits gave a line of at least apparent partial identity between 33,000 MW UK and extracts of human corneas (Fig. 14d) and ulcer tears (Fig. 14b); and antisera raised in goat and rabbit gave a line of identity between the 33,000 MW UK heavy chain and a component in normal human tears (Fig. 14a).

Discussion Although other biochemical systems are, no doubt, involved in ulceration, previous studies have provided

evidence for an important role of the PA/plasmin system in resorption of fibrin and fibronectin at the corneal surface,1 the generation of a persistent epithelial defect,12 and the stimulation of secretion and activation of collagenase in the stroma,3 contributing to corneal ulceration. The present studies were unCULTURE DAYS

MWX103 72 46 35

Fig. 12. PA species produced by ulcerating corneas in organ culture. MW species of 72,000, 46,000, and 35,000 daltons were detected. In agreement with results of the coupled, fluorescent assay (Fig. 6, - • -) highest activities occurred in harvests from days 2 to 4. UK, Calbiochem-Behring preparation contained 46,000 and 35,000 dalton species.

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INVESTIGATIVE OPHTHALMOLOGY b VISUAL 5CIENCE / April 1985

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CULTURE DAYS UK

ED-

UK Fig. 13. PA species produced by normal corneas in organ culture and by epithelial cells in cell culture. MW species of 72,000 and 46,000 daltons were produced by normal corneas and epithelial cells. In this experiment, peak activity on the gel occurred at day 5 of culture (day 2 sample was not assayed). UK, CalbiochemBehring preparation used as internal standard.

dertaken to extend our understanding of regulatory aspects of the PA/plasmin system of the cornea. PA from corneal cells, as from some other cell types,21"23 has been found to exist in both latent, protease-activatable form (plasminogen proactivator)

and in active form. Although it is not yet known if the latent form represents a complex of inhibitor and PA or a true zymogen in the corneal system, the existence of an activatable form demonstrates that, as with latent corneal collagenase, there is another

Fig. 14. Immunochemical relationships among plasnormol teots minogen activators, a, AnUK/55 tiserum made in goat (Tan^ o n t t - U K (Tonokol ULCER aka) and in rabbit (CollaboTEARS rative Research) to the 33,000 MW UK both give a UK(Tonako)*v J single precipitin band with a ( 33K) , Itair .normal tears component in normal huUK-33 man tears, which forms a line of identity with 33,000 ^ N . o o t i UK (Collab. Rev) MW UK. b, The precipitin UK-55 pattern with human ulcer »ean tears is complex, using antiserum (Ab) (Collaborative Research) made in rabbit to 33,000 MW UK. The cornUK—55 plexily might represent distinct, intact multiple species NORMAL or breakdown products. AnCORN€A tiserum does show (arrows) EXT. a line of identity between a 33,000 MW trace component in the 55,000 MW standard and a component UK-33 in tears, c, Antiserum (TanUK-55 . aka) made in goat against the 33,000 MW heavy chain of UK produced single precipitin bands against purified 33,000 MW UK (not shown) and against harvests from normal rabbit corneal epithelial cells, fibroblasts, and ulcer corneas, d, Antiserum, (Ab) (Collaborative Research) made in rabbit against the 33,000 MW heavy chain of UK did not form a precipitin band with rabbit ulcer harvest (not shown) but did form precipitin bands against both 33,000 MW and 55,000 MW UK (Collaborative Research) and a line of at least partial identity (arrow) between normal human cornea extract and the 33,000 MW chain of UK.

1

b

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PLA5MINOGEN ACTIVATOR IN CORNEAL ULCERATION/Wang er ol.

level of important regulation in the cascades that lead to stromal ulceration. Indeed, the existence of PA in a latent form is all the more significant because PA is thought to initiate the sequence that results in collagen destruction. Hence, successful inhibition of the conversion of plasminogen proactivator to activator would be expected to be important clinically. The observation in the current work that PA is detectable (within the limits of sensitivity of the assay) only as an active species early in the culture harvests of ulcerating corneas, but is present proportionately more in latent form in harvests of normal corneas, would suggest that ulceration is related to the conversion of latent to active PA. Alternatively, proteins (including inhibitors) that compete with proPA for added trypsin might be present in higher concentrations in early harvests of ulcer corneas than of normal corneas such that the early ulcer harvests only appear to contain no activatable PA (ie, proPA). The presence of active PA, however, is correlated with both high levels of active collagenase and the degradation of collagen in organ cultures of ulcerating rabbit corneas (Fig. 8). In normal corneas, in contrast, the presence of PA in mostly latent form is correlated with nearly undetectable levels of collagenase,15 all of which was in a latent form. The data suggest (Fig. 8) that the concentrations of latent PA (ie, proactivator) and of latent collagenase, and the concentrations of active PA and active collagenase, are under coordinate control. In previous work, a band with the mobility of plasmin, under various electrophoretic conditions, was found to elute out into the culture medium of ulcerating corneas with a typical "washout" profile.3 Also, the addition of plasminogen to organ culture resulted in an earlier peak of active PA in the medium as well as in raised levels of both latent and active collagenase and collagen degradation products in the medium. 3 These observations have been interpreted to mean that plasmin, generated from added plasminogen by active PA, stimulates fibroblasts to secrete PA (proPA) as well as latent collagenase and that the plasmin generated activates the latent collagenase to active collagenase, which attacks stromal collagen. The current results are compatible with the interpretation that, as endogenous plasmin elutes out from ulcerating corneas, both PA and collagenase become increasingly detectable in plasmin-activatable forms. Studies of the MW species of PA from ulcerating and normal corneas and from normal epithelial cells are consistent with the interpretation that corneal epithelial cells produce latent PA of 46,000 MW, which is converted proteolytically to an active species of PA, analogous to the known conversion of latent 55,000 MW UK (single chain) to active 55,000 UK (two chains) by plasmin.24 The basis for the variability

521

of the ratio of proPA to PA observed in normal epithelial cultures is not yet understood. It might reflect the production of different amounts of proPAactivating protease in different cultures or different levels of an inhibitor of activating protease in the different cultures. An earlier study25 had reported that rabbit corneal PA has a MW of 50,000 daltons; but no evidence for a latent form was presented. Experiments with 3 H-DFP 8 (diisopropylfluorophosphate) to label the active site of the enzyme as yet have not been done, so it is not yet possible to decide whether, like activated 55,000 MW UK, proteolytic activation of 46,000 MW PA is by scission of a single chain, analogous to the activation of plasminogen itself, by PA. It may be anticipated, however, that as in the processing of UK, the 46,000 MW band region on gels contains both latent and active PA. Demonstration of increased presence of a 3 H-DFP labeled33,000 MW heavy chain, active site fragment after activation, and electrophoresis under reducing conditions would constitute evidence for this hypothesis. The observation, moreover, that a PA species of 35,000 is present in harvests of ulcerating corneas is consistent with the subsequent proteolytic cleavage of active 46,000 MW corneal PA, as of UK, which latter cleavage has been reported by others.24 The observation in our previous work that immunoreactive, UK-like PA was co-linear with fibrin and fibronectin on the surface of the rabbit cornea after an alkali burn 1 suggested that PA, presumably from adjacent corneal epithelium, was regulated by adsorption to fibrin/fibronectin. Although UK species (ie, of 35,000 MW or 33,000 MW) frequently have been described as having very low affinity for fibrin2627 (unlike so-called "extrinsic, tissue activators"), recently high MW UK-like species with high affinity for fibrin have been described.28 Results of the current work suggest that 46,000 MW corneal PA does bind to fibrin. As reported for 35,000 MW UK, a form that is composed of the 33,000 MW active site-containing heavy chain disulfide bonded to a short fragment of the light chain of UK, the 35,000 MW corneal PA species does not appear to bind to fibrin, however (Fig. 5). Failure of a PA species to bind to fibrin and of dependence on fibrin for activation might have significant consequences for the cornea29 in that PA activity then would not be limited to the surface of fibrin. In the current work no fibrin was added to the reaction mixture for the coupled assay, and, unless soluble fibrin fragments were present in the ulcer harvests (which cannot be ruled out), the PA activity measured in harvests of both normal and ulcer corneas, as well as from cell cultures, was not fibrin dependent, as vascular PA activity is reported to be. 2730 As in the clinical use of 35,000 MW UK to

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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / April 1985

treat thromboembolic disease29 (but not with the use of fibrin-dependent plasminogen activators29), the ability of the 35,000 MW UK species to activate plasminogen in solution (ie, plasminogen not adsorbed to fibrin) can result in serious events—systemic bleeding due to the depletion of fibrinogen and clotting factors,29 in the case of thromboembolic therapy, and, hypothetically, in the alkali-burned cornea, in the activity of plasmin free in the stroma. In the cornea, therefore, a 35,000 MW species that does not adsorb to fibrin could be expected to generate plasmin in solution, which, as in organ3 or fibroblast culture,4 stimulates corneal fibroblasts in the cornea to secrete latent collagenase and then activates the collagenase1 to contribute to stromal destruction. Indeed, free plasmin has been found to stimulate collagenase secretion in a fibrin-free system apparently by degrading fibroblast cell surface fibronectin and causing depolymerization of the actin microfilament system.4 In addition to species of 46,000 MW and 35,000 MW, organ cultures and epithelial cultures contained relatively low levels of 72,000 MW PA. The MW of the latter species is consistent with that of extrinsic tissue activators (like "vascular PA"), which are reported to bind tightly to fibrin, to. require fibrin for activity, and to not cross-react with antibodies raised against UK.29 Although the ulcerating corneas contained blood vessels, vascular PA is reported to require dissociating agents (eg, thiocyanate), not used to treat cultures in the present work, in order for vascular PA to be released into solution.30 Thus, even though a vascular source of the 72,000 MW PA in ulcerating corneas cannot be ruled out, neither cultures of normal corneas nor of epithelial cells contained blood vessels, yet both type cultures demonstrated 72,000 MW PA. It is concluded, therefore, that corneal cells, themselves, can synthesize 72,000 MW PA. The presence of UK-like MW species of PA (46,000 and 35,000) and of the 72,000 MW species of PA would suggest that corneal cells can synthesize both UKlike and tissue activator-like PA. The production of both UK-like PA and tissue activator-like PA by a single tissue or cell type (even by vascular endothelial cells,31 formerly thought to make only tissue-type PA) agrees with what has been observed for other human cells32 and rat keratinocytes.33 Whether the same cell can synthesize both UK-like and tissue-like PA requires further study. The observation that corneal cells do synthesize both UK-like and tissue-like PA species (on the basis of MW and immunoreactivity) is an important observation in that it suggests the possibility of differential regulation based on what is known34 of the regulation of those species of PA. According to the model that has been proposed,1 epithelial cells, PMN's and fibroblasts all are thought

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to secrete PA as they participate in the events that follow an alkali burn (Fig. 10 in reference 1). Epithelium is thought to initiate the secretion of PA in response to fibrin/fibronectin on the surface of the stroma as required for plasmin-mediated debridement and/or epithelial migration. Abnormally high secretion and activation of PA are thought to result in the decreased adhesion of epithelium to fibrin/fibronectin, the generation of an epithelial defect, the continued secretion and activation of PA (proPA), and, eventually, the degradation of subepithelial basement membrane. Other workers have suggested that PA produced by PMNs, as by other migratory cells, is involved in the mechanism of migration through extracellular.matrix.35 PMNs that infiltrate the stroma after the burn and that appear to play an imortant role in tissue destruction3 do appear able to release PA to amplify PA-dependent destructive sequences. Although explants of normal corneal stroma have been found previously to lyse fibrin in a plasminogendependent manner 3 and corneal fibroblasts secrete collagenase,415 the role of fibroblasts in both organ and cell culture production of PA itself is not yet clear. In agreement with the results of others,36 the current results suggest that corneal epithelial cells alone in culture produce much more PA than do keratocytes (fibroblasts) alone, which, in our experience, produce little to no detectable PA activity. As in the case of stimulation by corneal epithelial cells of collagenase production by corneal fibroblasts, however,16 it is possible that, in vivo, fibroblasts become an important source of PA, in response to signals from epithelium or PMNs. In the current study, tears from ulcerating human corneas were examined for plasminogen activator, since UK-like PA has been detected by immunofluorescent methods on the ulcerating stromal surface1 and since tears from ulcer patients have been found to contain elevated levels of a collagenolytic protease37 and of antiproteases37'39 thought to be regulatory in corneal ulceration. As observed by others,40'41 normal human tears contain both PA activity and PA-immunoreactive material.40 As shown in this study (Fig. 14), PA in normal and in ulcer tears was immunoprecipitated by antibodies to UK, whereas, in other studies40 antiuterine PA (ie, antivascular, extrinsictype PA), but not anti-UK antibodies, has been reported to inhibit completely tear PA activity. The basis for the apparent inconsistency of results is not, at present, understood. Normal tears produced only a single precipitin band against anti-UK antibodies (Fig. 14a). The function(s) of PA in normal tears is not known. PA might act to prevent fibrin occlusion of the nasal puncta, a role in maintenance of duct patency suggested for PA elsewhere in the organism.42

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Also, PA might function normally in the desquamation (ie, degradation of intercellular attachments) of the corneal epithelium, as has been suggested to be its role also in turnover in vaginal and uterine epithelia.42 Ulcer tears produced a complex immunoprecipitation pattern (Fig. 14b) with anti-UK antibodies. The complex pattern might represent distinct species of UK-like PA or proteolytic breakdown of a single PA species, reflecting proteolysis related to ulceration. The interpretations made in the current work are based mostly on in vitro results and assume relevance of the observations made to processes that actually occur in vivo during ulceration. The interpretations, before being accepted as being true of the in vivo situation, require validation by identification, localization, and quantitation in situ during corneal ulceration. Where possible, studies have been done to localize to the cornea in situ components of the PA/ plasmin. system, in both normal and ulcerating corneas. 13 The further demonstration in the current work that UK-like PA is present in tears of human ulcer patients (as well as in normal human tears) would suggest that PA is present at the corneal surface in vivo, where it might have a role in tissue-destructive processes. Since conversion of latent PA (proactivator) to active PA occurs at the top of potentially important cascades leading to both tissue destruction and repair,1 understanding and regulation of that conversion would seem to be of great importance to the cornea. The mechanism(s) by which corneal PA species is (are) activated in vivo is not yet known. It is clear, however, that studies of the activation mechanism(s) involving proteolytic activity and fibrin and fibronectin should enhance the ability to modulate tissue destruction and repair in the cornea. Key words: corneal ulceration, plasminogen activator, plasminogen proactivator, collagenase, plasmin, fibrinolysis.

Acknowledgments The authors wish to thank Dr. Kenzo Tanaka, Kyushu University, Japan, for samples of 33,000 MW human urokinase and antibodies to urokinase, and Dr. Paul Kelley, Collaborative Research, Inc., Waltham, Massachusetts, for samples of 33,000 MW and 55,000 MW human urokinase.

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3. Berman M, Leary R, and Gage J: Evidence for a role of the plasminogen activator-plasmin system in corneal ulceration. Invest Ophthalmol Vis Sci 19:1204, 1980. 4. Berman M, Wang H-M, Wood J, and Law M: Plasmin regulates corneal collagenase secretion by degrading fibroblast cell surface/matrix fibronectin. ARVO Abstracts. Invest Ophthalmol Vis Sci 25(Suppl):6, 1984. 5. Wang H-M, Berman M, and Law M: Latent and active forms of plasminogen activator in corneal ulceration. ARVO Abstracts. Invest Ophthalmol Vis Sci 24(Suppl):44, 1983. 6. Chibber B, Deutsch D, and Mertz E: Plasminogen. Methods in Enzymology-Enzyme Purification, Part B: Affinity Methods. Vol. XXXIV, Jakoby W and Wilchek M, editors, New York, Academic Press, 1974, pp. 424-432. 7. Shaw E: Site-specific reagents for chymotrypsin, trypsin, and other serine proteases. In Methods in Enzymology, Enzyme ' Structure Part B, Vol. XXV, Hirs C and Timasheff S, editors. New York, Academic Press, 1972, pp. 655-671. 8. Kenyon K, Berman M, Rose J, and Gage J: Prevention of stromal ulceration in the alkali-burned cornea by glued-on contact lens. Ultrastructural evidence for a role of polymorphonuclear leucocytes in collagen degradation. Invest Ophthalmol Vis Sci 18:570, 1979. 9. Berman M, Cavanagh HD, and Gage J: 5' Adenosine monophosphate prevents collagen degradation in culture but does not prevent corneal ulceration. Exp Eye Res 24:391, 1977. 10. Sunderraj C, Freeman I, and Brown S: Selective growth of rabbit corneal epithelial cells in culture and basement membrane collagen synthesis. Invest Ophthalmol Vis Sci 19:1222, 1980. 11. Bigbee W, Weintraub H, and Jensen R: Sensitive fluorescent assays for urokinase using synthetic peptide 4-methoxy-/3naphthylamide substrates. Anal Biochem 88:114, 1978. 12. Christman J, Silverstein S, and Acs G: Plasminogen activators. In Proteinases in Mammalian Cells and Tissues, Barrett AJ, editor. New York, North-Holland Publishing, 1977, pp. 9 1 149. 13. Kocholaty W, Ellis W, and Jensen H: Activation of plasminogen by trypsin and plasmin. Blood 7:882, 1952. 14. Berman M, Manabe R, and Davison P: Tissue collagenase, a simplified, semiquantitative enzyme assay. Anal Biochem 54: 522, 1973. 15. Berman M, Leary R, and Gage J: Latent collagenase in the ulcerating rabbit cornea. Exp Eye Res 25:435, 1977. 16. Johnson-Muller B and Gross J: Regulation of corneal collagenase production. Epithelial-stromal cell interactions. Proc Natl Academy Sci 75:4417, 1978. 17. Laemmli V: Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680, 1970. 18. Grannelli-Piperno A and Reich E: A study of proteases and protease-inhibitor complexes in biological fluids. J Exp Med 148:223, 1978. 19. Dano K and Reich E: Inhibitors of plasminogen activation. In Proteases and Biological Control, Reich E, Rifkin D, and Shaw E, editors. Cold Spring Harbor Conference on Cell Proliferation, Cold Spring Harbor, New York, 1975, Cold Spring Harbor Laboratory, Vol 2,: pp.-357-366. 20. Ouchterlony O and Nilsson L: Immunodiffusion and immunoelectrophoresis. In Immunochemistry, Handbook of Experimental Immunology, Vol 1, Ch 19, Weir D, editor. Oxford, Blackwell, 1973, pp. 19-39. 21. Bernik M and Oiler E: Plasminogen activator and proactivator (urokinase precursor) in lung cultures. JAMWA 31:465, 1976. 22. Nolan C, Hall L, Barlow G, and Tribby I: Plasminogen activator from human embryonic kidney cell cultures—evidence for a proactivator. Biochem Biophys Acta 496:384, 1977. 23. Skriver L, Nielson I, Stephens R, and Dane K: Plasminogen

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33. Birkedal-Hansen H and Taylor R: Production of three plasminogen activators and an inhibitor in keratinocyte culture. Biochim Biophys Acta 756:303, 1983. 34. Levin E and Loskutoff D: Regulation of plasminogen activator production by cultured endothelial cells. Ann NY Acad Sci 401:184, 1982. 35. Reich E: Activation of plasminogen: A general mechanism for producing localized extracellular proteolysis. In Molecular Basis of Biological Degradation Processes, Berlin R, Herrmann H, Lepow I, and Tanzer J, editors. New York, Academic Press, 1978, pp. 155-170. 36. Lantz E and Anderson A: Release of fibrinolytic activators from the cornea and conjunctiva. Graefe's Arch Ophthalmol 19:263, 1982. 37. Prause J: Serum albumin, serum antiproteases and polymorphonuclear leucocyte neutral collagenolytic protease in the tear fluid of patients with corneal ulcers. Acta Ophthalmol 61:272, 1983. 38. Berman M, Barber J, Talamo R, and Langley C: Corneal ulceration and the serum antiproteases. I. a,-antitrypsin. Invest Ophthalmol 12:759, 1973. 39. Berman M, Gordon J, Garcia L, and Gage J: Corneal ulceration and the serum antiproteses. II. Complexes of corneal collagenases and a-macroglobulins. Exp Eye Res 20:231, 1975. 40. Rijken D, Wijngaards G, and Welbergen J: Immunological characterization of plasminogen activator activities in human tissues and body fluids. J Lab Clin Med 97:477, 1981. 41. Storm O: Fibrinolytic activity in human tears. Scand J Clin Lab Invest 7:55, 1955. 42. Astrup T: Fibrinolysis: An overview. In Prog Chem Fibrin Thrombolys 3. Davidson J, Rowan R, Samama M, and Desnoyers P, editors. New York, Raven Press, 1978, pp. 1-57.