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Gut, 1970, 11, 851-854
Histological localization of plasminogen activatorand
proteolytic activity in the human stomachand duodenum
P. ERAS, P. HARPEL, AND S. J. WINAWERFrom the Department
ofMedicine, The New York Hospital, Cornell Medical Center, and the
MemorialHospitalfor Cancer and Allied Diseases, New York
SUMMARY Plasminogen activator activity has been localized in
gastric and duodenal tissueusing a histological technique. This
activity could be separated from proteolytic activity
byincorporating E-aminocaproic acid into the fibrin substrate or by
heat treatment. Plasmin-ogen activator activity was found in
relation to mucosal and submucosal blood vessels in thetissues
studied. Proteolytic activity was identified in the surface
epithelium, the gastric antraland fundal glands, and in the antral
submucosal fat. Proximal duodenal tissue containedproteolytic
activity in relation to Brunner's glands.
Fibrinolytic activator activity (plasminogenactivator) has been
found in extracts of gastric andduodenal mucosa from patients with
duodenalulcer disease (Cox, Poller, and Thompson, 1967).The present
study reports on the histologicallocalization of this activity in
human gastric andduodenal tissue. Proteolytic activity could also
belocalized and distinguished from plasminogenactivator activity.
This was accomplished by theuse of E-aminocaproic acid and heat
treatmentof the fibrin substrate (Fig. 1).
EACAPlasmi nogen 1 lsioeactivators > Plasminogen
Heat +
Nonspecific . vNonesecific ---...~..---..> Fibrin film
lysisproteases 0Fig. 1. Outline of the pathway by which
c-amino-caproic acid (,-ACA) and heat treatment of the fibrinfilm
substrate distinguish plasminogen activatorfrom non-specific
proteolytic activity.
Patients and Methods
Tissue from the body of the stomach was obtainedat surgery from
three patients operated on forduodenal ulcer disease. In one
patient, additionalspecimens were obtained from the gastric
fundus,antrum, and duodenum. These specimens weretaken from areas
which appeared grossly normal.Microscopic examination of
haematoxylin andeosin-stained sections of these tissues showed
anormal appearance without evidence of in-flanmmation. Surgical
specimens half an inchsquare were washed in 0.15M sodium
chlorideand were quick-frozen in an International-Harriscryostat
microtome, and then stored at -25°Cand assayed within three
weeks.
Plasminogen-rich fibrinogen was prepared fromcitrated bovine
plasma according to the methodof Kekwick, MacKay, Nance, and Record
(1955).The Kekwick F. 1 W low-ionic strength, cold-insoluble
fibrinogen fraction was reconstitutedin citrate-saline buffer at a
concentration of3.0 g% and stored at -20°C for use in
preparingfibrin films.
FIBRIN FILMSTwenty NIH units of bovine thrombin (Parke-Received
for publication 9 March 1970.
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P. Eras, P. Harpel, and S. J. Winawer
Davis Co.) were added to 10 ml bovine fibrinogenwhich was then
diluted in Michaelis veronal buf-fer, pH 7.35, to a final
concentration of 1.5 g %.The fibrinogen was allowed to clot on a
levelcellophane strip 8 x 17 cm in a humid chamberat 4°C. Squares
of fibrin clot, each 2 cm, oncellophane were cut and inverted
(fibrin facedown) on a glass slide and the cellophane wasremoved.
Tissue sections from each specimenwerethen cut 8 microns thick on
the cryostat, placedon fibrin films and the fibrin slides were
in-cubated at 37°C in a moist chamber for 0, 1, 3, 5,10, 30, 45,
and 60 minutes. The reaction wasstopped by immersion in 10%
formaline-salinefor five minutes. The fibrin film was stained with
amodification of the haematoxylin and eosin stain.Excess fibrin was
trimmed with a sharp knife,and the preparation covered with
permount anda coverslip. The prepared slides were viewedwith a
stereoscopic microscope to determine thepresence of depressed
colourless areas in thefibrin film which represented areas of
fibrindigestion. The shortest incubation time requiredto produce a
clearly visible zone of lysis wasnoted and served as a
semi-quantitative index offibrinolytic activity.
PLASMINOGEN-FREE FIBRIN FILMSFibrin films were prepared in the
manner de-scribed, following which they were heated at85°C for 30
minutes in a moist chamber to destroyfibrin-associated plasminogen
(Lassen, 1952).
E-AMINOCAPROIC ACID-CONTAINING FIBRINFILMSFibrin films were
prepared as described with1 x 10-3M E-aminocaproic acid added to
thefibrinogen solution to inhibit selectively fibrinol-ysis
produced by plasminogen activator activity(Alkjaersig, Fletcher,
and Sherry, 1959).
Results
The gastric and duodenal surface epitheliumshowed proteolytic
activity which was notinhibited by E-aminocaproic acid or by
heat
treatment of the fibrin substrate. The data onlocation of
fibrinolytic activity in the tissuesstudied (other than surface
epithelial activity),the earliest time of onset of lysis, and the
effect ofadding E-aminocaproic acid and heating thefibrin
substrates are summarized in the Table.The gastric fundus showed
proteolytic activity inthe surface epithelium and glands.
Plasminogenactivator activity was found in mucosal and sub-mucosal
blood vessels (Fig. 2). Similar findingswere present in the gastric
antrum. In sectionstaken from the transitional zone, the
antralglands showed greater non-inhibitable proteolyticactivity
than did the fundal glands (Fig. 3). Inaddition, the antral
submucosal fat caused lysisof the fibrin films which was not
inhibited byE-aminocaproic acid or by heat treatment. Theproximal
duodenum showed proteolytic activityin the surface epithelium,
crypts, and Brunner'sglands. Activity of plasminogen activator
waslocalized to the mucosal and submucosal bloodvessels (Fig.
4).
Discussion
Fibrinolytic activity of tissue may be causedeither by proteases
which can directly hydrolysefibrin or by tissue activators which
convertplasminogen to plasmin, the fibrinolytic enzyme.Albrechtsen
(1959) found that most tissue con-tained plasminogen activator, but
he did notexamine gastrointestinal tissue. Histological
tech-niques, developed by Todd (1959 and 1964), madeit possible to
examine plasminogen-activatoractivity of tissue in relation to
histologicalstructure. His studies and those of others showedthat
the activity of plasminogen activator ismainly associated with the
blood vessels of thevarious tissues examined (Kwaan, 1966).
Studies of fibrinolytic activity in the gastro-intestinal tract
are few. Kwaan, Cocco, andMendeloff (1964) have shown activity of
plasmin-ogen activator at sites of crypt abscesses inpatients with
active ulcerative colitis. Cox et al(1967) found fibrinolytic
activity in gastric veinblood and no activity in samples obtained
fromthe gastric artery. Saline extracts of the gastricand duodenal
mucosa contained plasminogen
Patient Tissue Histological Location and Time of Earliest
LysisFibrin E-Aminocaproic Acid Fibrin Heated Fibrin
1 Gastric fundus Mucosal and submucosal blood No lysis (60 min)
No lysis (60 min)vessels (20 min)2 Gastric fundus Mucosal and
submucosal blood No lysis (60 min) No lysis (60 min)vessels (15
min)3 Gastric fundus Mucosal and submucosal blood No lysis (60 min)
No lysis (60 min)vessels (10 min)
Gastric antrum Mucosal and submucosal blood No lysis in blood
vessels at 60 No lysis in blood vessels at 60vessels, submucosal
fat (10 min) min; fat cells active min; fat cells activeDuodenum
Mucosal and submucosal blood No lysis in blood vessels at 30 No
lysis in blood vessels at 30vessels, Brunner's glands (5 min) min;
Brunner's glands active min; Brunner's glands active
Table Localization offibrinolytic activity in stomach and
duodenal tissue
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Histological localization ofplasminogen activator and
proteolytic activity in stomach and duodenum
Fig. 2. Specimen from gastric fundus following Fig. 3. Section
ofbody of the stomach (patient 3) at15-minute incubation with
fibrin film (patient 2). transition zone offundal and antral gland
incubatedRepresentative lytic zones, indicated by arrows, 60
minutes with E-ACA fibrin film. The clear lyticappear as light,
unstained areas in relation to mucosal zones in relation to mucosal
and submucosal bloodand submucosal blood vessels. vessels shown in
Fig. 2 are now not present (arrows
A and B) indicating that the lysis was produced byplasminogen
activator. Lytic zones are seen inrelationship to submucosal fat
cells (arrow C)indicating proteolytic activity. Antral glands on
leftshow greater proteolytic activity than do fundalglands on
right.
activator activity as shown by the ability to
lyseplasminogen-rich fibrin plates but not to lyseheated or
E-aminocaproic acid-containing plates.Cox, Poller, and Thomson
(1969) have also foundthat digital gastric compression at
laparotomyresulted in the development of plasmin-likeactivity in
peripheral venous and in gastricvenous blood. The highest
fibrinolytic activitywas found in patients with peptic ulcers.The
present study demonstrates that both
plasminogen activator activity and proteolyticactivity can be
identified in human gastric andduodenal tissue by a histochemical
technique.Although the tissues obtained were from
patientsundergoing surgery for duodenal ulcer diseaseno attempt can
be made in this preliminary studyto correlate the enzymatic
findings with thepresence of duodenal ulcer disease. In the
speci-mens examined, plasminogen activator activitywas found in
superficial mucosal and submucosalblood vessels in the stomach and
duodenum.That this is plasminogen activator was shown bythe
inhibition of this fibrinolytic activity by usinga heated fibrin
substrate or one containingE-aminocaproic acid in a concentration
whichwould inhibit plasminogen activator but notplasmin itself or
other proteases (Alkjaersiget al. 1959). In the specimen in which
comparison
Fig. 4. Lytic zones in relationship to mucosal bloodvessels (A)
in the proximal duodenum (patient 3)after five minutes' incubation.
Proteolytic activity, notinhibited on heated or E-ACA fibrin films,
is associatedwith surface (B) and Brunner's glands (C) ( x
100).
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854 P. Eras, P. Harpel, and S. J. Winawer
was possible, there appeared to be more plasmin-ogen activator
activity associated with the vesselsof the fundus than with the
antrum.
Epithelial proteolytic activity, which was notinhibited by
E-aminocaproic acid or heat treat-ment of the fibrin film, may be
due to residualacid-pepsin. Pepsin in the presence of hydro-chloric
acid has been shown to cause fibrin platelysis (Cox et al, 1967).
In the present study,gastric tissue was washed extensively before
assayin physiological saline, a procedure which mightbe expected to
eliminate most HCI and surfacepepsin. The assay was performed at
neutral pH,conditions which would favour the inactivationof both
pepsin and peptic enzymes. The epithelialsurface proteolytic
activity observed might there-fore be due to proteolytic enzymes
active in theneutral pH range. Taylor (1959) has identified
aprotease in gastric mucosal tissue which is activeat neutral pH.
The mucosal cells responsible forthe production of this enzymatic
activity have notbeen identified. It was of interest to find
pro-teolytic activity associated with both the fundaland antral
glands. Proteolytic activity wasassociated with the fundal, antral,
and theBrunner's glands of the duodenum. The activityof the
Brunner's and antral glands was greaterthan was the activity of the
fundal glands. Theproduction of pepsin occurs principally in
thechief cells of the fundal glands. Small amountsof peptic-like
activity have been reported induodenal juice and duodenal mucosal
extracts(Seijffers, Miller, and Segal, 1963; Cooke andGrossman,
1966) and in extracts from the pyloricgland area of the stomach
(Grossman andMarks, 1960). Linderstr0m-Lang, Holtzer, andOhlsen
(1935) found acid protease activity of thegastric mucosa in the
pig. Whereas highestactivity correlated with peptic cells of the
fundicglands, activity was found in mucosal cells of thepyloric and
antral glands as well. In the presentassay system the glands with
the greatest pro-teolytic activity have been reported to have
lowlevels of peptic activity. The activity observed maytherefore be
due to other as yet unidentifiedproteolytic enzymes.
Fat cells in the submucosa of the antrum alsohave proteolytic
activity. To our knowledge,proteolytic activity associated with
fat-containingcells has not been reported previously.The presence
of plasminogen activator activity
and proteolytic activity in the stomach andduodenum could have
implications in gastricand duodenal haemorrhage associated with
avariety of disease states.
This work was supported by a US Public HealthService traineeship
CST 562A67 from the CancerControl Program; a US Public Health
Servicespecial fellowship 2-F3-CA-3A, 497-02, fromthe NIH, National
Cancer Institute; by NIHgrant NB-0334605 (National Institute of
Neuro-logical Diseases and Blindness), and by anAmerican Cancer
Society research scholaraward.
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