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STUDIES ON A PROLACT IN - INACTIVATING
ENZYME SYSTEM IN BLOOD SERA

THOMAS F. HOPKINS

 

LIBRARY

Michigan State
University

 

 

 

Studies on a Prolactin-inactivating Enzyme System in Blood Sera

BY
Thomas P. HOpkina

An Abstract

Submitted to the College of Science and Arts
Michigan State University of Agriculture
and Applied Science in partial fulﬁllment
of the requirements for the degree of

Master of Science

Department of Physiology and Pharmacology

1961

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It has been demonstrated by several investigators that various protein
hormones can be inactivated by whole blood and by certain blood constituents
from a variety of species. The system(s) involved in protein hormone inac-
tivation by blood has not been clearly defined. However. some authors
attribute the action to that of fibrinolysin. The present experiments were
designed to determine whether blood serum would inactivate prolactin and to
attempt to establish some identity to the system.

Ovine prolactin was incubated two hours at 37. 5°C in human and guinea
pig serum. or euglobulins. human plasmin. plasminogen and streptokinase-
activated plasminogen. Prolactin activity was assayed in white Carneau
pigeons employing the method of Reece and Turner.

1. Incubation of prolactin in human blood serum of 10% and 45%
concentration resulted in mean losses of 36% and 54% respec-
tively. Incubation in human serum euglobulins of 50% concen-
tration caused a loss of 41% prolactin activity.

2. The mean loss of prolactin activity resulting from incubation
with 5. 7 caseinolytic units plasmin was 94%. Plasminogen at
the 5. 0 mg level was not appreciably effective (prolactin loss,

19%). however activation by 2000 U streptokinase caused a
loss of 74%.

3. Incubation in guinea pig serum or with serum euglobulins

5.

11

produced losses of 50% and 45% respectively. The albumin-
containing supernatant did not contain any demonstrable
protease activity. nor could it be produced by addition of
streptokinase.

Certain blood serum collections were devoid of protease and
fibrinolytic activities. This was found to be attributable to
the presence in serum of factors which inhibited the plasmin
system. Euglobulins prepared from such inactive sera were
contaminated with the inhibitors. but the concentration was
less than that of the serum.

The prolactine-inactivating capacity of streptokinase-activated
plasminogen was completely inhibited by antifibrinolysin. but
was enhanced by e-amino-N-caproic acid (Grade C). An AR
Grade of e-amino-caproic acid. reported to competitively
inhibit fibrinolytic activity of streptokinasec- activated plas-
minogen did not at the levels employed inhibit protease
activity. Below 0. 06 M concentration e-amino-caproic acid
enhanced fibrinolytic activity of streptokinase-activated plas-
minogen. Above 0. 06 M concentration protease but not fibrin-

olytic activity was enhanced.

W

It is well known that a deficiency as well as an excess of hormonal
activity can cause undesirable physiological effects; thus a hormonal
balance or equilibrium must be maintained for normal body functions. Varied
approaches have been applied in attempts to ascertain an understanding of
some of the mechanisms involved in maintaining a homeostatic condition.
As a result. considerable knowledge is available concerning the metab-
olism of steroid hormones. whereas knowledge of the metabolism of protein
hormones is quite scant.

It has been shown that protein hormones can be inactivated by various
biological tissues and tissue ﬂuids. and that the rate at which they dis-
appear from the circulating blood can be influenced by chemical poisoning
of or by surgical removal of specific organs or glands. However. an under-
standing of the mechanisms involved remains to be determined. Results of
some of the studies reported suggest the involvement of some yet unidenti-
fied enzyme system(s). Although the data are inconclusive. some authors
support the involvement of the blood fibrinolysin system in mammals.

To effectively study and elucidate such systems active in protein
hormone catabolism. it is necessary that the systems be isolated and
studied outside the animal body. However. when conducting such in E1119

studies as hormone inactivation by tissues or enzymes. it must be assumed

11

that the effects observed under in 11182 conditions are similar to those pro-

duced 13 115(2- Since hormones are generally secreted directly into and

transported throughout the body by the blood. it should be of first impor-

tance to investigate the fate of hormones while in the blood.

In the studies reported here. the protein hormone prolactin was chosen

for investigation. The studies were designed to determine:

b.

C.

the effects on prolactin activity of incubation in blood serum
whether prolactin activity would be inﬂuenced by the ﬁbrinolysin
system

the blood serum fraction containing the prolactin-inactivating
system

the relationship of the protease (prolactin-inactivating system)
activity to the fibrinolytic system. and

whether the fibrinolytic activity of streptokinase-activated plasmin-
ogen (profibrinolysin) could be influenced by specific inhibitors

without inﬂuencing the protease activity.

The results of these studies should give some information concerning

the blood enzyme involved in prolactin inactivation. and whether one or

more enzyme(s) are involved.

Aggngwledgemegtg

The author expresses thanks to Dr. I. Meites. Professor. Department
of Physiology and Pharmacology. for his interest throughout these studies.
for making available wherever possible materials and facilities needed.
for his assistance in parts of these studies and in preparation of this manu-
script. Grants to Dr. I. Meites from the Michigan Agricultural Experiment
Station and the United States Public Health Service provided financial sup-
port for this work.

Expressions of thanks are extended to Mr. Albert Rather. graduate
student. Department of Physiology and Pharmacology. for his assistance

in parts of these studies.

N r

Antifibrinolysin - antiplasmin.
Fibrinolytic activity - lysis of fibrin.
Plasmin - fibrinolysin (FL).

Plasminogen - profibrinolysin.

Proteolytic (protease) activity - inactivation of prolactin.

W

111111129. and 1.11m methods have been employed in studying the pos-
sible manner by which hormones are normally inactivated. Inactivation of
protein and peptide hormones by various biological tissues has been
reported.

The rate at which adrenocorticotropin (ACTH) disappears from the cir-
culating blood has been studied by Richards and Sayers (1951). Two hours
after administration only 2% of porcine ACTH activity could be detected in
the blood of men. After three hours ACTH activity was not detectable in
the blood. Similar results were obtained in the rat after injections of
porcine ACTH and sheep ACTH. Rat ACTH disappeared rapidly after being
injected intravenously into intact and adrenalectomized rats. Five minutes
after injection into intact rats 40% of the ACTH activity was found in the
extracellular fluid and 20% in the kidneys. After 15 minutes a negligible
amount was detectable in the extracellular fluid and 15% in the kidneys.
Less than 1/ 500th of the injected dose was estimated to be in the adrenals
after five minutes. There was no detectable activity in the liver or urine.
Raise. et al (1951) reported that ACTH was rapidly inactivated by heparin-
ized plasma from rats. rabbits. and humans.

Pincus. Hopkins and Hechter (1952) reported the presence of an ACTH
inactivating system in bovine. rat and human blood. which would inactivate

ACTH derived from pigs. sheep and whale. This system consisted of a

-2-

thermo-labila component and a thermo-stable component. both of which were
present in plasma. The inactivation was heat-dependent and could be re-
duced or completely inhibited by heating the blood to 50°C for one-half hour.
This suggested that an enzymatic process was involved. The inactivating
system waslater found to be concentrated in Cohn fraction 111.

Geschwind and Li (1952) reported that incubation of sheep ACTH with
rat plasma did not result in loss of activity. whereas incubation with liver
and kidney slices. diaphragm and homogenates of liver. kidney. and adrenal
tissues inactivated ACTH. White and Gross (1957) compared the effects of
bovine fibrinolysin with liver cathepsin. on the biological activity of corti-
cotrOpin-A. Since the structure of corticotropin-A was known it was pos-
sible to determine the positions and types of esters attacked by the enzymes
studied. These investigators found that bovine fibrinolysin at pH 6. 0-7. 5
splits corticotropin-A after arginine in position 8 and after lysine in position
15. causing a complete loss of biological activity. Bovine liver cathepsin
split corticotropin-A after leucine in position 31 and after phenylalanine
in position 35 without a resulting loss of ACTH activity. Their observations
on the action of fibrinolysin were in agreement with those of Troll and Sherry
(1954) who found that fibrinolysin attacks arginine and lysine esters.

Birmingham and Kurlents (1958) found that five-minute exposure of rat
adrenals to ACTH. in Krebs-Ringer bicarbonate buffer. stimulated the forma-

tion of corticoids in an ACTH-free medium and decreased the ACTH activity

-3-

in the medium in which the adrenals had been for a short time. Exposure

of one to one and one-half hours resulted in a complete loss of ACTH
activity in the medium and there was no corticoid production by the adrenals
subsequently placed in an ACTH-free medium. The interpretation was that
the ACTH had become bound or absorbed by the adrenals and thus was inac-
tivated.

Mirsky. Perisutti and Davis (1959) observed that somatotropin and
ACTH were hydrolyzed by streptokinase-activated human plasma or plas-
minogen. Meakin. Tingey and Nelson (1960) did not feel that the loss of
activity of exogenously administered ACTH was due to ”binding" by adrenals
of the dog. However. inactivation of porcine ACTH did occur when incubated
with heparinized blood. plasma and serum of the dog. Meakin and Nelson
(1950) reported that the ACTH-inactivating system of canine blood was heat
dependent and was inhibited by L-cysteine.

Hopkins. Tanner and Pincus (1951) isolated and concentrated from
human plasma a factor which when incubated with plasminogen and porcine
ACTH caused a marked loss of ACTH-activity. whereas the plasminogen
alone was ineffective. The purified "co-factor” was found to be a heat
stable. dialyzable substance of low molecular weight (less than 2000). it-
self devoid of activity.

Mersky. Perisutti and Davis (1959) reported that glucagon was hydro-

lysed 111.2122 by blood plasma from various species and found that the

-4...

highest rates of hydrolysis occurred with plasma from rat and monkey.
Lower rates were produced by plasma from chicken. man. guinea pig.
rabbit. cat. dog and cow. It was possible by addition of streptokinase
(SK) to the incubation mixture. to increase the glucagonolytic activity of
the plasma from man. monkey and cat. it was proposed that the inactive
precursor. plasminogen. was converted by $1: to the active enzyme plas~
min. which was responsible for the glucagonolytic activity of plasma.
Mirsky et a1 (1949) (1955) described a specific hydrolytic enzyme system
termed "insulinase" present in slices. homogenates and extracts of liver.
A rapid degradation of insulin resulted when incubated with this system.
Exogenously administered insulin was similarly rapidly inactivated by
intact mice.

Sonenberg. et a1 (1957) found that 835 labelled or acetylated thyro-
tmpin (TSH) did not cause enlargement of the thyroid of the rat or chick.
but these same preparations did inhibit endogenous TSH activity. Sonenberg
and Money (1957) prepared pituitary preparations having TSH and gonadotropic
activities. Acetylation of these preparations destroyed the TSH and gonado-
trepic activities and also inhibited the activity of exogenously administered
TSH.

Loeser (1934) observed that injected TSH disappeared from the blood
of intact rabbits in about one hour; whereas it was detectable in the blood

of thyroidectomized rabbits for‘as long as four hours. Seidelin (1940)

_5-

assayed TSH activity in the urine from intact and thyroidectomized guinea
pigs which had been given injections of TSH. TSH activity was found only
in the urine from thyroidectomized animals. Seidelin (1940) also observed
that the ovaries might similarly reduce the amount of activity in the urine
of rats following injections of gonadotropins.

Hall (1950) implanted pituitary tissue in various locations in immature
female rats. He observed that ovaries of rats bearing subcutaneous grafts
were larger than those of the rats bearing intrasplenic or intraperitoneal
implants. it was thought that the absence of ovarian stimulation in those rats
with intrasplenic or intraperitoneal pituitary grafts. was due to gonadotropin
inactivation by the liver. Hall (1950) also reported that incubation for one
hour at 37°C of human chorionic gonadotropin with homogenates of rat liver
caused a marked reduction in gonadotropic activity.

Beer and Tuzunkam (1952) studied the effects of liver from rats. pigeons
and frogs on melanocyte stimulating hormone (MSH). Rat liver was highly
effective in reducing MSH activity. The MSH-inactivating capacity was
reduced by about 50% in pregnancy. after adrenalectomy. and after produc-
tion of toxic lesions of the liver. The inactivation of MSH by liver from frog
or pigeon was considerably less than that caused by rat liver.

Shizume and Irie (1957) reported that exogenously administered MSH
rapidly disappeared from the blood of normal dogs. The rate of disappearance

from the blood of hepatectomized dogs was much less than that in intact

—6..

animals. Carbon tetrachloride poisoning of the liver also caused a reduced
rate of removal from the blood. Homogenates of liver inactivated MSH; where-
as kidney and muscle were much less effective. Long et a1 (1961) reported
that liver and muscle tissue did not inactivate a highly purified preparation

of MSH. but that brain tissue of cats greatly reduced MSH activity.

- Meites (1940) followed the rates at which known amounts of subcutane-
ously administered prolactin were detected in the urine of rabbits. Approx-
imately 1 as was detectable during the first 24 hours. only a trace during the
second 24 hour period. and none was detectable in the urine collected during
the third 24 hours. Intravenously administered prolactin could not be detected
in the urine collected during the following 72‘ hour period. About half of the
administered prolactin activity could be detected in the blood plasma five
minutes after intravenous injection; none was assayable thirty minutes after
injection.

Sonenberg et a1 (1951) administered 1131 labelled prolactin to normal
female rats. In studying the distribution of radioactivity in various organs
of the rats it was found that the greatest concentration was in the corpora
lutea. Essentially no activity was demonstrable in the mammary glands of
normal adult. immature. pregnant or lactating females. Cox (1951) injected
iodinated prolactin into mice and found that 20 minutes after the injection
the greatest concentration of activity was in the liver and kidneys. Lesser

amounts were found in the mammary glands. milk. ovaries and adrenals.

-7...

Sgouris and Meites (1952) incubated prolactin with various tissues
including liver. kidney. brain and muscle of female rats; mammary glands
from lactating and nonlactating guinea pigs and rabbits; pigeon crop glands;
corpora lutea and the remaining ovarian tissue from pseudOpregnant rabbits.
All tissues employed were effective inactivators of prolactin with the excep-
tion of muscle and brain. Lactating mammary tissue. pigeon crop and
ovarian tissues were the most effective. The inactivating system was shown
to be completely inhibited by boiling the tissue preparations for ten minutes.
Sgouris and Meites (1953) observed also that prolactin inactivation occurred
. more rapidly when incubated with lactating mammary tissue than when incu-

bated with mammary tissue from pregnant (non-lactating) rats.

ammun-
The ovine preparation used was generously supplied by the Endocrinology
Study Section. N. I. H. and had a reported activity of 15 International Units
(1. U.) per mg.
The prolactin was dissolved in distilled water to a concentration of 5. 0

mg prolactin per ml solution. A volume of 0. 1 ml (0. 5 mg) was used in each

experiment.

Wan
Collections of human blood were arranged and supervised by Mrs.

Margaret Shick at Olin Health Center. Michigan State University. The blood
was brought to the laboratory and after clotting had occurred. the blood was
centrifuged and the serum collected and pooled. Each pool consisted of
equal portions of serum from each of six to ten different blood samples. Each
pool was aliquoted into volumes of 5-10 ml. stored in a freezer and thawed

just prior to use. Serum concentrations for incubations were prepared by

diluting with Krebs-Ringer phosphate buffer 'of pH 7. 2.

W
The guinea pig blood was given essentially the same treatment as the

human blood. A 10 ml volume was collected from each guinea pig. The serum

pool consisted of equal volumes of serum from each of three female guinea pigs.

W

Human Plasmin (Fibrinolysin): The fibrinolysin solution was supplied
by The American Red Cross and Dr. I. T. Sgouris. Michigan State Health
Laboratories. Each ml of fibrinolysin solution contained 57 caseinolytic
units (C. U.) activity. Fibrinolysin solutions for incubation were prepared
by adding 0. 1 ml (5. 7 C. U.) fibrinolysin solution to 0. 8 ml Krebs-Ringer
phosphate buffer of pH 7. 2.
MW

Human plasminogen was supplied by Dr. I. T. Sgouris. Michigan State
Health Laboratories. Plasminogen was not readily soluble at neutral pH.

Therefore. a fine suspension was prepared in Krebs-Ringer phosphate buffer.

orsr lb 1 i

The author is indebted to Dr. Walter Seegers. Wayne State University.
Detroit. for the antifibrinolysin which was a product of Parke-Davis and Co. .
and was prepared from human blood. The antifibrinolysin was dissolved in
Krebs-Ringer phosphate buffer to a concentration of 500 Units per ml solution.
A volume of 0. 2 ml (100 Units) was used for inhibiting plasmin activity.
n-Aming- H—Qgpmjg Agid (Grade C) was purchased from California Biochemicals.
Los Angeles. California.
ﬁ-Amino-Qgprojg 591g (Analytical Reagent) was purchased from Nutritional

Biochemical Co. .‘ Cleveland, Ohio.

-10...

Ac or - a

Streptokinase (SK) was a gift from Mr. Albert Rather. Department of
Physiology and Pharmacology. Michigan State University. and was a
product of Lederle Laboratories.

We;

The method of Milstone (1941) was employed in preparing the euglobulin
fractions. Blood serum was diluted 20-fold; dilute acetic acid was added
and by aid of a Beckman Zeromatic pH meter was adjusted to pH 5. 3. After
storage overnight at 4°C the euglobulins were collected by centrifugation.
The euglobulin fractions were reconstituted in Krebs-Ringer phosphate buffer.

0 r In

A volume of 0. 1 ml (0. 5 mg) prolactin solution was added to each flask
containing the incubation medium. The total incubation volume was 1 ml.

As a control. standard prolactin was added to Krebs-Ringer phosphate buffer
and incubated simultaneously. The incubation ﬂasks were placed in a
Dubnoff Metabolic Shaking Incubator and after 2 hours’ incubation at 37. 5°C.
each sample was diluted 10-fold with 0. 9% saline solution to give an initial

prolactin concentration of 0. 05 mg/ml.

MW
Prolactin activity was assayed in mature White Carneau pigeons employ-

ing the method of Reece and Turner (1937). Each pigeon was given 0. 1 ml of

the assay solution daily for four days. On the fifth day the pigeons were

-11-

sacrificed. and the crop sacs were removed and rated. The highly sensi-
tive crop sac of the pigeon exhibits glandular proliferation in the presence
of small amounts of prolactin. The degree of response represents the
amount of prolactin activity present. By injecting incubated standard pro-
lactin over one cr0p sac. and comparing the response with that of an equiv-
alent amount of experimental sample injected over the other crop sac. each
bird served as its own control. To minimize subjectivity. ratings were
made by two independent coworkers who were unfamiliar with the assay
materials. but experienced in the assay technic.

sa -

Materials:

0. 1M phosphate saline buffer of pH 7. 4.

Pibrinogen: Human fibrinogen was supplied by Dr. I. T. Sgouris.
Michigan State Health Laboratories.

Thrombin: Bovine thrombin was supplied by Dr. Clyde Cairy. Depart-
ment of Physiology and Pharmacology. Michigan State Uni-
versity. and was a product of Parke. Davis & Co.

Preparation of Fibrin Plates and Assay of Pibrinolysin:

Petri dishes (9. 0 cm or 14. 0 cm diameter) were sterilized before being

used. Pibrinolytic activity was assayed on fibrin plates employing a modi-

fied method of Astrup and Mullertz (1952) and the heated plate method of

-12—

Lassen (1952).

A 10. 0 ml volume of 0. 2% solution of human fibrinogen. which had pre-
viously been dialyzed for approximately 18 hours against 0. 1M phosphate
saline buffer. was poured into the 9. 0 cm Petri dishes and was clotted by
addition of 0. 1 ml (10 units) bovine thrombin. When using the larger Petri
dishes. 15. 0 ml fibrinogen solution was clotted by 0. 15 ml (15 units)
thrombin. As many as four solutions could be assayed on the smaller
dishes. and as many as six on the larger dishes.

When heated plates were desired. they were heated. after clotting. for
15 minutes at 80°C in a Dubnoff shaking incubator. After cooling. single
drops (0. 03 ml or 10. 0 ,K) of each sample were placed on the plates and
incubated for 18 hours at 34°C. The area of the digested fibrin served as
an index of enzyme activity. The product of the two perpendicular diameters

was recorded in mmz.

_13..

A. Incubation of Prolactin in Human Blood Serum:

Human blood serum was diluted with Krebs-Ringer phosphate buffer.

A volume of 0. 1 m1 (0. 5 mg) prolactin solution was pipetted into the incu-
bation ﬂask which contained 0. 9 ml of the dilute serum and the preparations
were incubated for two hours at 37. 5°C. The effects on prolactin activity
of incubation with blood serum can be seen in Table 1. Incubation in
human serum of 10% concentration resulted in moderate but significant
losses (33. 36. and 40%) of prolactin activity. A somewhat greater loss
(41. 52. and 70%) of activity occurred when a similar amount of prolactin
was incubated in 45% serum.

These data show the presence. in human blood serum. of a system
capable of inactivating ovine prolactin. The activity of the system is tem-
perature dependent. since heating the blood for 30 minutes at 60° inhibited
the inactivating system. This would tend to eliminate the possibility of
inactivation due to surface reaction such as absorption or binding of pro-

lactin with serum proteins.

-14-

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-15..

B. The Effects of Plasmin on Prolactin Activity:

Plasmin (fibrinolysin. FL) is a naturally occurring fibrinolytic enzyme
present in some mammalian blood (Sherry. Fletcher. and Alkjaersig. 1959).
Sherry and his collaborators showed that plasmin is a proteolytic enzyme
with a more general action than fibrinolysis alone. Plasmin has been shown
to hydrolyze ﬁbrinogen, casein. gelatin. plasma accelerator globulins and
other proteins. and attack arginine and lysine esters (Troll. Sherry. and
M‘achman. 1954). The proteolytic activity of plasmin is further demonstrated
by its effect on the biological activity of prolactin. In Experiment 8. O. 1 ml
(0. 5 mg) prolactin was pipetted into each incubation ﬂask containing 5. 7
caseinolytic units (CU) (l4. 1 Loomis Units) fibrinolysin. The results are
presented in Table 2. where it can be seen that essentially a complete loss
of prolactin activity resulted after two hours' incubation with fibrinolysin.

White and Gross (1957) and Hopkins. Terner and Pincus (1961) reported
that adrenocorticotropin (ACTH) was readily inactivated by fibrinolysin. The
former authors (1957) reported that bovine fibrinolysin splits ACTH after
arginine in position 8. (Arginine-tryptophane) and after lysine in position
15. (lysine-lysine). resulting in a complete loss of the biological activity
(adrenal ascorbic acid depletion) of ACTH. White and Gross (1957) expressed
belief that the heat-labile component of the ACTH-inactivating factor reported
by Pincus. Hopkins and Hechter (1952) might be fibrinolysin since the latter

authors had traced the heat-labile component to fraction III which contains

-16-

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_ 17 -
plasminogen. (Kline. 1953).

C. Inactivation of Prolactin by Streptokinase-Activated Plasminogen:

The presence of plasmin in the blood depends on the conversion of the
enzymatically inactive precursor plasminogen into the active enzyme plasmin
(Astrup. 1958). The manner by which this is done is complicated and has
been defined by Alkjaersig. Fletcher and Sherry (1957) and by Astrup (1958).

A variety of agents known to activate plasminogen has been reported
by Tillet (1935). Astrup and Permin (1947). Astrup and Sterndorf (1952. 1953).
Sobel et a1 (1952) and Mullertz (1953). II-Iowever. streptokinase is the most
widely used activator of plasminogen for in 11119. studies on fibrinolytic activity.
Since prolactin was inactivated by plasmin. it was important to know what
effect streptokinase-activated plasminogen would have on prolactin activity.

Prolactin (0. 5 mg) was incubated with (a) plasminogen and (b) strepto-
kinase-activated plasminogen. The results are presented in Table 3. At
the 2. 0 mg level. plasminogen with or without 2000 U streptokinase did not
inactivate prolactin. Prolactin was not appreciably inactivated (19%) by
5. 0 mg plasminogen. whereas 5. 0 mg plasminogen activated by streptokinase
produced a marked increase in proteolytic activity. causing a 74% loss of

prolactin activity.

 

-18-

 

 

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-19..

D. (1) Incubation of Prolactin in Guinea Pig Serum and Euglobulins. and

in Human Euglobulins. (2) Demonstration of Anti-fibrinolysin in Some

Sara.

The proteolytic activity (prolactin inactivation) of blood is not limited
to human serum. In Table 4 it is shown that guinea pig serum or euglobulins
at 50% concentration have essentially the same degree of proteolytic activity.
and that precipitation of the euglobulin fraction removes all demonstrable
proteolytic activity. Human euglobulins prepared from serum pool 0 1 (Exper-
iment A) did not exhibit the same degree of activity as shown by the whole
serum. However. streptokinase activation increased the activity.

The proteolytic activity of human and animal serum shows a wide vari-
ation. This variation in activity may well be due to increased enzyme activ-
ators or inhibitors which have been shown to vary in different physiological
conditions (Macfarlane and Biggs. 1946; Pantl and Simon. 1958; Sherry.
Fletcher and Alkiaersig. 1959). Results in Table 5 show that guinea pig
serum from pool G 2 and human serum pool It 4 were ineffective as inactivators
of prolactin.

ﬁbrinolysin assays were done on unheated fibrin plates to determine
whether the proteolytically inactive human and guinea pig sera contained any
fibrinolytic activity. and also whether the absence of proteolytic activity
could be due to the presence of high levels of anti-ﬁbrinolysin. In each

experiment (Table 6) the sera or enzyme was prepared in 1. 0 ml Krebs- Ringer

-20..

 

 

 

 

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-23...

phosphate buffer. One drop (0. 03 ml) of each sample was put on fibrin plates
two minutes after preparation. The fibrin plates were incubated for 18 hours

at 34°C. after which the product of the two perpendicular lysed zones was
recorded. The area of fibrinolysis caused by fibrinolysin was 361 mmz. This
activity was more or less completelyinhibited by antifibrinolysin. I It can be seen
that the inactive human and guinea pig sera not only failed to cause fibrinolysis.
but also exalted an inhibitory action on plasmin. Antiplasmin activity of serum
has been reported by Milstone (1941); Macfarlane and Pilling (1946); Grob (1949);
and Sherry. Fletcher and Alkjaarsig (1959). Pibrinogen is contaminated with
plasminogen. therefore, lysis of fibrin occurs if plasminOgen activators. are
applied to the plate even in the absence of ﬁbrinolysin.

Table 7 shows results of experiments designed to determine whether the
antifibrinolytic action of human and guinea pig sera and euglobulins could be
overcome by streptokinase and fibrinolysin. 'l‘he sera and euglobulin fractions
were prepared in 0. 8 ml Krebs- Ringer phosphate buffer to which was added
0. 2 ml (2000 U) streptokinase. One drop (0. 03 ml) of each sample was put
on the fibrin plate before addition of the streptokinase to the solution. one
minute after adding streptokinase. 10 minutes after incubation with strepto-
kinase. and finally after 10 minutes' incubation with 5. 7 CU fibrinolysin. The
results are presented in Table 7. Pibrinolytic activity was produced one
minute after addition of streptokinase to the solution. The greatest amount of

activity was in the euglobulin fractions. It is not known if this activity was

-24..

due to streptokinase-activation of plasminogen present in the serum and
serum fractions. or to activation of plasminogen in the fibrin plate. Never-
theless. it is obvious that the sera contain greater concentrations of inhibf
itors of fibrinolysin; and that the guinea pig euglobulins contain the least
amount. The fibrinolytic activity is shown to have increased following a
ten-minute incubation period. It is believed that this increase was due to
a production of plasmin during incubation. The relative increase in activity
was approximately the same for human (75 mmz) and guinea pig (84 mmz) sen.
The smallest increase in activity. 18 mmz. was in the guinea pig euglobulin
fractions. whereas the increased lysis caused by human euglobulins was 47
mmz. Lysis caused by a similar amount of streptokinase which had been incu-
bated for ten minutes before application to the fibrin plate was 258 m2.
Addition of 5. 7 CU fibrinolysin to the incubation mixture did cause a
moderate increase in the lysis of fibrin. but the sum of the activities of fibrin-
olysin. streptokinase-activated plasminogen of serum. or euglobulins and
plasminogen present in the fibrin. were not sufficient to overcome the inhib-
itory actions of the antiplasmins. The area of lysis caused by control fibrin-
olysin which had been incubated for 10 minutes was 484 mmz; lysis caused
by fibrinolysin added to the streptokinase-activated human or guinea pig serum
was 196 mm2 each; for fibrinolysin added to streptokinase-activated human
and guinea pig euglobulins. 400 and 408 mm2 respectively. It is conceivable

that the greatest portion of lysis at this time was due to the action of the

-25..

added fibrinolysin; particularly in the euglobulin preparations. since the
activities were essentially the same for both species.

The data presented in Table 7 give a possible explanation for the failure
of these serum fractions to inactivate prolactin. (Table 5). It is still uncer-
tain whether any appreciable degree of plasmin was produced in the serum
samples. Lassen (1952) has shown that heating the fian plate denatures
the plasminogen. but does not appreciably reduce the sensitivity of fibrin to
lysis by plasmin. Therefore. any lysis of fibrin is due to fibrinolysin added to
the plate and not due to activators of plasminogen as in the unheated plate. By
use of the heated fibrin plates. it was possible to determine if any fibrinolytic
activity was produced in the serum or euglobulins.

One drop (0. 03 ml) of serum or euglobulin solution was put on the plate
before addition of. and after 10 minutes‘ incubation with streptokinase. It
can be seen from the data presented in Table 8 that the euglobulins caused
some lysis of fibrin. This experiment was run seven days after preparation of
the euglobulins and three days after the preceding experiment (Table 7). Plas-
minogen is known to undergo spontaneous activation on storage (Sherry. 1958).
Such a process could have occurred during storage of the euglobulins. and
would explain the presence of fibrinolytic activity without addition of strepto-
kinase to the mixture. The expected increase in fibrinolytic activity was
produced by streptokinase-activation of the euglobulins. however. the sera

were not activated by streptokinase. It must be assumed. therefore. that

-25..

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_28-

these sera from human and guinea pig blood contained inhibitors that inhib-

ited plasmin and probably activation of plasminogen.

E. Effects of Some Amino Acids on the Proteolytic and Pibrinolytic Activities
of Streptokinase-Activated Plasminogen.

Alkjaersig. Fletcher’and Sherry (1959) reported that e-amino-caproic
acid competitively inhibited activation of human or bovine plasminogen by
streptokinase; at low concentrations it enhanced the action of plasmin. where-
as. at higher (above . 06 M) levels it acted as a noncompetitive inhibitor of
plasmin. Salter (1958) reported that ovomucoid would inhibit the fibrinogeno-
lytic activity of plasmin. but did not effectively inhibit the fibrinolytic
activity. Conversely. e-amino-caproic acid inhibited fibrinolytic activity.
but did not inhibit fibrinogenolytic activity. These observations suggested
the possibility that the active enzyme. plasmin. has more than one active
site and that one of these can be blocked by specific inhibitors. while not
interfering with another enzymic property of the molecule. Streptoltinase-
conversion of plasminogen to plasmin was shown (Alltjaersig. Fletcher and
Sherry. 1959) to be blocked by e-amino-caproic acid. Caseinolytic activity
as well as fibrinolytic activity was inhibited. suggesting that these two
proteins. casein and fibrin. are hydrolyzed by the same site(s) of the plas-
min molecule. The activation of plasminogen by streptokinase involves an
enzymic process causing a release of approximately 17% of the nitrogen from

plasminogen (Sherry. Fletcher and Alkjaersig. 1959). It is not known if

-29...

e-amino-caproic acid alters the site at which plasminogen is split or just
what is involved in inhibiting the process.

Hopkins. Terner. and Pincus (1961) reported that plasminogen could be
activated by a factor prepared from human blood. The activated plasminogen
had peptidase (inactivation of ACTH) activity. but did not cause lysis in
the fibrin plate test. Streptokinase is a protein of relatively high molecular
weight. which can convert plasminogen to an enzyme (plasmin) with diverse
proteolytic activities. The "factor” employed by immune. Terrier and Pincus
(1961) was considerably smaller (molecular weight less than 2000). There-
fore. it differs from streptokinase as well as other known activators of plas-
minogen which with two exceptions are proteolytic enzymes (Sherry et a1. .
1959). It would appear in this case. that the type of activity produced

would be determined by the plasminogen activator employed. Such observe-v
' tions. if applicable to prolactin inactivation should make it possible to show
what relationship exists between the prolactin-inactivating system and the
fibrinolysin system.

In the following studies the effects on the activities of streptokinase-
activated plasminogen of two preparations of e-amino-caproic acid were
studied. In the first of these experiments e-amino-N-caproic acid (Grade C)
was incubated 10 minutes with plasminogen before addition of streptokinase.
After the 10 minute incubation period. 2000 U streptokinase and O. 5 mg pro-

lactin were added and the preparation was incubated for two hours. Following

-30..

the incubation period. one drop (10 A) of each preparation was put on the
heated fibrin plate to assay for fibrinolytic activity. A portion of the remain-
ing solution was diluted and assayed for prolactin activity.

Table 9 shows that the protease activity (prolactin inactivation) and
the fibrinolytic activity are markedly increased by e-amino-N-caproic acid.
Since it has been demonstrated that e-amino-caproic acid (of high purity
inhibits the fibrinolytic activity of streptokinase-activated plasminogen
(Alltjaersig et al. 1959). the next experiment was designed to see if fibrino-
lytic activity could be inhibited without interfering with the proteolytic
activity.

Plasminogen (5. 0 mg) was incubated for 10 minutes with 5. 0. 10. 0. or
50. 0 mg e-amino-caproic acid in O. 7 m1 Krebs-Ringer phosphate buffer. This
incubation period was to allow the amino acid to react with plasminogen
before adding streptoltinase. At the end of the incubation period 0. 2 ml
(2000 U) streptokinase and 0. 1 ml (0. 5 mg)’prolactin were added. and the
mixture was incubated for two hours. One drOp (10 )\) of each incubation
mixture was put on a heated fibrin plate and incubated 18 hours at 34°C.

Table 10 shows that the initial protease and fibrinolytic activities of
the plasminogen mixtures were increased by streptokinase-activation. As
was shown previously (Table 9). antifibrinolysin inhibits both activities.
Pibrinolytic activity was increased by 5. 0 mg (0. 026 M) e-amino-caproic

acid. but the protease activity was not influenced. The increase in fibrino-

-31..

 

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lytic activity is in agreement with reports of Alkjaersig. Fletcher. and Sherry
(1959) that e-amino-caproic acid below . 06 M concentration increases the
fibrinolytic activity of plasmin. Incubation with 10. 0 mg e-amino-caproic
acid (0. 052 M) caused a significant increase in protease activity. but the
degree of fibrinolytic activity was not correSpondingly increased. At higher
concentration (50. 0 mg; 0. 26 M) the protease activity was lower than that
produced by 10. 0 mg e-amino-caproic acid; whereas fibrinolytic activity was
the same.

It can be concluded from the data in Table 10 that fibrinolytic and pro-
tease activities of streptokinase-activated plasminogen are not directly
related. An increase in protease activity was not accompanied by a corres-
ponding increase in fibrinolytic activity. In working with such an impure en-
zyme preparation (proenzyme) as plasminogen. it is impossible to state
whether this represents more than one active site on the transformed product
(enzyme). since it is possible that other enzymes (trace) present were inﬂu-
enced by streptokinase as well as by e-amino-caproic acid.

The failure to effectively inhibit streptokinase conversion of plasminogen
to fibrinolysin may well be due to the excessive amount of activator employed
in the system (2000 U streptokinase). This would be of particular importance
if the following equation could be applied: E + 1 :5: 131; where the enzyme 12
(plasminogen) and inhibitor 1 complexing is reversible. The relative concen-

trations of e-amino-caproic acid and streptokinase would then determine the

-34-

rate at which plasmin would be formed.

In Table 10. mperiment 1. it can be seen that some plasmin was in
the system before activation by streptokinase. The increase in protease
activity caused by streptokinase was relatively small. but significant.
Approximately two-thirds of the activity was present before adding strepto-
kinase to the system. The fibrinolytic activity (initial) was approximately
50% of the activity present after streptokinase activation. E-amino-caproic
acid. at all levels employed here. increased fibrinolytic activity. but only
at the 10. 0 mg level was the proteolytic activity influenced. If the fibrino-
lysin system was the only enzyme system involved. then it is quite possible
that enzyme activity of activated plasminogen can be determined by the
activator employed and the type of inhibitor present. or that the plasmin
molecule possesses more than one active site. it is also shown that the
purity of e-amino-caproic acid is of importance. E-amino-N-caproic acid
at approximately ten times the molar concentration of the highest concentra-
tion of e-aminocaproic acid markedly enhanced the proteolytic activity of
streptokinase~activated plasminogen (Table 9). The results suggest that
e-amino-caproic acid at certain molar concentrations can enhance the prote-
olytic and fibrinolytic activity of streptokinase-activated plasminogen. but
that enhancement of the two activities does not occur at the same molar con-
centration.

From the data presented the following conclusions can be drawn. The

-35..

prolactin-inactivating system of blood serum is heat dependent and probably
enzymic in nature. All of the demonstrable protease (prolactin inactivating)
activity was present in the euglobulin fraction and could be increased by
addition of streptokinase. Since plasminogen. the precursor of the prote-
olytic enzyme plasmin. is concentrated in euglobulin fractions. it is pos-

sible that plasmin was involved in prolactin inactivation.

-35..

W

The methods employed previously in studying the effects of blood on
protein hormone activity have not been such that would elucidate the nature
of any enzyme system involved in protein hormone inactivation. It has been
shown that several of the protein hormones (ACTH. 8TH. and glucagon) can
be inactivated by blood serum or plasma from various species. and that the
degree of inactivation could in some instances be increased by addition of
streptokinase. a product of bacterial origin. Streptokinase has been exten-
sively studied and found to be a highly effective activator of the fibrinolysin
system. but has not been found to activate other enzyme systems. Thus.
some degree of specificity exists betwun streptokinase and fibrinolysin.

On this basis alone some investigators have assumed that the fibrino-
lysin system is responsible for the loss of protein hormone activity resulting
from incubation in blood. As a result of studies presented here no strong
dissension should result from such assumptions providing there be some
clarification of terminology. The terms "fibrinolysin" and "plasmin" are at
times used interchangeably. However. in the studies reported here reference
to the action of “fibrinolysin" has been restricted to the dissolution or lysis
of fibrin. Protease activity (inactivation of prolactin) has been attributed
to the action of "plasmin." which in certain instances may or may not have

fibrinolytic activity.

-37..

The present studies show that the two activities (fibrinolytic and protease)
coexist to some degree. but that one may be increased without a corresponding
increase in the other activity. Baker (1958) reported that fibrinolytic activity
of plasmin could be inhibited by e-amino-caproic acid without interfering
with fibrinogenolytic activity; ovomucoid inhibited fibrinogenolytic activity
but did not appreciably inhibit fibrinolytic activity. Hopkins. Terner and
Pincus (1961). by use of an activator prepared from human blood. converted
plasminogen to an enzyme possessing peptidase (ACTH-inactivating) activity.
but without fibrinolytic activity. The above observations support the multi-
fariousness of activated plasminogen.

The present observations are in agreement with reports of inactivation of
protein hormones by blood. plasmin and by streptokinase-activated plasmin-
ogen. The failure of certain samples of sera to inactivate prolactin was
shown here to be due to the presence of inhibitors which inhibited fibrinolytic
and protease activities of the serum and also inhibited the fibrinolytic activity
of plasmin. These inhibitors are therefore similar if not identical to anti-
fibrinolysin. which in these studies should more aptly be termed antiplasmin.
Additional studies employing specific inhibitors and varying amounts of strepto-
ltinase as well as other activators of plasminogen should be explored in

acquiring more details on the nature of these enzyme systems.

_38-

W

Alkjaersig. N.. Fletcher. A. P. and Sherry. S. E-aminocaproic Acid. an
Inhibitor of Plasminogen Activation. My}, 131:832-837. 1959..

Astrup. T. Activatcrs and Inhibitors of Pibrinolysis. in Proceedings of the
Fourth International Congress of Biochemistry. 195147-17 l. 1958.
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Astrup. T. and Mullertz. S. The Fibrin Plate Method for Estimating ﬁbrino-
1N0 ACHVICY. WW 595346-3510 1952-

Astrup. T. and Permin. P. M. Pibrinolysis in the Animal Organism. Hm
119:681-682. 1947.

Astrup. T. and Sterndorf. 1. An Activator of Plasminogen in Normal Urine.
WW 51:675-678. 1952.

Astrup. T. and Sterndorf. I. A Pibrinolytic System in Human Milk. 2:99.
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Birmingham. M. K. and Kurients. E. Inactivation of ACTH by Isolated Rat
Adrenals and Inhibition of Corticoid Formation by Adrenocortical Hormones.

Wham-60. 1958.

Cohn. E. 1.. Strong. 1.. 2.. Hughes. W. I... In. Mulford. D. 1.. Ashworth.
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Cost. 1.. Tim Preparation and Distribution of Iodo-Prolactin Labelled with I131.
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Beer. 3. and Tuzunkam. P. Concerning the Inactivation and Augmentation in
the Organism of the MSH at the Normal State and of Certain Pathological
Conditions. W 11:905-909. 1952.

Fantl. P. and Simon. S. B. Pibrinolysis Following Electrically Induced Con-
widen-o WWW 25:521-529. 1958.

Gesobmnd. I. E. and Li. C. H. inactivation of Adrenocorticotropic Hormone
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Glob. D. Further Studies on Protein. Polypeptide. and Other Inhibitors of

-39-

Serum Proteinase. Leucoproteinase. Trypsin and Papein. men.
21111191.. 11:103-124. 1949.

Hall. B. V. Inactivation of Gonadotmphins by Liver and Liver Homogenates.
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Hopkins. T. P. . Terner. C. and Pincus. G. Preparation and Properties of
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W 69:728-734. 1961.

Kline. D. L. The Purification and Crystallization of Plasminogen (Profibrin-
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Lasen. M. Heat Denaturation of Plasminogen in the Fibrin Plate Method.
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Loeser. A. Die Schilddrusenwirksame Substanz des Hypophysenvorderlappens.
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Long. 1. M. . Krivoy. W. A. and Guillemin. R. On a Possible Role of B-
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Tissue. 3W 19:176-181. 1961.

Macfarlane. R. G. and Biggs. R. Observations on Pibrinolysis: Spontaneous
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Mecfarlane. ll. G. and Pilling. I. Observations on Pibrinolysis: Plasminogen.
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Meakin. I. W. and Nelson. D. H. The Catabolism of Adrenocorticotropic
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indeednelm 51:73-79. 1960.

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-40—

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-41.-

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