Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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The present invention relates to the induction of
elevated levels of endogenous tissue plasminogen activator
(tPA). Specifically, the present invention relates to the
induction of these elevated tPA levels through the
administration of a basic placenta angiogenic factor (P~F).
Tissue plasminogen activator recently has been studied as
a potential therapeutic agent for the treatment of myocardial
infarctions and certain other blood clotting disorders In
particular, it has been postulated that tPA will dissolve the
blood clots which occur in the coronary arteries during a
myocardial infarction, thus re-opening the coronary arteries
and reestablishing blood circulation to the portions of the
heart muscle which otherwise would have been damaged during
the heart attack.
However, there are certain drawbacks to the use of tPA as
a therapeutic agent. In particular, tissue plasminogen
activator has an extremely short half life and no system has
yet been identified or developed which i8 capable of
sustaining the elevated tPA levels for the time needed to
dissolve not only the clots present in the coronary arteries
at the time of infarction but also the clots which may remain
circulating after the infarction and could form emboli in
other portions of the body. These emboli might be responsible
for various complications, including stroke.
To overcome this problem of achieving sustained, elevated
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tPA levels in the circulatory system, the present inventors
have discovered that the administration of basic placenta
angiogenic factor (PAF) will increase endogenous tPA levels.
The basic PAF may be administered over a period of time, such
as in the form of an intravenous drip, and will cause the
circulating tPA levels to remain elevated at least for the
duration of its administration.
SUMMARY 0~ THE INVENTION
An object of the present invention is to provide a method
for causing elevated levels of endogenous, circulating tPA
through the administration of a therapeutic agent. Another
object of the present invention is to provide a method for the
treatment of myocardial infarctions and other blood-clotting
disorders by administration of a therapeutic agent capable of
increasing the endogenous, circulating tPA levels.
Additional objects and advantages of the invention will
be set forth in part in the description which follows, and in
part will be obvious from the description or may be learned
~rom practlce of the invention. The objects and advantages
may be realized and attained by means of the instrumentalities
and combinations particularly pointed out in the appended
claims .
To achieve the objects and in accordance with the
purpose~ of the present invention, a method is disclosed which
causes elevated circulating levels of endogenous or naturally-
produced tPA. This method comprises administering basic
placenta angiogenic
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factor at a level and for a time sufficient to cause an
elevation in circulatory tPA levels. It is intended that
these levels remain elevated for 24-48 hours. In addition,
the basic PAF may be administered in conjunction with heparin,
which, as explained more fully hereinbelow, will increase its
efficacy.
Moreover, the present invention further achieves the
above objects by setting forth a method for the treatment of
myocardial infarctions involving at least a partial
dissolution of a portion of the blood clots formed in the
coronary arteries or in other blood-carrying vessels which
comprises:
(a) administering basic placenta angiogenic factor at a
dosage sufficient to result in a continuing elevated,
circulating tPA level capable thereby of increasing the
potential for plasminogen activation; and
(b) at least partially dissolving a portion of the blood
clots present in blood-carrying vessels by exposure to the
increased plasmin levels induced by the elevated tPA level.
Another embodiment of this invention includes the additional
step (c) of preventing re-occlusion of the blood-carrying
vessels by continued exposure to the elevated tPA levels.
The invention also provides a pharmaceutical composition
comprising a therapeutically-effective amount of PAF and
heparin in a pharmaceutically-acceptable carrier wherein the
heparin is present in an amount s~fficient to stabilize the
PAF.
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It is to be understood that both the foregoing general
description and the following detailed description are
exemplary and explanatory only and are not restrictive of the
invention, as claimed.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Reference will now be made in detail to the presently
preferred embodiments of the invention, which, together with
the following examples, serve to explain the principles of the
invention.
As noted above, the present invention relates to a method
of stimulating the production of elevated levels of endogenous
tPA in animals, humans and cultured cells. This method
involves, in part, the administration of basic placenta
angiogenic factor (PAF). This basic PAF is also known as
FGF~gjC or basic FGF. Basic PAF has been previously described
and methods for its isolation and recombinant-DNA methods for
its production have been provided in Canadian Patent
Application Serial No. 525,005 filed on December 11, 1986.
The compositions described by this patent application will be
referred to hereinafter as "basic PAF."
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In this method, basic PAF is administered at a dosage
sufficient to result in an elevation of circulating tPA
levels. These elevated tPA levels are capable of inducing
elevated plasmin levels. The exposure of clots in blood-
carrying vessels to the elevated plasmin levels caused bythese elevated tPA levels results in at least partial
dissolution of the clots. In addition, reocclusion of the
blood-carrying vessels may also be prevented by the elevated
tPA levels. The results obtained by this method, i.e., at
least partial dissolution of clots, are believed to be
obtained in part as a result of certain properties of the
basic PAF.
Basic PAF, isolated in a purified form or manufactured
through a recombinant-DNA method according to the procedures
set forth in the Moscatelli et al. applications, su~ra,
possesses at least three particular properties. These include
(1) the ability to cause cell migration, ~2) the ability, in
the presence of serum, to cause cell division ~the mitogenic
property), and ~3) the ability to induce the synthesis of
proteases by capillary endothelial cells.
The present inventors have discovered an additional
property of basic PAF. Specifically, when contacted with most
types of endothelial cells, low doses of basic PAF cause the
endothelial cells to produce, among other proteases, tissue
plasminogen activator. Tissue plasminogen activator has been
shown to be produced in vitro in accordance with this method
with a time-lag of several hours, and will be able to be
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produced in vivo by administration of PAF in the manner
described more fully hereinbelow.
In addition, the present investigators have discovered
that large doses of PAF administered to rabbits elicits a
rapid increase in circulating levels of tPA. This increase is
so rapid that it cannot be accounted for by de novo synthesis
but reflects a change in the distribution of existing tPA from
compartments of the vascular bed into the plasma.
The administration of basic PAF alone to a human or
animal by a method designed to bring the PAF into contact with
endothelial cells results in the production of tPA and is thus
embodied within the scope of the present invention. However,
it has been noted in some situations that administration of
basic PAF as the sole active ingredient in a pharmaceutical
preparation will stimulate not only tPA production but also
cell migration and mitogene6is, particularly in an area
denuded of live endothelial cells by myocardial infarction.
Thls additional result is also desirable because the migration
and division of new endothelial cells into the area of the
clot could restore endothelial monolayer in areas in the
occluded vessels where endothelial cells have died.
In addition, when combined with heparin, basic PAF
retains tPA inducing properties but loses its mitogenic
properties. Thus, administration of a pharmaceutical
preparation containing both basic PAF and heparin as active
ingredients results in a preparation which is contemplated for
use in alternative embodiments of this invention.
1 31 q61 0
Moreover, it is believed that the same effect, i.e., the
blocking of only the mitogenic properties of basic PAF, may be
achieved by combination of the basic PAF with various heparin
fragments. These heparin fragments are among those which are
capable of binding to the basic PAF.
It is postulated that the elimination of these properties
of basic PAF due to the presence of heparin or heparin frag-
ments might indicate that the protein which comprises the
basic PAF possesses at least two functions, one of which is
responsible for protease production and another of which pos-
sesses the mitogenic and migratory activity. Thus, it is
postulated that heparin is capable of inactivating the mito-
genic function without affecting the protease-production
function.
It is possible that these functions are imparted by
discrete and separable portions of the PAF molecule. In this
ca~e, it i5 also envisioned that the method of the present
invention could be practiced by administering a pharmaceutical
preparation whose active ingredient consists of the portion of
the basic PAF molecule which possesses an active protease
production function but which does not possess an active
mitogenic function.
The active composition of the various embodiments of the
present invention is preferably administered in a liquid form.
However, other administration formc, such as in inhalent mist,
are also envisioned. The preferred carrier is a physiologic
saline solution, but it is contemplated that other
pharmaceutically-acceptable liquid carriers may also be used.
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In one embodiment, it is preferred that the liquid carrier for
the basic PAF contain a "protein stabilizer." Preferably, the
protein stabilizer is albumin or heparin. A particularly
preferred stabilizer is plasma obtained from the patient who
is to receive the basic PAF.
The basic PAF may be formulated into a pharmaceutical
composition by combination of the basic PAF with a liquid
carrier as described above. Protein stabilizers and heparin
may be included in the initial formulation or may be added to
the preparation immediately prior to administration to the
patient.
Once the pharmaceutical preparation has been formulated,
it may be stored frozen or as a dehydrated or lyophilized
powder in sterile vials. It is preferred that a protein
stabllizer be added to the pharmaceutical preparation prior to
dehydration or lyophilization. Preferred storage is frozen at
at least -20C.
It i5 to be noted that it is preferred that the basic PAF
i~ both administered and ~tored in a formulation that has a
physiological pH. It i8 presently believed that storage and
administration at a high pH, i.e., greater than 10, or at a
low pH, i.e., less than 4, is undesirable.
It is presently pre~erred to administer the therapeutic
composition containing basic PAF via an intravenous route. A
preferred administration route includes the storage of basic
PAF at -20~C in sterile vials, either in the presence of
heparin or without. If without heparin, the heparin is added
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immediately subsequent to thawing and prior to administration
to the patient. In this preferred method, the frozen basic
PAF is thawed immediately prior to administration to the
patient. Upon thawing, a volume sufficient to suspend the
basic PAF, usually 1 ml, of the patient's plasma is added to
the basic PAF. This plasma will serve both to suspend the
basic PAF and to supply protein which will stabilize the
therapeutic material.
The desired dose of basic PAF may be administered by
bolus or by slow drip, either method intended to create a
predetermined concentration of the active ingredient in the
patient's blood supply. The specific dose is calculated
according to the body weight of the patient. It is noted that
the maintenance of circulating concentrations of PAF of less
than 0.5 nanograms (ng) per ml of plasma may not be an
e~ective therapy, while the prolonged maintenance of
circulating levels in excess of 5 micrograms (ug) per ml of
plasma may have undesirable side effects. Accordingly, it is
preferred that doses early in the therapy be administered on a
bolus such that circulating levels of PAF reach an initial
level of 1-2 micrograms per ml of plasma followed by doses
designed to keep the circulating level of PAF at or above
approximately 50 nanograms per ml of plasma. The time between
administration of the bolus and commencement of the
maintenance doses is dependent on the half-life of PAF in the
circulation. It is expected that the inclusion of heparin or
heparin fragments in the pharmaceutical composition will
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affect this parameter.
Further refinement of the calculations necessary to
determine the appropriate dosage for treatment involving each
of the above-me~tioned formulations are routinely made by
those of ordinary skill in the art and are within the ambit of
tasks routinely performed by them without undue
experimentation, especially in light of the dosage information
and assays disclosed herein. These dosages may be ascertained
through use of the established assays for determining dosages
utilized in conjunction with appropriate dose-response data.
It should also be noted that the basic PAF formulations
described herein may be used for veterinary applications, with
the dosage ranges being the same as those specified above for
humans.
It is understood that the application of teachings of the
present invention to a specific problem or environment will be
within the capab~lities of one having ordinary skill in the
art ln light of the teachings contained herein. Examples of
the products of the present invention and representative
processe~ for their preparation and use appear in the
~ollowing examples.
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EXAMPLE 1 - Induction of Tissue Plasminogen Activator Produc-
tion in Human Foreskin Capillary Endothelial
(HFCE) Cells by an Angiogenic Factor from a
Hepatoma Sonicate.
Isolation of human foreskin capillary endothelial (HFCE)
cells - Neonatal foreskins were obtained directly after
circumcision in the neonatal nursery. Each preparation was
incubated in Dulbecco's Modified Eagles Minimal Essential
Medium (DMEM) (obtained from Flow Laboratories (Medium Cat.
No. #10-331-22) p. 68 (1985)) with penicillin (100 U/ml) and
streptomycin (1 mg/ml) for 15 min, and the dermal tissue was
excised from the epidermis and minced using curved scissors
The tissue was digested with 0.75% (w/v) collagenase
(Worthington) in phosphate-buffered saline (PBS) containing
0.5% (w/v) bovine serum albumin (BSA) for 20 min at room
temperature. Medium with serum was added to stop the
digestion.
The digested tissue was gently aspirated with a 10 ml
pipette and passed through a Nitex* 110 micron mesh nylon-
covered funnel which allowed small aggregates of cells to pass
but retained larger pieces of tissue. The filtered material
was pelleted and gently resuspended in 3 ml of culture medium
consisting of 20% (v/v) heat-inactivated pooled human serum,
30% (v/v) medium conditioned by mouse sarcoma 180 cells, 50
ug/ml of endothelial cell growth supplement (ECGS)
(Collaborative Research), penicillin (10 U/ml), and
streptomycin (100 ug/ml) in DMEM (HFCE maintenance medium).
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Human serum samples were obtained from a hepatitis
testing laboratory. Serum samples from healthy donors taken
for routine screening were pooled and subjected to
centrifugation at 10,000 rpm in a Sorvall GSA rotor to remove
cells. When heat-inactivated serum was desired, the serum was
incubated at 56C for 30 min. The serum was then filtered
through a Nalgene 0.45 um filter and, if necessary, stored at
4~C until use.
A 1.5~ (w/v) solution of gelatin (Eastman Kodak) in PBS
was prepared and autoclaved. Aliquots of the gelatin solution
were added to tissue culture dishes several hours before
seed$ng the cells and allowed to incubate at room temperature.
The solution was aspirated and the dishes were washed with PBS
to remove excess gelatin.
The Nitex filtered material, resuspended in culture
medlum wlth inactivated human serum, was plated onto a 60 mm
gelatin-coated petri dish and cells were allowed to attach
overnlght. The surface was thoroughly washed with PBS and
fresh medium was added every other day thereafter. During
isolation, viable endothelial cells were recognized as
clusters of approximately 3-10 cells with characteristic
morphology which was easily distinguishable from fibroblasts.
Contaminating ~ibroblasts, wherever visible, were
mechanically scraped from the dish under direct microscopic
observation using a thin glass probe prepared by drawing a
glass pasteur pipette through a flame to produce a beaded
tip (approximately 0.1 mm). This technique was
l3lq6ln
carried out with the tissue culture dish on the stage of a
Wild inverted phase-contrast microscope in a laminar flow
hood. All cells at the periphery of the dish, i.e., out of
visual range, were removed by scraping with a silicone
spatula. The medium was then changed twice to remove floating
cells. This process was repeated occasionally as needed.
Colonies of endothelial cells were selected after several
weeks of culture using large cloning rings and the following
trypsinization techniques. Cells were washed with PBS and
incubated with 0.25~ (w/v) hog pancreas trypsin (ICN) in
0.14 M NaCl, 0.005 M KCl, 0.025 M Tris-HCl, pH 7.4, 0.002 M
EDTA for several minutes. The trypsinization was monitored by
phase contrast microscopy. When the cells became rounded and
detached from the dish (approximately 3 min.), the
trypsinization was stopped by the addition of equal or greater
volumes of medium with serum. Thereafter, the cells were
malntained as described above.
The cells were subsequently subcultured on 35 mm gelatin-
coated petri dishes at a dilution of 1:4.
~l~3~U3~h~l_f hepatoma sonicate - Cells from a
human hepatoma cell line, SK HEP-l cells (Accession No. HTB52,
American Type Culture Collection (ATCC), Rockville, Maryland),
were grown to confluence in 150 mm dishes in DMEM with 5%
fetal calf serum (FCS). The cells were then washed twice
with ice-cold PBS and scraped from the dish with a silicone
spatula. The cells were poole~ and sonicated for a total
of 3 min. on ice. The sonicate
131q~
was then clarified by centrifugation at 40,000 rpm in a
Beckman Ti50 rotor for 1 hr at 4C. The supernatant was
aliquoted and stored at -70OC until use.
Tetra decanoyl phorbol acetate (TPA~ treatment of cells -
Cell were grown to confluence in HFCE maintenance medium in 35
mm gelatin-coated dishes. Cultures were preincubated in DMEM
containing 15% human serum (i.e., without ECGS or S-180 cel
conditioned medium) for 24 hr. They were then incubated in
DMEM containing 15% human serum with the addition of 2 x 107 M
TPA for 18 hr. TPA-containing medium was prepared fresh by
diluting a 2 x 10 4 M stock solution in 100~ EtOH into the
medium and used immediately. For the measurement of
plasminogen activator (PA) in conditioned medium, TPA was
added to serum-free medium and the incubation and collection
were performed as described.
HePatoma sonicate treatment of cells - Cells were grown
to confluence in HFCE maintenance medium in 35 mm gelatin-
coated dishes. Cultures were preincubated in DMEM containing
15% human serum (i.e., without ECGS or S-180 cell conditioned
medium) for 24 hr. They were then incubated in DMEM
containing 15% human serum with the addition of 10% hepatoma
sonicate in PBS (final concentration 0.1 mg/ml) for 18 hr.
Hepatoma sonicate was prepared as described above and thawed
immediately before use.
Plasminoaen activator assav - Plasminogen activator was
assay by the ~2sI-fibrin plate method described by Unkeless et
al. in J. Exp. Med. 137: 85-111 (1973).
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The assays were preformed in 96-well Linbro trays with 3 cm2
surface area~well. Each well was coated with 30 ug of
plasminogen-free ~2sI-fibrinogen with a specific activity of
approximately 2000 cpm/ug. The plates were dried for 72 hr at
37C. Fibrinogen was converted to insoluble fibrin by the
addition of medium containing 2.5% serum as a source of
thrombin. After a 3 hr incubation, the wells were washed
twice with water and stored at 4C until use.
Conditioned medium and Triton* X-lOo detergent extracts
of cells were prepared and assayed essentially as described by
Gross et al. in J. Cell BIol. 9S: 974-981 (1982),
Dp~oi~ieally ineorporatod haroin b, rcfc~cc. Serum-free
conditioned medium was harvested and cellular debris was
removed by centrifugation at 2000 rpm in an IEC centrifuge
with a 284 rotor for 2 min. Monolayers were washed twice
with PBS, and the cells were scraped from the dish in 250 ul
of 0.5% triton in 0.1 M Tris-HCl, pH 8.1 using a silicon
spatula. Cell nuclei were removed by low-speed centrifugation
at 700 rpm for 10 min. Samples were stored at -20~C until
use.
Aliquots of cell extracts (l ug) or conditioned medium
(25 ul) were added to duplicate wells in 0.5 ml of 0.1 M Tris-
HCl buffer, pH 8.1, containing 4 ug of purified DFP-treated
human plasminogen (prepared from human plasma by the method of
DeutsCh and Mertz, as described in Science 170: 1095-1096
(1970)~ opcoiPio~y inoorporatcd hcrcin by rcfcrcncc~ and
0.025% BSA as
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carrier protein. The assay was incubated in a humidified atmos-
phere at 37C. One-hundred ul aliquots were removed from
duplicate wells at 2 hr and soluble 125I-fibrin degradation
products were counted in a Packard gamma scintillation counter.
Results are expressed as a percent of the total releasable
counts as measured by the addition of trypsin to duplicate wells
in each assay. Standard curves were prepared in each assay by
measuring the activities of a standard range of urokinase
samples. Samples were tested for plasminogen-independent
protease activity by the omission of plasminogen from the
incubation buffer.
Protein determinations - Protein determinations of cell
______________________
extracts were made by the Biorad Coomassie Blue staining
technique, using bovine serum albumin as a standard.
Plasmino~en activator production by HFCE cells - PA levels
________ __________________________________ _
were measured in both the cell extracts (cell-associated) and, in
some cases, serum-free conditioned medium of confluent cultures
Oe HFCE cells grown under various conditions. PA levels were
measured in many different isolates of HFCE cells, with the
results of 4 isolates shown in Fig. 1. Cell cultures were grown
to confluence under routine conditions as described above.
Before initiating an experiment, the cells were preincubated in
DMEM containing only 15% human serum for 24 hr. This was neces-
sary because the presence of ECGS in the growth medium elevated
levels of baseline PA activity in untreated cultures. The high
basal levels of PA resulted in a decrease in the calculated stim-
ulation by TPA and hepatoma sonicate to approximately 2-fold. A
24 hr preincubation in the absence of both ECGS and S-180 cell
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conditioned medium lowered the baseline PA activity. Therefore,at the start of the experiment, the culture medium was changed
to DMEM containing 15% human serum with or without TPA at 2 x
lO 7 M or hepatoma sonicate at 0.1 mg/ml. The cultures were
incubatea for 18 hours (overnight), and the next day the con-
ditioned medium was removed, and cell extracts were prepared for
PA assay as described above. The PA activity in l microgram of
cell protein was assayed in triplicate and routinely measured as
described above. Aliquots of the reaction mixture were measured
for soluble radioactivity after a 2 hr. incubation.
Numbers shown below the graph indicate different HFCE prim-
ary culture isolates. Human dermal fibroblasts, human umbilical
vein endothelial (HUVE~ cells, and bovine capillary endothelial
(BCE) cells were treated and assayed in a manner identical to the
lS HFCE cells. Open bar: control, untreated cultures. Hatched
bars: TPA-treated cultures. Closed bars: hepatoma sonicate-
treated cultures.
The concentrations of both TPA and hepatoma sonicate tested
were chosen because they were shown to give maximal stimulation
o~ PA in Bovine Capillary Endothelial (BCE) cell cultures as
described by Gross et al. in J. Cell Biol., supra; and Gross, et
, Proc Natl. Acad. Sci. 80: 2623-2627 (1983). Dose-response
assays showed that TPA at 2 x 107M and hepatoma sonicate at 0.1
mg/ml gave optimal stimulation in the HFCE cells.
2S Figure 1 shows the PA levels measured in extracts of cul-
tured HFCD cells. In each isolate tested, PA levels in untreated
cultures were relatively low compared to BCE cells. 30th TPA and
hepatoma sonicate produced an enhancement of PA activity
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over untreated control cultures in every isolate tested. The
degree of stimulation of PA activity varied between different
isolates but was always five- to fifteen- fold above the basal
levels of untreated cultures for both TPA and crude hepatoma
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sonicate-treated cells. The reason for this variation is
unknown. The increased fibrinolytic activity was plasminogen-
dependent; in the absence of plasminogen, no activity was
seen. BCE cells contained relatively high levels of PA in
untreated cultures and responded to treatment with TPA with
increased levels of PA.
The results of this experiment showed that HFCE cells
respond to both TPA and an angiogenic factor present in the
hepatoma sonicate with increases in cell-associated PA
activity. The present investigators have demonstrated that
the human hepatoma cells, SK HEP-1, produce an antiogenic
factor that is equivalent to PAF as characterized in the
pending Canadian application. It is concluded that the effect
of the hepatoma cell sonicate on the induction of PA in HFCE
cells could bs fully substituted by purified PAF.
EXAMPLE 2 Identification Of The PA Produced By HFCE
Cells In Response To Stimulation Of
Hepatoma Cell Sonicate As Tissue Type PA
~56-cvsteine labellina of cell cultures - Cells were grown
to confluence in standard maintenance medium. Control
cultures were also grown to confluence in standard medium.
These included RPMI-7272, a human melanoma cell line known to
produce high levels of tPA, as described by Rijkin, D.C. and
Collen, D., J. Biol. Chem. 256: 7035-7041 (1981), and human
embyronic lung cells, a cell strain known to produce high
levels o~ uPA and described by Rifkin in J. Cell Phys. 97:
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421-427 (1978). Cultures were preincubated with DMEM
containing 15~ human serum (i.e., ECGS and S-180 conditioned
medium were removed) for 24 hr. The cells were treated for 16
hr with or without TPA or hepatoma sonicate in DMEM containing
15% human serum. The cells were then preincubated in DMEM
without cysteine for 2 hr~ Finally, the confluent oultures of
HFCD cells were radiolabelled for 5 hr with 35S-cysteine (50
uCi/ml) in DMEM without cysteine containing 2% (v/v) dialyzed
pooled human serum.
10Immunopreci~itation - Cell extracts were prepared for
immunoprecipitation by a modification of the method described
by Stanley, J.R. et al. in Cell 24: 897-903 (1981),
~pcoifio llï inoorporae~ y~ ~. Conditioned
medium was harvested from the dishes and clarified by
15centrifugation at 2000 rpm for 5 min. The cell monolayexs
were washed 3 times with cold PBS, lysed in 250 ul of RIPA
buffer (0.05 M Tris-HCl, pH 7.2, containing 1% Triton X-100,
1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS),
0.15 M NaCl, 1 mM EDTA, 2 mM PMSF), scraped from the bottom of
the dish with a silicone spatula, left on ice for 10 min, and
clarified by centrifugation at 10,000 x g for 10 min. Before
specific immunoprecipitation, samples were preabsorbed using
non-immune rabbit serum and prctein A-Sepharose to reduce
nonspecific binding. Fifteen ul of normal rabbit serum were
incubated with the samples in a total of 1 ml in RIPA buffer
overnight at 4'C with mixing. Twenty ul of packed protein
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A-Sepharose beads were added with mixing for 90 min. at 4 C.
The pellets were collected by centrifugation in an Eppendorf
microfuge, and the supernatants were subjected to specific
immunoprecipitation. Immunoprecipitation was performed using
saturating amounts (20 ul) of rabbit antiserum to human
urokinase plasminogen activator (uPA), or to human tPA, for 16
hr at 4C. Twenty ul of packed protein A-Sepharose* beads
were added for 90 min at 4C and immune complexes on beads
were pelleted by centrifugation. The pellets were extensively
washed with RIPA buffer (five washes of one ml each) and H20
(twice), and boiled for 2 minutes in reducing sampl~ buffer
containing 5% 2-mercapoethanol and applied to 5-16% gradient
SDS-polyacrylamide gels as described by Laemmli, U.K. in
Nature 277: 6ao-685 (1970). Immediately after electro-
phoresis, the gels were fixed and processed for fluorography.
S~S-polvacrYlamide ~el electroPhoresis - SDS-
polyacrylamide gel electrophoresis was performed in a slab gel
apparatus using the discontinuous buffer system of Laemmli,
su~ra. The separating gel consisted of a linear 5 to 16%
acrylamide gradient: stac~ing gels were 3~ acrylamide.
Protein samples were mixed with equal volumes of 2X sample
buffer to a final concentration of 0.0625 M Tris-HCl, 10~
glycerol, 2% SDS, 0.001~ Bromophenol Blue, pH 6.8, and 5~ 2-
mercaptoethanol and boiled for 2 min. The following proteins
were used as molecular weight standards: B-galactosidase
(~r-130/000)/ phosphorylase A (~,=90,000), bovine
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serum albumin (Mr=68,000), aldolase (Mr=43,000), soybean
trypsin inhibitor (Mr=20,000), and lactalbumin (Mr=14,200).
Gels were fixed and stained using the silver stain method of
Wray et al., Anal. Biochem. 118: 197-20 (1981).
Fluoroqraphy - SDS-polyacrylamide gels of 35S-cysteine-
labelled proteins were processed according to the procedure of
Bonner and Laskey as described in Eur. J. Biochem. 46: 83-88
(1974). After PPO-DMSO impregnation, the dried gels were
exposed to preflashed Kodak XAR-5 film at -70C for 2 weeks.
Characterization of PA bv immuno~reci~itation - Two types
of PA have been described: tissue-type PA (tPA) and
urokinase-type PA (uPA). Each PA has a characteristic
molecular weight in SDS-polyacrylamide gels: tPA
approximately 66K daltons, uPA 50~ daltons.
Endothelial cells have been thought to be a source of
tPA. It has been shown to be produced by endothelial cells
cultured from human umbilical vein (Levin, E.G., Proc. Natl.
Acad. Sci. 80: 6804-6808 (1983) and bovine aorta (Levine and
Loskutof~, J. Cell Biol. 93: 631-635 (1982). However, these
cells were obtained from large vessels. Since the vast
majority of the endothelium is comprised of microvessel cells,
they may be an important source of tPA. Moscatelli, D.A., J.
Cell Biochem. 30: 19-29 (1986) characterized PA made by
bovine capillary endothelial cells under unstimulated
conditions as well as after
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1319610
22
stimulation with TPA or hepatoma sonicate. Using both
immunoprecipitation techniques and biochemical assays, he
showed the presence of tPA. Tissue-type PA was identified as
a broad band of fibrinolytically active material of Mr
approximately 66K to 93K daltons. After lengthy labelling and
incubation periods, a minor amount of uPA was identified.
Cell extracts and conditioned medium were also prepared from
cultures treated with TPA or hepatoma sonicate. It was shown
that there was a quantitative and not qualitative change in
the PA species produced. Levin and Loskutoff, (1982) supra
have also shown that bovine aortic endothelial cells produce
both types of PA, whose molecular weights are in agreement
with those described by Moscatelli.
Based on these results, there seems to be little
difference in the type of PA produced by bovine capillary
endothelial cells and bovine aortic endothelial cells. The
clrculating tPA ie proposed to be derived from both large
ves~el and microvessel endothelial cells, although the surface
area o~ the microvasculature is much larger and those cells
may be the ma~or source of tPA.
To characterize the type of PA produced by human
microvessel endothelial cells, ~FCE cells were grown to
confluence under standard maintenance conditions. They were
then treated with or without tPA or hepatoma sonicate.
Slxteen hours after treatment, the cells were radiolabelled
for 5 hrs with 35S-cysteine at 50 uCi/ml in the presence of tPA
or hepatoma sonicate as described above. Conditioned medium
131q610
23
was harvested and cell extracts were prepared and subjected to
specific immunoprecipitation with antiserum to tPA. Samples
were first preabsorbed with normal rabbit serum.
Immunoprecipitates were subjected to SDS-polyacrylamide gel
electrophoresis and fluorography as described.
As previously noted by the fibrin-plate assay, there
appears to be little PA in untreated HFCE cultures as seen by
only a faint band with an Mr in the range of 66K daltons. the
band was not present on the immunoprecipitate obtained with
preimmune serum. However, tPA is easily detected in the
immunoprecipitates of TPA-treated cultures as a broad band at
the molecular weight range of the RPMl-7272 standard (66K-
93R). There is also an increase in the amount of immuno-
precipitatable tPA in the hepatoma sonicate-treated cultures.
Thus tPA is present only in low amounts on the cell extracts
of untreated HFCE cells. There i~ an increase in the amount
of tPA ln TPA- or hepatoma sonicate-treated cultures as judged
by immunoprecipitation with antiserum raised against tPA.
Tissue-type PA was also immunoprecipitated from the
conditicned medium of untreated, TPA-treated, and hepatoma
sonicate-treated HFCE cells.
HFCE cell conditioned medium from untreated cultures
contains little or no discernible tPA as measured by
immunoprecipitation. However, tPA is seen as a broad band in
the molecular weight range of 66K to 93K daltons in
immunoprecipitated material from TPA-treated HFCE cell
conditioned medium. Hepatoma sonicate
1 31 961 0
also produced an increase in the amount of
immunoprecipitatable tPA in the conditioned medium o~ HFCE
cultures.
The tPA immunoprecipitated from both the cell extracts
and conditioned media showed the presence of a broad band
corresponding to an MrOf approximately 66K to 93K. The broad
range is similar to that obtained by Moscatelli su~ra for BCE
cells, Levin and Loskutoff, J. Cell Biol. 94: 631-636 (1982)
for bovine aortic endothelial cells, and Levin E.G., Proc.
Natl. Acad. Sci. 80: 6804-6808 (1983) for HUVE cells. These
high molecular weight forms of tPA have been shown to be due
to complexes formed between the tPA and an inhibitor of PA
which is also produced by the HWE cells (Levin, 1983 su~ra)
and bovine aortic endothelial cells (Loskutoff, et al., Proc.
Natl. Acad. Sci. 80: 2956-2960 (1983)). It is likely that the
high molecular weight forms of tPA seen here are also enzyme-
inhibitor complexes.
Immunopxecipitation of HFCE cell extracts and conditioned
medium was also performed using antiserum prepared against
urokinase-type PA (uPA). No radiolabelled proteins were
speci~ically immunoprecipitated from either the cell extracts
or conditloned medium of either untreated or treated cultures.
Human embryonic lung cells, known to produce urokinase, were
sub~ected to immunoprecipitation as a control.
The results were confirmed by fibrin autography according
to the method of Granelli-Piperno and Reich, J. Exp. Med. 148:
223-224 (1978.).
~ ,.
1 3 1 ~6 1 0
Aliquots of cell extracts and conditioned medium of both sti-
mulated and unstimulated HFCE cell cultures were subjected to
SDS-polyacrylamide gel electrophoresis and laid over
plasminogen-containing fibrin-agar gels. Lysis zones were
observed only at the molecular weight range of tPA, in the
range of ~6K to 93K daltons. No lysis was seen at lower
molecular weights, even with prolonged incubation times.
Therefore, it appears that tPA is produced by human
endothelial cells in culture in low amounts. Stimulation of
the cells by either TPA or hepatoma sonicate resulted in an
increase in PA in both the cell extract and conditioned
medium.
Urokinase-type PA activity in HFCE cells could not be
detected either by immunoprecipitation or by biochemical
assays of PA activity in fibrin-agar gels.
EXAMPLE 3 The effect of heparin on tissue plasminogen
acti~ator (tPA) stimulation in bovine capillary
endothelial (BCE) cells by human placental
angiogenic factor (hPAF).
Bovine capillary endothelial (BCE) cells were isolated
~rom the bovine adrenal cortex and grown as described pre-
viously by Gross et al., supra, specifically incorporated
herein by reference. The cells were grown in alpha modified
minimal essential medium (MEM) supplemented with 10% ~v/v)
cal~ serum and antibiotics (penicillin 10 U/ml and strep-
tomycïn, 100 ug/ml). Before assay, cells were passaged with
trypsin-EDTA as described in Example 1 onto 35 mm dishes and
allowed to grow to confluency.
131~610
Human placental angiogenic factor was isolated as
described in the pending Canadian application discussed above
with the following modification. After elution from heparin-
Sepharose, the active fractions were dialyzed against 0.2 M
NaCl, 20 mM MES pH 6.0, clarified by centrifugation at loO,000
g for 60 min and loaded on a FPLC-mono S column eqllilibrated
with the same buffer. The active protein was eluted with a
gradient of 0.2 to 0.7 M NaCl in 20 mM MES, pH 6Ø The
active fractions were determined by bio assay on BCE cells as
described previously by Moscatelli et al. in Proc. Natl. Aca.
Sci. 83: 2091-2095 (1986).
Plasminoqen Activator Assay - Confluent cultures of BCE
cells that had been maintained for at least two days in alpha
MEM in 5g6 calf serum were changed to fresh medium containing
different amounts of basic PA~, as determined by protein
assay, in the absence or presence of heparin (50 ~Ig/ml).
Heparin, porcine intestinal mucosa, grade II, 176 units/mg,
was purchased from Sigma tSt. Louis). After incubation at 37
for 16 hours in a humidified atmosphere of 10% C02, 90g6 air,
the cell layers were washed twice with cold phospate-buffered
saline (PBS) pH 7.5 and were extracted with 0.5% (v/v) Triton
X-100 in 0.1 M sodium phosphate pH 8.1 and the cell extracts
assayed for plasminogen activator (PA) activity as described
in Example 1.
In the absence of heparin, an increase in the
levels of PA in cell extracts was seen at doses
oî basic PAF of 0.3 ng/ml and
-- 26 --
r-~
1319610
10 ng/ml. The ED50, the half maximal response, was seen at
approximately 3 ng/ml. This corresponds to an increase from
approximately 4 ~inutes of urokinase activity to approximately
30 minutes of urokinase activity. In the presence of 50 ug/ml
heparin, there was a similar increase in the levels of PA with
increasing levels of basic PAF starting at O. 3 ng/ml and
continuing to 30 ng/ml. At the lowest doses of heparin
tested, even in the absence of basic PAF, there was an
increase in the base levels of PA production. However, there
was no substantial change in the ED50 nor was there any
significant difference in the amount of stimulation in the
presence of heparin once background levels were subtracted
when compared to the stimulation seen in the absence of
heparin.
Therefore, the inventors have concluded that heparin does
not block the ability of basic PAF to stimulate PA activity in
BCE cells. This contrasts with the reports of heparin
blocking the mitogenic effect of bovine basic fibroblast
growth factor, a molecule which is 98% homologous to basic
PAF. Bovine basic fibroblast growth factor is reported to
have virtually no mitogenic activity in the presence of
heparin when tested on several different types of endothelial
cells. Massoglia, et al., J. Cell Physiol. 27: 121-136
(1986).
- 27 -
1319610
EXAMPLE 4 Induction of Circulating Concentrations of
Tissue Plasminogen Activator by Intravenous
ABministration Of Placental Angiogenic Factor
Rabbits weighing 3.5 - 5.0 kg were anesthetized with a
combination of ketamine and xylazine during the course of the
experiments. ~lacental angiogenic factor (PAF) was
administered in a volume of 0.5-1.0 ml by injection into an
ear vein. Arterial blood samples were taken from the opposite
ear. Blood samples were collected immediately prior to
administration of the peptide and at various time points
thereafter. In a typical assay, blood samples would be
collected at 30 second intervals from the time of injection to
5 minutes following injection. Additional blood samples at
approximately 10 minutes post-injection were routinely taken.
Inhibitors of tissue plasminogen activator and of plasminogen
were prevented from associating with their target proteases by
acldi~ication of the blood samples immediately upon collection
u~ing a modlfication of the protocol described by B. Wiman,
3., et al. in Clinica Chimica Acta 127: 279-288 (1983).
3rie~1y, a 0.5 ml sample o~ blood was mixed immediately upon
drawing witn 0.4 ml of 1 . O M acetate buf f ered to pH 3.9 with
NaOH, and 0.05 ml of 3.2% w/v trisodium citrate. Samples were
centri~uged to separate cells from plasma and 0.1 ml of plasma
was added to an additional 0.11 ml of acetate buffer and 0.1
ml o~ 0.12 M Tris bu~er pH 8.7. This solution was then
incubated at 37-C for 20 minutes to
- 28 -
1 3 1 96 1 0
inactivate inhibitors of plasmin and plasminogen activator.
Twenty microliters of this solution was used in subsequent
plasminogen activator assays.
Tissue plasminogen activator was measured essentially as
described by Ranby M., et al, in Thrombosis Research 27: 743-
749 (1982). The chromogenic plasmin substrate S-2251 was
replaced in this assay by D-norleucyl-hexahydrotyrosyl-lysine-
para-nitroanilide, (Spectrozyme PL, from American Diagnostica
Inc., Greenwich, CT). Acidified and heat treated plasma
samples were diluted 1:40 in 0.12 M Tris buffer pH 8.7
containing purified plasminogen and the chromogenic plasmin
substrate. The reaction was initiated by the addition of des
A fibrin and continued at 37C for 1-20 hours depending on the
sensitivity required. Fibrin and plasminogen dependence of
the reaction were criteria for tPA activity.
Using this assay protocol, the in vivo effect of human
PAF was tested in rabbits. A single dose of 0.6 mg in
phosphate buffered saline was given. Circulating levels of
tPA rose in response to this dose to approximately twice the
unstimulated level within 3 minutes and remained elevated for
several minutes thereafter. This demonstrates that PAF can
act in vivo to elevate circulating levels of tPA. Further,
human PAF was, on a molar basis, more active in this regard
than either des-aminos-D-arginine8-vasopressin (DDAVP) or
bradykinin. Control rabbits were injected with phosphate-
bu~fered saline and showed no increase in circulating tPA
levels.
- 29 -
, .
1 3 1 q6 1 0
This demonstration that PAF is interacting with target
cells in the vascular bed and promoting a biological response
is consistent with the contention that the peptide is
biologically active in the circulation at least long enough to
stimulate its natural receptor. Furthermore, the in vitro
data show that capillary endothelial cells have a second,
sustained response in which tPA synthesis and secretion is
induced and continues even after the stimulus (PAF) is
removed. Therefore, it is e~pected that the present in vivo
demonstration of tPA release in response to PAF predicts the
ability of PAF to mediate the second longer term response as
well. The second response does not require the continued
presence of PAF but is sensitive to the dose and length of the
time that PAF is maintained in the circulation.
It will be apparent to those skilled in the art that
Various modi~ications and variations can be made to the
processes and products o~ the present invention. Thus, it is
intended that the present invention cover these modifications
and variations of this invention provided they come within the
scope o~ the appended claims and their equivalents.
