CA1319610C - Method for inducing endogenous production of tissue plasminogen activator (tpa) - Google Patents

Abstract

ABSTRACT OF THE INVENTION

A method for achieving elevated circulating plasmin levels and thus causing the dissolution of blood clots is disclosed.
This method is particularly suited for the treatment of myocardial infarctions and other clotting disorders. This method involves in two phases the elevation of endogenous, circulating tissue plasminogen activator (tPA) levels through the administration of basic placenta angiogenic factor (PAF). In the first phase, a relatively high circulating concentration of basic PAF effects the release of tPA from sites in the vascular bed into the circulation. In the second phase, lower circulating concentrations are maintained for a period of time necessary to induce the synthesis and secretion of tPA. This induction will elevate the circulating concentrations of tPA, thus causing increases in the circulating levels of plasmin, an enzyme capable of dissolving blood clots. A method for accomplishing similar purposes is also disclosed which involves the administration of basic PAF in conjunction with heparin as the active ingredients in a pharmaceutical preparation.

Description

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 ',.~

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 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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131q610 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."

131q610 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 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 1 3 1 q6 1 0 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 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.

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 * TRADE MARK

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131q610 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 A

131q610 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 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 -17a-,'~
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131q610 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:

1 3 1 q6 1 0 421-427 (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 13196~0 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 * TRADE MARK

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 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 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 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.).

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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 r-~

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).

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 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.

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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.

Claims

Claim:

1. 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 sufficient to stabilize the PAF.