WO1988003030A1 - METHOD FOR INDUCING ENDOGENOUS PRODUCTION OF TISSUE PLASMINOGEN ACTIVATOR (tPA) - Google Patents

Abstract

Method for achieving elevated circulating plasmin levels and thus causing the dissolution of blood clots. 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

MEDTHOD FOR INDUCING ENDOGENOUS PRODUCTION

OF TISSUE PLASMINOGEN ACTIVATOR (tPA) BACKGROUND OF THE INVENTION The present invention relates to the induction of elevated levels of endogenous tissue plasminogen activator (tPA). Specifically, the present invention relates to the in¬ duction of these elevated tPA levels through the administration of a basic placenta angiogenic factor (PAF) .

Tissue plasminogen activator recently has been stud¬ ied as a potential therapeutic agent for the treatment of myocardial infarctions and certain other blood clotting disor¬ ders. In particular, it has been postulated that tPA will dis¬ solve the blood clots which occur in the coronary arteries dur¬ ing a myocardial infarction, thus re-opening the coronary arteries and re-establishing 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 is 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 the 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 com¬ plications, including stroke.

To overcome this problem of achieving sustained, ele¬ vated tPA levels in the circulatory system, the present inven¬ tors 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 OF 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 from practice 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 purposes 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 intend¬ ed that these levels remain elevated for 24-48 hours. In addi¬ tion, the basic PAF may be administered in conjunction with heparin, which, as explained more fully hereinbelow, will in¬ crease 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 cornary 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 poten¬ tial 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 levels Another embodiment of this invention includes the additional step (c) of preventing re-occlusion of the blood-carrying ves¬ sels by continued exposure to the elevated tPA levels.

It is to be understood that both the foregoing gen¬ eral 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 not 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, Dα_S_1.•C_ 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 two United States patent applications. These applications are U.S. Patent Application Serial No. 809,873, filed December 17, 1986, by Moscatelli et al. entitled "Human Placenta Angiogenic Factor Capable Of Stimulating Capillary Endothelial Cell Protease Synthesis, DNA Synthesis and Migration" and United States Patent Application Serial No. 888,554, filed on July 16, 1986, by Moscatelli et al. , entitled "Human Placenta Angiogenic Factor Of Stimulating Capillary Endothelial Cell Protease Synthesis, DNA Synthesis and Migration." Both of these United States Patent Applica¬ tions are specifically incorporated herein by reference. The compositions described by these patent applications will be referred to hereinafter as "basic PAF."

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 ves¬ sels to the elevated plasmin levels caused by these elevated tPA levels results in at least partial dissolution of the clots. In addition, reocclusin 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 cer¬ tain properties of the basic PAF.

Basic PAF, isolated in a purified form or manufac¬ tured through a recombinant-DNA method according to the

SUBSTITUTE SHEET procedures set forth in the Moscatelli et al. applications, supra, possesses at least three particular properties. These include (1) the ability to cause cell migration, (2) the abil¬ ity, in the presence of serum, to cause cell division (the mitogenic property), and (3) the ability to induce the synthe¬ sis 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 actiator 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 pro¬ duced in vivo by administration of PAF in the manner described more fully hereinbelow.

In addition, the present investigators have dis¬ covered 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 synthe¬ sis 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 mitogenesis, particularly in an area denuded of live endothelial cells by a myocardial infarction. This additional result is also desirable because the migration and division of new 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 prop¬ erties. 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 alterative embodiments of this invention. 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 prop¬ erties of basic PAF due to the presence of heparin or heparin fragments .might indicate that the protein which comprises the basic PAF possesses at least two functions, one of which is re¬ sponsible for protease production and another of which pos¬ sesses the mitogenic and migratory activity. Thus, it is pos¬ tulated that heparin is capable of inactivating the mitogenic 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 case, it is 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 pro¬ duction function but which does not possess an active mitogenic f nction.

The active composition of the various embodiments of the present invention is preferably administered in a liquid form. However, other administration forms, such as an 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. 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 pre¬ ferred 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 car¬ rier 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 stabilizer be added to the pharmaceutical preparation prior to dehydration or lyophilization. Preferred storage is frozen at least -20°C

It is to be noted that it is preferred that the basic PAF is both administered and stored in a formulation that has a physiological pH. It is 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 preferred 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 immediately subsequent to thawing and prior to adminis¬ tration 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 pre¬ determined concentration of the active ingredient in the pa¬ tient' 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 effec¬ tive 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 in 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 depen¬ dent 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-mentioned formulations are routinely made by those of ordinary skill in the art are within the ambit of tasks rou¬ tinely performed by them without undue experimentation, espe¬ cially 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 conjunc¬ tion with appropriate dose-response data.

It should also be noted that the basic PAF formula¬ tions 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 capabilities of one having ordinary skill in the art in light of the teachings contained herein. Examples of the products of the present invention and representative pro¬ cesses for their preparation and use appear in the following examples .

EXAMPLE 1 - Induction of Tissue plasminogen Activator

Production 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 in¬ cubated 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 ( orthington) in phosphate-buffered saline (PBS) containing 0.5% (w/v) bovine serum albumin (BSA) for 20 min at room tem¬ perature. 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) .

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 56°C for 30 min. The serum was then filtered through a Nalgene 0.45 urn 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 solu¬ tion were added to tissue culture dishes several hours before seeding 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 medium with inactivated human serum, was plated onto a 60mm gelatin-coated petri dish and cells were allowed to attach overnight. The surface was thoroughly washed with PBS and fresh medium was added every other day thereafter. During iso¬ lation, viable endothelial cells were recognized as clusters of approximately 3-10 cells with characteristic morphology which was easily distinguishable from fibroblasts. Contaminating fibroblasts, 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 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 spat¬ ula. 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 fol¬ lowing 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 KC1, 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 main¬ tained as described above.

The cells were subsequently subcultured on 35 mm gelatin-coated petri dishes at a dilution of 1:4.

Preparation of hepatoma sonicate - Cells from a human hepatoma cell line, SK HEP-1 cells (Accession No. HTB52, Ameri¬ can 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 pooled and sonicated for a total of 3 min on ice. The sonicate was then clarified by centrifugation at 40,000 rpm in a Beckman Ti50 rotor for 1 hr at 4°C. The supernatant was aliquoted and stored at -70°C until use.

Tetra decanoyl phorbol acetate (TPA) treatment of cells - Cells were grown to confluence in HFCE maintenance me¬ dium 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 2 x 10 -7 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 easurement of plasminogen activator (PA) in conditioned medi¬ um, TPA was added to serum-free medium and the incubation and collection were performed as described.

Hepatoma soniate treatment of cells - Cells were grown to confluence in HFCE maintenance medium in 35 mm gelatin—coated dishes. Cultures were preincubated in DMEM con¬ taining 15% human serum (i.e., without ECGS or S-180 cell con¬ ditioned 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.

Plasminogen activator assay - Plasminogen activator was assayed by the 125I-fibrin plate method described by

Unkeless et al. in J. Exp. Med. 137: 85-111 (1973), specifical¬ ly incorporated herein by reference. The assays were performed in 96-well Linbrotrays with 3 cm surface area/well. Each well was coated with 30 ug of plasminogen-free 125I-fibπnogen with a specific activity of approximately 2000 cpm/ug. The plates were dried for- 72 hr at 37°C. Fibrinogen was converted to in¬ soluble 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 4°C until use.

Conditioned medium and Triton X-100 detergent extracts of cells were prepared and assayed essentially as described by Gross et al. in J. Cell Biol. 95: 974-981 (1982), specifically icorporated herein by reference. Serum-free con¬ ditioned 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 silicone 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 (1 ug) or conditioned medi¬ um (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 -li¬

the method of Deutsch and Mertz, as described in Science 170: 1095-1096 (1970), specifically incorporated herein by refer¬ ence) and 0.025% BSA as carrier protein. The assay was incu¬ bated in a humidified atmosphere at 37°C. One-hundred ul ali- quots were removed from duplicate wells at 2 hr and soluble

125 I-fibrin degradation products were counted in a Packard gamma scintillation counter. Results are expressed as a per¬ cent 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.

Plasminogen 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 of 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 as described above. Before initiating an experiment, the cells were preincubated in DMEM containing only 15% human serum for 24 hr. This was nec¬ essary because the presence of ECGS in the growth medium ele¬ vated levels of baseline PA activity in untreated cultures. The high basal levels of PA resulted in a decrease in the cal¬ culated stimulation by TPA and hepatoma sonicate to approxi¬ mately 2-fold. A 24 hr preincubation in the absence of both ECGS and S-180 cell conditioned medium lowered the baseline PA activity. Therefore, at the start of the experiment, the cul¬ ture medium was changed to DMEM containing 15% human serum with or without TPA at 2 X 10 M or hepatoma sonicate at 0.1 mg/ml. The cultures were incubated overnight, and the next day the conditioned medium was removed, and cell extracts were prepared as described above.

The concentrations of both TPA and hepatoma sonicate tested were chosen because they were shown to give maximal stimulation of PA in Bovine Capillary Endothelial (BCE) cell cultures as described by Gross et al. in J. Cell Biol., supra; and Gross, et al. Proc. Natl. Acad. Sci. 80: 2623-2627 (1983), specifically incorporated herein by reference. Dose-response

_7 assays showed that TPA at 2 x 10 M and hepatoma sonicate at

0.1 mg/ml gave optimal stimulation in the HFCE cells.

Figure 1 shows the PA levels measured in extracts of cultured HFCE cells. In each isolate tested, PA levels in untreated cultures were relatively low compared to BCE cells. Both TPA and hepatoma sonicate produced an enhancement of PA activity over untreated control cultures in every isolate test¬ ed. 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 sonicate-treated cells. The reason for this variation is unknown. The increased fibrinolytic activity was plasminogen-dependent; in the absence of plasminogen, no activ¬ ity 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 activ¬ ity. The present investigators have demonstrated that the human hepatoma cells, SK HEP-1, produce an angiogenic factor that is equivalent to PAF as characterized in the two U.S. Pat¬ ent applications of Moscatelli et al. discussed above. It is concluded that the effect of the hepatoma cell sonicate on the induction of PA in HFCE cells could be 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

35S-cysteine labelling 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 lev¬ els of uPA and described by Rifkin in J. Cell Phys. 97: 421-427 (1978), specifically incorporated- herein by reference. Cul¬ tures 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 cultures of HFCE cells were radio- labelled for 5 hr with 35_g-cysteine (50 uCi/ml) in DMEM without cysteine containing 2% (v/v) dialyzed pooled human serum.

Immunoprecipitation - 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), specifically incorporated herein by reference. Conditioned me¬ dium was harvested from the dishes and clarified by centrifugation at 2000 rpm for 5 min. The cell monolayers 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 dodecylsulfate (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 protein 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 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 4°C. Twenty ul of packed protein A-Sepharose beads were added for 90 min at 4^βC 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 sample buffer containing 5% 2-mercaptoethanol and applied to 5-16% gradient

SDS-polyacrylamide gels as described by Laemmli, U.K. in Nature 277: 680-685 (1970), specifically incorporated, herein by refer¬ ence. Immediately after electrophoresis, the gels were fixed and processed for fluorography.

SDS-polyacrylamide gel electrophoresis - SDS—polyacrylamide gel electrophoresis was performed in a slab gel apparatus using the discontinuous buffer system of Laemmli, supra. The separating gel consisted of a linear 5 to 16% acrylamide gradient; stacking 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 molecu¬ lar weight, standards: B-galactosidase (M =130,000), phosphorylase A (M =90,000), bovine serum albumin (M =68,000), aldolase (M =43,000), soybean trypsin inhibitor (M =20,000), and lactalbumin (M =14,200). Gels were fixed and stained using the silver stain method of Wray et al. , Anal. Biochem. 118 197-20 (1981).

Fluorography - SDS-polyacrylamide gels of

35 S-cysteme-labelled proteins were processed according to the procedure of Bonner and Laskey as described in Eur. J. Biochem. 46: 83-88 (1974), specifically incorporated herein by refer¬ ence. After PPO-DMSO impregnation, the dried gels were exposed to preflashed Kodak XAR-5 film at -70°C for 2 weeks.

Characterization of PA by immunoprecipitation - Two types of PA have been described: tissue-type PA (tPA) and urokinase-type PA (uPA). Each PA has a characteristic molecu¬ lar weight in SDS-polyacrylamide gels: tPA approximately 66K daltons, uPA 50K 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 Loskutoff, J. Cell Biol. 93: 631-635 (1982). However, these cells were obtained from large vessels. Sice 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 cap¬ illary 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 M approxi¬ mately 66K to 93K daltons. After lengthy labelling and incuba¬ tion periods, a minor amount of uPA was identified. Cell extracts and conditioned medium were also prepared from cul¬ tures 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) supr 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 dif¬ ference in the type of PA produced by bovine capillary endothelial cells and bovine aortic endothelial cells. The circulating tPA is proposed to be derived from both large ves¬ sel and microvessel endothelial cells, although the surface area of the microvasculature is much larger and those cells may be the major source of tPA.

To characterize the type of PA produced by human microvessel endothelial cells, HFCE cells were grown to conflu¬ ence under standard maintenance conditions. They were then treated with or without tPA or hepatoma sonicate. Sixteen hours after treatment, the cells were radiolabelled for 5 hr with 35S-cysteine at 50 uCi/ml in the presence of tPA or hepatoma sonicate as described above. Conditioned medium was harvested and cell extracts were prepared and subjected to spe¬ cific 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 assays, there appears to be little PA in untreated HFCE cultures as seen by only a faint band with an M in the range of 66K daltons. The band was not present in 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 RPM1-7272 standard (66K-93K) . There is also an increase in the amount of immunoprecipitatable tPA in the hepatoma sonicate—treated cultures. Thus tPA is present only in low amounts in the cell extracts of untreated HFCE cells. There is an increase in the amount of tPA in TPA-or hepatoma sonicate-treated cultures as judged by immunoprecipitation with antiserum raised against tPA.

Tissue-type PA was also immunoprecipitated from the conditioned 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 condi¬ tioned medium. Hepatoma sonicate also produced an increase in the amount of immunoprecipitatable tPA in the conditioned medi¬ um of HFCE cultures.

The tPA immunoprecipitated from both the cell extracts and conditioned media showed the presence of a broad band corresponding to an M of approximately 66K to 93K. The broad range is similar to that obtained by Moscatelli supra 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 HUVE cells (Levin, 1983 supra) 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. Immunoprecipitation of HFCE cell extracts and condi¬ tioned medium was also performed using antiserum prepared against urokinase-type PA (uPA). No radiolabelled proteins were specifically immunoprecipitated from either the cell extracts or conditioned medium of either untreated or treated cultures. Human embryonic lung cells, known to produce urokinase, were subjected 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), specifically incorporated herein by reference. Aliquots of cell extracts and conditioned medium of both stimulated and unstimulated HFCE cell cultures were sub¬ jected to SDS-polyacrylamide gel electrophoresis and laid over plasminogen-containing fibrin-agar gels. Lysis zones were ob¬ served only at the molecular weight range of tPA, in the range of 66K 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 in¬ crease 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 activator (tPA) stimulation in bovine capillary endothelial (BCE) cells by human placental angiogenic factor (hPAF) .

Bovine capillary endothelial (BCE) cells were iso¬ lated from the bovine adrenal cortex and grown as described previously 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) calf serum and antibiotics (penicillin 10 U/ml and streptomycin, 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.

Human placental angiogenic factor was isolated as described in the two U.S. Patent applications of Moscatelli e_t al. 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 100,000 g for 60 min and loaded on a FPLC-mono S column equilibrated 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 6.0. 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), specifically incorporated herein by reference.

Plasminogen Activator Assay - Confluent cultures of BCE cells that had been maintained for at least two days in alpha MEM in 5% calf serum were changed to fresh medium con¬ taining different amounts of basic PAF, as determined by pro¬ tein assay, in the absence or presence of heparin (50 mg/ml) . Heparin, porcine intestinal mucosa, grade II, 176 units/mg, was purchased from Sigma (St. Louis) . After incubation at 37° for 16 hours in a humidified atmosphere of 10% CO«, 90% air, the cell layers were washed twice with cold phosphate-buffered sa¬ line (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 of basic PAF of 0.3 ng/ml and 10 ng/ml. The ED__n, the half maximal response, was seen at approximately 3 ng/ml. This corresponds to an increase from approximately 4 munits of urokinase activity to approxi¬ mately 30 munits 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 0.3 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 substan¬ tial change in the ED-.- nor was there any significant differ¬ ence 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 activ¬ ity 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 vir¬ tually 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. Placental angiogenic factor (PAF) was adminis¬ tered 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 typi¬ cal assay, blood samples would be collected at 30 second inter¬ vals from the time of injection to 5 minutes following injec¬ tion. 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 acidification of the blood samples immediately upon collection using a modification of the protocol described by B. Wiman, B., et al . in Clinica Chimica Acta 127: 279-288 (1983), specifically incorporated herein by reference. Briefly, a 0.5 ml sample of blood was mixed immediately upon drawing with 0.4 ml of 1.0 M acetate buffered to pH 3.9 with NaOH, and 0.05 ml of 3.2% w/v trisodium citrate. Samples were centrifuged 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 of 0.12 M Tris buffer pH 8.7. This solution was then incubated at 37^βC 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), specifically incorporatead herein by reference. 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 reac¬ tion was initiated by the addition of des A fibrin and con¬ tinued at 37°C for 1-20 hours depending on the sensitivity re¬ quired. 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-amino-D-arginine -vasopressin (DDAVP) or bradykinin. Control rabbits were injected with phosphate-buffered saline and showed no increase in circulating tPA levels.

This demonstration that PAF is interacting with tar¬ get cells in the vascular bed and promoting a biological re¬ sponse 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 expected that the present in vivo demonstration of tPA release in response to PAF predicts the ability of PAF to medi¬ ate the second longer term response as well. The second re¬ sponse does not require the continued presence of PAF but is sensitive to the dose and length of the time that PAF is main¬ tained in the circulation. It will be apparent to those skilled in the art that various modifications and variations can be made to the pro¬ cesses and products of the present invention. Thus, it is in¬ tended that the present invention cover these modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Claims

WHAT IS CLAIMED IS

1. A method for elevating endogenous tissue plas¬ minogen activator (tPA) levels in the blood stream comprising administering a basic placenta angiogenic factor (PAF) at a level and for a time sufficient to cause an increase in the circulating tissue plasminogen activator levels.

2 . The method of claim 1 wherein said circulating tissue plasminogen activator levels are elevated to a concen¬ tration of at least 15 nanograms per milliliter of plasma.

3. The method of claim 2 wherein said circulating tissue plasminogen activator levels are elevated to a concen¬ tration of at least 50 nanograms per milliliter of plasma.

4. The method of claim 1 wherein the basic PAF is administered in conjunction with heparin.

5. The method of claim 2 wherein the basic PAF is administered in conjunction with heparin.

6. The method of claim 3 wherein the basic PAF is administered in conjunction with heparin.

7. A method for the treatment of myocardial infarctions involving at least a partial dissolution of a por¬ tion of a blood clot formed in a coronary artery or in other blood-carrying vessel comprising:

(a) administering basic placenta angiogenic (PAF) factor at a dosage sufficient to result in an elevated, circulating tissue plasminogen activator (tPA) level capable of increasing plasmin formation; and

(b) at least partially dissolving a portion of the blood clot present in blood-carrying vessel by exposure to the increased plasmin levels produced by the elevated tPA level.

8. The method of claim 7 which further comprises:

(c) preventing re-occlusion of the blood- carrying vessels by continued exposure to the increased circulating plasmin levels.

9. The method of claim 8 wherein circulating basic PAF levels are maintained at at least 50 nanograms PAF per milliliter plasma.

10. The method of claim 9 wherein the circulating basic PAF levels are maintained at at least 1-2 micrograms PAF per milliliter plasma.

11. The method of claim 7 wherein the basic PAF is administered in conjunction with heparin.

12. The method of claim 8 wherein the basic PAF is administered in conjunction with heparin.

13. The method of claim 7 wherein the basic PAF is administered in conjunction with a protein stabilizer.

14. The method of claim 8 wherein the basic PAF is administered in conjunction with a protein stabilizer.

SUBSTITUTESHEET