Gene encoding an inhibitor of tissue-type and urokinase-type plasminogen activators

A biological reagent and diagnostic system for the detection and quantitation of endothelial plasminogen activator inhibitor (PAI) are disclosed, as are substantially pure, recombinant human endothelial PAI, its biologically pure gene and a vector for cloning the gene and expressing a gene product.

TECHNICAL FIELD 
The present invention relates to a biochemical reagent system including a 
receptor and an indicating means that recognizes and selectively binds to 
a plasminogen activator inhibitor, and more particularly relates to 
biochemical reagent and diagnostic systems for the detection and 
quantitation of beta-migrating endothelial cell plasminogen activator 
inhibitor in blood and other biological samples, as well as to the human 
beta-migrating, endothelial cell plasminogen activator inhibitor in 
substantially pure form, and its gene. 
BACKGROUND OF THE INVENTION 
Endothelial cells line the luminal surface of the vascular bed and are 
thought to play an active role in the specific proteolytic breakdown of 
locally deposited fibrin, Todd, J. Pathol. Bacteriol., 78, 281 (1959); 
Astrup, in Progress in Chemical Fibrinolysis and Thrombolysis, Davidson et 
al. eds., vol. 3, pp. 1-57, Raven Press, New York (1978). The potential of 
endothelium to initiate and control this process is emphasized by its 
capacity to synthesize and release plasminogen activators (PAs), Loskutoff 
et al., Proc. Natl. Acad. Sci. (USA), 74, 3903 (1977); Shepro et al., 
Thromb. Res., 18, 609 (1980); Moscatelli et al., Cell, 20, 343 (1980); 
Laug, Thromb. Haemostasis, 45, 219 (1981); Booyse et al., Thromb. Res., 
24, 495 (1981), including both tissue-type and urokinase-type molecules, 
Levin et al., J. Cell Biol., 94, 631 (1982); Loskutoff et al., Blood, 62, 
62 (1983). Endothelial cells can also produce inhibitors of fibrinolysis, 
Loskutoff et al., Proc. Natl. Acad. Sci. (USA), 74, 3903 (1977); Levin et 
al., Thromb. Res., 15, 869 (1979); Loskutoff et al., J. Biol. Chem., 256, 
4142 (1981); Dosne et al., Thromb. Res., 12, 377 (1978); Emeis et al., 
Biochem. Biophys. Res. Commun., 110, 392 (1983); Loskutoff et al., Proc. 
Natl. Acad. Sci. (USA), 80, 2956 (1983); Levin, Proc. Natl. Acad. Sci. 
(USA), 80, 6804 (1983). 
Although these inhibitors probably serve important regulatory roles in 
controlling the fibrinolytic system of the vascular wall, little is known 
about their specificity, mode of action, or biochemical nature. The 
conclusion that these inhibitors are actually synthesized by endothelial 
cells is obscured somewhat by recent reports that cultured cells can bind 
and internalize protease inhibitors from serum-containing culture medium, 
Cohen, J. Clin. Invest., 52, 2793 (1973); Pastan et al., Cell, 12, 609 
(1977); Rohrlich et al., J. Cell Physiol., 109, 1 (1981); McPherson et 
al., J. Biol. Chem., 256, 11330 (1981). 
The possibility of producing relatively unlimited amounts of tissue-type 
plasminogen activator (t-PA) by recombinant DNA technology as described in 
British patent application GB No. 2,119,804 A, published Nov. 23, 1983, 
has generated much interest, both clinically and commercially. The 
conversion of the relatively inactive molecule into an extremely efficient 
thrombolytic agent by fibrin itself, suggests that t-PA can exist as an 
active enzyme only when localized to the fibrin-platelet thrombus itself. 
Thus, t-PA is considered to be a much more specific thrombolytic agent 
than urokinase-type plasminogen activator and streptokinase. 
The interactions between t-PA and fibrin have raised the argument that 
natural inhibitors of t-PA are not necessary to regulate this system; 
i.e., regulation is achieved through the formation/dissolution of fibrin, 
and, thus, do not exist. It is clear that the existence of such inhibitors 
in human blood would complicate attempts to design a specific, efficient, 
and safe thrombolytic program based upon natural and genetically 
engineered t-PA. At the very least, calculations such as those of dose, 
treatment time and efficacy of treatment would be difficult to predict 
and/or monitor. This problem would be especially acute if inhibitor levels 
varied from individual to individual. 
The existence of specific inhibitors of t-PA in plasma is a matter of some 
dispute, Collen, Thromb. Haemostas., 43, 77 (1980). In fact, it has been 
reported, Korninger et al., Thromb. Haemostas., 46, 662 (1981), that the 
activity of t-PA added to plasma had an in vitro half-life of 90 minutes 
as compared to an in vivo half-life of 2 minutes, Korninger et al., 
Thromb. Haemostas, 46, 658 (1981). Based upon these observations, those 
authors concluded that t-PA inhibition by plasma was physiologically 
unimportant. 
That conclusion has recently been challenged in Kruithof et al., Prog. in 
Fibrinolysis, 6, 362 (1983). In Chmielewska et al., Thromb. Res., 31, 427 
(1983), direct evidence was recently reported for the existence of a rapid 
inhibitor of t-PA in plasma. In all cases, this anti-t-PA activity was 
detected in the plasma of patients with or at risk to develop thrombotic 
problems; i.e., the very individuals most likely to receive t-PA therapy. 
This finding may account for the failure of Korninger et al., Thromb. 
Haemostas., 46, 662 (1981), to detect such an activity since they only 
examined the plasma of "normal" individuals. These reports on t-PA 
inhibitors represent little more than qualitative descriptions of an 
"activity" detected in the blood of some individuals. 
Recently, an antifibrinolytic agent in cultured bovine endothelial cells 
was detected, Loskutoff et al., Proc. Natl. Acad. Sci. (USA), 80, 2956 
(1983). This inhibitor is a major endothelial cell product and is an 
inhibitor of plasminogen activator since it can neutralize the activity of 
both fibrin-independent (urokinase-type) and fibrin-dependent 
(tissue-type) plasminogen activators (PAs). The observation that human 
platelets contain an immunologically similar inhibitor, Erickson et al., 
Haemostasis, 14 (1), 65 (1984) and J. Clin. Invest. 74, 1465 (1984), that 
is released by them in response to physiologically relevant stimuli, e.g., 
thrombin, and in parallel with other platelet proteins, e.g., Platelet 
Factor 4, emphasizes the potential importance of this inhibitor in human 
biology. Antiserum to the plasminogen activator inhibitor (PAI) from 
bovine aortic endothelial cells (BAEs) has been employed to show that the 
human endothelial PAI as well as that from plasma, serum and platelets are 
related, i.e., immunologically similar. Erickson et al., Proc. Natl. Acad. 
Sci. USA, 82, 8710 (1985). 
The inhibitor found by Loskutoff et al., Proc. Natl. Acad. Sci. (USA), 80, 
2956 (1983), was purified from bovine aortic endothelial cell conditioned 
media by a combination of concanavalin A affinity chromatography and 
preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis 
(SDS-PAGE), and was shown to be a single chain glycoprotein of a molecular 
weight of 50,000 daltons, having an isoelectric point of 4.5-5 [van Mourik 
et al., J. Biol. Chem. 259, 14914 (1984)]. 
Recent evidence indicates that there are three immunologically distinct 
plasminogen activator inhibitors (PAIs). The first is that discussed above 
that is derived primarily from endothelial cells. The second, reported by 
Astedt et al., Thromb. Haemostasis, 53, 122 (1985) was isolated from 
placenta. The third, reported by Scott et al., J. Biol. Chem., 260 7029 
(1985) is protease nexin. 
The endothelial cell type PAI differs, in addition to immunologically, from 
placental PAI and protease nexin in that it inhibits both single chain and 
two chain tissue-type plasminogen activator (t-PA) as well as 
urokinase-type plasminogen activator (u-PA), while protease nexin and the 
placental PAI exhibit substantially no t-PA inhibition at physiological 
concentrations. Those latter two inhibitors do inhibit u-PA activity at 
physiological concentrations. Still further, endothelial PAI exhibits 
beta-mobility when analyzed by agarose zone electrophoresis while the 
other two PAIs do not. In addition the endothelial cell PAI is stable to 
low pH values (e.g. pH 3) and SDS (0.1%), while the other two inhibitors 
are rapidly inactivated by either of these treatments. [van Mourik et al., 
J. Biol. Chem., 259, 14914 (1984)]. 
The results discussed hereinafter illustrate that the endothelial cell type 
PAI exhibiting beta-mobility is also present in human placental extracts 
as is the placenta PAI reported by Astedt et al., Thromb. Haemostasis, 53, 
122 (1985). Since two types of PAI are obtainable from placenta, the human 
PAI hereinbefore referred to as of endothelial cell origin will usually be 
referred to as beta-PAI or endothelial PAI, or endothelial cell type PAI 
while the PAI first isolated from placenta is referred to as 
placental-type PAI or placental PAI. 
SUMMARY OF THE INVENTION 
The present invention contemplates a biochemical reagent system and methods 
of preparing and using same, diagnostics utilizing the reagent system, a 
method for detecting PAI, a substantially pure recombinant proteinaceous 
molecule that is immunologically similar to human endothelial cell type 
plasminogen activator inhibitor and has the binding and inhibiting 
activities of human beta-migrating, endothelial cell plasminogen activator 
inhibitor, and its gene. 
The biochemical reagent system comprises (a) a receptor such as an antibody 
raised in an animal host to endothelial cell plasminogen activator 
inhibitor; i.e., an anti-plasminogen activator inhibitor, and (b) an 
indicating means. In one aspect of the invention, the biochemical reagent 
system is comprised of (a) a receptor that can be a polyclonal antibody 
raised in an animal host and (b) an indicating means. The indicating means 
and receptor can be a single molecule or can be composed of a plurality of 
individual molecules. The receptor binds to endothelial cell 
(beta-migrating) plasminogen activator inhibitor that itself binds to and 
inhibits tissue-type or urokinase-type plasminogen activators. The 
indicating means labels the receptor, and in so doing indicates the 
presence of the inhibitor in a sample to be assayed such as serum of 
patients having thrombotic disease. The receptor of the reagent system of 
the present invention selectively binds to endothelial cell plasminogen 
activator inhibitor bound to either tissue-type (t-PA) or to 
urokinase-type (u-PA) plasminogen activators. 
In another aspect of the present invention, a method of forming a 
polyclonal receptor for use in a biochemical reagent system is 
contemplated. The method comprises the steps of: (a) administering to an 
animal host endothelial cell type plasminogen activator inhibitor (PAI) in 
an amount sufficient to induce the production of antibodies to the 
inhibitor, the antibodies being a receptor for the inhibitor; (b) 
collecting antisera containing the antibodies from the immunized host; and 
(c) recovering the receptor from the antisera. 
Yet another aspect of the present invention relates to a method of forming 
a biochemical reagent system. The method comprises the steps of forming 
the polyclonal receptor described above as steps (a)-(c) with an 
additional step (d) of combining the receptor with an indicating means. 
Both of the above methods can also include the step of administering to the 
host after step (a) and a sufficient period of incubation (maintenance) of 
the host, e.g., 1-2 weeks, but before step (b), a second injection of the 
same inhibitor to boost the production of antibody. 
The present invention also contemplates polyclonal receptors produced by 
the above-described method. 
In a further aspect of the present invention, a solid phase assay method of 
detecting the presence and quantity of endothelial cell type plasminogen 
activator inhibitor in a sample to be assayed is contemplated. The method 
comprises the steps of: (a) providing a solid matrix on which to assay the 
sample; (b) affixing on the solid matrix a binding reagent that binds to 
(complexes with) the inhibitor to form a solid phase support, the binding 
reagent being a plasminogen activator selected from the group consisting 
of t-PA and u-PA or the above-described polyclonal receptor; (c) admixing 
an aliquot of a liquid sample to be assayed with the solid phase support 
to form a solid-liquid phase admixture; (d) maintaining the admixture for 
a predetermined time sufficient for the binding reagent to bind to 
(complex with) any of the plasminogen activator inhibitor present in the 
sample; (e) separating the solid and liquid phases; and (f) determining 
the presence of inhibitor that bound to (complexed with) the binding 
reagent. 
In preferred practice, the quantity of inhibitor bound to the binding 
reagent is determined by (i) admixing an aqueous liquid solution of second 
binding reagent that binds to the inhibitor bound on the solid support 
with the solid phase obtained after step (e) above to form a second 
solid-liquid phase admixture, the second binding reagent complexing with 
the inhibitor; (ii) maintaining the second solid-liquid admixture for a 
predetermined time sufficient for the second binding reagent to bind (form 
a complex) with the inhibitor (typically about 2 to about 4 hours); (iii) 
separating the solid and liquid phases of the second solid-liquid phase 
admixture; and (iv) determining the quantity of the second binding reagent 
that bound to the inhibitor, and thereby determining the quantity of 
inhibitor. 
The present invention further includes a mammalian diagnostic system such 
as a kit. The kit includes at least one package containing as an active 
ingredient the biochemical reagent system of this invention and t-PA or 
u-PA. The biochemical reagent system comprises a polyclonal receptor in 
dry, solution, or dispersion form, that, when admixed with an indicating 
means and a sample to be assayed, binds selectively to endothelial 
plasminogen activator inhibitor (PAI) present in the sample and indicates 
the presence and amount of the inhibitor. Indicating groups that can be 
contained in the system include a radioactive element, a biologically 
active enzyme, or an NMR-active element. 
The diagnostic system can also include a solid matrix that can be a 
microtiter strip such as that containing twelve wells in a row. The t-PA 
or u-PA present is preferably bound to the solid matrix. 
The diagnostic system can further include a standard against which to 
compare the assay results, as well as various buffers in dry or liquid 
form, for, inter alia, washing the wells, diluting the sample or diluting 
the labeled reagent. 
The use of a biochemical reagent system of this invention includes the 
detection and quantitation of a specific plasminogen activator inhibitor 
that is bound to (complexed with) a plasminogen activator such as 
tissue-type or urokinase-type plasminogen activator. An especially 
preferred use of such a reagent system relates to the detection of 
plasminogen activator inhibitor in an in vitro protocol. 
Still another aspect of the present invention is a substantially pure, 
recombinant proteinaceous molecule, antibodies to which immunoreact with 
human endothelial cell type plasminogen activator inhibitor. More 
preferably, that recombinant molecule also binds to and inhibits the 
activities of at least t-PA, and most preferably binds to and inhibits the 
activities of both t-PA and u-PA. The recombinant molecule is 
immunologically different from protease nexin and placental PAI. In one 
embodiment, the recombinant molecule is substantially free of 
polypeptide-linked glycosyl groups, while in another embodiment the 
inhibitor contains polypeptide-linked glycosyl groups. In one 
non-glycosylated embodiment, the recombinant molecule exhibits an apparent 
relative molecule mass (M.sub.r) of about 180 kildoaltons (kda) in 
SDS-PAGE analysis as a fusion polypeptide, while in another 
non-glycosylated embodiment the M.sub.r is about 40 kda. 
It is seen from the above discussion that the recombinant molecule need not 
have the full biological activity of the naturally occurring (native) 
human endothelial cell type PAI. While it is preferred that the molecule 
have biological activity of its native homolog and bind to as well as 
inhibit at least t-PA, the recombinant molecule is also useful because of 
its immunological similarity to the native protein and thus its ability to 
induce secretion of receptor molecules that cross-react with the native 
protein, as is discussed hereinafter. 
Another aspect of the present invention is a biologically pure DNA molecule 
containing about 1140 to about 3000 nucleotides and including a 
nucleotide sequence that consists essentially of a nucleotide sequence, 
from left to right and in the direction from 5'-terminus to 3'-terminus, 
corresponding to the sequence represented by the formula in FIG. 22 from 
nucleotide position 13 to about 1153, and in a consistent reading frame 
coding for human endothelial cell type plasminogen activator inhibitor. I 
another embodiment, the DNA molecule sequenc corresponds to that from 
position 1 to about position 1960. In still another embodiment, the DNA 
molecule sequence corresponds to that from position 1 to about position 
1153. Yet another embodiment the DNA sequence corresponds to the entire 
DNA sequence shown in FIG. 22. 
A non-chromosomal vector for cloning DNA in a replication/expression medium 
comprising a replicon compatible with the medium and containing a 
before-described DNA molecule in such a manner that the vector can 
propagate the DNA molecule. The vector preferably further includes a 
transcriptional promoter that is operatively linked to the contained DNA 
molecule adjacent to the 5'-terminus of the DNA molecule, and compatible 
with the replication/expression medium for expressing a product coded for 
by the DNA molecule that includes a recombinant, human endothelial cell 
type plasminogen activator inhibitor. More preferably, the vector still 
further includes a translation initiating codon and a translation 
terminating codon, both of which are operationally linked to the contained 
DNA molecule adjacent to the 5'-terminus and the 3'-terminus thereof, 
respectively, and compatible with the replication/expression medium for 
expressing a product coded for by the DNA molecule. 
A solid phase assay method of detecting the presence and quantity of 
endothelial cell type human plasminogen activator inhibitor in a sample to 
be assayed constitutes yet another aspect of this invention. Here, a solid 
phase support is provided comprising a solid matrix to which the 
substantially pure, recombinant proteinaceous molecule (described before) 
is affixed. An aliquot of a liquid sample to be assayed is admixed with a 
predetermined amount of a binding reagent that binds to (complexes with) 
both the recombinant molecule of the solid support and the inhibitor to be 
assayed. That admixture is maintained under biological assay conditions 
and for a predetermined period of time sufficient for the binding reagent 
to bind to any of the inhibitor present in the sample. That admixture is 
admixed with the solid support to form a solid-liquid phase admixture. 
That admixture is maintained under biological assay conditions for a 
predetermined period of time sufficient for any binding reagent of the 
admixture not bound to inhibitor molecules of the sample to bind to the 
recombinant inhibitor of the solid support. The solid and liquid phases 
are thereafter separated, and the amount of binding reagent bound to the 
recombinant inhibitor of the solid support is determined. In one 
embodiment, the binding reagent is a receptor molecule that immunoreacts 
with both the recombinant proteinaceous molecule and human endothelial 
cell type PAI. In another embodiment, the binding reagent is t-PA or u-PA. 
The present invention provides several benefits and advantages. 
One benefit of the present invention is that the biochemical reagent system 
and diagnostic system of the invention are highly specific. Biological 
samples frequently contain numerous fibrinolytic inhibitors. It is 
difficult to distinguish among them by existing assays since those assays, 
in general, measure the capacity of a sample to decrease the activity of 
plasminogen activators or plasmin. In contrast, a preferred diagnostic 
system of the present invention detects only inhibitor bound to particular 
plasminogen activators, and furthermore, detects only those recognized by 
the specific antiserum utilized. 
Another benefit of the present invention is that the reagent system of the 
invention is quantitative, providing in one embodiment, a measure of the 
quantity of functionally active inhibitor bound to PAs, and not inhibitor 
activity. Therefore, the reagent system may not be as influenced by 
changes in salt or pH, for example, as are enzymatic assays. It is the 
functionally active form that is likely to change in various diseases. 
The inhibitor, as released by endothelial cells and platelets, exists in 
two forms, one active, and one inactive. The inactive form can be 
activated by treatment with denaturants, such as SDS and guanidine. Thus, 
an advantage of the reagent and diagnostic systems of the present 
invention is that they can be used to measure the relative amount of both 
active and inactive inhibitor in various samples. 
Still another benefit of the present invention is that it provides a means 
for assaying the total amount of endothelial cell type plasminogen 
inhibitor present, whether active or inactive. 
Another advantage of the present invention is that a diagnostic system of 
the invention can employ tissue-type plasminogen activator (t-PA) or 
urokinase-type plasminogen activator (u-PA) bound to wells of microtiter 
plates, and thus readily lends itself to screening large numbers of 
samples in a rapid and reproducible manner. 
Other advantages and benefits of the present invention will become readily 
apparent to those skilled in the art from the following description of the 
invention, the drawings and the appended claims.

DETAILED DESCRIPTION OF THE INVENTION 
Definitions 
The term "plasminogen activator inhibitor" (PAI) as used herein is meant to 
indicate a protein that inhibits or checks the action of a plasminogen 
activator. The PAI useful herein can be from a number of sources such as 
bovine aortic endothelial cells.(BAEs), human sources such as endothelial 
cells, placental extracts, platelets, plasma and serum, a transformed or 
neoplastic cell line (e.g. HG 1080), or that proteinaceous molecule 
prepared by recombinant techniques such as a fusion polypeptide as 
described herein. It is preferred that the PAI utilized at least bind to 
and inhibit the activity of tissue-type plasminogen activator, and thus 
the endothelial or beta-PAI described herein is the PAI of particular 
interest and choice as compared to the so-called placental PAI or protease 
nexin that primarily inhibit u-PA. More preferably, the useful inhibitor 
binds to and inhibits both t-PA and u-PA, as is also discussed herein. 
"Plasminogen activator" is a protein that activates plasminogen, 
particularly in plasma, and converts it into plasmin in the fibrinolytic 
system of blood, cells, tissues and bodily fluids. Plasminogen activators 
useful in the present invention include tissue-type plasminogen activator 
(t-PA) and urokinase-type plasminogen activator (u-PA). As used herein, 
"urokinase-type" is meant to indicate urokinase and its homologous 
proteins as found in mammals other than humans. 
The phrase "immunologically different" is used herein to mean that 
antibodies raised to one molecule do not immunoreact (cross-react) with 
another molecule. For example, antibodies raised to the recombinant 
proteinaceous molecule discussed hereinafter do not cross-react with 
either protease nexin or placental plasminogen activator inhibitor. 
Conversely, the phrase "immunologically similar" is used herein to describe 
a molecule that is capable of inducing the secretion of antibodies that 
cross-react with another molecule, and thus, the two molecules are 
immunologically similar. For example, antibodies raised to the recombinant 
proteinaceous molecule discussed hereinafter cross-react with human and 
bovine endothelial cell type plasminogen activator inhibitors, and as a 
consequence, the three molecules are immunologically similar. 
The phrase "proteinaceous molecule" is utilized herein to denote a 
relatively large polypeptide that has a relative apparent molecular mass 
of at least about 40,000 daltons. That phrase is meant to include both the 
recombinant molecule discussed hereinafter as a fusion between a portion 
of a beta-galactosidase protein derived from a vector and also the 
molecule translated from a denominated genomic sequence such as a sequence 
of FIG. 22. 
The term "binding reagent" is used herein to mean a biologically active 
molecule that binds to or complexes with another molecule. A binding 
reagent can therefore be a receptor and its ligand such as an antibody or 
antigen that immunoreacts with its respective ligand (antigen) or receptor 
(antibody). Beta-PAI along with t-PA or u-PA also constitute binding 
reagents for each other, as do S. aureaus Cowan strain protein A and an 
antibody Fc portion. Specific binding reagents and the moieties to which 
they bind are exemplified further hereinafter. However, receptors are 
utilized in this section to exemplify most of the terms defined that 
relate to binding reagents. 
The term "receptor" as used herein is meant to indicate a biologically 
active molecule that binds to an antigen ligand. A receptor molecule or 
receptor of the present invention is an antibody, a substantially intact 
antibody in substantially purified form, such as is found in ascites fluid 
or serum of an immunized animal, or an idiotype-containing polypeptide 
portion of an antibody such as Fab and F(ab').sub.2 antibody portions as 
are described hereinafter. 
Biological activity of a receptor molecule, or other binding reagent, is 
evidenced by the binding of the receptor to its antigenic ligand upon 
their admixture in an aqueous medium and maintenance under biological 
assay conditions for a predetermined time period of from minutes to hours 
such as about 10 minutes to about 16-20 hours that is sufficient to form 
an immunoreactant (complex). 
Biological assay conditions are those that maintain the biological activity 
of the ligand and receptor molecules, or other binding and bound entities. 
Such assay conditions include a temperature range of about 4.degree. C. to 
about 45.degree. C. and physiological pH values and ionic strengths. 
Preferably, the receptors and other binding reagents also bind to the 
antigenic ligand within a pH value range of about 5 to about 9, and at 
ionic strengths such as that of distilled water to that of about one molar 
sodium chloride. Methods for optimizing such conditions are well known in 
the art. 
Idiotype-containing polypeptide portions (antibody combining sites) of 
antibodies are those portions of antibody molecules that include the 
idiotype and bind to the ligand, and include the Fab, Fab' and F(ab')2 
portions of the antibodies. Fab and F(ab')2 portions of antibodies are 
well known in the art, and are prepared by the reaction of papain and 
pepsin, respectively, on substantially intact antibodies by methods that 
are well known. See for example, U.S. Pat. No. 4,342,566 to Theofilopolous 
and Dixon. Fab' portions of antibodies are also well known and are 
prepared by the reduction of F(ab').sub.2 disulfide bonds as by 
mercaptoethanol followed by alkylation of the reduced cysteine residues so 
produced with a reagent such as iodoacetamide. Intact antibodies are 
preferred receptors, and are utilized as illustrative of the receptor 
molecules of this invention. 
Antibodies and receptor molecules are discussed herein as being "raised" to 
a particular immunogen. While idiotype-containing antibody portions are 
products of man's action on antibodies, and are thus not "raised" as such, 
the term "raised" is used in conjunction with such receptors for 
convenience of expression. 
The receptors utilized as illustrative in the present invention are 
polyclonal receptors. A "polyclonal receptor" (Pab) is a receptor produced 
by clones of different antibody-producing (-secreting) cells that produce 
(secrete) antibodies to a plurality of epitopes of the immunizing 
molecule. Monoclonal receptors as are secreted by clones of a hybridoma 
cell that secretes but one kind of antibody moleucle are also 
contemplated. The hybridoma cell is fused from an antibody-producing 
(secreting) cell and a myeloma or other self-perpetuating cell line. Such 
receptors as whole antibodies were first described by Kohler and Milstein, 
Nature, 256, 495-497 (1975), which desciption is incorporated herein by 
reference. 
Non-human, warm blooded animals usable in the present invention as hosts in 
which the polyclonal receptors are raised can include poultry (such as a 
chicken or a pigeon), a member of the ratitae bird group (such as an emu, 
ostrich, cassowary or moa) or a mammal (such as a dog, cat, monkey, goat, 
pig, cow, horse, rabbit, guinea pig, rat, hamster or mouse). Preferably, 
the host animal is a rabbit. 
Receptors, and other binding reagents, are utilized along with an indicator 
labeling means or "indicating group" or a "label". The indicating group or 
label is utilized in conjunction with the receptor as a means for 
signalling (determining) that a specific inhibitor has bound to the 
receptor. 
The terms "indicator labeling means", "indicating group" or "label" are 
used herein to include single atoms and molecules that are linked to the 
receptor or used separately, and whether those atoms or molecules are used 
alone or in conjunction with additional reagents. Such indicating groups 
or labels are themselves well-known in immunochemistry and constitute a 
part of this invention only insofar as they are utilized with otherwise 
novel receptors, methods and/or systems. 
The signal-providing label utilized is typically linked to another molecule 
or part of a molecule, as discussed hereinafter. As such, the label is 
operationally linked to that other molecule or molecule part such as a 
receptor so that the binding of the molecule to which the label is linked 
is not substantially impaired by the label and the desired signalling 
provided by the label is not substantially impaired. 
The indicator labeling means can be a reactive fluorescent labeling agent 
that chemically binds to antibodies or antigens without denaturing them to 
form a fluorochrome (dye) that is a useful immunofluorescent tracer. 
Suitable reactive fluorescent labeling agents are fluorochromes such as 
fluorescein isocyanate (FIC), flourescein isothiocyanate (FITC), 
dimethylamino-naphthalene-S-sulphonyl chloride (DANSC), 
tetramethylrhodamine isothiocyanate (TRITC), lissamine rhodamine B200 
sulphonyl chloride (RB 200 SC) and the like. A description of 
immunofluorescence analysis techniques is found in DeLuca, 
"Immunoflourescence Analysis," in Antibody As A Tool, Marchalonis et al. 
eds., John Wiley & Sons Ltd., p. 189-231 (1982), which is incorporated 
herein by reference. 
The indicator labeling means can be linked directly to a receptor of this 
invention, to a useful antigen or binding reagent such as t-PA or u-PA, or 
can comprise a separate molecule. It is particularly preferred that the 
indicator means be a separate molecule such as antibodies that bind to a 
receptor of this invention. Staphylococcus aureus Cowan strain protein A, 
sometimes referred to herein as protein A, can also be used as a separate 
molecule indicator or labeling means where an intact or substantially 
intact antibody receptor of this invention is utilized. In such uses, the 
protein A itself contains a label such as a radioactive element or a 
fluorochrome dye, as is discussed hereinafter. 
The indicating group can also be a biologically active enzyme, such as 
horseradish peroxidase (HRP) or glucose oxidase, or the like. Where the 
principal indicating group is an enzyme such as HRP or glucose oxidase, 
additional reagents are required to visualize the fact that a 
receptor-ligand complex has formed. Such additional reagents for HRP 
include hydrogen peroxide and an oxidation dye precursor such as 
diaminobenzidine. An additional reagent useful with glucose oxidase is 
2,2'azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). 
Radioactive elements provide another class of label, and are used herein as 
exemplary of useful labels. An exemplary radiolabeling agent that can be 
utilized in the invention is a radioactive element that produces gamma ray 
emissions. Elements which themselves emit gamma rays, such .sup.124 I, 
.sup.125 I, .sup.128 I, .sup.131 I, .sup.132 I, and .sup.51 Cr represent 
one class of gamma ray emission-producing radioactive element indicating 
groups. Particularly preferred is .sup.125 I. Another class of useful 
indicating groups are those elements such as .sup.11 C, .sup.18 F, .sup.15 
O, and .sup.13 N that themselves emit positrons. The positrons so emitted 
produce gamma rays upon encounters with electrons present in the analysis 
medium. Also useful is a beta ray emitter, such as .sup.111 indium. 
Radioactive monoclonal receptors can be made by culturing an appropriate 
hybridoma in a medium containing radioactive amino acids, as is well 
known. Both monoclonal and polyclonal receptors can be prepared by 
isolating the receptors and then labeling them with one of the above 
radioactive elements. Radiolabeling of proteins is well known in the art 
and will not be discussed further herein. 
The following abbreviations and symbols are used herein. 
______________________________________ 
bp base pair(s) 
kb 1000 bp 
kda kilodalton(s) 
M.sub.r apparent relative molecular mass 
DNA deoxyribonucleic acid 
cDNA complementary DNA 
RBS ribosome binding site 
(Shine-Dalgarno sequence) 
RNA ribonucleic acid 
replicon the unit that controls 
individual acts of replication; it 
has an origin at which 
replication is initiated and it 
may have a terminus at which 
replication stops. 
______________________________________ 
The term "corresponds" in its various grammatical forms is used herein in 
relation to nucleotide codon sequences to mean the nucleotide codon 
sequence described containing only conservative codon substitutions that 
encode for particular amino acid residues along the protein sequence. 
The term "conservative codon substitution" as used above is meant to denote 
that one codon has been replaced by another leading to translation of a 
protein in which an amino acid residue has been replaced by another, 
biologically similar residue. Examples of conservative substitutions at 
the amino acid residue (translation or expression) level include the 
substitution of one hydrophobic residue such as Ile, Val, Leu or Met for 
another, or the substitution of one polar residue for another such as 
between Arg and Lys, between Glu and Asp or between Gln and Asn, and the 
like. 
In some instances, the replacement of an ionic residue by an oppositely 
charged ionic residue such as Asp by Lys has been termed conservative in 
the art in that those ionic groups are thought to merely provide 
solubility assistance. In general, however, replacement of an ionic 
residue by another ionic residue of opposite charge is considered herein 
to be "radical replacement", as are replacements between nonionic and 
ionic residues, and bulky residues such as Phe, Tyr or Trp and less bulky 
residues such as Gly, Ile and Val. 
The terms "nonionic" and "ionic" residues are used herein in their usual 
sense to designate those amino acid residues that normally either bear no 
charge or normally bear a charge, respectively, at physiological pH 
values. Exemplary nonionic residues include Thr and Gln, while exemplary 
ionic residues include Arg and Asp. 
At the nucleotide level, the term "corresponds" is also meant to denote 
that the third nucleotide of a codon can be replaced by another nucleotide 
so that the codon containing the substitution encodes for the same amino 
acid residue as did the unsubstituted codon. Such redundancy of different 
codons being translated to the same amino acid residue is well known in 
the art. 
The term "substantially pure" as used herein refers to a biological 
substance substantially free from all heterogeneous or extraneous matter. 
When used in a context describing or depicting nucleotide sequences, the 
purine or pyrimidine bases forming the nucleotide sequence are depicted as 
follows: 
A--deoxyadenyl 
G--deoxyguanyl 
C--deoxycytosyl 
T--deaxythymidyl 
In describing a nucleotide sequence each three-letter triplet constituted 
by the bases identified above represents a trinucleotide of DNA (a codon) 
having a 5'-end on the left and a 3'-end on the right. 
The amino acid residues and amino acid residue sequences of the proteins 
described herein are depicted by their three-letter or single-letter 
symbols that are identified and correlated in the Table of Correspondence 
below: 
______________________________________ 
Table of Correspondence 
SYMBOLS FOR AMINO ACIDS 
Three-Letter 
Single-Letter 
______________________________________ 
Alanine Ala A 
Arginine Arg R 
Asparagine Asn N 
Aspartic acid Asp D 
Cysteine Cys C 
Glutamic acid Glu E 
Glutamine Gln Q 
Glycine Gly G 
Histidine His H 
Isoleucine Ile I 
Leucine Leu L 
Lysine Lys K 
Methionine Met M 
Phenylalanine Phe F 
Proline Pro P 
Serine Ser S 
Threonine Thr T 
Tryptophan Trp W 
Tyrosine Tyr Y 
Valine Val V 
______________________________________ 
Amino acid residues of proteins and polypeptides are in their natural, L, 
configurations. 
I. GENERAL DISCUSSION 
The present invention is directed to a biochemical reagent system and to 
methods of preparing and using same, as well as to diagnostics utilizing 
the reagent system. The reagent system comprises (a) a receptor raised in 
an animal host to endothelial cell type plasminogen activator inhibitor or 
to a substantially pure, recombinant molecule described hereinafter, and 
(b) an indicating means. 
The present invention also contemplates methods of forming a polyclonal 
receptor and a biochemical reagent system of the invention. 
The method of forming a polyclonal receptor for use in a biochemical 
reagent system of the present invention comprises administering to an 
animal host, preferably a mammal (e.g., a rabbit, goat or horse) an 
endothelial (beta-) plasminogen activator inhibitor (PAI) or substantially 
pure, recombinant molecule described herein in an amount sufficient to 
induce the production of antibodies to the inhibitor. The resulting 
antibodies constitute receptor molecules for the inhibitor. Antisera 
containing the antibodies are then collected from the immunized host and 
the receptor so produced is recovered. 
The biochemical reagent system of the invention is formed by combining the 
receptor molecules formed as described above with an indicating means. 
Suitable indicating means are those previously described hereinbefore. It 
is particularly preferred that the indicating means be a separate 
molecule. 
A further embodiment of the invention is a solid phase assay method for 
directly detecting the presence, and if desired, quantity of endothelial 
cell type plasminogen activator inhibitor in a sample to be assayed. One 
aspect of the method comprises the steps of: (a) providing a solid matrix 
on which to assay a sample; (b) affixing on the solid matrix a binding 
reagent that binds (complexes with) to the inhibitor to form a solid phase 
support; (c) admixing an aliquot of a liquid sample to be assayed with the 
solid phase support to form a solid-liquid phase admixture; (d) 
maintaining the admixture under biological assay conditions for a 
predetermined time (typically about 2 to 4 hours) sufficient for the 
binding reagent to bind to (complex with) endothelial cell type 
plasminogen activator inhibitor present in the sample; (e) separating the 
solid and liquid phases; and (f) determining the presence of inhibitor 
that bound to (complexed with) the binding reagent. 
The presence of the inhibitor that complexed with the binding reagent can 
be determined in a number of ways that depend upon the type of assay 
direct or indirect/competitive (hereinafter), used. In one preferred 
embodiment for a direct assay, that determination is made by the steps of 
(i) admixing an aqueous liquid solution of second binding reagent that 
binds to the inhibitor of the sample bound on the solid support with the 
solid phase obtained after step (e) above to form a second solid-liquid 
phase admixture, the second binding reagent complexing with the inhibitor; 
(ii) maintaining the second solid-liquid admixture under biological assay 
conditions for a predetermined time sufficient for the second binding 
reagent to bind (form a complex) with the inhibitor (typically about 2 to 
about 4 hours); (iii) separating the solid and liquid phases of the second 
solid-liquid phase admixture; and (iv) determining the quantity of the 
second binding reagent that bound to the inhibitor, and thereby 
determining the quantity of inhibitor. 
The amount of second binding agent that binds to or complexes with the 
inhibitor is typically determined by an indicating means, as described 
hereinbefore. The indicating means can be linked to the second binding 
reagent so that the second binding agent and indicating means are one 
molecule. More preferably, the second binding reagent and indicating means 
are separate molecules. 
Thus, where the indicating means is linked to the second binding reagent, 
the above method for determining the presence of inhibitor complexed with 
the first-named binding reagent can be carried out using the steps of (i) 
admixing an aqueous, liquid solution of second binding reagent containing 
a linked indicating means with the solid phase obtained after step (e) 
above to form a second solid-liquid phase admixture, the second binding 
reagent binding to (complexing with) the inhibitor, and the indicating 
means providing a means of determining the quantity of the second binding 
reagent that bound to the inhibitor; (ii) maintaining the admixtures under 
biological assay conditions for a predetermined time sufficient for the 
second binding reagent to bind to (complex with) the inhibitor; (iii) 
separating the solid and liquid phases of the second solid-liquid phase 
admixture; and (iv) determining the quantity of second binding reagent 
that bound to the inhibitor. 
The indicating means is a separate molecule in particularly preferred 
practice. In such situations, the bound (complexed) inhibitor can be 
determined by the steps of (i) admixing a liquid solution of second 
binding reagent with the solid phase obtained after step (e) above to form 
a second solid-liquid phase admixture, the second binding reagent binding 
to (complexing with) the inhibitor; (ii) maintaining the admixture so 
formed under biological assay conditions for a predetermined period of 
time sufficient for the second binding reagent to bind to (complex with) 
the inhibitor; (iii) separating the solid and liquid phases of the second 
solid-liquid phase admixture; (iv) admixing a separate molecule indicator 
labeling means (as discussed hereinbefore) to form a third solid-liquid 
phase admixture; (v) maintaining the third solid-liquid phase admixture 
under biological assay conditions for a predetermined period of time 
sufficient for the second binding reagent and indicator labeling means to 
bind (typically about 2 to about 4 hours); (vi) separating the solid and 
liquid phases of the third solid-liquid phase admixture; and (vii) 
determining the amount of separate molecule indicator labeling means that 
bound to the second binding reagent. 
Details for the above embodiment are given hereinafter wherein the first 
binding reagent is t-PA or u-PA, the second binding reagent is rabbit 
anti-inhibitor antibody and the separate molecule indicator means is a 
goat anti-rabbit IgG antibody. 
In yet another method, the amount of inhibitor reacted or complexed with 
the first binding reagent can be determined without the use of a second 
binding reagent. In this embodiment, the indicator labeling means is 
linked directly to the inhibitor, and the amount of inhibitor is 
determined by that label. 
For example, the proteins present in a sample to be assayed can be 
radiolabelled with 125-iodine following one of the procedures described 
hereinafter. After separation of the solid and liquid phases of step (e), 
hereinbefore, the radiolabelled, but unbound, proteins are removed from 
the admixture thereby leaving radiolabelled, bound inhibitor on the solid 
support. The presence and amount of that bound, radiolabelled inhibitor 
can then be determined using a gamma counter. A similar result can be 
obtained using a reactive fluorescent molecule as the indicator labeling 
means such as fluoroscein isocyanate to react with the components of the 
assayed sample in place of the radioactive element. 
Preferred first and second binding reagents include tissue-type and 
urokinase-type plasminogen activators or the before described receptor of 
the invention. If the first binding reagent utilized is tissue-type or 
urokinase-type plasminogen activator, then the second binding reagent is 
the receptor. Alternatively, if the first binding reagent utilized is the 
receptor, then the second binding reagent is one of the above plasminogen 
activators. Thus, the first binding reagent is (a) a plasminogen activator 
selected from the group consisting of t-PA and u-PA, or (b) a receptor of 
this invention that binds to the inhibitor, and the second binding reagent 
is (a) a plasminogen activator selected from the group consisting of t-PA 
and u-PA, or (b) a receptor of this invention. However, the first and 
second binding reagents are different. Thus, the second binding reagent 
can also be refered to as the other of (a) and (b) not used as the first 
binding reagent. 
The separate molecule indicator labeling means is preferably used where the 
second binding reagent is an intact or substantially intact antibody 
receptor of this invention that binds to the inhibitor. As such, the 
separate molecule indicator labeling means is preferably an antibody such 
as goat anti-rabbit IgG or protein A having a linked indicating group such 
as a radioisotope, enzyme or fluorochrome dye. 
A competitive assay is utilized in another aspect of the invention. Here, a 
solid support is provided that comprises a solid matrix having the 
substantially pure recombinant, proteinaceous molecule (described 
hereinafter) affixed thereto. An aliquot of a liquid sample to be assayed 
for natural (native) endothelial cell type PAI is admixed with a 
predetermined amount of binding reagent that binds to both (1) the 
recombinant inhibitor affixed to the matrix as part of the solid support 
and (2) the inhibitor to be assayed. Exemplary of such binding reagents 
are the aforementioned receptor molecules and also t-PA and u-PA. The 
admixture so formed is maintained under biological assay conditions for a 
predetermined period of time sufficient for the binding reagent to bind to 
any inhibitor present in the sample to form a complex. 
That admixture is then admixed with the before-described solid support to 
form a solid-liquid phase admixture. The solid-liquid phase admixture is 
maintained under biological assay conditions for a predetermined period of 
time sufficient for any binding reagent of the admixture not bound to 
inhibitor molecules of the sample to bind to the recombinant inhibitor 
molecules of the solid support. 
The solid and liquid phases are then separated, as by decantation and 
rinsing. The liquid phase and its contents can be retained and assayed as 
by SDS-PAGE or reverse fibrin autography to provide a direct determination 
of the presence of beta-PAI. More preferably, an indirect determination is 
made in which the presence (amount) of human endothelial PAI in the sample 
is determined by assaying the amount of binding reagent complexed with the 
solid phase recombinant human endothelial PAI, and comparing that amount 
with known standards to arrive at a qualitative, and if desired, 
quantitative determination by difference. 
The determination of the amount of binding reagent bound to the solid phase 
is carried out using the general methods discussed before, except that the 
second binding reagent is a different molecule from those utilized above. 
Thus, where the first binding reagent is t-PA or u-PA, the second binding 
reagent typically is a label-linked antibody raised to either appropriate 
protein. Where the first binding reagent is a receptor, the second binding 
reagent typically is a label-linked antibody raised in a host animal 
different from that in which the first binding reagent receptor molecules 
were raised, such as goat anti-rabbit antibodies where rabbit 
anti-beta-PAI antibodies are the first binding reagent. Label-linked 
protein A is also useful in this indirect, competition assay. It is noted 
that label-linked receptor molecules can also be used as the first binding 
reagent, thereby eliminating the need for use of second binding reagent. 
The before-described solid phase assay methods are particularly preferred. 
However, it is to be understood that liquid phase (homogeneous) assays are 
also contemplated. Such systems are well known in the art and need not be 
described in detail. 
Briefly, however, liquid systems can also utilize a receptor, u-PA or t-PA 
as the first binding agents of the assay. The indicating means for such 
systems typically is an antibody that is linked to a signalling enzyme in 
such a way that binding of the antibody to its substrate in the complex 
formed inhibits the activity of the linked enzyme, thereby indicating the 
presence of a complex ingredient to which the antibody binds. Exemplary 
labeling means for such assays are illustrated in U.S. Pat. Nos. 
3,817,837, 3,996,345. A plasminogen activator such as t-PA can also be 
utilized as a labeled first binding agent when linked to an enzyme whose 
activity is inhibitable by the formation of a t-PA/human beta-PAI complex. 
Such a labeling system is thus in some ways analogous to the enzyme-hapten 
systems described in U.S. Pat. No. 3a,875,011. 
The present invention further contemplates a diagnostic system, that can be 
in the form of a kit, for detecting the presence and quantity of 
beta-migrating, human plasminogen activator inhibitor in a sample. The kit 
includes at least one package containing (1) as an active ingredient, an 
effective amount of the biochemical reagent system of the invention in 
dry, solution, or dispersion form, and (2) t-PA or u-PA. 
The diagnostic system can also include a solid matrix that can be a 
microtiter strip or plate having a plurality of wells. The t-PA or 
urokinase present is preferably bound to the solid matrix. 
Suitable solid matrices useful in the diagnostic system and methods 
described hereinbefore include 96 well microtiter plates sold under the 
designation Falcon Microtest III Flexible Assay Plates (Falcon Plastics, 
Oxnard, Calif.) and microtiter strips sold under the designation Immulon I 
and II (Dynatech, Alexandria, VA). The microtiter strip or plate is made 
of a clear plastic material, preferably polyvinyl chloride or polystyrene. 
Alternative solid matrices for use in the diagnostic system and methods 
include polystyrene beads, about 1 micron to about 5 millimeters in 
diameter, available from Abbott Laboratories, North Chicago, Ill.; 
polystyrene tubes, sticks or paddles of any convenient size; and 
polystyrene latex whose polystyrene particles are of a size of about 1 
micron and can be centrifugally separated from the latex. 
The solid matrix can also be made of a variety of materials such as 
cross-linked dextran, e.g. Sephadex G-25, -50, -100, -200 and the like 
available from Pharmacia Fine Chemicals of Piscataway, New Jersey, agarose 
and cross-linked agarose, e.g. Sepharose 6B, CL6B, 4B, CL4B and the like 
also available from Pharmacia Fine Chemicals. 
The agarose or Sepharose matrices are typically activated for linking using 
cyanogen bromide. The activated matrix is then washed with one molar 
glycine and linked to the biochemical reagent system of the invention, 
t-PA or u-PA without drying of the activated matrix (solid support). The 
matrix-linked reagent system, t-PA or u-PA is then washed and is ready for 
use. Further details of use of these solid matrices are provided in 
Section III, A2. 
The diagnostic system can further include a standard against which to 
compare the assay results and various buffers in dry or liquid form. 
An indicating means such as those described hereinbefore is preferably 
supplied along with the receptor in the biochemical reagent system of the 
invention, and can be packaged therewith when linked to the receptor or 
more preferably is packaged separately when a separate molecule indicating 
means is used. Additional reagents such as hydrogen peroxide and 
diaminobenzidine can also be included in the system when an indicating 
group such as HRP is utilized. Such materials are readily available in 
commerce, as are many indicating groups, and may not be supplied along 
with the diagnostic system. In addition, some reagents such as hydrogen 
peroxide can decompose on standing, or are otherwise short-lived like some 
radioactive elements, and are better supplied by the end-user. 
The data from several studies, discussed hereinafter, were performed to 
assess the nature of the plasminogen activator inhibitor and the ability 
of the biochemical reagent diagnostic systems of the present invention to 
detect and quantify plasminogen activator inhibitor bound to plasminogen 
activator in human serum. 
The diagnostic system of the invention is based upon the ability 
endothelial cell type PAI to bind to t-PA or u-PA immobilized on plastic 
microtiter wells. After washing, the extent of binding is quantified by 
admixing and maintaining (incubating) the complex first with rabbit 
antiserum to the inhibitor and then with .sup.125 I-goat anti-rabbit IgG. 
Using a diagnostic system and assay method of the present invention, it 
was found that the reaction between t-PA and the inhibitor was rapid 
(greater than 78 percent binding within 1 hour), time-and 
concentration-dependent, and sensitive over a broad range of inhibitor 
concentrations [1-100 nanograms per milliliter (ng/ml)]. Exogenously added 
t-PA and u-PA were found to compete with the immobilized t-PA for the 
inhibitor with a 50 percent reduction in binding obtained with 12 ng/ml of 
t-PA and 6 ng/ml of u-PA. 
It is to be understood that the results discussed hereinafter are 
illustrative of embodiments utilizing the biochemical reagent and 
diagnostic systems of the present invention and the present invention is 
not intended to be so limited. 
A biologically pure DNA molecule that codes for human endothelial cell type 
PAI or a fusion polypeptide containing that PAI, i.e., a proteinaceous 
molecule that is immunologically similar to that PAI, constitutes a 
further aspect of the invention. That DNA molecule contains about 1140 to 
about 3000 nucleotides or nucleotide base pairs (bp) and includes a 
nucleotide sequence that consists essentially of the nucleotide sequence 
corresponding to a nucleotide sequence represented by the formula of FIG. 
22, from left to right and in the direction from 5'-terminus to 
3'-terminus (5'-end to 3'-end). The reading frame of a nucleotide sequence 
of the invention must, of course, be consistent with that of human 
endothelial cell type PAI or a fusion polypeptide containing endothelial 
cell type PAI. Thus, the reading frame must not shift, but must be the 
same as that in which the nucleotide residue at position 13 shown in FIG. 
22 is the first nucleotide residue of a triplet codon. 
A suitable DNA molecule includes a nucleotide sequence that corresponds to 
the depicted sequence from nucleotide position 13 to about position 1153. 
Other suitable DNA molecules include DNA sequences corresponding to the 
depicted sequences from nucleotide position 1 to about nucleotide position 
1153, from nucleotide position 1 to about nucleotide position 1960, as 
well as the entire sequence shown in FIG. 22 from nucleotide position 1 to 
about nucleotide position 2995. 
A nucleotide sequence of this invention consists essentially of one of the 
before-described sequences. Thus, a nucleotide sequence of the invention 
excludes additional nucleotides that affect the basic and novel 
characteristics of a nucleotide sequence that codes for human endothelial 
PAI. 
A nucleotide sequence of the invention can include one or more 
transcriptional promoter sequences operationally linked to the sequence 
adjacent to the 5'-terminus thereof. Where translation of the DNA and 
protein expression are desired, the DNA also includes a translation 
initiating codon (ATG) and a translation terminating codon (TAA or TAG or 
TGA), each operationally linked adjacent to the 5'-terminus and 
3'-terminus, respectively, of the sequence, with the translation 
initiating codon being located between the promoter sequence and the 
5'-terminus. 
A DNA sequence that codes for all or a portion of another molecule can also 
be included in the DNA molecule so that the translated (expressed) 
proteinaceous molecule is a fusion polypeptide that includes an amino acid 
residue sequence of all or a portion of that other molecule fused (linked 
by a peptide bond) to the expressed, human endothelial PAI. An exemplary 
fusion polypeptide is the proteinaceous molecule discussed hereinafter 
that contains a portion of the beta-glactosidase molecule fused to the 
amino-terminus of human endothelial cell PAI. That molecule also includes 
four amino acid residues of the PAI leader peptide fused by peptide bonds 
between the beta-galactosidase portion and the PAI sequence. 
All of the above nucleotide sequences can be present so long as an 
enumerated DNA molecule remains replicable, where only replication is 
desired. Where replication and translation (proteinaceous molecule 
expression) are desired, those nucleotide sequences are present so long as 
the DNA molecule remains replicable and the proteinaceous molecule 
containing the amino acid residue sequence of human, endothelial cell PAI 
expressed exhibits immunological cross-reactivity (discussed hereinafter) 
with the naturally occurring bovine and human endothelial PAI described 
herein. The expressed proteinaceous molecule also preferably binds to t-PA 
and exhibits inhibition of at least t-PA in a reverse fibrin autography 
assay as discussed herein. Most preferably, that expressed molecule also 
binds to and exhibits inhibition of u-PA activity in the reverse fibrin 
autography assay. 
Since a nucleotide sequence of the invention contains a nucleotide sequence 
that corresponds to a sequence whose formula is represented in FIG. 22, 
conservative codon substitutions as well as conservative nucleotide 
substitutions are contemplated, as those phrases are defined herein. 
A nucleotide sequence of the invention can be single-stranded as is shown 
in FIG. 22. Most preferably, however, a DNA sequence is linked by hydrogen 
bonds to a second DNA molecule, the second DNA molecule having a 
nucleotide sequence that is complementary and in antiparallel orientation 
to the first-named DNA. Thus, most preferably, a DNA of the invention is 
double-stranded and contains a before-described DNA sequence corresponding 
to all or an enumerated portion of the sequence shown in FIG. 22 along 
with a complementary sequence. 
A non-chromosomal vector for propagation and expression of a desired DNA 
nucleotide sequence as defined hereinabove in a replication/expression 
medium, e.g., a unicellular organism or the like such as E. coli, S. 
cerevisiae or mammalian cells such as COS cells, is also contemplated. 
That vector comprises a replicon that is compatible with the 
replication/expression medium and contains therein the DNA molecule to be 
replicated in a manner such that the vector can propagate the DNA 
molecule. 
In addition, the non-chromasomal vector also includes those sequence 
components that are utilized for transcription and translation. To that 
end, a transcriptional promoter can be operationally linked to the DNA 
molecule present adjacent to the 5'-terminus thereof, as already noted. 
The transcriptional promoter can be endogenous to the vector or exogenous 
to the vector. A transcriptional promoter endogenous to the vector such as 
the lac Z promoter-operator utilized herein is preferred. A translational 
terminator can also be operationally linked adjacent to the 3'-terminus of 
the DNA molecule in some instances, although the nucleotide sequence 
represented by the formula of FIG. 22 contains such a terminator sequence. 
A before-defined DNA of FIG. 22 molecule lacks an initiation codon (ATG) 
adjacent to the 5'-terminus of the sequence that begins translation in a 
replication/expression medium. Such a codon can be ligated to a defined 
DNA molecule in frame, as discussed hereinbefore, or can be a portion of 
the vector nucleotide sequence as exemplified herein. 
Human endothelial PAI is an excreted protein, and thus as expressed 
naturally, contains a so-called polypeptide leader or signal sequence that 
assists in migration of the protein through the plasma membrane, and that 
is excised in a post-translational event. Only a portion of the encoded 
polypeptide leader sequence of human endothelial PAI is included in a 
before-defined DNA molecule sequence. The examplary DNA molecule 
illustrated herein is expressed within the cytoplasm of 
replication/expression medium such as E. coli, as compared to the 
periplasmic space of the cells. Expression into the cytoplasm of E. coli 
is usual for that replication/expression medium. 
The before-discussed transcription promoter, translation initiating and 
translation terminating codons and sequence coding for a polypeptide 
leader sequence, where used, are normally parts of the non-chromosomal 
vector as compared to a DNA molecule of the invention. For use in 
expression of the proteinaceous molecule, the vector normally also 
includes a ribosome binding site (Shine-Delgardo sequence) adjacent to the 
5'-terminus of the DNA molecule and located upstream from the initiation 
codon, as is well known. The vector's promoter such as the lacZ promoter 
utilized herein typically contain a ribosome binding site. 
Thus, the nucleotide sequence of the vector, aside from those nucleotides 
needed for the replication and general vector function include, in frame 
and from 5'-termi-nus to 3'-terminus, a ribosom binding site operationally 
linked adjacent to the 5'-terminus of a transcription promoter; that 
promoter operationally linked to the 5'-terminus of the translation 
initiating codon; that codon operationally linked to the 5'-terminus of: 
(a) a leader sequence for expression in eukaryotes, or (b) a sequence of a 
portion of another molecule that is expressed as a fusion polypeptide with 
the desired human endothelial PAI, or (c) a DNA molecule of this 
invention; where (a) or (b) is present, that sequence is operationally 
linked to the 5-terminus of a DNA molecule of this invention. The DNA 
molecule of this invention, however linked adjacent to its 5'-terminus, is 
linked adjacent to its 3'-terminus to a translation terminating codon. As 
is apparent from examination of FIG. 22, additional nucleotides can also 
be present operationally linked to the 3'-terminus of the terminating 
codon so long as those additional nucleotides do not interfere with 
transcription and/or translation, as those events are desired, or can 
interfere with the immunological similarity of an expressed proteinaceous 
molecule to human or bovine endothelial PAI. Most preferably, any 
additional nucleotides also do not interfere with the biological activity 
toward plasminogen activators exhibited by an expressed proteinaceous 
molecule. 
It is to be understood that all of the DNA sequences of the vector must be 
compatible with the replication/expression medium utilized for replicating 
the DNA, and more preferably expressing a product coded for (encoded by) a 
DNA molecule of this invention. A vector of the invention is at least 
capable of replicating (propagating) a DNA molecule of the invention. More 
preferably, the vector is capable of not only replicating a DNA molecule, 
but is also capable of expressing or translating the genomic information 
of that DNA into a recombinant proteinaceous molecule that is 
immunologically similar to human endothelial PAI, as defined herein. 
A non-chromosomal vector of this invention need not be limited to those 
vectors useful for replicating and translation (expression) in E. coli as 
host replication/expression medium. Substantially any vector useful for 
replicating (propagating) a DNA sequence can be utilized for replicating 
the DNA, e.g. in mammation or eukaryotic cells. 
A wide range of such vectors is commercially available as are appropriate 
host replication media. Exemplary vectors, both plasmids and 
bacteriophages and hosts are available from the American Type Culture 
Collection of Rockville, Md., and are listed in its CATALOGUE OF BACTERIA, 
PHAGES AND rDNA VECTORS, sixteenth ed., 1985. In addition, plasmids, 
cosmids and cloning vectors are listed as being available in catalogues 
from Boehringer Mannheim Biochemicals of Indianapolis, Ind.; Bethesda 
Research Laboratories, Inc. of Gaethersberg, Md., and New England Biolabs, 
Inc. of Beverly, Mass. 
Another aspect of the invention is a substantially pure, recombinant 
proteinaceous molecule that is immunologically similar to human 
endothelial cell type plasminogen activator inhibitor; that is, antibodies 
raised to the recombinant molecule immunoreact with native human 
endothelial cell type plasminogen activator inhibitor. As already noted, 
the recombinant molecule need only have the amino acid residue sequence of 
the native molecule to be useful in inducing the secretion of 
cross-reactive antibodies that are useful in assays. As such, this 
molecule can be translated from a replication/expression medium containing 
a vector having an appropriate translational start and preferably stop 
codons, as already described. The above-described immunological similarity 
can be demonstrated by inducing antibodies in rabbits following the 
procedure described hereinafter in section III A 3 for bovine endothelial 
PAI. 
The substantially pure recombinant proteinaceous molecule also preferably 
possesses some, if not all, of the biological activity of human, 
endothelial cell type PAI. Such a molecule constitutes another embodiment 
of this aspect of the invention. 
Thus, substantially pure, recombinant human endothelial cell type PAI is 
also contemplated herein. The recombinant molecule binds to and inhibits 
the activity of human t-PA as determined by reverse fibrin autography as 
described herein. The molecule also preferably binds to and inhibits the 
activity of u-PA. 
The recombinant molecule is immunologically different from protease nexin 
and human placental PAI. An immunological difference between molecules can 
be assessed in a number of manners and from an antigenic or immunogenic 
viewpoint. Most easily, however, the immunological difference between the 
three molecules is shown by a specific binding study using polyclonal 
antibodies that bind to the recombinant molecule. Those antibodies are 
substantially free from specific binding with either protease nexin or 
with human placental PAI. 
The recombinant human endothelial cell type PAI is immunologically related 
and similar to bovine endothelial cell type PAI and naturally occurring 
human endothelial cell type PAI as is evidenced by the fact the polyclonal 
antibodies raised to the bovine PAI specifically immunoreact with botht he 
recombinant and naturally occurring PAIs. In addition, polyclonal 
antibodies or other receptors raised to naturally occurring human 
endothelial PAI immunoreact with bovine and the expressed recombinant PAI 
molcules. Similarly, polyclonal antibodies or other receptors raised to 
the recombinant molecule immunlogically bind specifically to both 
naturally occurring bovine and human endothelial cell type PAI molecules. 
Thus, the expressed, recombinant human endothelial cell type PAI exhibits 
immunological cross-reactivity with naturally occurring bovine and human 
endothelial PAI molecules. 
The substantially pure recombinant human endothelial cell type PAI is 
substantially free from extraneous proteins and polypeptides as can be 
ascertained by SDS-PAGE analysis followed by staining with Coomassie 
Brilliant blue dye. The desired purity can be achieved, for example, by 
use of an affinity column or other sorbant containing affixed polyclonal 
antibodies to bovine endothelial PAI as the sorbing moiety, followed by 
standard elution and protein purification techniques. 
In a particular embodiment, the recombinant human endothelial cell type PAI 
is substantially free from polypeptide-linked glycosyl groups as is the 
exemplary fusion polypeptide described herein. Such molecules are 
typically expressed using a procaryotic replication/expression medium such 
as E. coli. 
The recombinant human endothelial cell type PAI can also contain 
polypeptide-linked glycol groups. The glycosylated molecule is typically 
expressed from eukaryotic cells is replication/expression medium such as 
mammalian Chinese hamster ovary (CHO) cells or COS cells using for example 
an SV40-derived or other vector, as is well known. 
An eukaryotic replication/expression medium and appropriate vector such as 
CHO cells and an SV40-derived vector or a yeast replication/expression 
medium such as S. cerevisiae and appropriate vector such as a vector 
derived from pTDTl are also particularly useful where the expressed 
recombinant molecule is desired to be excreted into the culture medium. A 
nucleic acid sequence that encodes a leader or signal peptide sequence is 
used for expression of the recombinant molecule into the culture medium. 
As discussed previously, other genetic signals can be included in the 
constructs to facilitate secretion of PAI out of the cell and into the 
culture medium. This improves the purification procedure. In both 
prokaryotic and eukaryotic systems a "leader peptide" at the 
amino-terminus of the protein acts as a signal for secretion. This leader 
peptide is cleaved off by host cellular protease to form the mature 
protein. 
To generate such a protein containing a leader sequence, "fusion" 
polypeptides are constructed in which the nucleotide coding region for the 
leader peptide of a known secreted protein is ligated in the same reading 
frame to the 5'-terminus of the coding region of the PAI to be expressed 
and secreted. Because the cleavage signals are resident on the leader 
peptide and the cleavage dependent protease is in the host, normal 
secretion results. 
This fusion protein strategy has been applied successfully in both yeast 
and in CHO cells. For example, Filho et al., Biotechnology 4:311, 1986 
describe a construct for yeast expression in which the leader peptide from 
yeast alpha factor including the protease cleavage signal is fused to the 
eukaryotic protein mouse alpha-amylase, is driven by the alpha factor 
promoter and results in secretion into the medium of mature protein. Other 
similar constructs using the alpha factor gene promoter have been reported 
to express foreign proteins in yeast Bitter et al., Proc. Natl. Acad. Sci. 
USA., 81:5530 (1984); Brake et al., Proc. Natl. Acad. Sci. USA., 4642, 
(1984); and Singh et al., Nucleic Acid Res. 12:8927 (1984)]. 
In mammalian cell expression systems, the leader peptide from Herpes 
Simplex Virus glycoprotein D has been fused to portions of the envelope 
glycoprotein from HTLV III. The fusion polypeptide was secreted in CHO 
cells (Lasky et al., Science, 233:209 (1980). 
Methods to increase production have been reported in which the copy number 
of the transfected plasmid is maintained at a high number per cell. This 
is accomplished by including a selectable marker such as a dihydrofolate 
reductase (dhfr) on the expression plasmid and culturing the transformed 
cells that are deficient in dhfr in the presence of medium that requires 
high levels of dhfr for growth [Simonsen et al., Proc. Natl. Acad. Sci. 
USA, 80:2495 (1983)]. Similarly, an SV40 based vector such as pKSV-10 that 
contains an SV40 origin of replication can be maintained in COS cells at a 
high copy number because the COS cells also contain a defective SV40 viral 
genome that preferentially stimulates the expression vector's SV40 origin 
to replicate autonomously in the host cell's cytoplasm [Gluzman, Cell, 
23:175 (1981)]. 
In addition, as already noted, the recombinant protein can be expressed in 
several forms. In one embodiment, it is expressed as a protein whose amino 
acid residue sequence is that represented by the inferred formula shown in 
FIG. 22 from about amino acid residue position 1 (DNA nucleotide numbers 
13-15) through amino acid residue position 379 (DNA nucleotide numbers 
1154-1157). When so expressed and substantially free of polypeptide-linked 
glycosyl groups, the recombinant protein exhibits an apparent relative 
molecular mass (M.sub.r) of about 40,000 daltons [40 kilodaltons (kda)] as 
determined by SDS-PAGE analysis. In another embodiment, the PAI is 
expressed as part of a fusion polypeptide having an M.sub.r of about 180 
kda by SDS-PAGE analysis, and containing a portion of the 
beta-galactosidase molecule operationally linked by a peptide bond to the 
amino-terminal amino acid residue whose formula is shown at amino acid 
residue position -4 (nucleotide positions 1-3) in FIG. 22. Both of those 
exemplary molecules exhibited biological activity similar to that of human 
endothelial cell plasminogen activator inhibitor in that they were capable 
of binding to and inhibiting the activity of u-PA in reverse fibrin 
autography, as is discussed hereinafter and illustrated in FIG. 21. The 
recombinant protein can also be expressed with an amino-terminal leader 
polypeptide sequence, as already described. 
As is apparent from the previous discussion, the substantially pure, 
recombinant human endothelial cell type PAI is useful in solid phase 
competitive assay systems when affixed to a solid matrix as a part of a 
solid support. That recombinant PAI is also useful as an immunogen used to 
raise antibodies for use as receptor molecules in other assay systems. 
This latter use is similar to the use of the substantially pure 
recombinant proteinaceous molecule that need not have biological activity. 
When so used, and particularly when used affixed to a solid matrix as part 
of a solid support in an assay, it is not of great import that the protein 
or fusion polypeptide be substantially free of bacterial 
lipopolysaccharide (LPS) or other bacterial cellular products as are known 
to often contaminate proteinanceous materials prepared by recombinant 
techniques. However, where substantial freedom from LPS is desired, as 
where the material is utilized as an immunogen, well known techniques can 
be utilized to prepare the PAI substantially free of bacterial LPS and 
other bacterial cellular debris. See for example, Issekutz (1983), J. Imm. 
Methods, 61, 271-281 and Sofer, BIO/TEC-NOLOGY, December, 1984, 1035-1038 
and the citations therein. The replication/expression medium can also be 
adjusted as by use of the yeast S. cerevisiae or mammalian cells as 
discussed before as the expression medium along with a suitable vector as 
is known. 
II. RESULTS 
A. Purification of the Bovine Endothelial Cell (BAE) Inhibitor 
It had previously been shown that CM (as described in Section III A 1 and 
2, hereinafter, and in the following papers) from BAEs contained both 
tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activators, Levin 
et al., J. Cell Biol., 94, 631 (1982), as well as an inhibitor of 
fibrinolysis, Loskutoff et al., Proc. Natl. Acad. Sci. (USA), 80, 2956 
(1983). Fractionation of this CM by affinity chromatography on 
concanavalin A-Sepharose revealed that the u-PA and t-PAs could be 
separated from each other, Loskutoff et al., Blood, 62, 62 (1983), and 
suggested that this approach also would be useful for the purification of 
the inhibitor. 
One liter of CM was applied to a concanavalin A-Sepharose column and the 
column was processed as described in Section III hereinafter. The peak 
fractions were pooled, fractionated by SDS-PAGE, and analyzed for protein 
by staining with Coomassie Brilliant Blue, and for the presence of 
fibrinolytic activators and inhibitors by reverse fibrin autography. As 
shown in FIG. 1, more than 85 percent of the protein applied to the column 
was recovered in the run-through effluent (pool I). This fraction 
contained both albumin and u-PA, but no inhibitor. Some inhibitor was 
detected in Pool III, the fraction containing the majority of recovered 
t-PA activity, Loskutoff et al., Blood, 62, 62 (1983) but represented less 
than 20 percent of the total inhibitor as judged by the relative size of 
the lysis-resistance zones, Erickson et al., Anal. Biochem., 137, 454 
(1984). The majority of detectable inhibitor activity was recovered in the 
concanavalin A, pool II fraction, as shown in the inset in FIG. 2, a 
fraction containing only 5 percent of the total protein. It appeared to 
comigrate with one of the major stained proteins. 
The concanavalin A pool II also was analyzed by SDS-PAGE in tube gels and 
results are shown in FIG. 2. After electrophoresis, the gel was sliced and 
extracts of the slices tested for their ability to inhibit u-PA-mediated 
lysis of .sup.125 I-fibrin. Again, inhibitor activity was detected in a 
single region of the gel, and migrated with a relative mobility (R.sub.f) 
that was indistinguishable from that of the lysis-resistant zone shown in 
the inset of FIG. 2 (i.e., R.sub.f =0.6). Few other proteins were detected 
in this region of the gel, suggesting that the purification could be 
completed by extracting the inhibitor out of such gels. 
The extracts with the highest inhibitor activity (FIG. 2, slices 49-52) 
were pooled and reanalyzed on 7.5-20 percent gradient gels and the results 
shown in FIG. 3. A single protein was detected when the gel was stained 
with Coomassie Brilliant Blue (FIG. 3, lane 1) or periodic acid-Schiff 
reagent (FIG. 3, lane 2), and it comigrated with the inhibitor as revealed 
by reverse fibrin autography (FIG. 3, lane 3). The amount of inhibitor 
antigen present in the starting CM and in the various pooled fractions was 
determined by the rocket technique of Laurell, Scand. J. Clin. Lab. 
Invest, 29, 21 (1977), using antisera developed to the purified inhibitor. 
These screenings indicated that CM contained 0.6 micrograms/ml (ug/ml) of 
inhibitor, that 600 micrograms of inhibitor were applied to the 
concanavalin A column (FIG. 1), and that 90 microgams were recovered from 
the final gel extracts (FIGS. 2 and 3). Thus, this purification protocol 
yielded a recovery of approximately 15 percent of the starting antigen. 
The purified inhibitor had an apparent relative molecular mass (M.sub.r) 
of about 50,000.+-.2,500 daltons under both reducing and non-reducing 
conditions when compared directly to M.sub.r standards. 
B. Preliminary Characterization of the Purified Inhibitor 
PAs convert single chain plasminogen into two-chain plasmin by cleavage of 
a single arginine-valine bond, Summaria et al., J. Biol. Chem., 242, 4279 
(1967). This process can be monitored by SDS-PAGE in the presence of 
reducing agents, Mussoni et al., Thromb. Res., 34, 241 (1984); Summaria et 
al., J. Biol. Chem., 242, 4279 (1967); Dano et al., Biochim. Biophys. 
Acta, 566, 138 (1979). To determine whether the inhibitor was an 
anti-activator, its ability to inhibit this cleavage was assessed and the 
results shown in FIG. 4. The purified inhibitor blocked the ability of 
both u-PA and t-PA to cleave .sup.125 I-plasminogen into its 
characteristic heavy and light chains, and did so in a dose-dependent 
manner. Inhibition of t-PA was associated with the formation of an 
enzyme-inhibitor complex that was still apparent after SDS-PAGE as shown 
in FIG. 5. 
The inhibitor activity of the purified molecule, like that detected in CM 
collected from confluent BAEs, Loskutoff et al., Proc. Natl. Acad. Sci. 
(USA), 80, 2956 (1983), was not destroyed upon incubation at pH 2.7 for 60 
minutes at 37.degree. C., or upon exposure to SDS as shown in FIG. 6. In 
contrast, the inhibitor activity of purified protease nexin was abolished 
by these same treatments. The inhibitor activity of these proteins was not 
affected by incubation for 30 minutes at 37.degree. C. in the presence of 
5 percent 2-mercaptoethanol. 
C. Purification of the Inhibitor from BAEs Cultured in the Presence of 
L[3,4,5-.sup.3 H] Leucine 
Both plasma and serum contain inhibitors of fibrinolysis, Loskutoff, J. 
Cell Physiol., 96, 361 (1978); Mullertz, in Progress in Chemical 
Fibrinolysis and Thrombolysis, Davidson et al. eds., vol. 3, pp. 213-237, 
Raven Press, New York (1978); Collen, Thromb. Haemostas., 43, 77 (1980). 
Cultured endothelial cells may internalize or bind these serum proteins 
and subsequently release them back to the serum-free medium, Cohen, J. 
Clin. Invest., 52, 2793 (1973); Pastan et al., Cell, 12, 609 (1977); 
Rohrlich et al., J. Cell Physiol., 109, 1 (1981); McPherson et al., J. 
Biol. Chem., 256, 11330 (1981), during the preparation of CM. To determine 
whether the inhibitor actually was synthesized by BAEs, or was simply a 
contaminating serum inhibitor, the inhibitor was purified from the CM of 
cells cultured in the presence of L[3,4,5-.sup.3 H] leucine, employing the 
same protocol as that developed for the purification of the inhibitor from 
unlabeled CM. Two peaks of radiolabeled proteins were recovered when the 
concanavalin A-Sepharose column was eluted with alpha-methyl mannoside in 
the presence of low and high salt. The peak II fractions containing 
inhibitor were pooled and subjected to further analysis by SDS-PAGE and 
the results shown in FIG. 7. Both inhibitor activity and the majority of 
the radioactivity were recovered in the same fractions. These two 
activities also comigrated when the peak inhibitor fractions (fractions 
52, 53 in FIG. 7) were pooled, dialyzed, and subjected to subsequent 
analysis by alkaline PAGE (FIG. 8). Taken together, these data indicated 
that the inhibitor was a biosynthetic product of the cells, and not a 
contaminating serum protein. 
Immunoprecipitation screenings were performed both to confirm the above 
results and to quantitate inhibitor synthesis by cloned BAEs. The results 
are shown in FIG. 9 and in Table I below. 
TABLE I 
______________________________________ 
Inhibitor Synthesis by BAEs 
CPM Recovered.sup.a 
Cell Gel 
Isolated.sup.b 
CM Pool II Extract 
Immunoprecipitate 
______________________________________ 
BAE.sub.26 
9.3 .times. 10.sup.6 
2.9 .times. 10.sup.6 
1.2 .times. 10.sup.6 
-- 
(100%) (30%) (12%) 
Clone A 3.2 .times. 10.sup.7 
-- -- 8.2 .times. 10.sup.5 
(100%) (2.5%) 
Clone B 4.5 .times. 10.sup.7 
-- -- 1.5 .times. 10.sup.6 
(100%) (3.4%) 
______________________________________ 
.sup.a The total, TCAprecipitable radioactivity in the various fractions 
and in the immunoprecipitates was determined by standard procedures well 
known in the art. The data are normalized to the percent (shown in the 
parenthesis) of the cpm in the starting material (CM) recovered at each 
step. 
.sup.b In each case, approximately 1.5 .times. 10.sup.7 cells were labele 
with L[3,4,5-.sup.3 H] leucine (20 Ci/ml) for 24 hours as described in 
Section III hereinafter. The serumfree CM (15 ml) was collected and 
fractionated as indicated. 
In these immunological screenings, radiolabeled CM collected from cloned 
BAEs was incubated (maintained under bilogical assay conditions) with 
admixed antibody to the purified inhibitor. The bound material was 
extracted from the antibody protein A-Sepharose beads, fractionated by 
SDS-PAGE, and analyzed by autoradiography (FIG. 9). A single radiolabeled 
polypeptide of an approximate M.sub.r of 50,000 daltons was revealed, and 
it had inhibitor activity when analyzed by reverse fibrin autography. This 
protein did not adsorb to protein A-Sepharose beads prepared with 
preimmune serum. The total radioactivity recovered from the various CMs 
analyzed in these immunoprecipitation screenings, and the recovery of 
radiolabeled protein at each step of the purification (FIGS. 7-8; Table 
I), indicates that the inhibitor accounts for between 2.5-12 percent of 
the total protein synthesized and secreted by the cells in a 24 hour 
period (Table I). 
D. Development and Evaluation of a Functional Assay for Inhibitor 
(Inhibitor Binding Assay) 
Polyvinyl chloride (PVC) plastic wells were coated overnight at 4.degree. 
C. with varying concentrations of t-PA to affix the t-PA to the polyvinyl 
chloride solid matrix, and to determine the optimal concentrations of t-PA 
for the assay of the present invention as shown in FIG. 10. The wells were 
washed, blocked with BSA to remove non-specific protein binding sites, and 
maintained (incubated) for 2 hours at 37.degree. C. with three different 
concentrations of purified inhibitor (20, 50 and 100 ng/ml) to form 
complexes. After washing, the extent of binding was quantified by 
incubating (maintaining under biological assay conditions) the complexes 
so formed first with admixed rabbit anti-inhibitor receptor (diluted 
1:100) followed by admixed .sup.125 I-goat anti-rabbit IgG 
(1.5.times.10.sup.5 cpm/well). As the t-PA concentration used to coat the 
PVC. wells was increased from 0.1 to 1.0 microgram/ml, the detection of 
bound inhibitor increased at all three concentrations (FIG. 10). 
Increasing the t-PA coating concentration above 1 microgram/ml did not 
increase the detection of bound inhibitor. Thus, subsequent screenings 
employed a t-PA concentration of 1 microgram/ml for coating the PVC wells 
to affix the t-PA thereto. 
The kinetics of the interaction of the inhibitor with immobilized t-PA were 
determined in order to optimize the maintenance (incubation) period for 
inhibitor-containing solutions. Purified inhibitor (100 ng/ml) was 
incubated at 37.degree. C. for various times on either t-PA or BSA coated 
wells. The bound inhibitor was then quantified with the rabbit 
anti-inhibitor receptor (1:100) followed by .sup.125 I-goat anti-rabbit 
IgG (1.5.times.10.sup.5 cpm/well). The reaction between the inhibitor and 
immobilized t-PA was a fast reaction with over 75 percent binding 
occurring within 30 minutes (FIG. 11). During this period, the inhibitor 
did not bind to control, BSA coated wells. For convenience, a 1 hour 
incubation time for inhibitor-containing solutions was used in subsequent 
screenings. 
The maintenance (incubation) time for the polyclonal receptor and 
indicating means binding times were similarly optimized. t-PA- or 
BSA-coated wells were incubated for 1 hour at 37.degree. C. with the 
inhibitor (50 ng/ml) and then incubated with the rabbit anti-inhibitor 
receptor for various periods of time. Bound receptor was detected by a 2 
hour incubation with the indicator (.sup.125 I-goat anti-rabbit IgG). 
Alternatively, the wells were incubated for 2 hours with the receptor and 
the incubation time for the indicator was varied. Both the receptor and 
indicator associated rapidly with their respective antigen in the assay 
with over 80 percent binding occurring after 1.5-2 hours (FIG. 12). 
Therefore, subsequent screenings employed a 2 hour incubation period for 
both the receptor and indicator. 
The effect of varying dilutions of rabbit anti-inhibitor receptor on the 
detection of inhibitor was determined to optimize the assay's sensitivity. 
t-PA-coated wells were incubated for 1 hour at 37.degree. C. with various 
concentrations of inhibitor (1-100 ng/ml). After washing, the wells were 
incubated with various dilutions of rabbit anti-inhibitor receptor 
(1:50-1:500) and the bound antibody was detected with .sup.125 I-goat 
anti-rabbit IgG (1.5.times.10.sup.5 cpm/ml). Optimal detection of 
inhibitor occurred at a 1:50-1:75 dilution of the antisera (FIG. 13). 
Subsequent screenings employed a 1:75 dilution of the antisera. The effect 
of varying-concentrations of .sup.125 I-goat anti-rabbit IgG 
(2.5.times.10.sup.4 -3.times.10.sup.5 cpm/well) was similarly screened to 
optimize the assays's sensitivity. Optimal detection of inhibitor occurred 
at 2.5-5.times.10.sup.4 cpm/well of .sup.125 I-goat anti-rabbit IgG (FIG. 
14). 
A typical standard dose-response curve of purified inhibitor as detected in 
this assay is shown in FIG. 15. The assay was sensitive to 1 ng/ml, 
demonstrated a linear response to inhibitor between 10 and 100 ng/ml and 
saturated at inhibitor concentrations above 250 ng/ml. A dose-response 
using bovine aortic endothelial cell conditioned media (CM) is also shown 
in FIG. 15. Comparison of this curve with the standard curve indicates 
that this CM sample contained approximately 100 ng/ml of functionally 
active inhibitor. For convenience, the standard curve was routinely 
plotted on a log vs. log plot for the purpose of calculating inhibitor 
concentrations in unknown samples (FIG. 16). It can be seen that plotting 
in this way gave a straight line. 
E. Comparison of the Inhibitor Binding Assay to Reverse Fibrin Autography 
The sensitivity of the functional assay (inhibitor binding assay) of the 
present invention was compared with the sensitivity of another assay, 
reverse fibrin autography, commonly used for the detection and 
quantitation of PA inhibitor. Various concentrations of inhibitor (0.5 
ng-10 ng/lane) were fractionated by SDS-PAGE and then analyzed by reverse 
fibrin autography. The results are shown in FIG. 17. In this technique, 
the washed polyacrylamide gel was layed on an indicator gel containing 
fibrin, plasminogen and a PA. Plasmin was slowly formed, resulting in the 
general lysis of the gel except in areas where inhibitors were present in 
the corresponding polyacrylamide gel. The sensitivity of reverse fibrin 
autography was 2.5 ng/lane (FIG. 17) and since 0.1 ml was applied to each 
lane, its sensitivity was 25 ng/ml, or 25 times less sensitive than the 
inhibitor binding assay. 
F. Applications of the Inhibitor Binding Assay 
The inhibitor binding assay of the present invention was used to study the 
interaction of purified enzymes with the inhibitor. Three purified PAs 
(t-PA, u-PA and streptokinase) were preincubated for 1 hour at 37.degree. 
C. with the purified inhibitor (50 ng/ml) and the ability of the inhibitor 
to subsequently bind to t-PA was quantitated in the inhibitor binding 
assay. Exogenously added t-PA and u-PA were found to compete with the 
immobilized t-PA for binding to the inhibitor, with a 50 percent reduction 
in binding obtained at 12 ng/ml of t-PA and 6 ng/ml of u-PA, as shown in 
FIG. 18. Neither streptokinase, nor DFP-inactivated t-PA (data not shown) 
affected the binding of the inhibitor to immobilized t-PA. 
The inhibitor binding assay of the invention was also used to detect 
inhibitor in human plasma and serum. Plasma and serum were prepared from 
blood collected from 16 healthy human donors and the inhibitor activity in 
each sample measured in the inhibitor binding assay. Normal human plasma 
contained low or undetectable levels of inhibitor, as shown in FIG. 19. In 
contrast, serum from these donors contained high levels of inhibitor 
activity, as also shown in FIG. 19. 
Finally, the assay was employed to determine and compare inhibitor levels 
in plasma from normal donors and donors with suspected abnormalities in 
their hemostatic system. The results are shown below in Table II. 
TABLE II 
______________________________________ 
Detection of Inhibitor in Normal and Patient Plasma 
Inhibitor 
Sample Dilution cpm Bound (ng/ml) 
______________________________________ 
Normal plasma 
1:5 1500 N.D..sup.1 
1:10 700 N.D. 
Patient Plasma 
1:5 4700 25 
1:10 2300 25 
______________________________________ 
.sup.1 "N.D." indicates no inhibitor detected (less than 2 ng/ml). 
These samples were kindly provided by Dr. B. Wiman. It can be seen from 
this screening that no inhibitor was detected in normal plasma, while the 
patient plasma had approximately 25 ng/ml. This same patient was shown to 
have elevated inhibitor when studied with a different assay in Wiman, 
Thrombosis Research, 31, 427 (1983). 
G. Identification of endothelial cell type beta-PAI activity in placenta 
Two inhibitor zones having an M.sub.r of about 50-55 kilodaltons (kda) were 
revealed when 20 microliters (ul) of a crude human placental extract were 
analyzed for PAI activity by SDS-PAGE [Laemmli, (1970) Nature (London), 
227, 680-685] and reverse fibrin autography (RFA) [Erickson et al., (1984) 
Analytical Biochemistry 137, 454-463]. Immunoprecipitation studies 
demonstrated that the two inhibitor zones resulted from the presence of 
both the placental-type PAI [Astedt et al., (1985) Thromb. Haemostasis. 
53, 122-125] and the endothelial cell-type PAI [Loskutoff et al., (1983) 
Proc. Nat. Acad. Sci. U.S.A., 80:2956-2960; Emeis et al., (1983) Biochem. 
Biophys. Res. Commun. 110:392-398; Thorsen et al. (1984) Biochim. Biophys. 
Acta 802, 111-118; van Mourik et al., (1984) J. Biol. Chem. 259, 
14914-14921]. Quantitation using a radioimmune assay [Schleef et al. 
(1985) J. Lab. Clin. Med. 106, 408-415] indicated that the extract 
contained 270 nanograms per milliliter (ng/ml) of endothelial (beta-) PAI. 
Since the placental tissue had been extensively washed prior to extraction, 
this beta-PAI was most likely synthesized by cells contained in placenta 
and not a serum contaminant. Placenta was therefore employed as a source 
for the isolation of a cDNA for beta-PAI. 
H. Isolation of Human Beta-PAI cDNA 
Approximately 7.times.10.sup.5 recombinant phages from a .lambda.gt.sub.11 
expression library containing cDNA inserts prepared from human placental 
mRNA were obtained from Dr. Jose Millan of Cancer Research Center, La 
Jolla Cancer Research Foundation, La Jolla, Calif. That expression library 
is disclosed in Millan, (1986) J. Biol. Chem. 261, 3112-3115 whose 
disclosures are incorporated herein by reference, as containing 
1.times.10.sup.6 independent recombinant phages. 
Cytoplasmic-extracts from the phage-infected E. coli were screened 
immunologically to identify those phages containing cloned cDNAs that 
expressed the beta-PAI or a fusion polypeptide including that PAI fused at 
its amino-terminus to a portion of the beta-galactosidase molecule encoded 
by the .lambda.gt.sub.11 vector. Thirty-four positive clones were 
obtained, half of which continued to be positive through a second 
screening. Three positive clones were randomly selected and plaque 
purified, and phage DNA was prepared. 
The phage DNA from the three clones denominated .lambda.1.2, .lambda.3 and 
.lambda.9.2 was digested with EcoRI and the cDNA inserts were determined 
to be 1.9, 3.0 and 1.9 kilobase pairs (kb) in length, respectively. The 
EcoRI 3.0 kb cDNA insert from .lambda.3 was subcloned into a 
pBR322-derived plasmid vector, pGEM-.sub.3, to form the recombinant 
plasmid pPAI.sub.3 using E. coli MC1061 as a unicellular 
replication/expression medium as described by Bolivar et al., (1977) Gene 
2, 95-113. The subcloned cDNA insert was excised with EcoRI, and purified 
using an agarose gel. The cDNA insert was nick-translated and shown to 
hybridize with .lambda.1.2 and .lambda.9.2 DNAs at high stringency, 
indicating that the DNA inserts in the three clones were related. 
Three lines of evidence support the conclusion that the three isolated 
clones code for a proteinaceous material that is or includes the human 
endothelial cell type, (beta-migrating) PAI. 
(i) Induction of an E. coli lysogenic strain prepared by infecting a high 
frequency of lysogeny strain (Y1089; ATCC 37196) with .lambda.9.2 resulted 
in the expression of a recombinant fusion polypeptide having an apparent 
relative mass of about 180,000 daltons (M.sub.r =180 kda) that was 
recognized by an affinity-purified IgG from antisera raised against the 
purified BAE beta-PAI (FIG. 20, lane B). An E. coli strain lysogenic for 
.lambda.gt.sub.11, and thus lacking the cDNA insert, did not produce such 
an immunoreactive protein (FIG. 20, lane C). 
(ii) The 180 kda recombinant fusion polypeptide and the BAE beta-PAI share 
antigenic epitopes, since affinity purification of the antiserum to BAE 
beta-PAI on the recombinant fusion polypeptide yielded antibodies that 
recognized the purified BAE beta-PAI in Western blots (FIG. 20, lane D). 
(iii) Analysis of E. coli extracts by SDS-PAGE followed by RFA revealed 
that the .lambda.9.2 lysogen containing the 1.9 kb insert, but not the 
.lambda.gt.sub.11 lysogen, expresses PAI activity (FIG. 21, lane B). 
Surprisingly, two recombinant proteinaceous PAIs with M.sub.r s of 180 kda 
and 40 kda, respectively, were present in the .lambda.9.2 extracts. To 
investigate the relationship of these PAIs, proteins from the lysogens 
were fractionated by SDS-PAGE, and were again analyzed by Western blotting 
but this time the autoradiograms were developed after a longer exposure 
(FIG. 21, lanes E and F). Two polypeptides were detected, and these 
co-migrated with the inhibitor activities (compare lanes B and E). The 
great majority of the beta-PAI antigen produced by the .lambda.9.2 lysogen 
was detected at an M.sub.r of approximately 180 kda; however, a small 
amount of antigen was also detected at M.sub.r 40 kda (FIG. 21, lane E). 
Since the two PAIs share immunologic and biological properties, the 
smaller is most likely derived from the larger through proteolytic 
processing of the beta-galactosidase-PAI fusion polypeptide. The specific 
activity of the released 40 kda protein may be higher than that of the 
larger fusion protein because it is no longer sterically hindered by a 
fused polypeptide fragment. However, even though of lower specificity 
activity, possibly caused by steric hinderance, the 180 kda fusion 
polypeptide did exhibit plasminogen activator inhibitory activity and was 
shown to be immunologically related (similar) to BAE PAI. 
The observed biological activity (binding to and inhibiting of) t-PA 
activity was surprising inasmuch as the replication/expression medium was 
a procaryotic cell and the proteinaceous molecule is native to mammals. In 
addition, the native molecule is glycosylated while the expressed fusion 
polypepide and smaller 40 Kda protein were substantially free of 
glycosylation. Thus, although the expressed proteinaceous molecules could 
be expected to be folded differently from the native molecule and were 
free of glycosylation while the native moleacule is glycosylated, the 
expressed proteinaceous molecules exhibited the biologic activity of the 
native, mammalian protein. 
Recombinant plasmid pPAI.sub.3 contained in host E. coli strain MC1061 was 
deposited at the American Type Culture Collection, 12301 Parklawn Drive, 
Rockville, Md., 20852. It was received on Aug. 19, 1986, and was given the 
designation ATCC 67188. 
The present deposit was made in compliance with the Budapest Treaty 
requirements that the duration of the deposit should be for 30 years from 
the date of deposit for 5 years after the last request for the deposit at 
the depository or for the enforceable life of a U.S. patent that matures 
from this application, whichever is longer. The recombinant 
plasmid-containing cells will be replenished should they become non-viable 
at the depository. 
I. Nucelotide Sequence of DNA Coding for Human Beta-PAI and Assignment of 
Protein Sequence 
The DNA sequence from both strands of the 3.0 kb cDNA insert of clone 
.lambda.3 was established by sequencing deletion subclones constructed by 
the method of Dale et al. (1985) Plasmid 13, 31-40. FIG. 22 shows the DNA 
sequence along with the inferred amino acid residue sequence. This is not 
a full-length cDNA since the untranslated region, the initiation codon, 
and most of the signal peptide are missing from the 5'-terminus end of the 
molecule. 
In order to identify the codon coding for the amino-terminus of the mature 
protein, the deduced amino acid residue sequence was aligned with that of 
partially sequenced bovine beta-PAI and partially sequenced human 
endothelial PAI isolated by other means in our laboratory. The 
amino-terminal residue of mature human endothelial cell type PAI was 
determined to be valine (Val; V). Based on that alignment the valine 
designated number 1 in FIG. 22 is the amino-terminal residue of the human 
endothelial (beta-) PAI. 
Two originally obtained clones contained different codons at the -4 amino 
acid residue position. One of those codons codes for a serine (Ser; S) 
residue (.lambda.3) while the other (.lambda.9.2) codes for a glutamic 
acid residue (Glu; E). Glutamic acid has been found to be the correct 
residue at that position and is so shown in FIG. 22. 
The naturally occurring human beta-PAI is secreted and is therefore likely 
to contain a signal peptide [Blobel et al., (1975) J. Cell Biol. 67, 
852-862]. The signal peptidase normally cleaves to the carboxyl side of 
residues with small neutral side chains such as glycine, alanine and 
serine [von Heijne, (1984) J. Mol. Biol. 173, 243-251]. Thus, the alanine 
(ala) at the amino-terminal side of the valine (val) designated number 1 
may represent the terminal residue of the signal peptide. 
The reading frame shown in FIG. 22 is the only one without multiple 
termination codons, and codes for 383 residues followed by a TGA stop 
codon. Removal of the putative signal peptide by cleavage between alanine 
at position -1 and the valine designated as number 1 results in a mature 
beta-PAI that is 379 residues long and has a calculated molecular weight 
for the carbohydrate-free molecule of 42,770 daltons. This calculation 
agrees well with the molecular weight of the unglycosylated form of the 
BAE beta-PAI as determined by in vitro translation of its mRNA [Sawdey et 
al. (1986) Thromb. Res. 41, 151-160]. Human beta-PAI is glycosylated [van 
Mourik et al., (1984) J. Biol. Chem. 259, 14914-14921] and the amino acid 
residue sequence in FIG. 22 contains 3 putative glycosylation sites 
conforming to the canonical asn-x-ser/thr sequence [Marshall (1974) 
Biochem. Soc. Symp. 40, 17-26] at positions 209-211, 265-267, and 329-331. 
The 3'-untranslated region of the 3.0 kb cDNA is 1788 base pairs (bp), 
excluding the poly (A) tract. The consensus polyadenylation sequence 
AATAAA is found sixteen bp upstream from the poly (A) attachment site, 
which is in agreement with previous reports that this sequence is 
generally located 15-25 nucleotides upstream from the polyadenylation site 
[Proudfoot, (1976) Nature (London) 263, 211-214]. 
The two clones carrying cDNA inserts of 1.9 kb were partially sequenced and 
appear to be identical. These clones are also identical to the 3.0 kb cDNA 
except that they are truncated and lack much of the 3'-untranslated region 
(i.e., they lack the region 3'from nucleotide 1960). 
Northern blot analysis of total RNA prepared from the human fibrosarcoma 
cell line HT 1080 (ATCC CCL 121), using the 3.0 kb cDNA as probe, 
indicated the presence of two distinct transcripts, 3.0 and 2.2 kilobases 
in length (FIG. 23). This observation suggests that the 1.9 kb cDNAs may 
have been copied from the shorter RNA transcript. A similar size 
heterogeneity at the 3'-termini of mRNAs has been observed in other 
systems. It may result from expression of more than one gene, from 
alternative splicing events, or from the use of multiple polyadenylation 
signals [Crabtree et al., (1982) Cell 31, 159-166; King et al., (1983) 
Cell 32, 707-712; Hickok et al., (1986) Proc. Natl. Acad. Sci. USA., 83, 
594-598]. The mechanism in this case is not clear. 
Although the only polyadenylation consensus signal (AATAAA) found in this 
cDNA sequence is at the 3'-terminus of the 3.0 kb cDNA (FIG. 22), a 
similar but slightly modified sequence (AATAAT) was found at nucleotide 
positions 1998-2003. If this sequence were used as a signal for poly (A) 
addition it could explain the presence of the shorter transcript. In other 
systems, polyadenylation has been found to take place in the absence of 
the AATAAA sequence [Ohkubo et al., (1983) Proc. Natl. Acad. Sci. USA., 
80, 196-2200]. 
Comparison of the deduced amino acid sequence with other proteins using the 
Fast Protein Analysis homology program [Lipman et al. (1985) Science 227, 
1435-1441] revealed that the beta-PAI was 25-30% homologous with 
antithrombin III (AT III), alpha.sub.1 -antitrypsin (alpha.sub.1 AT), 
alpha.sub.1 -antichymotrypsinogen and ovalbumin, and therefore is a member 
of the serine proteinase inhibitor super family of proteins (serpins). The 
serpins have diverged from an ancestral molecule over a 500 million year 
period [Carrell et al., (1985) Trends Biochem. Sci. 10, 20-24] and now 
represent a diverse group of related proteins that control the major 
proteolytic cascades of the body (e.g., the coagulation, complement, 
fibrinolytic, and inflammatory cascades). 
The inhibitory specificity of the serpins appears to be defined primarily 
by a single amino acid residue in the reactive center, the so-called 
P.sub.1 residue [Carrell et al., (1985) Trends Biochem. Sci. 10, 20-24]. 
In general, this amino acid residue reflects the known specificity of the 
target proteinase. The reactive center of the serpins is located near the 
carboxyl-terminus, and because it appears to protrude from the rest of the 
molecule [Carrell et al. (1985) Trends Biochem. Sci. 10, 20-24], may 
represent the ideal substrate or "bait" for the protease. 
Protease inhibition is associated with the formation of 1:1 complexes 
between inhibitor and enzyme. The amino acid residue sequences of the 
reactive centers of beta-PAI, alpha.sub.1 AT and AT III are aligned in 
FIG. 24, using single letter amino acid residue designations. 
In this alignment, the R (arg) residue at position 346 is the P.sub.1 
residue of the PAI. Plasminogen activators convert plasminogen into 
plasmin by cleavage of a single arg-val bond [Collen, (1980) Thromb. 
Haemostasis 43, 77-89]. Thus, this alignment is consistent with the known 
arg-specificity of PAs. The finding that the P.sub.17 residue is glutamic 
acid (E) also supports this alignment, since this glutamic acid acts as 
the `hinge` in serpins and is conserved in all serpins sequenced to date 
[Carrell et al., (1985) Trends Biochem. Sci. 10, 20-24]. 
Human beta-PAI is unusually sensitive to oxidants and rapidly loses its 
activity in the presence of low concentrations of chloramine T. Since 
there are no cysteines in the deduced protein sequence (FIG. 22), and 
since the activity of the oxidatively inactivated beta-PAI can be restored 
by treatment with methionine sulfoxide peptide reductase, the loss of 
activity appears to reflect the oxidation of a critical methionine. The 
methionine in the reactive center of beta-PAI (i.e., at position 347, the 
inferred P.sub.1 ' position) is a likely candidate, since alpha.sub.1 AT 
also is sensitive to oxidation and its loss of activity has been related 
to the oxidation of the P.sub.1 methionine [Carrell et al., (1985) Trends 
Biochem. Sci. 10, 20-24]. In both cases, the resulting methionine 
sulphoxide is a bulkier residue and may not readily fit into the pocket of 
its substrate proteinase. 
It has been suggested that the ability to selectively inactivate 
alpha.sub.1 AT by oxidation of its active site methionine is an important 
and unique regulatory feature of this system. Activated neutrophils may 
neutralize the inhibitor by the secretion of oxygen free radicals [Carrell 
et al., (1985) Trends Biochem. Sci. 10, 20-24] at inflammatory sites. This 
additional level of regulation may provide the means by which essential 
tissue breakdown can take place, even in the presence of inhibitors which 
normally inhibit neutrophil elastase. Elevated PA activity has been 
correlated with tissue destruction, tissue remodelling, and with the 
formation of new organs [for review, Dano et al., (1985) Adv. Cancer Res. 
44, 139-266]. The ability to oxidatively inactivate the beta-PAI present 
in these tissues may also be an important regulatory feature of these 
systems, enabling PAs to function in the presence of their inhibitor. 
Thus, the local generation of oxidants may inactivate both alpha .sub.1 AT 
and beta-PAI, and in the process unleash a cascade of proteolytic enzymes 
including elastase, plasmin, and collagenase [Moscatelli et al., (1980) in 
Proteases and Tumor Invasion, ed. P. Struli (Raven Press, New York) pp. 
143-152]. 
III. Materials and Methods 
A. Assay-Related 
1. Plasminogen Activator 
Tissue-type plasminogen activator (t-PA) was isolated from human melanoma 
cell conditioned media as described in Rijken et al., J. Biol. Chem., 256, 
7035 (1981). Briefly, human melanoma cells were grown to confluent 
monolayers in plastic tissue culture flasks (Falcon, Oxnard, CA) at 
37.degree. C. in atmospheric air supplemented with 6 percent of CO.sub.2. 
The growth medium consisted of 100 ml of modified Eagle's essential medium 
supplemented with sodium bicarbonate (16 ml of a 7.5 percent solution per 
liter of medium), L-glutamine (10 ml of a 200 mM solution per liter of 
medium), and heat-inactivated newborn calf serum (final concentration, 10 
percent). The cells were washed with medium without calf serum and 
incubated with 25 ml of serum-free medium. The resulting conditioned 
medium (CM) was harvested and replaced on 3 consecutive days, centrifuged 
at 7000.times.g for 30 minutes and stored at -20.degree. C. until use. 
When indicated, Aprotinin (Calbiochem-Behring, La Jolla, Calif.) was 
added, both to the serum-containing and to the serum free medium (20 
KIU/ml, final concentration). 
Commercially available urokinase (5.times.10.sup.5 CTA units of WINKINASE, 
Sterling-Winthrop, Rensselaer, N.Y.) was purified further by affinity 
chromatography as described in Holmberg et al., Biochim. Biophys. Acta, 
445, 215 (1976). 
2. Plasminogen Activator Inhibitor 
Bovine aortic endothelial cells (BAEs) employed for the purification of the 
inhibitor were isolated from the aorta of cows by the method of Booyse et 
al., Thromb. Diathes. Haemorrh., 34, 825 (1975), whose teachings are 
incorporated herein by reference, and cultured in 150 cm2 flasks (Falcon 
Plastics, Oxnard, CA) in 15 ml of modified Eagle's medium supplemented 
with 10 percent fetal calf serum (Irvine Scientific, Santa Ana, CA) as 
described in Levin et al., Thromb. Res., 15, 869 (1979). The cells for the 
screenings had been passaged 16-22 times at a 1:5 ratio, and in general 
had been confluent for at least one week prior to the preparation of 
conditioned media (CM) as described below. 
Cloned BAEs were employed for some of the metabolic labeling screenings. 
These clones were developed from single cells that grew out of a primary 
cell preparation. Briefly, freshly isolated cells were seeded into 60 mm 
dishes and allowed to attach overnight. The cells were washed with 
pre-warmed medium, released from the culture dish with trypsin (GIBCO, 
Long Island, N.Y.), dispersed gently with a pipette, and diluted to 
approximately 20 cells per ml in growth medium. Four to five aliquots (50 
microliters each) of the diluted cells were then placed on the inverted 
sterile underside of Cooper dish lids (Falcon Plastics, Oxnard, Calif.) 
and incubated for 60 minutes at room temperature to allow cell attachment. 
After the position of each of the cellular droplets was marked with a pen, 
the lids were inverted back onto Cooper dish bottoms containing confluent 
BAEs in 6.7 ml of growth medium. The confluent BAEs had been maintained in 
this medium for 24 hours, presumably elaborating growth factors, Gajdusek 
et al., J. Cell Biol., 85, 467 (1980). The marked areas were examined in 
the microscope, and those areas containing single cells were monitored on 
consecutive days for cell growth. When these clones had grown to a few 
thousand cells, the cells were removed by ring cloning in the presence of 
trypsin, distributed into 0.5 cm microtiter wells (Falcon) containing 100 
microliters of growth medium, and allowed to grow to confluency. Empty 
lids also were inverted onto Cooper dish bottoms containing confluent 
BAEs, and served as controls for this method. These lids remained free of 
cells throughout the incubation period indicating that cells from bottoms 
did not detach and reattach on the lids. The clones developed by this 
procedure were positive for Factor VIII-related antigen indicating that 
they consisted of endothelial cells, Jaffe et al., J. Clin. Invest., 52, 
2757 (1973). 
Confluent monolayers were then washed twice with 15 ml of PBS and 
subsequently incubated with 15 ml of serum-free medium. After 24 hours, 
the resulting CM was collected, pooled, centrifuged for 5 minutes at 
400.times.g and, after adding NaN.sub.3 and Tween 80 (Sigma Chemicals, St. 
Louis, Mo.) to concentrations of 0.02 percent and 0.01 percent 
respectively, stored at -30.degree. C. until further use. Approximately 1 
liter of CM was passed over a 10 ml concanavalin A-Sepharose (Sigma 
Chemicals, St. Louis, Mo.) column (1.5.times.5 cm) previously equilibrated 
with phosphate-buffered saline (PBS) containing 0.02 percent NaN.sub.3 and 
0.01 percent Tween 80 [polyoxyethylene (80) sorbitan monooleate], at a 
speed of 10 ml/h at 4.degree. C. 
After collecting the flow-through material, the column was washed with at 
least 10 column volumes of PBS containing 1 M NaCl, 0.01 percent Tween 80 
and 0.02 percent NaN.sub.3 (pH 7.4) to remove non-specifically adsorbed 
proteins. The column was washed with approximately the same volume of this 
buffer but without the added NaCl, and then eluted in 2 steps. In the 
first, protein was eluted with 0.01 M sodium phosphate, pH 7.2, containing 
0.5 M alpha-methyl-D-mannoside (Sigma Chemicals, St. Louis, Mo.), 0.02 
percent NaN.sub.3 and 0.01 percent Tween-80, at a speed of 2.5 ml/h. The 
column was eluted a second time with the same buffer but containing 1 M 
NaCl. 
The second step in the purification involved preparative SDS-PAGE. The 
inhibitor-containing fractions (identified by slab gel electrophoresis and 
reverse fibrin autography) were pooled and aliquots (225 microliters) were 
subjected to SDS-PAGE in tube gels. When the tracking dye reached the 
bottom of the gel, the gels were frozen and cut into 1 mm slices. Every 
two slices were combined and extracted for 24 hours at 4.degree. C. with 
0.2 ml of PBS containing 0.01 percent Tween. Each extract was then tested 
for inhibitor activity by the .sup.125 I-fibrin plate assay (described 
below). The fractions containing the peak of inhibitor activity were 
pooled and stored at -70.degree. C. until further use. 
The inhibitor also was purified from CM collected from cells cultured in 
the presence of L-[3,4,5-.sup.3 H] leucine. In this case, the cultures 
were washed twice with 15 ml of leucine-free MEM (GIBCO, Long Island, 
N.Y.) and then were incubated in the presence of 15 ml of leucine-free MEM 
containing 20 microCi/ml of L-[3,4,5-.sup.3 H] leucine (158 Ci/mmol; New 
England Nuclear, Boston, Mass.). After 24 hours, the media were collected 
as described above, combined with 55 ml of unlabeled CM, and passed over a 
1 ml concanavalin A-Sepharose column (0.6.times.3.5 cm) at a speed of 4 
ml/h. The column was washed and eluted at 1 ml/h. Again, the 
inhibitor-containing fractions were pooled and subjected to preparative 
tube gel electrophoresis. The resulting inhibitor containing gel extracts 
were stored at -70.degree. C. until further use. 
Polyclonal receptors to the purified inhibitor were raised in rabbits as 
described in detail hereinafter. Protein A-Sepharose CL-4B (Pharmacia Fine 
Chemicals, Piscataway, N.J.) was rehydrated in PBS containing 0.02 percent 
NaN.sub.3, 0.05 percent Tween 20 [polyoxyethylene (20) sorbitan 
monolaurate] and 0.1 percent bovine serum albumin, and washed 3 times with 
a ten-fold excess of this buffer. The IgG fraction of the antisera was 
coupled to the washed beads as specified by the manufacturer at a ratio of 
approximately 80 micrograms protein A-Sepharose per 40 microliters of 
either anti-inhibitor reagent or pre-immune serum. The IgG-coated beads 
were added to 1 ml of CM collected from cloned BAEs cultured in the 
presence of [3,4,5-.sup.3 H] leucine. The samples were incubated for 1 
hour at room temperature, the beads were washed by centrifugation (3 times 
with 1 ml of PBS-Tween buffer) and extracted for 1 hour at 37.degree. C. 
with 0.25 M Tris-HCl (pH 6.8) containing 2.2 percent SDS, 20 percent 
glycerol, 0.025 percent bromophenol blue and 2.5 percent (v/v) 
2-mercaptoethanol. The resulting supernatant was analyzed by SDS-PAGE in 
slab gels or by liquid scintillation counting. 
SDS-PAGE in slab (15.times.10.times.0.15 cm) and tube (10.times.0.5 cm) 
gels was then performed according to Laemmli, Nature (Lond.), 227, 680 
(1970), whose illustrative teachings are incorporated herein by reference. 
The stacking gel consisted of 4 percent polyacrylamide and the separation 
gel of 9 percent polyacrylamide (both gels had a cross linkage of 3 
percent). Slab gels consisting of a 7.5-20 percent gradient of 
polyacrylamide in the separation gel also were prepared. After 
electrophoresis, the gels were fixed and stained either with 50 percent 
tricholoracetic acid containing 1 percent Coomassie Brilliant Blue 
(BioRad, Richmond, Calif.), or with periodic acid Schiff reagent, as in 
Ginsburg et al, in Methods in Hematology, Harker et al. eds., vol. 8, pp. 
158-176, Churchill Livingstone, New York (1983). 
Molecular weight standards employed to determine the apparent molecular 
weight of the purified inhibitor included phosphorylase B (92,500), human 
plasminogen (90,000), transferrin (77,000), bovine serum albumin (66,200), 
human serum albumin (66,000), ovalbumin (43,500), carbonic anhydrase 
(31,000), soybean trypsin inhibitor (21,500), lysozyme (14,400) and the 
66,000, 52,300, and 46,500 subunits of human fibrinogen. 
To localize radiolabeled proteins, the stained slab gels were dried and 
processed for autoradiography as described in Bonner et al., Eur. J. 
Biochem., 46, 83 (1974). The positions of the radiolabeled protein in tube 
gels was determined by slicing the gels into 1 mm pieces, extracting each 
gel slice into buffer as described above, and determining the 
radioactivity in each fraction. 
Alkaline (SDS-free) continuous PAGE was performed as in Hjerten et al., 
Anal. Biochem., 11, 219 (1965), using 0.37 M Tris-glycine (pH 9.5) as both 
gel- and running buffer. Tube gels were 10 percent polyacrylamide with a 
cross linkage of 2.5 percent. Samples were brought to 40 percent sucrose, 
applied to the gel, and subjected to electrophoresis, first for 0.5 hours 
at 2.5 mA/cm.sup.2 and then for 1-1.5 hours at 5 mA/cm.sup.2. 
Isoelectric focusing gels were prepared in glass tubes (2.5 mm) as in 
O'Farrell, J. Biol. Chem., 250, 4007 (1975). The resulting pH gradient was 
determined by cutting the gels into 1 mm slices. Every two slices were 
combined and extracted into 0.2 ml H.sub.2 O for 18 hours at 4.degree. C., 
and the pH and radioactivity in each of these extracts was determined. 
Slices from parallel gels also were extracted into 0.2 ml PBS/Tween and 
assayed for inhibitor activity and radioactivity. 
Inhibitor activity in polyacrylamide gels was localized either by direct 
measurement of the ability of the gel extracts to inhibit u-PA-mediated 
lysis of .sup.125 I-fibrin [fibrin-plate method of Loskutoff et al., Proc. 
Natl. Acad. Sci. (USA), 74, 3903 (1977)], or by reverse fibrin autography, 
as in Erickson et al., Anal. Biochem., 137, 454 (1984). In the latter 
technique, the white lysis-resistant zones in the indicator film resulted 
from the presence of inhibitors in the slab gel. 
To determine the stability of the inhibitor under denaturing conditions, 
the purified molecule (20 micrograms/ml) was incubated for 1 hour at 
37.degree. C. in 0.02M glycine, pH 2.7, containing 25 micrograms/ml of 
human serum albumin. The sample was neutralized by the addition of three 
volumes of assay buffer (pH 8.1) and subsequently tested at various 
dilutions made in assay buffer for residual activity by the .sup.125 
I-fibrin plate assay. Inhibitor (20 micrograms/ml) also was incubated for 
1 hour at 37.degree. C. in PBS containing 0.025 percent SDS and albumin 
(25 micrograms/ml). The SDS was neutralized by the addition of three 
volumes of assay buffer containing 0.18 percent Triton X-100 
[polyoxyethylene (9) octyl phenyl ether], and residual inhibitor activity 
was measured. Samples treated with PBS instead of glycine and SDS served 
as controls for these screenings. The effect of acid glycine and SDS on 
the inhibitor activity of purified protease nexin (160 micrograms/ml) was 
determined in a similar manner. 
3. Formation of Polyclonal Receptors 
Antisera to the inhibitor were raised in New Zealand rabbits by 
subcutaneous injections of 20 micrograms of purified inhibitor dissolved 
in 1 ml of saline and emulsified with 1 ml of Freund's complete adjuvant 
(Miles Laboratories, Naperville, Ill.). Booster injections employing 10 
micrograms of purified inhibitor in 0.5 ml of saline and emulsified with 
an equal quantity of incomplete Freund's adjuvant (Miles Laboratories, 
Naperville, Ill.) were administered at 2 week intervals. Serum containing 
polyclonal receptors to the inhibitor was collected 10 days after the 
third and fourth immunizations and pooled. 
4. Inhibitor Binding to t-PA Assay 
Purified t-PA (50 microliters/well, 1 microgram/ml) in phosphate-buffered 
saline (PBS) was incubated overnight at 4.degree. C. in U-bottom 
microtiter plates (PVC. plastic, Falcon 3911, Microtest III, Falcon, 
Oxnard, Calif.). At this and every subsequent step, the plates were washed 
with SPRIA buffer (PBS supplemented with 0.1 percent BSA, 0.05 percent 
NaN.sub.3 and 0.05 percent Tween 20). To "block" any remaining sites on 
the plastic, 3 percent BSA (200 microliters/well) was incubated in the 
wells for 1 hour at 37.degree. C. Test samples and standard curves of 
purified inhibitor were prepared in dilution buffer (PBS supplemented with 
3 percent BSA, 5 mM EDTA, 0.1 percent Tween 80, and 0.02 percent 
NaN.sub.3) and 50 microliters/well were incubated for 1 hour at 37.degree. 
C. Bound inhibitor was detected by incubation for 2 hours at 37.degree. C. 
with rabbit anti-inhibitor receptor (1:75 dilution in dilution buffer, 50 
microliters/well). The bound antibody-inhibitor-t-PA complex then was 
quantitated by incubation for 2 hours at 37.degree. C. with .sup.125 
I-labeled goat anti-rabbit IgG (5.times.10.sup.4 cpm/well, Cappel 
Laboratories, Cochranville, Pa.). The wells were cut individually and the 
radioactivity in each well determined in a gamma counter (CT (80-800) 
CT/T, General Electric, Milwaukee, Wis.). 
5. Miscellaneous 
Plasminogen was purified from outdated human plasma by affinity 
chromatography on lysine-Sepharose as described in Deutsch et al., 
Science, 170, 1095 (1970). Protein was determined by the method of 
Bradford, Anal. Biochem., 12; 248 (1976), using bovine serum albumin as 
the standard. PA activity was assayed on .sup.125 I-fibrin coated 
multiwell tissue culture dishes as described by Loskutoff et al., Proc. 
Nat. Acad. Sci. (USA), 74, 3903 (1977). Proteins were enzymatically 
labeled with .sup.125 I using solid-state lactoperoxidase/glucose oxidase 
reagents (Bio-Rad Laboratories, Richmond, Calif.) and carrier-free Na 
.sup.125 I (Amersham, Arlington Heights, Ill.), or, alternatively by the 
Iodo-gen procedure of Fraker et al., Biochem. Biophys. Res. Commun., 80, 
849 (1978), modified so that the labeling interval was only 5 minutes and 
the temperature was 4.degree. C. A typical specific activity of the final 
product was 1-4.times.10.sup.6 cpm/microgram protein. Bovine fibrinogen 
(fraction II, Calbiochem-Behring, La Jolla, CA) was purified as suggested 
in Mosesson, Biochim. Biophys. Acta, 57, 204 (1962) to remove plasminogen. 
Protease nexin was purified from cultured human fibroblasts as in Scott et 
al., J. Biol. Chem., 258, 10439 (1983) and kindly provided by Dr. J. 
Baker, University of Kansas, Lawrence, Kans. The .sup.125 I-plasminogen 
cleavage assay was performed as described in Loskutoff et al., J. Biol 
Chem., 256, 4142 (1981) and Mussoni et al., Thromb. Res., 34, 241 (1984). 
B. Beta-PAI-Related 
1. Reagents 
Restriction enzymes, alkaline phosphatase, T4 DNA ligase, E. coli DNA 
polymerase I, Klenow fragent of DNA polymerase I, and T4 DNA polymerase 
were purchased from Boehringer Mannheim GmbH. Alpha-.sup.32 p dGTP (3000 
Ci/mmol) and 35SdATP-alpha-S (600 ci/mmole; 1 Ci =37 GBq) were purchased 
from Amersham. Human alpha-thrombin was a generous gift of J. Fenton 
(Albany, New York, N.Y.), while fibrinogen was purchased from 
Calbiochem-Behring, La Jolla, Calif. The purified human urokinase [W. H. 
O. Urokinase Standard (preparation 66-46)] was obtained from the National 
Institute for Biological Standards and Control, Hollyhill, Hampstead, 
London, Great Britain. Human plasminogen was obtained and purified, 
according to the procedures of Deutsch et al., (1970) Science, 170, 
1095-1096. The purification of BAE beta-PAI and the development of 
antibodies to it were as described by van Mourik et al., (1984) J. Biol. 
Chem., 259, 14914-14921. Antiserum to the placental PAI was a gift from 
Dr. James Wun of The Rockerfeller University, New York, N.Y. Phage 
.lambda.gt.sub.11 is available from the ATCC as ATCC 37194. 
2. Preparation and analysis of crude placental extract 
Frozen human placenta (3.5 g) was washed with PBS and extracted, into 15 ml 
of PBS containing 0.5% Triton X-100 [polyoxyethylene (9) octyl phenyl 
ether] at 4.degree. C. The tissue was homogenized using a Dounce 
homogenizer, and cellular debris was removed by centrifugation at 
10,000.times.g for 10 minutes. The extracts were analyzed for inhibitor 
activity by reverse fibin autography [Erickson et al., (1984) Analytical 
Biochemistry, 137, 454-463]. Monospecific antisera against human 
placental-type PAI and bovine beta-PAI were coupled to protein A Sepharose 
(Pharmacia, Uppsala, Sweden) and employed as described [van Mourik et al., 
(1984) J. Biol. Chem., 259, 14914-14921; Sawdey et al. (1986) Thromb. 
Res., 41, 151-160] to immunoprecipitate the PAIs present in the extract. 
3. Immunological Screening of a .lambda.gt.sub.11 cDNA library 
A human .lambda.gt.sub.11 cDNA library derived from a premature (34 week 
old) human placenta and consisting of 1.times.10.sup.6 independent 
recombinant phages [Millan, (1986) J. Biol. Chem. 261, 3112-3115] was 
screened immunologically [Young et al., (1983) Proc. Natl. Acad. Sci. USA, 
80, 1194-1198; Young et al., (1983) Science, 222, 778-782; Huynh et al., 
(1984) in DNA Cloning Techniques: A Practical Approach, ed. Glover, D. 
(IRL Press, Oxford)] for beta-PAI, using the affinity purified IgG 
fraction [Cuatrecasas (1969) Biochem. Biophys. Res. Commun. 35, 531-537] 
of antibodies to the purified BAE beta-PAI as antibody probe [van Mourik 
et al., (1984) J. Biol. Chem. 259, 14914-14921]. To visualize antibody 
binding, .sup.125 I-labeled protein A [55 milliCuries per milligram 
(mCi/mg)] was employed. Autoradiography was performed by exposing the 
filters to Kodak XAR5 film with an intensifying screen at -80.degree. C. 
4. Western blot analysis of E. coli lysates 
Lambda gt.sub.11 and recombinant lysogens were induced and crude extracts 
of infected E. coli were prepared as described [Huynh et al., (1984) in 
DNA Cloning Techniques: A Practical Approach, ed. Glover, D. (IRL Press, 
Oxford)]. For Western blot analysis of the expressed PAI, 50 microliters 
(ul) of crude extract were fractionated by SDS-PAGE [Laemmli, (1970) 
Nature (London) 227, 680-685]. The proteins were electrophoretically 
transferred to nitrocellulose paper and immunoblotted as described [Lammle 
et al., (1986) Thromb. Res. 41, 747-759; Johnson et al., (1984) Gene Anal. 
Techn. 1, 3-8], using the immunoglobulin fraction of antiserum purified on 
either beta-PAI affinity columns (above) or on the isolated substantially 
pure fusion polypeptide. 
For the affinity purification of antisera on the isolated, substantially 
pure fusion polypeptide, 900 ul of crude extract from induced E. coli 
lysogens [Huynh et al., (1984) in DNA Cloning Techniques: A Practical 
Approach, ed. Glover, D. (IRL Press, Oxford)] were fractionated by 
SDS-PAGE and transferred to nitrocellulose paper. Strips containing 
proteinaceous materials of M.sub.r 150-200 kilodaltons (kda) were excised 
from the nitrocellulose sheets and used for the affinity purification of 
antisera. Blocking of the nitrocellulose filter strips, binding of 
specific antibodies, and washings were performed as described for the 
screening of .lambda.gt.sub.11 libraries with antibody probes [Huynh et 
al. (1984) in DNA Cloning Techniques: A Practical Approach, ed. Glover, D. 
(IRL Press, Oxford)]. To elute bound antibody, the filter strips were 
incubated twice with 200 ul 0.1 M glycine-HCl buffer, pH 2.5, containing 
0.02% fetal calf serum for 3 minutes. The eluted material was neutralized 
by the addition of 140 ul 0.5 M Tris-HCl, pH 8.0, dialyzed overnight, and 
used as the primary antibody in Western blotting analysis. 
5. Nucleic acid methods 
(a) Cloning and Sequencing of PAI Genes 
Phage particles prepared by the plate-lysate method and purified by CsCl 
equilibrium centrifugation were used for the purification of phage DNA 
[Maniatis et al., (1982) in Molecular Cloning:A Laboratory Manual, Cold 
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.]. Plasmid DNA was 
isolated by the method of Birnboim et al., (1979) Nucleic Acids Res. 7, 
1513-1522, followed by two consecutive ethidium bromide/CsCl equilibrium 
centrifugations. Enzyme reactions were carried out according to the 
conditions suggested by the suppliers. Total RNA was prepared by the 
method of Berger et al., (1979) Biochemistry 18, 5143-5149 from cultured 
HT 1080 cells (ATCC CCL 121), fractionated by agarose gel electrophoresis 
in the presence of formaldehyde [Fellous et al., (1982) Proc. Natl. Acad. 
Sci. USA., 79, 3082-3086], and subjected to Northern blot analysis 
[Thomas, (1980) Proc. Natl. Acad. Sci. USA., 77,5201-5205]. 
DNA from .lambda.gt.sub.11 clones was digested with EcoRI endonuclease and 
the excised cDNA insert was subcloned into bacteriophage M13 cloning 
vector mp9 Messing, (1982) Gene 19, 269-276] or plasmid vector pGEM-3 
(Promega Biotec, Madison, Wis.). M13 clones containing the cDNA insert in 
both orientations were isolated and deletion libraries of both strands 
were constructed using the single-stranded M13 method of Dale et al., 
(1985) Plasmid 13, 31-40. Before sequencing, the size of the M13 templates 
was determined by electrophoresis on 0.7% agarose gels, and selected 
templates were sequenced by the dideoxy-chain-termination method [Sanger 
et al., (1977) Proc. Natl. Acad. Sci. USA., 74, 5463-5487]. Both DNA 
strands were sequenced, and over 80% of each strand was sequenced two or 
more times. 
Processing of DNA sequence data was accomplished using the Staden program 
[Staden, (1982) Nucl. Acid. Res. 10, 4731-4751]. Homology searches were 
done utilizing the Pearson Fast Protein homology program [Lipman et al., 
(1985) Science 227, 1435-1441]. 
(b) Expression in Eukaryotes 
(i) Production of PAI by Recombinant 
DNA Expression in Mammalian Cells 
A recombinant DNA vector capable of expressing the PAI gene in mammalian 
Chinese hamster ovary (CHO) cells is constructed in the following manner 
using procedures that are well known in the art and are described in more 
detail in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold 
Springs Harbor Laboratories (1982). 
Lambda 3 clone DNA or plasmid pPAI3 DNA is first subjected to restriction 
endonuclease digestion with restriction endonuclease EcoRI, and the 
resulting 3 kilobase pair (kb) fragment is purified by size fractionation. 
The 3'-recessed termini of the 3 kb restriction endonuclease digested DNA 
are filled in using the Klenow fragment of DNA Polymerase I. A synthetic 
oligonucleotide fragment having the sequence: 
EQU GCATCGGATCCGATGC 
is produced according to the methods of Caruthers et al., J. Am. Chem. 
Soc., 103:3185 (1981), and Gait et al., Cold Spring Harbor Symp. Quant. 
Biol., 47:393 (1983) and is blunt-end ligated to the filled in 3 kb 
fragment using T4 DNA ligase. The resulting ligated fragment comprising 
the 3 kb fragment and the synthetic oligonucleotide is further subjected 
to BamHI restriction endonuclease digestion. 
The simian virus (SV40) based expression vector, pKSV-10 (Pharmacia Fine 
Chemicals, Piscataway, N.J.), is subjected to BglII restriction 
endonuclease digestion. The above prepared BamHI digested fragment 
containing PAI coding sequences is combined with the BglII digested vector 
and ligated using T4 DNA ligase which results in the formation of a 
circular recombinant expression plasmid denominated pSV-PAI. 
The expression plasmid pSV-PAI contains an intact E. coli ampicillin 
resistance gene. E. coli RR101 (Bethesda Research Laboratories, 
Gaithersburg, Md.) when transformed with pSV-PAI can thus be selected on 
the basis of ampicillin resistance for those bacteria containing the 
plasmid. Plasmid-containing bacteria are then cloned and the clones are 
subsequently screened for the proper orientation of the inserted PAI 
coding gene into the expression vector. 
Screening for orientation is accomplished by taking into account the 
location of PstI restriction endonuclease sites on both the inserted gene 
and the expression vector. In pSV-PAI, PstI digestion generates fragments 
of about 3.9, 3.1, 3.0 and 0.5 kb if the PAI-containing insert is 3'to the 
SV40 transcriptional promoter when read from left to right as shown in 
FIG. 22, and generates fragments of about 3.9, 3.5, 2.6 and 0.5 kb when 
the insert is in the reverse orientation. Therefore, identification of 
pSV-PAI constructs with the proper orientation requires size analysis of 
the fragments generated after PstI restriction endonuclease digestion, and 
the selection of those wherein the fragments are about 3.9, 3.1, 3.0 and 
0.5 kb. The construct with the insert in the appropriate beforementioned 
orientation is selected for further use in expression, and is referred to 
hereinafter as pSV-PAI. 
The above obtained plasmid, pSV-PAI, containing the gene that encodes PAI 
is propagated by culturing E. coli containing the plasmid. The plasmid DNA 
is isolated from E. coli cultures using the alkaline lysis method 
described in Maniatis et al., i Molecular Cloning: A Laboratory Manual, 
Cold Spring Harbor Laboratories (1982). 
Expression of PAI is accomplished by the introduction of pSV-PAI into the 
mammalian cell line, CHO, using the calcium phosphate-mediated 
transfection method of Graham et al., Virol., 52:456 (1973). To ensure 
maximal efficiency in the introduction of pSV-PAI into all CHO cells in 
culture, the transfection is carried out in the presence of a second 
plasmid, pSV2NEO (ATCC #37149) and the cytotoxic drug G418 (GIBCO 
Laboratories, Grand Island, N.Y.) as described by Southern et al., J. Mol. 
Appl. Genet., 1:327 (1982). Those CHO cells that are resistant to G418 are 
cultured and have acquired therein both plasmids, pSV2NEO and pSV-PAI, and 
are designated CHO/pSV-PAI cells. By virtue of the genetic architecture of 
the pSV-PAI expression vector, PAI is expressed in the resulting 
CHO/pSV-PAI cells and can be detected in and purified from the cytoplasm 
of these cells. 
Expressed PAI is conveniently detected by the reverse fibrin autography 
(RFA) assay described before. To that end, CHO/pSV-PAI cells are cultured 
and subsequently lysed in SDS-PAGE sample buffer as described by Laemmli, 
Nature, 227:680 (1970). The resulting solution containing cellular protein 
is centrifuged at 15,000 rpm for 10 minutes and the supernatants collected 
therefrom. The resulting supernate is subjected to SDS-polyacrylamide gel 
electrophoresis and biologically active PAI is analysed by RFA. 
(ii) Production of PAI by Recombinant DNA Expression in Yeast 
The recombinant vector capable of expressing the PAI gene in the yeast 
Saccharomyces cerevisiae (S. cerevisiae) is constructed in the following 
manner using procedures referred to in Section III B5 (b) (i). Many of the 
steps are identical to those used above except as is necessary to 
accomodate insertion of the PAI gene into a yeast-compatible expression 
vector and the manipulation of yeast cells. 
After isolation of the 3 kb fragment from the .lambda.3 clone or pPAI3 and 
the filling in of the 3'-recessed termini, a similarly prepared synthetic 
oligonucleotide having the sequence: 
EQU GCATCGATGC 
is blunt-end ligated thereonto the resulting fragment. The yeast expression 
vector, pTDTl (ATCC #31255), and the oligonucleotide ligated fragment are 
both ClaI restriction endonuclease digested, combined and further ligated 
together using T4 DNA ligase to form a circular recombinant expression 
plasmid denominated pY-PAI. Following transformation of E. coli with the 
plasmid so prepared, the selection of ampicillin resistant, plasmid 
containing bacteria follows as above relying upon the ampicillin 
resistance gene present on pY-PAI. 
Plasmid-containing bacteria are cloned and screened for the proper 
orientation of the inserted PAI gene by essentially following the method 
above. In pY-PAI, BamHI restriction endonuclease digestion generates 
fragments of about 7.4 and 3.3 kb if the PAI-containing insert is 3' to 
the TRPl yeast promoter of pTDTl when read from left to right as shown in 
FIG. 22. The construct with the insert in the beforementioned appropriate 
orientation is selected for further use in expression, and is referred to 
hereinafter as pY-PAI. 
Expression of PAI in yeast is accomplished by transformation of strain SHY3 
S. cerevisiae (ATCC #44771) using pY-PAI plasmid DNA propagated and 
purified as described before. After transformation by the spheroplast 
procedure of Hinnen et al., Proc. Natl. Acad. Sci. U.S.A., 75:1929 (1978), 
SHYU3 cells are cultivated in nutrient selection medium comprising 0.67 
percent yeast nitrogen base without amino acids (Difco Laboratories, 
Detroit, Mich.), 2 percent glucose and 50 micrograms per milliliter each 
of adenine, leucine and uracil as described by Miyajima et al., Mol. Cell 
Biol., 4:407 (1984). By virtue of the genetic architecture of the pY-PAI 
expression vector, PAI is expressed in the resulting pY-PAI transformed 
SHY3 cells, hereinafter SHY3/pY-PAI cells, and can be detected in and 
purified from the cytoplasm of these cells. 
Expressed PAI is conveniently detected by the RFA assay as described 
before. To that end, SHY3/pY-PAI cells are exponentially grown to a 
density wherein the optical density at 600 nanometers (nm) (OD600) is 
equal to 1, and then the yeast cells are washed with water and suspended 
in three volumes of a solution containing 50 mM potassium phosphate (pH 
7.0), 1 mM ethylenediamine tetra-acetic acid (EDTA), 5 mM 
2-mercaptoethanol, 0.5 mM phenylmethylsulfonylflouride and 1 microgram 
each per milliliter of leupeptin and pepstatin. Yeast cells are then 
disrupted by manual shaking with glass beads and centrifuged at 12,000 rpm 
for 30 minutes in a Sorval SS34 rotor. The supernatant from centrifugation 
is diluted 1:1 with a twice-concentrated yeast cellular proteins also 
containing the SDS-PAGE sample buffer described before. This solution is 
centrifuged and analysed by RFA after SDS-PAGE as also described. 
In each of the above illustrations, the resulting expressed PAI contains 
the entire amino acid residue sequence and further includes the sequence 
as follows located at the amino-terminal end of the mature PAI: 
EQU Met-Gln-Phe-Gly-Glu-Gly-Ser-Ala. 
The foregoing is intended as illustrative of the present invention but not 
limiting. Numerous variations and modifications can be effected without 
departing from the true spirit and scope of the invention.