Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
6387
BT-0028
Title
HIG~ LEVEL EXPRESSION ~F
FUNCTIONAL HUMAN PLASMINOGEN ACTIVATOR
INHIBITOR (PAI-1) IN E. COLI
Backqround of the Invention
Human t~ssue-type plasminogen actlvator (t-PA), a
single chain serine protease of Mr 68,000, is a key
physlological regulator of fibrinolysls. It converts
the zymogen plasminogen into plasmin, the enzyme which
degrades the fibr~n network of the thrombus (Collen
(1980) Thromb. Haemosta. 43:77-82; Ri~ken and Collen
(1981) J. Biol. Chem. 256:7035-7041). Apparently, in
the presence of a clot, both t-PA and plasmlnogen bind
to fibrin and form a ternary complex in which
plasminogen is efficiently activated (Hoylaerts et al.
(1982) J. Biol, Chem. 257:2912-2919; Ranby (1982)
Biochim. BioDhvs. Acta 704:461-469). The affinity for
fibrin makes t-PA clot-specif~c, and useful as a
therapeutic agent for fibrinolytic therapy in man (Van
de Werf et al. (1984) Circulation 69:605-810).
The principal physiological regulator of t-PA
appears to be a specific, fast-acting, plasminogen
activator inhibitor (PAI-1). PAI-1 ~s a protein of Mr
50,000 which b~nds to t-PA in a 1:1 complex, and
inactivates the serine protease (Van Mourik et 3~1
(1984) J. Biol Chem. 259:14914-14921; Colucci et al.
(1985) J. Clin. Invest. 75:818-824; Almer and Ohlin
(1987) Thromb. Research 47:335-339). A number of
recent clin~cal studies suggest that elevated levels of
PAI-1, by reducing the net endogenous fibrinolytic
capacity, may contribute to the pathogenesis of various
thrombotic disorders, including myocardial infarction,
deep ve~n thrombosis, and dissem~nated intravascular
coagulation (Hamsten et al. (1985) New En~land Journal
~63~7
of Medlclne 313:1557-1563; Wlman et al. (1985) ~. Lab.
Clin. Med. I05:265-270). These observatlons suggest
that the t-PA:PAI-1 lnteract~on may be a sultable
target for pharmacologlc lnterventlon aimed at
enhancing endogenous fibrinolytic activlty. On the
other hand, induction of endogenous PAI-1 by
admlnlstratlon of endotoxin ln rabblts fa~led to alter
thrombolysls by lnfuslon of t-PA. M. Coluccl et al.
(1986) J. Cl~n. Invest. 78:138-144. Thls suggests that
administration of exogenous PAI-1 would not alter
fibrinolysis in vivo.
The lsolatlon of human PAI-1 cDNA has prev1Ously
been reported (Ny et al. (1986) Proc. Natl. Acad. Scl,
USA 83:6776-6780; Pannekoek et al, (1986) EMBO ~.
5:2539-2544; Glnsburg et al. (1986) J. Clln. Invest.
78:1673-1680; Wun and Kretzmer (1987) FEBS Letters
210:11-16).
Ny et al. (suDra) disclosed the expression of
functional rPAI in E. coli, using a phage lambda
gtll-derlved vector. rPAI was expressed as a beta-
galactosidase-PAI fusion proteln of about 180 kDa, with
the PAI codlng sequence fused to the E. coll beta-
galactosldase codlng sequence. A PAI-derived fragment
of about 40 kDa was also detected by lmmunoblot and by
reverse fibrin autography assay. This 40 kDa form of
rPAI was not characterlzed by Ny et al., but likely
arises by translatlon lnitlatlon at Met/aa #3, slnce an
E. coll translation lnitlatlon start slgnal (AGGA)
fortultously occurs 5' to thls Met (see Table 1). Wun
and Kretzmer (supra) simllarly used a lambda gtll
expression vector to express functional rPAI ln E.
coli. Pannekoek et al. (supra) reported the expression
of a functional 43 kDa form of rPAI in E. coll using a
pUC8-derlved vector, as assayed by reverse flbrln
autography.
2~6~7
Although Ny et al. (suDra), Pannekoek et al.
(suDra), and Wun and Kretzmer (suPra) report the
expression of functlonal rPAI-1 in E. coli, they do not
report the purif~cation of the E. coli-expressed rPAI
and do not quantitate the specific activ~ty of the
crude preparations of rPAI. It is, therefore, not
clear from these references whether the rPAI could be
purified from E. coli in a fully functional form, or
whether, alternatively, the purified E. coli-expressed
PAI would have reduced activity relatlve to mammalian
cell-expressed PAI or would require an actlvation step
to attain full activity. PAI-1 isolated from many
mammalian cell types is obtained in a partlally active
latent form: the latent form can be converted to a
fully act~ve form, with about 6-fold increased specific
activity relative to the latent form, by treatment w1th
denaturants such as sod~um dodecylsulfate (SDS) (Hekman
and Loskutoff (1985) J. Biol. Chem. 260:11581). In
fact, a recent report from the Pannekoek lab states
that rPAI-1 is expressed in E. coli almost exclusively
in an inactive, latent form (Lambers et al.
FibrinolYsls (1988) 2, Supp. 1:33). Apparently the 43
kDa form reported by Pannekoek et al. (suDra) is
predominantly inactive. Another recent report states
that rPAI-1 expressed in E. coli has biological
activity toward urokinase almost equal to that of human
fibrosarcoma PAI-1 (Lawrence et al. Fibrinolvsis (1988)
2, Supp.1:54).
Brief Summarv of the Invention
The invention provides substantially pure,
biologically functional, nonfused mature form E. coli-
expressed human PAI-1 protein having a specific
activity of about 0.3 unlt/ng, where a unit is defined
as the amount of protein required to neutralize 1
2~ ~3~7
interndtional unit of t-PA in an S2288 chromogenic
assay, where the amidolytic activity of t-PA is
measured .
The inventlon also relates to substantially pure,
biologlcally functional, nonfused mature form E. coli-
expressed human PAI-1 having a specific actlvity, as
defined above, the biological activity of which is not
slgnificantly enhanced by treatment with proteln
denaturants.
The invention relates further to a recombinant
plasmid expresslon vector capable of expression in E.
coli of soluble, biologically functional human PAI-1,
wherein the DNA codlng sequence for mature PAI-1 is
operatively linked to a phage lambda PL promoter or to
a tac promoter, preferably to a phage lambda, PL,
promoter, whlch gives particularly high level
expression. Preferred is a plasmid which is or which
has the identifying characteristics of pCE1200. The
invention also relates to an E. coli cell line
transformed with such a plasmid, preferably an E. coli
cell line that is or that has the identifying
characterlstics of E. coli strain TAP 106. The
inventlon relates further to biologically active
recombinant mature human PAI-1 expressed by such a cell
line.
The invention also provides a method for
identifying inhibitors of the binding of t-PA and PAI-
1, comprising the steps of:
1) providing in a container, under conditions
which permit the b~nding of t-PA and PAI-1:
a) a surface on which is immobilized a known
quantity of PAI-1 protein, the PAI-1
protein being attached to the surface by a
monoclona) antibody specific for PAI-1,
said monoclonal antlbody characterized in
~163~7
that lt blnds to PAI-1 at a doma~n not
lnvolved ln the lnteractlon of t-PA and
PAI-l;
b) a known quantlty of labeled t-PA; and,
c) a known quantlty of a compound to be
evaluated for lnhlbitlon of t-PA-PAI-1
b~nding; and
2) measur~ng the amount of labeled t-PA bound to
PAI-1 and determ1ning the extent of inhlb~tlon
of t-PA-PAI-1 blnding.
Preferred ls a method for ldentlfylng lnhlbltors of
the blnding of t-PA and PAI-l wherein the surface on
which the PAI-l is 1mmobilized is a m~crotiter plate.
Also preferred is a method wherein the labeled t-PA is
radiolabeled.
The lnvention relates further to a pharmaceut1cal
compositlon comprising, in a pharmaceutically
acceptable veh~cle, an amount of rPAI-1 of the
inventlon, that ~s therapeutically effect~ve to
counteract excessive or inapproprlate flbrinolysls.
Also lncluded ls a method of treatlng inapproprlate or
excess~ve flbrlnolysis ln a mammal suffering from such
a condltion, comprising administering to the mammal an
effective dose of E. coli-expressed rPAI of the
inventlon.
The ~nvention relates further to a recombinant
human PAI-l peptide having an amino acid sequence
correspond~ng to that of Table 1, with an N-terminal
sequence of Met-Ser-Ile-Val-His or Ser-Ile-Val-H~s,
where Val is amlno acid ~24 of the PAI-l precursor
proteln, and to a recombinant human PAI-1 peptlde
havlng an amino acid sequence correspondlng to that of
Table 1, and having an N-term~nal sequence of Met-Val-
Hls or Val-His, where Val is amino acld #24 of the PAI-
1 precursor protein.
Brief Descrlption of the Drawinq
Figure 1 - Scatchard analysls of blnding data for
the bindlng of 125I-t-PA to PAI-1 in the assay of the
inventlon, characterized ln that PAI-1 is bound to d
solid support using a PAI-1-speclflc monoclonal
antibody. PAI-1-containlng wells, prepared as
described ln Example 5 were incubated with 100 fmoles
of 125I-t-PA and lncreasing concentration of unlabeled
t-PA ~rdnging from 10 fmoles to 10 nmoles). The total
t-PA bound in each well was determined, and was plotted
versus the bound/free ratio. Each point represents the
averdge value of trlplicate samples.
Detailed DescrlDtion of the Invent1On
We have expressed ln E. coll two forms of rPAI-1 of
about 43 kDa that closely correspond to the two mature
forms of PAI-1 (Andreasen et al. (1986) FEBS Letters
209:213-218). It was found that mature form rPAI could
be expressed at high levels ln E. coli, i.e., at levels
of 10 to 15% of total E. coll proteln uslng a plasmld
vector employing the phage lambda PL promoter. Use of
the PL promoter-contalning plasmid resulted ln
significantly higher levels of expression in E, coli
than d plasmld vector employlng the "tac" promoter.
Whereas the PL promoter-containing vector resulted in
rPAI accumuldtion of 10 to 15% of total cell protein,
the "tac" promoter-conta~n1ng expression vector only
produced rPAI at levels of about 1% of total cell
proteln. We have in addition found that E. coli-
expressed nonfused mature form rPAI purlfled tohomogenelty exhiblts full biological activity.
Speclfically, the purified E. coli-expressed rPAI
exhiblted specific functional activity exceedlng that
of purlfled human flbrosarcoma cell PAI by S to
10-fold, depending on the assay employed. Moreover, E.
3 ~ 7
coli-expressed pur~fied rPAI d~d not require an
act~vation step to convert the proteln from a less
active to a more active form.
Our results contrast with those of prevlous
researchers. Lawrence et al. (F~brinolvsis (1988) 2,
Supp.1 54) report E. coli-expressed rPAI with actlvity
equal to that of human fibrosarcoma PAI. In contrast,
rPAI obtained using our process exhibits functional
activity exceeding that of human fibrosarcoma PAI by 5
to 10-fold. Other recent reports (Lambers et al.
(1988) Fibrinolysis 2, Supp.1:33) state that rPAI ~s
obtained from E. coli almost exclusively ~n an ~nactive
latent form. As discussed above, rPAI obtained by the
methods of the invention exhibits full functional
activity that is not enhanced by and does not require
any actlvation procedures.
A further aspect of the invention involves a
process for detecting inhibitors of the interaction of
PAI and t-PA. Thls process employs rPAI which ~s
lmmobilized to a solid support using a PAI-spec~f~c
monoclonal antibody which is first attached to the
solid support. The binding of radiolabeled t-PA to the
immobilized PAI is quantitated by separating the bound
and free t-PA. Previous assays for the quantitation of
PAI and t-PA binding which have used immobilized PAI
(PCT WO 88/01273) did not utilize a PAI-specific
monoclonal antibody to capture the PAI on the solid
support and present it for binding to t-PA. We have
found that the use of such a capture antibody markedly0 improves the sensltivity of the binding assay.
Examele 1
Isolation of Human PAI-1 cDNA
A human umbilical cord endothelial cell (Gimbrowne
et al. (1974) J. Cell B~ol. 60:673) cDNA lambda gtll
library was constructed using published methods (Gubler
~16~
and ~offman (1983) Gene 25:263). Oligonucleot~de
probes to screen the library were designed based on
published PAI-1 sequences (Ny et al (1986) Proc. Natl.
Acad. Sci. USA 83:6776-6780; Pannekoek et al. (1986)
EMB0 J. 5:2539-2544). A mixture of the following
probes was used:
1. 5' GAATTCCTGCAGCTCAGCAGC 3'
2. 5' TGGACCAGCTGACACGGCTGGTGC 3'
3. 5' TCTGAGGCCAGGTGGCCACTAGATGGGGGATGG 3'
4. 5' AGAGAGGCACCTCTTTTTCATAAGGGGCAGC M 3'
Approximately 3 X 106 recombinant lambda plaques
were screened for PAI-1 sequences. Four strongly
reactive poslt1ves and 9 weaker posltives were
isolated. The insert size, removed from lambda vector
by EcoRI digest, of the stronger positives was found to
be approx~mately 2100 to 3100 bp long and was
therefore, consistent with the si2e expected for a PAI-
1 cDNA (Ny et al,, suDra; Ginsburg et al. (1986) J.
Clin. Invest. 78:1673-1680).
The 2.1 kbp cDNA insert from one of the lambda
isolates, destgnated ECE3-1, was inserted into the
EcoRI site of the pTZ19R vector (Pharmacia, Piscataway,
NJ) to yield plasmid pECE3-1. Restriction analyses and
DNA sequenclng of pECE3-1 revealed that it contained a
human PAI-1 gene cDNA and coded for a 44 kDa protein of
identical amino acid sequence to that previously
described (Table 1; Ny et al., suPra; Pannekoek et al.,
suDra; Ginsburg et al., suPra). The nucleic acid
sequence of PAI-1 clone ECE3-1 only differed from that
of Pannekoek et al. within the untranslated region of
the cDNA (Table 2). A translat~on stop signal (TGA)
terminates translatlon with amino acid #402, prol~ne.
2~3~7
Table 1
PAI-1 cDNA Clone ~CE3-1 DNA Sequence
and Encoded Amlno Ac~d Sequence:
_. ~
EcoRI
1AATTCCTGCAGCTCAGCAGCCGCCGCCAGAGCAGGACGAACCGCCAATCGCAAG&CACC
1+ + + + + 60
CTTAAGGACGTCGAGTCGTCGGCGGCGGTCTCGTCCTGCTTGGCGGTTAGCGTTCCGTGG
TCTGAGAACTTCAGGATGCAGATGTCTCCAGCCCTCACCTGCCTAGTCCTGGGCCTGGCC
61 120
AGACTCTTGAAGTCCTACGTCTACAGAGGTCGGGAGTGGACGGATCAGGACCCGGACCGG
M Q M S P A L T C L V L G L A
lpaLl
CTTGTCTTTGGTGAAGGGTCTGCTGTGCACCATCCCCCATCCTACGTGGCCCACCTGGCC
121+ + + ----+---------+---------+ 180
GAACAGAAACCACTTCCCAGACGACACGTGGTAGGGGGTAGGATGCACCGGGTGGACCGG
L V F G E G S A Y H H P P S Y V A H L A
22 24
TCAGACTTCGGGGTGAGGGTGTTTCAGCAGGTGGCGCAGGCCTCCAAGGACCGCAACGTG
181 + 240
AGTCTGAAGCCCCACTCCCACAAAGTCGTCCACCGCGTCCGGAGGTTCCTGGCGTTGCAC
S D F G V R V F Q Q V A Q A S K D R N V
GTTTTCTCACCCTATGGGGTGGCCTCGGTGTTGGCCATGCTCCAGCTGACAACAGGAGGA
241+ -_- _+_ __ __ +_ + _ + + 300
CAAAAGAGTGGGATACCCCACCGGAGCCACAACCGGTACGAGGTCGACTGTTGTCCTCCT
V F S P Y G V A S V L A M L Q L T T G G
GAAACCCAGCAGCAGATTCAAGCAGCTATGGGATTCAAGATTGATGACAAGGGCATGGCC
301+ + 360
CTTTGGGTCGTCGTCTAAGTTCGTCGATACCCTAAGTTCTAACTACTGTTCCCGTACCGG
E T Q Q Q I Q A A M G F K I D D K G M A
'7
RsaI
CCCGCCCTCCGGCATCTGTACAAGGAGCTCATGGGGCCATGGAACAAGGATGAGATCAGC
361 + + - +---------+---------+---------+ 420
GGGCGGGAGGCCGTAGACATGTTCCTCGAGTACCCCGGTACCTTGTTCCTACTCTAGTCG
P A L R H L Y K E L M G P W N K D E I S
ACCACAGACGCGATCTTCGTCCAGCGGGATCTGAAGCTGGTCCAGGGCTTCATGCCCCAC
421 480
TGGTGTCTGCGCTAGAAGCAGGTCGCCCTAGACTTCGACCAGGTCCCGAAGTACGGGGTG
T T D A I F V Q R D L K L Y Q G F M P H
TTCTTCAGGCTGTTCCGGAGCACGGTCAAGCAAGTGGACTTTTCAGAGGTGGAGAGAGCC
481 + + 540
AAGAAGTCCGACAAGGCCTCGTGCCAGTTCGTTCACCTGAAAAGTCTCCACCTCTCTCGG
F F R L F R S T V K Q V D F S E V E R A
AGATTCATCATCAATGACTGGGTGAAGACACACACAAAAGGTATGATCAGCAACTTGCTT
541 + + + ------+---------+---------+ 600
TCTAAGTAGTAGTTACTGACCCACTTCTGTGTGTGTTTTCCATACTAGTCGTTGAACGAA
R F I I N D W V K T H T K G M I S N L L
GGGAAAGGAGCCGTGGACCAGCTGACACGGCTGGTGCTGGTGAATGCCCTCTACTTCAAC
601 +- -------+---------+---------+---------+ + 660
CCCTTTCCTCGGCACCTGGTCGACTGTGCCGACCACGACCACTTACGGGAGATGAAGTTG
G K G A V D Q L T R L V L V N A L Y F N
GGCCAGTGGAAGACTCCCTTCCCCGACTCCAGCACCCACCGCCGCCTCTTCCACAAATCA
661 720
CCGGTCACCTTCTGAGGGAAGGGGCTGAGGTCGTGGGTGGCGGCGGAGAAGGTGTTTAGT
G Q W K T P F P D S S T H R R L F H K S
GACGGCAGCACTGTCTCTGTGCCCATGATGGCTCAGACCAACAAGTTCMCTATACTGAG
721 + 780
CTGCCGTCGTGACAGAGACACGGGTACTACCGAGTCTGGTTGTTCMGTTGATATGACTC
D G S T V S V P M M A Q T N K F N Y T E
TTCACCACGCCCGATGGCCATTACTACGACATCCTGGAACTGCCCTACCACGGGGACACC
781 840
AAGTGGTGCGGGCTACCGGTAATGATGCTGTAGGACCTTGACGGGATGGTGCCCCTGTGG
F T T P D G H Y Y D I L E L P Y H G D T
ll
CTCAGCATGTTCATTGCTGCCCCTTATGAAAAAGAGGTGCCTCTCTCTGCCCTCACCAAC
~41 + + + + 900
GAGTCGTACAAGTAACGACGGGGAATACTTTTTCTCCACGGAGAGAGACGGGAGTGGTTG
L S M F I A A P Y E K E V P L S A L T N
ATTCTGAGTGCCCAGCTCATCAGCCACTGGAAAGGChACATGACCAGGCTGCCCCGCCTC
901 + + + + 960
TAAGACTCACGGGTCGAGTAGTCGGTGACCTTTCCGTTGTACTGGTCCGACGGGGCGGAG
I L S A Q L I S H W K G N M T R L P R L
CTGGTTCTGCCCAAGTTCTCCCTGGAGACTGAAGTCGACCTCAGGAAGCCCCTAGAGAAC
961 + + + + + + 1020
GACCAAGACGGGTTCAAGAGGGACCTCTGACTTCAGCTGGAGTCCTTCGGGGATCTCTTG
L V L P K F S L E T E V D L R K P L E N
CTGGGAATGACCGACATGTTCAGACAGTTTCAGGCTGACTTCACGAGTCTTTCAGACCAA
1021 - -+ --------+---------+--______ +_________+ _______+ 1080
GACCCTTACTGGCTGTACAAGTCTGTCAAAGTCCGACTGAAGTGCTCAGAAAGTCTGGTT
L G M T D M F R Q F Q A D F T S L S D Q
GAGCCTCTCCACGTCGCGCAGGCGCTGCAGAAAGTGAAGATCGAGGTGAACGAGAGTGGC
1081 + + + + 1140
CTCGGAGAGGTGCAGCGCGTCCGCGACGTCTTTCACTTCTAGCTCCACTTGCTCTCACCG
E P L H V A Q A L Q K V K I E V N E S G
ACGGTGGCCTCCTCATCCACAGCTGTCATAGTCTCAGCCCGCATGGCCCCCGAGGAGATC
1141 1200
TGCCACCGGAGGAGTAGGTGTCGACAGTATCAGAGTCGGGCGTACCGGGGGCTCCTCTAG
T V A S S S T A V I V S A R M A P E E
ATCATGGACAGACCCTTCCTCTTTGTGGTCCGGCACAACCCCACAGGAACAGTCCTTTTC
1201 + + + + + 1260
TAGTACCTGTCTGGGAAGGAGAAACACCAGGCCGTGTTGGGGTGTCCTTGTCAGGAAAAG
I M D R P F L F V V R H N P T G T V L F
ATGGGCCAAGTGATGGAACC ~ CCCTGGGGAAAGACGCCTTCATCTGGGACAAAACTG
1261 + + ~ + + + 1320
TACCCGGTTCACTACCTTGGGACTGGGACCCCTTTCTGCGGAAGTAGACCCTGTTTTGAC
M G Q V M E P *
402
3 ~ '7
Nsl I
GAGATGClTCGGGAAAGAAGAAACTCCGAAGAAAAGAATTTTAGTGTTAATGACTCTTTC
1321+ + + + + + 1380
CTCTACGTAGCCCTTTCTTCTTTGAGGCTTCTTTTCTTAAAATCACAATTACTGAGAAAG
BglII
TGAAGGAAGAGAAGACATTTGCCTTTTGTTAAAAGATGGTAAACClGATCTGTCTCCAAG
1381+ + + 1~40
ACTTCCTTCTCTTCTGTAAACGGAAAACAATTTTCTACCATTTGGTCTAGACAGAGGTTC
ACCTTGGCCTCïCCTTGGAGGACCTTTAGGTCAAACTCCCTAGTCTCCACCTGAGACCCT
1~41+ + ~ + + + lS00
TGGAACCGGAGAGGAACCTCCTGGAAATCCAGTTTGAGGGATCAGAGGTGGACTCTGGGA
GGGAGAGAAGTTTGAAGCACAACTCCCTTAAGGTCTCCAAACCAGACGGTGACGCCTGCG
1501+ + + + 1560
CCCTCTCTTCAAACTTCGTGTTGAGGGAATTCCAGAGGTTTGGTCTGCCACTGCGGACGC
GGACCATCTGGGGCACCTGCTTCCACCCGTCTCTCTGCCCACTCGGGTCTGCAGACCTGG
1561+ + 1620
CCTGGTAGACCCCGTGGACGAAGGTGGGCAGAGAGACGGGTGAGCCCAGACGTCTGGACC
TTCCCACTGAGGCCCTTTGCAGGATGGAACTACGGGGCTTACAGGAGCTTTTGTGTGCCT
1621+ + + + + + 1680
AAGGGTGACTCCGGGAAACGTCCTACCTTGATGCCCCGAATGTCCTCGAAAACACACGGA
RsaI
GGTAGAAACTATTTCTGTTCCAGCTCACATTGCATCACTCTTGTACTGCCTGCCACCGCG
1681 1740
CCATCTTTGATAAAGACAAGGTCGAGTGTAACGTAGTGAGAACATGACGGACGGTGGCGC
GAGGAGGCTGGTGACAGGCCAAAGCGAGTGGAAGAAACACCCTTTCATCTCAGAGTCCAC
1741+ + + + + + 1800
CTCCTCCGACCACTGTCCGGTTTCGCTCACCTTCTTTGTGGGAAAGTAGAGTCTCAGGTG
RsaI
TGTGGCACTGGCCACCCCTCCCCAGTACAGGGGTGCTGCAGGTGGCAGAGTGAATGTCCC
1801+ + + + + + 1860
ACACCGTGACCGGTGGGGAGGGGTCATGTCCCCACGACGTCCACCGTCTCACTTACAGGG
3 ~ 7
CCATCATGTGGCCCAACTCTCCTGGCCTGGCCATCTCCCTCCCCAGAAACAGTGTGCATG
1861 1920
GGTAGTACACCGGGTTGAGAGGACCGGACCGGTAGAGGGAGGGGTCTTTGTCACACGTAC
GGTTATTTTGGAGTGTAGGTGACTTGTTTACTCATTGAAGCAGATTTCTGCTTCCTTTTA
1921+ t + + + + 1980
CCAATAAAACCTCACATCCACTGAACAAATGAGTAACTTCGTCTAAAGACGAAGGAAAAT
TTTTTATAGGAATAGAGGAAGAAATGTCAGATGCGTGCCCAGCTCTTCACCCCCCAATCT
1981 2040
AAAAATATCCTTATCTCCTTCTTTACAGTCTACGCACGGGTCGAGAAGTGGGGGGTTAGA
RsaI
CTTGGTGGGGAGGGGTGTACCTAAATATTTATCATATCCTTGCCCTTGAGTGCTTGTTAG
2041+ - ----+---------+---------+---------+----~ 2100
GAACCACCCCTCCCCACATGGATTTATAAATAGTATAGGAACGGGAACTCACGAACAATC
EcoRl
AGAGAAAGAGAACTACTAAGGAAAAGGAATTC
2101 + - + + 2132
TCTCTTTCTCTTGATGATTSCTTTTCCTTAAG
2~387
14
Table 2
Comparison of the Sequence of PAI 3' Untranslated Reglon ~n
PAI-1 cDNA Clone ECE3-1 (upper ro~) and that of
5Pannekoek et al. (EMB0 ~. (1986) 5:2537) (lower row):
1667 GCTTTTGTGTGCCTGGTAGAAACTATTTCTGTTCCAGTCACATTGCCATC 1716
1111111111111111111111111111111111111 1 1111
1751 GCTTTTGTGTGCCTGGTAGAAACTATTTCTGTTCCAGCTCACATTGCATC 1800
101717 ACTCTTGTACTGCCTGCCACCGCGGAGGAGGCTGGTGACAGGCCAAAGGC 1766
1801 ACTCTTGTACTGCCTGCCACCGCGGAGGAGGCTGGTGACAGGCCAAA.GC 1849
1767 CAGTGGAAGAAACACCCTTTCATCTCAGAGTCCACTGTGGCACTGGCCAC 1816
11111111111111111111111111111111111111111111111~1
1850 GAGTGGAA~AAACACCCTTTCATCTCAGAGTCCACTGTGGCACTGGCCAC 1899
ExamDle 2
Expression of Recomb~nant PAI-1 ~n Bacter~a
ExDression o~ Full Lenqth PAI-1 in E.coli
Immunoblots and functlonal assay by reverse f~brin
autography (Loskutoff et al. (1983) Proc. Natl. Acad.
Sci. USA 80:2956) of lysates of heat-induced lambda
gtll lysogens demonstrated the presence of PAI-1.
Functional PAI-1 expressed us~ng the lambda gtll vector
had an est~mated molecular welght of approx~mately 50
kDa on SDS-polyacrylamlde gel electrophoresls (SDS-
PAGE) gels. The 2.1 kbp PAI-1-contalning EcoRi to
EcoRI fragment was inserted at the EcoRI s~te and 3' to
the trD promoter in a plasmid derivative of pKGP36-trp
(Ivanoff et ~1~ (1986) Proc. Natl. Acad. Sci. USA
83:5392-5396; ATCC No. 39413) which contains a unique
EcoRI site 3' to the trD promoter. The resultant
plasmid, which contains the PAI coding sequence
lnserted ~n the correct orientation w~th respect to the
3 ~ ~7
promoter, is designated pPAI2.1. E. coli
containing pPAI2.1 expressed an approximately 50 kDa
form of rPAI-1 which was detected by reverse fibrin
autography assay (Loskutoff et al~, suDra). However,
rPAI-1 proteln levels produced in E. coli containing
pPAI2.1 were not detectable (estimated to be less than
0.1% of total E. coli protein) by Coomassie blue
staining of SDS-PAGE gels. A fortuitous E. coli
translation start signal (AGGA) 5' to the start of the
PAI coding sequence is likely the start signal of
translation, with the second ATG (Met aa #3; Table 1)
in the coding sequence acting as the start site of
translation. Thus, the approximately 50 kDa rPAI-1
product expressed by pPAI2.1 appears to closely
correspond to full length pro-form PAI-1, only
differing by the deletion of aa residues #1 (Met) and
#2 (Gln) (see Table 1). This form of PAI-1 is not
mature form PAI-1 since it contains most of the leader
sequence.
Expression of Mature PAI-1 in E. coli
The N-terminal leader peptide of the PAI-1 coding
region was removed by a partial ApaLI and NsiI digest
of HaeIII methylase treated pECE3-1 DNA. The
approximately 1800 bp fragment containing the PAI
coding sequence was modified by adding a synthetic DNA
adaptor to the 5' end of the fragment as shown below:
Svnthetic AdaDtor PAI-1 Gene Codinq Sequence
Bam Eco Nco ApaL
5' GGATCCGAATTCC ATG G TGCAC CAT
3' CCTAGGCTTAAGG TAC CACGT G GTA
Met Val His His
This fragment, following digestion with the
restriction enzyme NcoI, was lnserted at the NcoI and
2~3~7
16
PstI sites within the polylinker of the E. coli
expression vector pKK233-2 (Amann and Brosius (1985)
Gene 40:183; Straus and Gilbert (1985) Proc. Natl.
Acad. Sci. USA 82:2014), to yield plasmid ptac-PAI.
In ptac-PAI mature form rPAI-1 is expressed in E. coli
under control of the strong "tac" promoter (Amann and
Brosius (1985) Gene 40:183). E. coli strains HB101 or
JM109 (mcrB~) containing ptac-PAI produced functional
mature PAI-1 (i.e., PAI-1 lacking N-terminal leader
peptide that is normally removed during translocation
in the eukaryotic cell) as determined by SDS-PAGE,
reverse flbrin autography, amidolyt~c assay, t-PA
binding activity, and immunoblot analysis (see below
for details of these assays). In e~ther host, cells
conta~ning this plasmid produced recomblnant mature
form PAI-1 at a level of approximately 1% of total E.
coli proteln, as determined by Coomassie brilliant blue
sta~ning of SDS-PAGE gels. The apparent molecule
weight on SDS-PAGE gels was 45 kDa. The PAI-derived
product of ptac-PAI corresponds to one of the two forms
of mature PAI-1 (the two-residue shorter form) found in
mammalian cells. In mammalian cells, two forms of
mature PAI-1 are found in approximately equal amounts:
a form with an N-term~nus of Ser-Ala-Val-His-Hls and a
two-resldue shorter form with N-terminus Val-His-Hls
(Andreasen et al. (1986) FEBS Letters 209: 213-218)
(see Table 1). Thus, the two forms of mature PAI-1
begin with aa #22 (Ser) and #24 (Val) (see Table 1).
The leader sequence, amino acids #1-21 or #1-23, is
removed in mammalian cells to y~eld the mature form
PAI-1.
We also constructed a plasmid pCE1200 designed to
express in E. coli the mature form of PAI with the N-
terminus Ser-Ala-Val-H~s-His. The construction of
plasmid pCE1200 ~s described below. The 2.1 kbp PAI
17
EcoRI to EcoRI fragment, isoldted from the plasmld
pECE3-1, WdS digested wlth restrlctlon enzyme ApaLI and
the ends were fllled-in wlth DNA polymerase I Klenow
fragment and the four deoxyrlbonucleotides. This DNA
was then dlgested wlth BglII and the 1206 bp fragment
contalning a 5' filled-ln ApaLI slte (blunt end) WdS
recovered. The plasmid expression vector pCE31
(described below) was digested with ClaI and the
overhang was filled-in with DNA polymerase I Klenow
fragment and the four deoxyribonucleotides. The vector
was digested with Bam~I and the resulting large
fragment containing the lambda PL promoter was puri~ed
from an agarose gel. The 1206 bp PAI DNA fragment WdS
then llgated lnto pCE31 at the fllled-ln ClaI and BamHI
sites, to yield plasmld pCE1200.
The nucleotlde sequence of pCE1200 encoding the N-
and C-termlnal amino acld sequence of the PAI-1-derlved
protein WdS determined and is shown below.
5' ATG AGT ATC GTG ...... CCC TAG
M S I V .............. P END
#22 ~402
The predicted molecular welght of the proteln
expressed from pCE1200 is approximdtely 42 kDa based on
an average of 110 Da/amino acld. The product expressed
by pCE1200 dlffers from the longer mature form of PAI
by the conservatlve substitution of Ala residue ~23
with Ile.
The expresslon of rPAI-1 encoded by pCE1200 ls
under the control of the phage lambda PL promoter. The
plasmid pCEl200 ln E. coli host TAP106 (deposited with
the American Type Culture Collectlon, Rockvllle, MD;
ATCC accession number 67911) contains a defectlve
lambda prophage lncludlng a temperature sensitive
mutant repressor gene (cI857). At low temperatures
2 ~ ~ ~ 3 ~ r7
18
(32C), the mutant repressor is active and
transcr~ption from PL is shut off; however, when the
temperature ~s raised to 42C the repressor is
inactlvated and transcription lnitiating at the PL
promoter can proceed (Re~nikoff and Gold, Maximizina
Gene ExPression, Butterworths, 1986). The genotype of
the TAP106 host is leu~, bio~, gal K (am), bl-,
~lacU169, rpsL, Sup E44, ~def [~BamHI, N-, Kanr, CI857,
~blo].
Production of PAI-1 involves growing the culture
under conditlons at which the repressor ls actlve,
preferably at temperature of 32C. The culture may
then be induced to produce PAI-1 by changing to
cond~tions whlch inact~vate the repressor and allow
expression from the PL promoter, e.g., raislng the
temperature to 42C. Other conditlons can be used to
inactivate the repressor, e.g., adding nalidixic acld
or mitomycin C.
Typical large scale induction conditions require
growth at 32C in LB media supplemented with ampic~llin
(100 ~g/mL) and glucose (3%) until mid-log growth phase
and then the culture is induced by rais~ng the
temperature of the fermenter to 42C (Sisk et al.
(1986) Gene 48:183-193). The culture is grown at the
elevated temperature for 4 to 8 hours, after which time
the cells are harvested by centrifugation and fro~en at
-70C prior to PAI-1 puriflcatlon. The yields of
recomblnant PAI-1 in E. coli us~ng the CE1200
expression vector are about 10 to 15~ of total E. coli
proteln, as estimated by Coomassie blue staining of
SDS-PAGE gels. As defined herein, "high level"
expression of recomb~nant PAI-1 means yields of at
least 5%, and preferably 10~ or greater. The E. coli-
expressed PAI-1 protein of the invention was not only
produced at high levels, but was also in a soluble,
easily purified form.
2~387
19
Plasm1d pCE1200 in E. coli K12, TAP 106 (host) was
deposited on March 15, 1989 in the American Type
Culture Collection (ATCC), Rockville, MD, in accordance
with the provisions of MPEP 60~3.01 (p) (C) (1) (2) and
(3) and the Budapest Treaty. The ATCC accession number
is 67911. Access to the culture will be available
during pendency of the patent application to one
determ~ned by the Commiss~oner to be entitled thereto
under 37 CFR 1.14 and 35 USC 122. Upon grant~ng of a
patent all restrictlons on the availability of the
culture to the public will be irrevocably removed.
Plasmid pCE31 was derived from plasmid pJL6, which
is described by Lautenberger et al. (Gene 23:75-84,
1983). Plasmid pJL6 was digested with restriction
enzymes BstXI and ClaI and a synthetlc DNA sequence was
inserted containing a consensus ribosome b~nd~ng site
(SD) and an ATG initiation codon. The sequence of the
synthetic ollgonucleotide used was as follows:
BstXI ClaI
SD M S
S' CCAACCTCTGGACATTGCAAGGAGTTTATAAATG AGT ATC GAT 3l
3' GGTTGGAGACCTGTAACGTTCCTCAAATATTTAC TCA TAG CTA 5'
As shown above, there is a three amino acid-encoding
sequence 5' to the ClaI site (Met-Ser-Ile). P1asmid
pCE31 provides the ClaI site 3' to a translation start
site allowing DNA coding sequences to be inserted and
operatively linked to the phage lambda (PL) promoter.
ExamDle 3
Purification of PAI ExDressed in E. coli
E, coli cells conta~ning the PAI protein were
suspended ~n ten volumes of 50 mM Na phosphate pH 6 and
2 ~
dlsrupted using d sonlcdtor (Heat Systems Ultrasonlcs).
The sonlcatlon WdS carrled out for about 5 mln uslng a
medlum tip probe. The sample WdS kept cold ln an lce
bath dur~ng the son1cat1On. The sonlcated sample was
then centr1fuged at 10,000 rpm for about 20 mln and the
supernatant saved. The pellet obtained was resuspended
1n 5 volumes of 50 mM Na phosphate buffer and
resonlcated using slmllar condltlons dS for the flrst
sonlcation. The material obtalned after the second
son~cation was centrifuged at 10,000 rpm dS before.
The supernatants obtalned after the two centrlfugatlon
steps were pooled, flltered through a 5 mlcron f11ter
and pumped onto d 4.4 x 30 cm Q Sepharose fast flow
(Pharmac1a) column, equ111brated w1th 50 mM Na
phosphate pH 6.0, at a flow rate of about lO mL/m1n.
The absorbance of the effluent was mon1tored at 280 nm,
the column was then washed w1th 50 mM Nd phosphate
buffer untll there was very 11ttle 280 nm absorbing
material 1n the effluent (about 0.5 column volumes).
The effluent from the Q Sepharose column WdS lOdded
onto a S Sepharose column equil~brated w1th 50 mM Na
phosphate pH 6 at about 10 mL/min. The absorbance of
the effluent WdS monitored at 280 nm. The column WdS
washed at 10 mLtm1n with 50 mM Na phosphate buffer
until the 280 nm absorbance was close to the basel~ne
obtained w1th buffer alone (about 1 column volume).
The prote1n was then eluted from the column at the same
flow rate us1ng a 0 to 1 M NaCl grad1ent 1n 50 mM Na
phosphate pH 6. The fractions were assayed for PAI
uslng sod1um dodecyl sulfate polyacrylam1de gel
electrophores1s (SDS-PAGE) and analyt1cal HPLC, using
the methods outl1ned below. The PAI was found to be
about 85 to 90% pure by SDS-PAGE and by analyt1cal
HPLC. The fract1Ons containlng PAI were pooled,
diluted about 2.5 fold w1th water and loaded onto
21
another 4.4 x 30 cm S Sepharose coluMn equilibrated
with 50 mM Na phosphate buffer pH 6. The column was
washed wlth about 2 column volumes each of starting
buffer and 50 mM Na phosphate buffer pH 8.0
successively. The PAI was eluted using 150 mM Na
phosphate pH 8.6. The PAI obtained at this stage was
found to be about 98% pure by SDS-PAGE and by
analytlcal HPLC. PAI of greater than about 95~ purity
is cons~dered "substantlally pure".
Other standard proteln purification procedures may
be used, as known to those skilled in the art.
Generally speaking, the cells are disrupted, preferably
by sonicat~on. The soluble supernatant is then
isolated, and the PAI-1 is purified from the soluble
supernatant using column chromatography. In the method
descr~bed above, Q Sepharose is an anion exchange
column; S Sepharose is a cation exchange column. The
described purification procedure takes advantage of the
finding that rPAI-1 does not bind to the anion exchange
column and does bind to the cation exchange column.
Those of skill in the art can devise other purification
methods without undue experimentation.
The fractlons were assayed for PAI by SDS-PAGE and
by analytical reverse phase HPLC. The SDS-PAGE was
carried out using the Pharmacia Phast system
(Pharmacia, Piscataway, NJ) utilizing the procedures
outlined in the users manual. An 8 to 25%
polyacrylamide gradlent gel was used and the proteins
were visualized by staining with Coomassie blue.
Analytical HPLC was carried out using an HP 1090 M HPLC
system. A 4.6 x 50 mm C4 Vydac column was used. The
column was equilibrated in 0.1% TFA and the samples
were applied to the column in the same solvent. The
samples were eluted using an acetonitrile gradient.
Purlfied PAI was used to establish the retention time
under the conditions of the assay.
22
The N-termlnal sequence of pur~fled E. coli-
expressed rPAI-1 expressed from plasmld pCE1200 was
determined to begln Ser-Ile-Val-His-Nis. Thus, the
N-terminal Met residue of the nascent proteln appears
to be efficiently removed by amino peptidases ~n
E. coli.
It w~ll be recognized that short form mature PAI-1
(aa #24-402) or long form mature PAI-1 (aa#22-402)
described above may be produced In the same or similar
manner by introduclng the desired modifications in the
PAI-1 DNA coding sequence, as is known to those skilled
in the art. Mature PAI-1 as def~ned herein includes
both long form and short form prote~ns, from wh~ch the
N-term1nal leader sequence has been removed. Also
included are mature PAI-l proteins havlng substantially
the same amino acid sequence and substantially the same
activity.
ExamDle 4
Functional Assavs for PAI-l
The relative activities of purif~ed recomblnant E.
coli-expressed human rPAI-l products of both pCE1200
and ptac-PAI (98% pure, as descr~bed in Example 3), and
purified nonrecomb~nant human PAI-1 expressed ln a
human f~brosarcoma cell line, des~gnated hPAI-1~ were
compared using the assays descr~bed below. Purified
hPAI-l was obtained from Amer~can Diagnostica Inc. (New
York, NY), with a pur~ty of about 98%, as estlmated by
SDS-PAGE and Coomassie stainlng of SDS-PAGE gels.
The activities of the purif~ed recombinant and
nonrecomb~nant PAI-1 preparations were compared ~n
three d~fferent assays:
1) The S2251 assay measures the enzymatic ability
of tissue plasm~nogen act1vator (t-PA) to generate
plasmin from its plasminogen precursor by determining
the level of amidolyt~c act~v~ty of generated plasm~n
23
on the chromogenic substrate D-Val-Leu-Lys-p-
nitroanillde (S2251); lnhibit~on of the act~vity by
PAI-1 is determined;
2) The S2288 assay measures the am~dolytic activity
of t-PA on the chromogenic substrate D-Ile-Pro-Arg-NH-
nitroanilide (S-2288) and inhibition of this activity
by PAI-1 is determined;
3) The binding of iodlnated t-PA (125I-t-PA) by
PAI-1 captured onto m~crot~ter wells w~th a specif~c
anti-PAI-1 monoclonal ant~body.
The rPAI-1 products of both pCE1200 and ptac-PAI
were pur~fied, as detailed in Example 3, and shown to
have slmilar specif~c actlvitles ln the assays here
descr~bed.
In the S2251 assay, 50% inhlbition of 10
international units (IU) of t-PA act~vity was observed
wlth 2.5 x 10-4 mg/mL of hPAI-1, and w~th 5.3 x 10-5
mg/ml of rPAI-1. The activ~ty of t-PA is expressed ~n
IU by comparison with the International Reference
Preparat~on for t-PA. Account~ng for an assay volume
of 100 ~l, it ~s calculated that 25 ng of hPAI-1
reduces the activlty of 10 IU of t-PA by one-half.
Defining a unit of PAI-1 as the amount of protein
needed to neutralize 1 IU of t-PA, we determlned the
specific act~vity for the hPAI-1, in terms of units/ng,
of 5/25 or 0.2 units/ng. A sim~lar calculation with
the rPAI-1 y~elds a spec~f~c activity of approx~mately
1 unit/ng. The rPAI 1 product of either pCE1200 or
ptac-PAI was, therefore, approximately 5-fold more
active than the hPAI-1 ~n th~s particular assay. The
substant~ally pure E. coli expressed rPAI-1 of the
invention will have a specific actlvity in the S2251
chromogenic assay of about lu/ng. Those skilled ~n the
art w~ll recogn~ze that some var~at1On in speclfic
3 8 ~1
24
actlvlty, e.g., ln the range of +10%l, ls to be
expected.
In the S2288 assay, 50% inhlbitlon of 10 IU of t-PA
actlvity was observed with 1.6 x 10-3 mg/ml of hPAI-1,
and wlth 1.7 x 10-4 mg/ml of rPAI-1. These values
yleld specific activities of 0.03 and 0.3 unlts/ng for
the hPAI-1 and rPAI-1, respectlvely. The rPAI-1 is
therefore approximately 10-fold more active than the
hPAI-1 in this assdy. The lower specific actlvities
for the PAI-1 in the S-2288 assay, in comparison with
the S-2251 assay, most llkely reflect the fact that
t-PA's actlvlty in the S-2251 assay is dependent upon
and greatly enhanced by f~brin.
In the t-PA binding assay (see below, Example 5),
0.002 mg/ml of rPAI-1 bound approximately 6-fold more
iodinated t-PA than did 0.002 mg of hPAI-1. Thus,
again, rPAI-1 exhlblted a signiflcantly greater
specific activlty than did hPAI-1.
The PAI-1 lsolated from many cell types ls obtained
~n a partially ~nactive or latent form; the actlvlty
can be induced or increased by treatment with protein
denaturants such as sodium dodecylsulfate (SDS) (Hekman
and Loskutoff (1985) J. Biol. Chem, 260:11581). In
the S2251 chromogenic assay, the actlvity of hPAI-1
from human fibrosarcoma cells was noted to be increased
approximately 5.6-fold following an actlvatlon
procedure which involved incubation of the proteln for
1 h at 37 ln the presence of 0.1% SDS, and dlalysls of
the material overnight against 150 mM Na2HP04. This
result indicates that the hPAI-1 contains a certain
content of inactive or latent spec~es. In contrast,
sim~lar treatment of the E. coll-expressed rPAI-1 of
the invent~on did not increase its activity. E. coli
expressed rPAI-1, the activ~ty of which ~s not
significantly increased by treatment with protein
2Q~387
denaturants, e.g., less than about 10%, is cons~dered
to be withln the lnvention. Our results establish an
advantage of our rPAI-1 composition in comparison with
the hPAI-1, based on its higher specific activity in
all three funct~onal assays and on the absence of any
latent specles or need for an activation step to obtain
fully active PAI-1. The present results are surprising
since they contradict those of Lambers et al. (the
Pannekoek lab) who reported that rPAI-1 expressed in E.
coli was expressed almost exclusively in an inactive,
latent form (Fibrinolvsis (1988) 2, Supp.1, 33).
Examole 5
PAI:t-PA Bindina Assav
To ldentify potential compounds capable of
prevent~ng the bindlng of t-PA to PAI-1, and to better
understand the nature of the lnteraction between these
two proteins, we have developed a bindlng assay
utilizing recombinant PAI-1 and radiolabeled t-PA,
125I-t-PA, for measuring the interaction between these
two proteins. Th1s assay relies on the use of a PAI-
1-specific monoclonal antibody to capture the
recombinant PAI-1 onto m~crotiter wells, and measuring
the amount of radioactivlty (125I-t-PA) bound to the
well following incubation with 125I-t-PA. Our studies
with this assay indicate it is specific, it mimics the
physiological reaction between t-PA and PAI-1, and is
suitable for screening large numbers of compounds for
their activity ln inhlbiting the bindlng of t-PA to
PAI-1. Furthermore, the assay will prove useful in
identifying specific domains on the t-PA and PAI-1
involved in thelr interaction, an important first step
in the process of ratlonally designing ~nhibitors of
binding of the t-PA and PAI-1. Human, one chain t-PA,
iodinated using the iodogen method, was purchased from
3 ~ '~
26
Du Pont-New England Nuclear (Billerica, MA), and is
des~gnated 125I-t-PA.
PAI:t-PA Blnding Assay: 50 ~l of a murine
monoclonal antibody specific for PAI-1 (#379, Americdn
Diagnostica Inc.), resuspended ~n 0.1 M Na2C03 buffer
to a concentration of 2 ng/~l, was added to wells of
96-well microtlter plates made using Removawell strip
holders (Dynatech Labs). The plates were incubdted at
4C overnlght, after which time the wells were washed
with phosphate buffered sallne (PBS), dried by patting
on a paper towel, and incubated with 5% bovine serum
albumin in PBS for 2 h at room temperature, or
overnight dt 4C. Plates may be stored in this
condition for weeks at 4C. Following three washes
with PBS-Tween 10 (0.05%) and 1 wash wlth PBS, 50 ~l of
the PAI-1-conta~ning lysate from E. coll cells
(containing approximately 100 ng of rPAI-1) and 30 ~l
of 0.01 M Tris buffer, pH 8.0, was added to each well
for a 2 h incubation at room temperature, or an
overnight incubation at 4C. Following another 3
washes with PBS-Tween 20 and 1 with PBS, 50 ~l of Tris
buffer containing S0,000 cpm of 125I-t-PA
(approximately 100 fmoles), and test compounds, where
appropriate, were added to the wells contalning
immobilized PAI-1, for a 1 h incubation at room
temperature. Wells were then washed 3 times with
PBS-Tween 10, patted dry on a paper towel, transferred
to 12 x 75 mm test tubes, and counted for radloact~vity
in a gamma counter. Data is expressed as the percent
of added 125I-t-PA which WdS bound to the wells.
We analyzed the effects of varying the
PAI-1-specific monoclonal ant~body concentration used
in the PAI:t-PA bindlng assay. As mentioned above, the
purpose of th~s antibody is to selectively capture the
rPAI-1 onto the wells in such a way as to facilitate
3 8 ~
27
PAI-t-PA blnding, i.e., to appropriately present the t-
PA binding site for PAI. Optimal binding, expressed dS
the % of the total t-PA input bound by the PAI-l, was
obtained with 100 ng of the antlbody per well (Table
3). This antibody concentration was therefore utilized
routinely. As is also evident from Table 3, binding
WdS sUbstdntldlly lower when this cdpturing dntibody
WdS omitted. Thls antibody therefore captures PAI-l on
the plastic well in d conformation which allows t-PA to
bind to it. The use of a monoclonal antibody specific
to PAI-l to capture PAI-l distinguishes this assay from
other t-PA-PAI-l binding assays that have been
descrlbed. While the capture monoclonal antibody
utilized in the described procedure ~s commerc~ally
available (#379 from American Diagnostica, NY, NY), the
state of the art of hybridoma technology is such that
generation of other PAI-l monoclonal antibodies which
have substantially the same characteristlcs and which
can be used to serve the same purpose as #379 is a
straightforward exercise. The major cr1terion for
identifying such antibodies is that they recognize and
bind determinants on PAI-l not involved in its
interaction with t-PA.
-
Table 3
Effect of Increasing the PAI-l-capturlng
Monoclonal Antlbody on the Bindlng
of 125I-t-PA to PAI-l
nq of AntibodY/Well c~m 125I-t-PA bound
0 297
4768
S0 6603
100 10334
250 9740
500 9489
As shown in Table 3, the binding of t-PA to
immobilized PAI was substantially lower when the
capturing antibody was om~tted. The antibody therefore
captures PAI-1 in a conformation which permits t-PA to
bind to it. The use of a monoclonal antibody to
capture PAI-l in a conformation which allows it to bind
t-PA dist~ngu~shes the assay from other t-PA-PAI-1
binding assays which have been described. The PAI-
specif~c capture antibody utilized in th1s Example ls
commercially available (#379 from American
Diagnostica). The state of the art of hybridoma
technology is such that production of other PAI-
l-specific monoclonal antibodies to serve the same
purpose is a straightforward exercise. For example,
Table 4 shows the % of the total t-PA input bound by
immobilized PAI-1, where the PAI-l was captured with
different PAI-l monoclonal antibodies. Six dlfferent
monoclonal antibodies were tested, the first three of
which were from the commercial vendor American
Diagnostica (379, 380, and 3783) and the last three of
3 ~ 7
which were generated in our laboratory uslng hybridoma
technology known to those of skill in the art (FCD10,
BBA4 and FAG9). All signlficantly increased the
binding of t-PA to PAI-l in comparison with a control
test where no antibody WdS utilized. All the
monoclonal antibodies in this experiment were utilized
at a concentration of 1 ~9 per well.
Table 4
X Bindlng of 125I-t-PA to PAI-l
Captured on Mlcrotiter Wells wlth Dlfferent
PAI-l Monoclonal Antlbodles
15Monoclonal Antibodv % t-PA CaDtured
tnone) 2
379 42
380 66
3783 31
FCD10 19
BBA4 49
FAG9
Increasing the amount of PAI-1-containing lysate up
to 50 ~1 per well resulted in increased t-PA blndlng.
ELISA studies revealed that approximdtely 100 ng of
PAI-l is the maxlmum amount that is immobllized by the
100 ng of capturing antlbody.
The speciflcity of the t-PA-PAI-1 interactlon was
demonstrated ln an experiment where unlabeled t-PA, in
increasing concentrations, was found to compete with a
fixed amount of 125I-t-PA for PAI-1 blnding (Table 5).
3 ~ 7
Table 5
Effect of Increaslng the t-PA
Concentration on the B~nd~ng of 125I-t-PA to PAI-1
-
rt-PAl. ~q/well % Bound
0.0 100.0
0.01 62.7
0.10 56.5
101.0 34.4
10.0 11.9
50.0 4.8
lhe k~netics of t-PA binding to PAI-1 were exam~ned
by incubating 125I-t-PA for various times on PAI-l
coated wells, and examinlng the total cpm bound at each
time point. As shown in Table 6, this time-course
experiment ylelded a blphaslc curve displaying a very
rapid initial phase of binding, followed by a second,
slower phase of b~nding. This kinetic analysis ls
consistent with the physiological react10n between t-PA
and PAI, which has been characterized as involving a
two-step reaction: a very fast reversible phase
followed by a slower irreversible phase (Hekman and
Loskutoff (1988) Arch. Biochem. BioDhys. 262:199-210).
3 ~ 7
Table 6
Klnetlcs of 125I-t-PA Blndlng to PAI-1
5 tlme (mln) cDm
0.25 893
0.50 627
0.75 532
1.00 592
1.5 927
2.0 1146
2.5 968
3 0 1125
3.5 1191
4.0 1273
5.0 1954
7.5 2416
10.0 2189
12.5 2508
15.0 2931
25.0 3595
30.0 3777
40.0 5392
50.0 3840
60.0 5063
70 - 0 4503
80.0 4935
90.0 5699
120.0 5924
135.0 5499
150.0 5489
~ ~ h ~ ~$ f
Scatchard analysis (Scatchard (1949) Ann. N.Y.
Acad Sci. 51:660-668) of binding dal:a for the binding
of t-PA to PAI revealed a curvillnear plot (Flgure 1).
This data supports the notion of a two-site interactlon
between t-PA and PAI: one high afflnlty, low capacity
component; and a second lower affinity, high capacity
component. That two s~tes on t-PA are involved in its
~nteraction w~th PAI-l in our assay system is further
supported by the data in Table 7 and Figure 1. Table 7
indicates that compounds directed to the catalytic slte
of t-PA (the tripeptide D-Phe-L-Pro-L-Arg
chloromethylketone and the monoclonal antibody CD2), as
well as compounds directed to lysine binding sltes on
the kringle 2 domain of t-PA (lysine and its analog
epsilon-amino caproic ac~d), are both capable of
lnh~bit~ng the b1nding of t-PA to immob~l~zed PAI-l.
An irrelevant amlno acid, glycine, had no lnhibitory
effect.
L~ 7
33
Table 7
Effect of Compounds Directed to D~fferent
Domains of t-PA on the Blnd~ng 125I-t-PA to PAI-1
ComPound % Inhibition
CD2 89
D-Phe-L-Pro-L-Arg
Chloromethylketone 85
Lysine 65
t -amlno caproic acid 55
Glycine 0
Compounds were lncubated w~th 125I-t-PA on
PAI-1-conta~ning wells for 1 h, as described above.
CD2 and chloromethylketone were used at 20 ~9 per well,
whlle lysine, ~-amino caproic acid, and glyclne were
used at 0.1 M per well.
2 ~ 7
34
Table 8 compares the inhlbitory effect of the
purified heavy and light chalns of t-PA (prepared by
reduction of two-chain t-PA with dithiolthreltol) with
that of intact t-PA on the blndin~q of 125I-t-PA to PAI-1.
5 The purified light chain, which contains the catalytic
moiety of the t-PA, was a potent inhibltor of bindlng as
would be predicted based on our knowledge that the
catalytlc site of a serlne protease is lnvolved in
binding to its speciflc inhibitor. However, the purified
heavy chain also displayed inhibitory properties
suggestlng the presence of a domaln on this chain that
also interacts with PAI-1.
-
Table 8
Effects of Intact 2-Chaln t-PA,
Purlfled Heavy Chaln, and Pur~fled Llght
Chaln on the Blndlng of 125I-t-PA to PAI-l
% Bound ~ Bound % Bound
t-PA ~q A chain B chain intact 1-chain
0.0 100 100 100
0.005 90 62 50
0.01 94 ~8 53
0.05 95 40 31
0.1 83 37 30
0.5 70 30 26
1.0 57 32 22
A growing body of clinical data (Hamsten et al.
(1985) New Enqland Journal of Medicine 313:1557-1563;
Wlman et al. (1985) J. Clin. Med. 105:265-270) suggests
that elevated levels of PAI-1, by binding to and
35 inactivating t-PA, contribute to the pathogenesis of
~13~3~7
various thrombotic dlsorders including myocardial
infarction, deep vein thrombosls and stroke. Thls
recognition of PAI-1's pathological significance has
led to lnterest in identifying novel compounds which
prevent PAI's binding to, and inactivation of, t-PA.
The microtiter well envlronment of the assay will
permit the screenlng of large numbers of test compounds
for the desired activity, that is, inhibition of the
b~nding of t-PA to PAI-1. PAI-1-coated wells may be
prepared weeks in advance, thereby reducing the time,
and increasing the efficiency of drug screening.
Furthermore, our data lndicates that in the assay of
the ~nvention the interact~on between immobilized PAI-1
and 125I-t-PA mimics the physiological reactlon between
these two proteins. This evidence includes the
following: 1) binding of 125I-t-PA to immobilized PAI-1
is inhibited by increasing concentrations of unlabeled
t-PA; 2) the kinetics of binding in our assay system
yielded a biphasic curve consistent with the
physiological reaction between t-PA and PAI-1 (Hekman
and Loskutoff (1988) Arch, Biochem. Biophvs.
262:199-210); and 3) Scatchard analysis of binding
data, as well as studies utilizing various binding
inhibitors, suggest that two sites on t-PA are involved
in its interaction with PAI-1, as has been described in
the interaction of other serine proteases with their
specific inh~bitors (W~man and Collen (1978) Eur. J.
Blochem. 84:573-578). In summary, our results support
the validity of utilizing this t-PA-PAI-1 blnding assay
as a first step for identifying compounds wh1ch may
ultlmately be active, ~n vivo, in enhancing
fibrinolytic activity.
36
ExamPle 6
Therapeutic Use of rPAI-l ln the Control of
Fibrinol~sis
The destruction of blood clots is medlated by
plasmin breakdown of fibrin. Normally thls process of
flbrinolysis is terminated by the lnhlbitory action of
antl-pldsmln. However, there exist clinlcal conditior,s
characterized by excessive flbrlnolysis or bleedlng
caused by elther defects in fibrinolytic inhibitors, or
as a side-effect of therapeutic admlnistration of
fibrinolytic agents such as t-PA, urokinase or
streptoklnase. One speclflc clln1cal example is
increased flbrinolysis and bleedlng which occurs in
patlents during liver transplantatlon and wh~ch has
been related to increased clrculating plasma levels of
t-PA (Dzlk et al. Blood 71:1090 (1988)).
Pharmacologlcal inhibitors of t-PA may represent
lmportant therapeutic agents for controlling excesslve
fibrlnolysis during liver surgery. On the other hand,
Coluccl et al. (1986) J. Clin. Invest. 78:138-144,
report that lnductlon of endogenous PAI-1 ln rabbits
did not alter thrombolysls by lnfuslon of t-PA. This
su~gests that adminlstratlon of exogenous PAI-1 would
not inhiblt fibrinolysis in vivo. The lack of
pharmacokinetic data lends further uncertainty as to
the ln vivo effect of exogenously administered PAI~ It
is also unclear whether unglycosylated rPAI-1 expressed
by a prokaryotlc cell would exhlbit diminlshed actlv1ty
in a mammal relatlve to the endogenous glycosylated
peptlde.
We therefore investigated the activity of rPAI-1 ln
inhibltlng flbrinolysis in vlvo for the purpose of
evaluating its therapeutic potential for eventual use
in humans as a t-PA inhibitor. For this study, human
flbrin clots were lnjected lnto rabblts (24 mg/kg~,
37
which were infused with either sallne or purifled
rPAI-1 at 10 ~g/kg/mln. At various times, serum
samples were collected and assayed for speciflc fibrin
degradatlon products (D-dimer) by means of a D-dimer
ELISA (American Diagnostica). As shown in Table 9,
fibrln degradation, as indicated by the D-dimer
concentration, was significantly reduced in those
rabblts infused with rPAI-1 in compar1son with those
animals infused with saline. Th~s study reveals the in
vivo activity of rPAI-1 in inhibitlng fibrinolysls, and
suggests an important role for the protein as an
inhibitor of fibrlnolysis during liver transplant
surgery and other clinical conditions characterized by
excesslve flbrlnolysls.
Table 9
Effect of rPAI-l on Flbrln Degradatlon In ~llvo
D-dlmer Concentratlon
time (min) Saline infuslonPAI-1 lnfuslon
0 15 + 2.7 14 + 3.9
15 + 2.6 14 + 3.0
19 + 0.6 13 + 2.8
40 + 3.5 13 + 0.6
48 + 3.3 16 + 1.2
105 80 + 6.5 18 + 0.1
