A plasminogen analogue activatable by thrombin to have plasmin activity which contains the cleavage site sequence (SEQ ID NO: 23): ##STR1## where Xaa at position 1 represents P4; Xaa at position 2 represents P3; Xaa at position 5 represents P1'; and Xaa at position 6 represents P2' where P3 is a basic amino acid residue, P4 is a hydrophobic amino acid residue and each of P1' and P2' is independently a non-acidic amino acid residue, said site being cleavable by thrombin between Arg and P1'.

This invention is a improvement of the invention disclosed in our copending 
paint application WO-A-9109118, and relates to plasminogen analogues which 
are activated by thrombin to have fibrinolytic activity or to inhibit 
blood clot formation. It also relates m nucleic acid (DNA and RNA) coding 
for all or part of such compounds. The invention also relates to their 
preparation, pharmaceutical compositions containing them and their use in 
the treatment of thrombotic disease. 
BACKGROUND OF THE INVENTION 
Plasminogen is a key component of the fibrinolytic system which is the 
natural counterpart to the clotting system in the blood. In the process of 
blood coagulation, a cascade of enzyme activities is involved in 
generating a fibrin network which forms the framework of a clot, or 
thrombus. Degradation of the fibrin network (fibrinolysis) is accomplished 
by the action of the enzyme plasmin. Plasminogen is the inactive precursor 
of plasmin and conversion of plasminogen to plasmin is accomplished by 
cleavage of the peptide bond between arginine 561 and valine 562 of 
plasminogen. Under physiological conditions this cleavage is catalysed by 
tissue-type plasminogen activator (tPA) or by urokinase-type plasminogen 
activator (uPA). 
If the balance between the clotting and fibrinolytic systems becomes 
locally disturbed, intravascular clots may form at inappropriate locations 
leading to conditions such as coronary thrombosis and myocardial 
infarction, deep vein thrombosis, stroke, peripheral arterial occlusion 
and embolism. In such cases, the administration of fibrinolytic agents has 
been shown to be a beneficial therapy for the promotion of clot 
dissolution. Antithrombotic agents are also useful for the prevention of 
clot formation. 
However, the problem with the majority of agents used for fibrinolytic 
treatment is that at clinically useful doses they are not thrombus 
specific, as they activate plasminogen in the general circulation. An 
alternative approach to enhancing fibrinolysis is disclosed in our 
copending patent application WO-A-9109118, and is based on the use of 
molecules activatable to have fibrinolytic activity or to inhibit clot 
formation. The activation is catalysed by one or more endogenous enzymes 
involved in blood clotting. An advantage of this approach is that thrombus 
selectivity of fibrinolysis or inhibition of clot formation activity is 
achieved by way of the thrombus-specific localisation of the activating 
enzyme. In particular, WO-A-9109118 discloses, inter alia, plasminogen 
analogues activatale to plasmin by cleavage by thrombin. 
Thrombin (E. C. 3.4.21.5) is a serine protease which catalyses the 
proteolysis of a number of proteins including fibrinogen (A alpha and B 
beta chains), Factor XIII, Factor V, Factor VII, Factor VIII, protein C 
and antithrombin IIl. The structure required for recognition by thrombin 
appears to be partially determined by the local amino acid sequence around 
the cleavage site, but is also determined to a variable extent by 
sequence(s) remote from the cleavage site. For example, in the fibrinogen 
A alpha chain, residues P2 (Val), P9 (Phe) and P10 (Asp) are crucial for 
.alpha.-thrombin-catalysed cleavage at the Arg(16)-Gly(17) peptide bond 
(Ni, F. et al 1989, Biochemistry 28 3082-3094). WO-A-9109118 discloses 
that optimum for alpha-thrombin may have the structure (i) (SEQ ID NO: 23) 
##STR2## 
where Xaa at position 1 represents P4; Xaa at position 2 represents P3; 
Xaa at position 5 represents P1'; and Xaa at position 6 represents P2' 
where each of P3 and P4 is independently a residue of a hydrophobic amino 
acid (such as valine) and each of P1' and P2' is independently a 
non-acidic amino acid residue, or structure (ii) (SEQ ID NO:24): 
##STR3## 
where Xaa at position 1 presents P2 and Xaa at position 3 represents P1'; 
where P2 or P1' is a glycine residue. Accordingly, the thrombin 
activatable plasminogen analogue compounds disclosed as preferred in 
WO-A-9109118, and all those specifically exemplified therein, have 
cleavage sites conforming to the foregoing structures (i) or (ii). The 
data reported in WO-A-910918 suggest that, of the specifically exemplified 
thrombin-cleavable plasminogen analogues, that designated T19 is the most 
effective in the assay systems used. It has a cleavage site based on 
Factor XIII, Pro (559) and Gly (560) of wild-type plasminogen having been 
replace by (SEQ ID NO:25): 
##STR4## 
The thrombin cleavage site of T19, the most effective of the compounds 
specifically exemplified, therefore conforms with the requirements of 
structure (i) above-referenced according to WO-A-9109118. 
In the absence of cofactors, Factor XI has been reported not to be cleaved 
by thrombin (Naito, K. and Fujikawa, K (1991), J. Biol. Chem. 
266:7353-7358), or to be only slowly cleaved with a k.sub.cat /K.sub.m 
=1.6.times.10.sup.5 M-1 min-1 (Gailani, D. and Broze, G. J. 1991, Science 
253, 909-912). The cleavage site sequence of this thrombin substrate 
differs from the preferred general formulae i) and ii) of WO-A-9109118. In 
factor XI, a basic amino acid, Lys, is in the P3 position. However, 
although thrombin cleavage activity at this site in factor XI is very low 
compared to that of Factor XIII (k.sub.cat /K.sub.m =1.4.times.10.sup.5 
M-1sec-1; Naski et al;., 1991, Biochemistry 30 934-941), it has now been 
found in accordance with this invention, that plasminogen analogues having 
a thrombin cleavage sequence with a basic amino acid residue in the P3 
position have surprisingly increased activity compared to T19. Such novel 
analogues are cleaved more rapidly by thrombin, and exhibit increased 
activity in a linked chromogenic assay. More significantly, such analogues 
are active in a plasma clot lysis assay, which more closely resembles 
conditions in vivo. 
A further example of a thrombin cleavage site where P3 is a basic amino 
acid residue is found in single-chain urokinase, where P3 is arginine. 
Cleavage at this site produces inactive two-chain urokinase (Ichinose et 
al., (1986) J. Biol. Chem. 261 3486-9). In the absence of cofactors, 
cleavage of single-chain urokinase by thrombin is also moderately slow, 
with a k.sub.cat /K.sub.m =3.8.times.10.sup.4 M-1 sec-1;(de Munk et al., 
1990 Fibrinolysis 4 161); however, plasminogen analogues bearing the 
urokinase cleavage site have now been shown to have increased activity 
compared to T19. 
SUMMARY OF THE INVENTION 
Accordingly, the present invention is an improvement of the invention 
disclosed in WO-A-9109118 in that it provides a plasminogen analogue which 
is activatable by thrombin to have plasmin activity (as generally 
disclosed in WO-A-9109118) but specifically characterised in that it 
comprises a thrombin-cleavable site sequence (SEQ ID NO:23) 
##STR5## 
where Xaa at position 1 represents P4; Xaa at position 2 represents P3; 
Xaa at position 5 represents P1'; and Xaa at position 6 represents P2'; 
wherein P3 is a residue of a basic amino acid, P4 is a residue of a 
hydrophobic amino acid, and each of P1' and P2' is independently a 
non-acidic amino acid residue, said site being cleavable by thrombin 
between Arg and P1'.

DETAILED DESCRIPTION OF THE INVENTION 
Plasminogen has been numbered according to the protein sequencing studies 
of Sottrup-Jensen et al. (in: Atlas of Protein Sequence and Structure 
(Dayhoff, M. O., ed.) 5 suppl. 3, p.95 (1978)) which indicated that 
plasminogen was a 790 amino acid protein and that the site of cleavage was 
the Arg(560)-Val(561) peptide bond. However, a suitable plasminogen cDNA 
useful in this embodiment of the invention and that isolated by Forsgren 
et al (FEBS Letters 213 254-260 (1987)) code for a 791 residue protein, as 
shown in SEQ ID NO: 29, with an extra Ile at position 65. In this 
specification, the numbering of the amino acids in plasminogen corresponds 
to that of the cDNA used. There may be polymorphism in the structure of 
plasminogen and there may be forms of plasminogen in which the numbering 
of the cleavage site differs but it is intended that such variants be 
included in the embodiment. 
Therefore the term "plasminogen analogue", as used in this specification, 
means a molecule differing from wild type plasminogen and having the 
ability to be cleaved or otherwise acted on to form a molecule having 
plasmin activity. 
Plasminogen analogues within the scope of this embodiment of the invention 
retain the fibrin binding activity of wild type plasminogen to an adequate 
degree but may have altered inhibition characteristics; preferred 
plasminogen analogues have a plasma half life which is comparable with 
that of wild type plasminogen, but this property is not essential. 
In the thrombin-cleavable site sequence present in plasminogen analogues 
according to the invention, the basic amino acid residue P3 may be a 
lysine or arginine residue; the hydrophobic amino acid residue P4 may be a 
valine, isoleucine or leucine residue; and each of the non acidic amino 
acid residues P1' and P2' may independently be a valine or isoleucine 
residue. 
Particular plasminogen analogues within the scope of the invention contain 
the cleavage site (SEQ ID NO:26): 
##STR6## 
where Xaa at position 8 represents P1' and Xaa at position 9 represents 
P2';, or the cleavage site 
##STR7## 
where each of P1' and P2' is independently a non-acidic amino acid 
residue. As mentioned, P1' and P2' may be isoleucine or valine residues. 
Specific and preferred compounds according to the invention are plasminogen 
analogues: 
a) in which Pro(559), Gly(560) are replaced by Thr, Thr, Lys, Ile, Lys, Pro 
and Val (562) is replaced by Ile. In this analogue, amino acid 563 is 
valine as in the wild type. This mutant has been designated BB10151 (SEQ 
ID NO:1). 
b) in which Cys(558), Pro(559), Gly(560) are replaced by Ala, Gly, Gln, 
Lys, Thr, Leu, Arg, Pro; and Cys(566) is replaced with Ala. In this 
analogue, amino acids 562 and 563 are valine as in the wild type. This 
mutant has been designated BB10156 (SEQ ID NO:10). 
c) in which Cys(558), Pro(559), Gly(560) are replaced by Ala, Leu, Arg, 
Pro; and Cys(566) is replaced with Ala. In this analogue, amino acids 562 
and 563 are valine as in the wild type. This mutant has been designated 
BB10170 (SEQ ID NO:12). 
d) in which Pro(559), Gly(560) are replaced by Val, Glu, Leu, Gln, Gly, 
Leu, Arg, Pro. In this analogue, amino acids 562 and 563 are valine as in 
the wild type. This mutant has been designated BB10171 (SEQ ID NO:18). 
e) in which Cys(558) Pro(559), Gly(560) are replaced by Ala,Thr, Thr, Lys, 
Ile, Lys, Pro; Val (562) is replaced by Ile; and Cys(566) is replaced with 
Ala This mutant has been designated BB10158 (SEQ ID NO:11). 
Plasminogen analogues in accordance with the invention have been defined by 
particular reference to the nature of their thrombin-cleavable site 
sequence, since it is the surprising rapidity of cleavage at that site 
which underlies the improved thrombolytic activity of the compounds. 
However, it is likely that plasminogen analogues in accordance with the 
invention (i.e. containing the now identified novel cleavage site 
sequences) may contain other modifications (as compared with wild type 
plasminogen) which may be one or more additions, deletions or 
substitutions at sites more or less remote from the cleavage site, without 
losing the benefit of rapid cleavage. An example of such a modification 
would be the addition, removal, substitution or alteration of one or more 
kringle domains to alter fibrin binding activity or reduce 
.alpha.2-antiplasmin binding. A specific example would be mutation of the 
lysine-binding site located on Kringle 1 to interfere with the binding of 
.alpha.2-antiplasmin to this site. Such variants may be resistant to 
inhibition by .alpha.2-antiplasmin. Preferred embodiments include BB10189, 
BB10190 and BB10192, which are the plasminogen analogues BB10153, BB010170 
and BB10171 respectively with the additional mutations of Asp 137&gt;Ser and 
Asp 139 &gt;Ser. 
Another example would be mutation to prevent disulphide bond formation 
between Cys (558) and Cys (566), for example by replacing one or both of 
the cysteines with alanine residues, in order to remove the constraint put 
on the cleavage site by the disulphide bond formed between the cysteine 
residues. Of the preferred embodiments described above, variants BB10156, 
BB10158 and BB10170 have such open-loop modifications. 
An example of a modification involving deletion would be lys-plasminogen 
variants of a plasminogen analogue in which the amino terminal 68, 77 or 
78 amino acids have been deleted. Such variants may have enhanced fibrin 
binding activity as has been observed for lys-plasminogen compared to 
wild-type glu-plasminogen (Bok, R. A. and Mangel, W. F. 1985, Biochemistry 
24 3279-3286). A further example involving deletion would be variants of a 
plasminogen analogue in which the kringle 1 or kringle 1-4 domains have 
been deleted to impair .alpha.2-antiplasmin binding. Such variants may be 
resistant to inhibition by .alpha.2-antiplasmin. Deletion of kringles 1-4 
would also alter the fibrin binding and pharmokinetic properties of the 
molecule. 
For the highly clot selective analogue of plasminogen of the present 
invention it may be preferred to introduce a mutation in the serine 
protease domain that interferes with plasmin inhibitor binding (SEQ ID 
NO:2 depicts the serine protease domain of wild-type plasmin, and 
references herein to that domain, where numbered, use the numbering of SEQ 
ID NO: 2). This mutation could be in a position analogous to that shown to 
prevent inhibitor binding to tissue plasminogen activator (Madison, E. L. 
et al 1989 Nature 339 721-724) or could be in another position which 
prevents inhibitor binding to plasminogen; such modifications are 
described in co-pending patent application PCT/GB 9301632, which discloses 
endopeptidases of the chymotrypsin superfamily which exhibit resistance to 
serine protease inhibitors. Such reisistance is provided by a modification 
in the endopeptidase or its precursor which induces one of the following 
a) a conformational change in the local fold of the protease; 
b) a change in the relative orientations of the protease and inhibitor on 
forming a complex; 
c) a change in the steric bulk of the protease in the region of the 
inhibitor, 
d) a change in the electrostatic potential field in the region of the 
inhibitor binding site; or 
e) any combination of the above. 
A preferred embodiment is BB10158 with the A4 mutation (Glu606 to Lys) 
described in PCT/GB 9301632 (BB10199). This mutation is designed to 
interrupt ionic interactions on the surface of plasminogen, interfering 
with binding to antiplasmin. Mutagenesis was carried out using a 24 base 
oligonucleotide (SEQ ID NO:3) CTTGGGGACT TCTTCAAGCA GTGG, designed to 
convert Glu606 to Lys. Other preferred embodiments have, either singly or 
in combination, mutations at Glu606, Glu623, Phe583, Met585 or Lys 607. 
The Glu606 and Glu623 mutations were exemplified in PCT/GB 9301632. An 
example of this embodiment is BB10153 which is the plasminogen analogue 
BB10151 with the additional mutations of Glu606 to Lys and Glu623 to Lys. 
Other plurally-modified plasminogen analogues in accordance with the 
invention may include one or more modifications to prevent, reduce or 
alter glycosylation patterns. Plasminogen analogues incorporating such 
modifications may have a longer half-life, reduced plasma clearance and/or 
higher specific activity. 
Preferred features of plasminogen analogues within the scope of the 
invention also apply, where appropriate, to other compounds of the 
invention, mutatis mutandis. 
The plasminogen analogues of the first aspect of the invention can be 
synthesised by any convenient route. According to a second aspect of the 
invention there is provided a process for the preparation of such a 
plasminogen analogue, the process comprising coupling successive amino 
acid residues together and/or ligating oligopeptides. Although proteins 
may in principle be synthesised wholly or partly by chemical means, it is 
preferred to prepare them by ribosomal translation, preferably in vivo, of 
a corresponding nucleic acid sequence. The protein may be glycosylated 
appropriately. 
It is preferred to produce proteins of the invention by using recombinant 
DNA technology. DNA encoding a naturally occurring plasminogen may be 
obtained from a cDNA or genomic clone or may be synthesised. Amino acid 
substitutions, additions or deletions are preferably introduced by 
site-specific mutagenesis. DNA sequences encoding plasminogen analogues 
may be obtained by procedures familiar to those skilled in the art of 
genetic engineering. 
The process for producing proteins using recombinant DNA technology will 
usually include the steps of inserting a suitable coding sequence into an 
expression vector and transferring the vector into a host cell. Therefore, 
according to a third aspect of the invention, there is provided synthetic 
or recombinant nucleic acid coding for a proteinaceous compound as 
described above. The nucleic acid may be RNA or DNA and may be in the form 
of a vector, such as a plasmid, cosmid or phage. The vector may be adapted 
to transfect or transform prokaryotic (for example bacterial) cells and/or 
eukaryotic (for example yeast or mammalian) cells. A vector will comprise 
a cloning site and usually at least one marker gene. An expression vector 
will have a promoter operatively linked to the sequence to be inserted in 
the cloning site, and, preferably, a sequence enabling the protein product 
to be secreted. 
The plasminogen analogues of the invention may be expressed using a vector 
of the type described in WO-A-9109118, which comprises a first nucleic 
acid sequence coding for a protein or embodying a cloning site, 
operatively linked to a second nucleic acid sequence containing a strong 
promoter and enhancer sequence derived from human cytomegalovirus, a third 
nucleic acid sequence encoding a polyadenylation sequence derived from 
SV40 and a fourth nucleic acid sequence coding for a selectable marker 
expressed from an SV40 promoter and having an additional SV40 
polyadenylation signal at the 3' end of the selectable marker sequence. 
It is to be understood that the term "vector" is used in this specification 
in a functional sense and is not to be construed as necessarily being 
limited to a single nucleic acid molecule. So, for example, the first, 
second and third sequences of the vector defined above may be embodied in 
a first nucleic acid molecule and the fourth sequence may be embodied in a 
second nucleic acid molecule. 
This vector enables the plasminogen analogues to be expressed and secreted 
into the cell culture medium in a biologically active form without the 
need for any additional biological or chemical procedures. 
According to a third aspect of the invention, there is provided a process 
for the preparation of nucleic acid encoding the plasminogen analogues 
described above, the process comprising coupling successive nucleotides 
together and/or ligating oligo- and/or poly-nucleotides. 
In a further aspect of the invention, there is provided a cell or cell line 
transformed by nucleic acid and/or a vector as described above. Suitable 
cells or cell lines to be transformed include both prokaryotic (for 
example Escherichia coli) and eukaryotic cells, such as yeast cells 
(including Saccharomyces cerevisiae and Pichia pastoris) and mammalian 
cells. Mammalian cells which grow in continuous culture and which can be 
transfected or otherwise transformed by standard techniques are preferred. 
Examples of suitable cells include Chinese hamster ovary (CHO) cells, 
mouse myeloma cell lines such as NS.O slashed. and P3X63-Ag8.653, COS 
cells, HeLa cells, BHK cells, melanoma cell lines such as the Bowes cell 
line, mouse L cells, human hepatoma cell lines such as Hep G2, mouse 
fibroblasts and mouse NIH 3T3 cells. CHO cells are particularly preferred 
as hosts for the expression of plasminogen and plasminogen analogues. 
Transformation may be achieved by any convenient method; electroporation is 
particularly suitable. 
Plasminogen analogues of the present invention may be used for the 
prophylaxis and/or treatment of conditions caused by an imbalance between 
clotting and fibrinolysis, the method comprising administering to a 
patient an effective amount of the plasminogen analogue. Therefore, 
according to a further aspect of the invention, there is provided a 
plasminogen analogue as disclosed herein, for use in medicine, 
particularly in the prophylaxis and/or treatment of conditions caused by 
an imbalance between clotting and fibrinolysis, where it is desired to 
produce local fibrinolytic and/or anticoagulant activity. Such conditions 
include myocardial and cerebral infarction, arterial and venous 
thrombosis, thromboembolism, post-surgical adhesions, thrombophlebitis and 
diabetic vasculopathies and coagulation imbalances associated with cancer. 
The invention also provides the use of a plasminogen analogue as disclosed 
herein in the preparation of a thrombolytic, antithrombotic or 
thrombolytic antithrombotic agent. 
Furthermore, there is also provided a pharmaceutical or veterinary 
composition comprising one or more plasminogen analogues as disclosed 
herein and a pharmaceutically or veterinarily acceptable carrier. Such a 
composition may be adapted for administration orally, by intravenous or 
intramuscular injection or by infusion. Suitable injectable compositions 
include preparations of sterile plasminogen analogue(s) in isotonic 
physiological saline and/or buffer and may also include a local 
anaesthetic to alleviate the pain of injection. Similar compositors may be 
used for infusion. Where the compound is to be administered as a topical 
treatment, it may be formulated as a cream, ointment or lotion in a 
suitable base. 
The compounds of the invention may be supplied in unit dosage form, for 
example as a dry powder or water-free concentrate in a hermetically sealed 
container such as an ampoule or sachet. 
The quantity of material to be administered will depend on the amount of 
fibrinolysis or inhibition of clotting required, the required speed of 
action, the seriousness of the thromboembolic position and the size of the 
clot. The precise dose to be administered will, because of the very nature 
of the condition which compounds of the invention are intended to treat, 
be determined by the physician. As a guideline, however, a patient being 
treated for a mature thrombus will generally receive a daily dose of a 
plasminogen analogue of from 0.01 to 10 mg/kg of body weight either by 
injection in for example up to 5 doses or by infusion. 
The following Examples of the invention are offered by way of illustration, 
and not by way of limitation. 
EXAMPLE 1 
Construction Expression and Purification of BB10151 
The isolation of plasminogen cDNA and construction of the vectors pGWH and 
pGWHgP have been described in WO-A-91/09118. In pGWHgP, transcription 
through the plasminogen cDNA can initiate at the HCMV promoter/enhancer 
and the selectable marker gpt is employed. 
The techniques of genetic manipulation, expression and protein purification 
used in the manufacture of the modified plasminogen example to follow, are 
well known to those skilled in the art of genetic engineering. A 
description of most of the techniques can be found in one of the following 
laboratory manuals: "Molecular Cloning" by T. Maniatis, E. F. Fritsch and 
J. Sambrook published by Cold Spring Harbor Laboratory, Box 100, New York, 
or "Basic Methods in Molecular Biology" by L. G. Davis, M. D. Dibner and 
J. F. Battey published by Elsevier Science publishing Co Inc, New York. 
Additional and modified methodologies are detailed in the methods section 
below. 
BB10151 is a plasminogen analogue in which the amino acid residues 
Pro(559), Gly(560) are replaced by Thr, Thr, Lys, Ile, Lys, Pro and 
Va1(562) is replaced by Ile to produce a cleavage loop clearable by 
thrombin (SEQ ID NO:1). This site is based on a potential thrombin 
cleavage site in factor XI. The procedures used in this example are 
essentially as described in WO-A-9109118 Examples 2 and 3, with the 
mutagenesis reaction carried out on the 1.87kb KpnI to HincII fragment of 
plasminogen cloned into the bacteriophage M13mp18. Single stranded 
template was prepared and the mutation made by oligonucleotide directed 
mutagenesis. In this case a 48 base long oligonucleotide (SEQ ID NO:4) 
CACCCCCCTA CGATTCTAGG TTTAATTTTA GTTGTACATT TCTTCGGC; was used to direct 
the mutagenesis. 
Plasmid DNA was introduced into CHO cells by electroporation using 800 V 
and 25 .mu.F. Selective medium containing 250 .mu.l/ml xanthine, 5 
.mu.g/ml mycophenolic acid, 1x hypoxanthine-thymidine (HT)) was added to 
the cells 24 hours post transfection and the medium was changed every two 
to three days. Plates yielding gpt-resistant colonies were screened for 
plasminogen production using an ELISA assay. Cells producing the highest 
levels of antigen were re-cloned and the best producers scaled up into 
flasks with production being carefully monitored. Frozen stocks of all 
these cell lines were laid down. Producer cells were scaled up into roller 
bottles to provide conditioned medium from which plasminogen protein was 
purified using lysine SEPHAROSE 4B. (The word SEPHAROSE is a trade mark.) 
The cell line used to produce this mutant protein was 123.C6. 
EXAMPLE 2 
Construction of BB10153 
BB10153 is a derivative of BB10151 (SEQ ID NO:1) containing two additional 
mutations (Glu606 to Lys and Glu623 to Lys) to impair binding of 
.alpha.2-antiplasmin. The 663bp EcoRV to Sph I fragment of BB10151 (SEQ ID 
NO:1) (cloned in pUC - see Example 1) was removed and replaced with the 
equivalent 663bp fragment from the antiplasmin resistant mutant A3A4. 
Construction of this is described in example 5 of PCT/GB9301632. The 24 
base oligonucleotide 5'CTT GGG GAC TTC TTC AAG CAG TGG3'(SEQ ID NO: 3), 
was used to convert Glu-606 to Lys and the 27 base oligonucleotide (SEQ ID 
NO:5): GTTCGAGATT CACTTTTTGG TGTGCAC; was used to convert Glu623 to Lys. 
The full length plasminogen was then cloned into the expression vector 
pGW1H prior to the insertion of the gpt selection marker as described in 
WO-A-9109118 Example 2. 
EXAMPLE 3 
Construction of BB10156(SEQ. ID NO10), BB10158 (SEQ ID NO:11) & BB10170 
(SEQ ID NO:12) 
The DNA encoding plasminogen mutant BB10150 was used as the template for 
production of BB10156 (SEQ. ID NO10), BB10158 (SEQ ID NO:11) & BB10170 
(SEQ ID NO:12). BB10150 is a plasminogen analogue in which the amino acid 
residues Pro(559), Gly(560) are replaced by Val, Val, Pro and has the 
additional mutations Cys(558) to Ala and Cys(566) to Ala to prevent 
disulphide bond formation. The opened cleavage loop sequence of BB10150 is 
shown in SEQ ID NO:6. BB10150 was made by mutagenesis using two 
oligonucleotide primers (SEQ ID NO:7): CTAGGTACAA CCGCTTTCTT CGGCT and 
(SEQ ID NO:8): GGTGGGCCAC CGCCCCCCCC AC and the 1.87kb; KpnI to HincII 
fragment of mutant T13 (see patent application WO-A-9109118, Example 13 
and SEQ ID NO:9) cloned into M13. 
Plasminogen analogues BB10156(SEQ. ID NO:10), BB10158 (SEQ ID NO:11) & 
BB10170 (SEQ ID NO:12) were all constructed by site specific mutagenesis 
using the previously described mutant BB10150 in M13 as the template so 
that they all have the same Cys to Ala mutations at residues 558 and 566. 
BB10156 (SEQ ID NO:10) is a plasminogen analogue in which the amino acid 
residues Pro(559), Gly(560) are replaced by Gly, Gln, Lys, Thr, Leu, Arg, 
Pro (SEQ ID NO:10). BB10158 is a plasminogen analogue in which the amino 
acid residues Pro(559), Gly(560) are replaced by Thr, Thr, Lys, Ile, Lys, 
Pro and Va1(562) is replaced by Ile (SEQ ID NO:11). BB10170 is a 
plasminogen analogue in which the amino acid residues Pro(559), Gly(560) 
are replaced by Leu, Arg, Pro (SEQ ID NO:12). The oligonucleotides used to 
prime each mutagenesis are shown below: 
______________________________________ 
MUTANT OLIGONUCLEOTIDE SEQUENCE 
______________________________________ 
BB10156 SEQ ID NO:13 
CAACCCTAGG TCTAAGTGTT TTCTGACCCG CTTTCTTCG 
BB10158 SEQ ID NO:14 
CAACCCTAGG TTTGATCTTC GTTGTCGCTT TCTTCG 
BB10170 SEQ ID NO:15 
CACAACCCTA GGTCTAAGCG CTTTCTTCGG 
______________________________________ 
In each case, following DNA sequencing, the mutation was cloned directly 
into the pGW1Hg.plasminogen expression vector using the restriction 
enzymes HindIII and SplI. These sites had previously been introduced at 
the extreme 5'end of plasminogen and at 1850 respectively via mutagenesis; 
the plasminogen amino acid coding sequence was not affected by this 
procedure. 
EXAMPLE 4 
Construction of BB10169 and BB10171 
BB10169 is a plasminogen analogue in which the amino acid residues 
Pro(559), Gly(560) are replaced by Val, Glu, Leu, Gln, Gly, Ile, Lys Pro 
and Val (562) is replaced by Ile (SEQ ID NO:16). BB10169 was made by 
oligonucleotide directed mutagenesis of BB10151 in M13 using the 42 base 
oligonucleotide (SEQ ID NO:12) GATTCTAGGT TTAATGCCCT GCAGTTCCAC ACATTTCTTC 
GG; followed by cloning into pGW1Hg plasminogen using HindIII and SplI. 
BB10171 is a plasminogen analogue in which the amino acid residues 
Pro(559), Gly(560) are replaced by Val, Glu, Leu, Gln, Gly, Leu, Arg, Pro 
(SEQ ID NO:18). The single stranded M13 from BB10169 was used as the 
template for the construction of BB10171. Mutagenesis was primed using the 
39 base oligonucleotide (SEQ ID NO:19) CACCCCCCTA CCACTCTGGG TCTCAGGCCC 
TGCAGTTCC; and, following DNA sequencing, was cloned into the expression 
vector using HindIII and SplI. 
EXAMPLE 5 
Construction and Expression of BB10189, BB10190 and BB10191 
Plasminogen analogues BB10189, BB10190 and BB10191 have the kringle 1 
double mutation Asp(137) to Ser, Asp(139) to Set to disable the lysine 
binding site. The mutation was performed in the BB10153, BB10170 and 
BB10171 backgrounds (see examples 2, 3 & 4) using the 28 base long 
oligonucleotide (SEQ ID NO:20) CCCTGCGGAG AGTTGGATGG ATTCCTGC; The 
mutation was then cloned into pGW1Hg.plasminogen using the restriction 
enzymes HindIII and SplI. 
EXAMPLE 6 
Construction of BB10181 
BB10181 has the cleavage site sequence of BB10170 and an additional 
mutation of Phe(583) to Arg to interfere with the binding of 
.alpha.2-antiplasmin. BB10181 was constructed via an intermediate 
BB10150-based construct using M13 containing full length BB10150 as the 
template and the oligonucleotide (SEQ ID NO:21) GAAGTGCATT CCTCTCCTCG 
TACGAAG as the primer; The mutated BB10150 gene was then cloned into 
pGW1Hg using the restriction enzymes HindIII and SmaI. The BB10181 
expression vector was then made from this intermediate by replacing the 
HindIll to SplI fragment from this plasmid with the corresponding portion 
from BB10170 (see example 3). 
EXAMPLE 7 
Construction of BB10186 
BB10186 has the cleavage site sequence of BB10171 and an additional 
mutation of Met(585) to Arg to interfere with the binding of 
.alpha.2-antiplasmin. BB10186 was made in a similar manner to that 
described in example 6 above for BB10181. The intermediate BB10150 
construct was made using the 25 base long oligonucleotide (SEQ ID NO:22) 
CCACAGAAGT GTCTTCCAAA CCTCG followed by cloning; into pGW1Hg. BB10186 was 
then made by a fragment switch using the HindIII to SplI fragment from 
BB10171 (see example 4). 
EXAMPLE 8 
Construction of BB10199 
BB10199 has the cleavage site sequence of BB10158 and an additional 
mutation of Glu(606) to Lys to interfere with the binding of 
.alpha.2-antiplasmin. BB10199 was made essentially as described in PCTGB 
9301632 examples 2 & 3. The KpnI to EcoRV fragment of a BB10158 (see 
example 3, above) was used to replace the corresponding fragment of a 
BB1051 Glu(606) to Lys construct cloned into pUC and was then cloned into 
the final expression vector as described in PCTGB 9301632. 
EXAMPLE 9 
Cleavage of BB10151 
Plasminogen mutants (12.5 .mu.g) were incubated with 2.8 .mu.g thrombin as 
described in Method 1. The time course of cleavage of the plasminogen 
mutants was determined by quantitative gel scanning; 50% cleavage times 
for T19 and BB10151 were 9 and 3 minutes respectively. Gel scan data for 
cleavage of BB10151 are shown in FIG. 1. 
EXAMPLE 10 
Activation of BB10151 
Purified BB10151 protein was assayed for activation using the linked 
chromogenic assay (see Method 2.1). Results of this assay are shown in 
FIG. 2 in which the increase in absorbance at 405 nm with time 
demonstrates that plasmin activity is generated upon incubation of BB10151 
with thrombin. T19 is shown for comparison and was found to be 
approximately 2 times less potent than BB10151 in this assay. 
EXAMPLE 11 
Plasma Clot Lysis 
The ability of BB10151 and T19 to lyse a plasma clot was determined as 
described in Method 2.2 and the results of such an assay are shown in FIG. 
3. BB10151 (20 .mu.g/ml) was able to cause complete lysis of the clot 
whereas T19 did not lyse the clot at concentrations up to 150p, g/ml. Thus 
BB10151 was found to be at least 7 times more active than T19 at inducing 
lysis of a plasma clot. Representative examples of other plasminogen 
analogues of the invention were compared for plasma clot lysis activity at 
a concentration of 40 .mu./ml and the results in Table 1 show that they 
possess similar activity to BB10151 
TABLE 1 
______________________________________ 
Plasminogen Analogue 
Time for 50% clot lysis (min) 
______________________________________ 
BB10151 4.5 
T19 not lysed 
BB10158 12.5 
BB10199 6 
BB10153 4.1 
BB10171 4.4 
BB10156 6.6 
BB10170 3.1 
______________________________________ 
Methods 
1. Cleavage Analysis 
Plasminogen analogues are assessed for susceptibility to cleavage by 
thrombin using SDS PAGE under reducing conditions. Typical incubation 
volumes of 0.125 ml in 100mM Tris HCl pH 7.4 consist of plasminogen 
analogue, at the concentration shown in the examples, and thrombin, at the 
concentration shown in the examples. Incubations are performed at 
37.degree. C. Control incubations are performed under the same conditions 
in the absence of thrombin. The activation reactions were stopped by 
precipitating the protein by the addition of trichloroacetic acid to a 
final concentration of 20% and standing at 4.degree. C. for &gt;4 hours. The 
precipitates were then pelleted, washed with acetone and resuspended in 
SDS PAGE sample buffer (0.1 m Tris pH6.8, 10% glycerol, 1% SDS, 0.5% 
mercaptoethanol and 0.05% bromophenol blue). The samples were analysed 
either on 8-25% gradient gels or 12% gels. The resulting gels were 
analysed using a SHIMADZU Gel Scanner which scans the gel and calculates 
the concentration of protein in bands by determining the area under the 
peaks. (The word SHIMADZU is a trade mark.) The rate of cleavage of 
plasminogen was thus determined by measuring the disappearance of the 
plasminogen band at approximately 92kDa and the appearance of the plasmin 
heavy chain band at approximately 66kDa. 
2. Activation Analysis 
2.1 Linked Chromogenic Assay 
Plasminogen analogue and thrombin are incubated together in the presence of 
the chromogenic substrate S2251 and plasmin produced by activation 
directly cleaves the S2251 leading to an increase in absorbance at in a 
total volume of 880 .mu.l in a buffer containing 50 mM Tris HCl, 0.1 mM 
EDTA, 0.005% Triton X100 and 0.1% HSA. S2251 is added to a final 
concentration of 0.35 mg/ml and the plasminogen analogue concentration 
used is 3 .mu.g/ml. The thrombin concentration used is 4.55 NIHU/ml. 
Aliquots of 100 .mu.l of the reaction are removed during incubation at 
37.degree. C. and added to 25 .mu.l d 4% acetic acid, in microtitre 
plates, to stop the reaction. At the completion of the time course the 
plates are read on a microplate reader at a wavelength of 405 nm. 
2.2 In Vitro Plasma Clot Lysis Assay 
A mixture of 50 .mu.l rabbit plasma (anticoagulated with 3.8% trisodium 
citrate), 50 .mu.l APTT reagent (Instrumentation Labs) and an appropriate 
volume of plasminogen analogue in 0.1M Tris HCl pH 7.4 is made up to 200 
.mu.l with the same buffer in a well of a 96 well microtitre plate. A 
separate well contains 4.4 .mu.l 500 mM CaCl.sub.2 mixed with 50.6 .mu.l 
of the same buffer. The plate is incubated at 37.degree. C. for 30 minutes 
and clotting is initiated by transferring 50 .mu.l of the CaCl.sub.2 to 
the well containing plasminogen analogue. Progress of clot formation and 
dissolution is followed by measuring the absorbance at 405nm (620nm 
reference) at timed intervals during continued incubation at 37.degree. C. 
for 1 hour. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 29 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: BB10151 
(B) LOCATION: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ThrThrLysIleLysProArgIleXaa 
15 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 230 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Domain 
(B) LOCATION: 1-230 
(D) OTHER INFORMATION: Serine Protease Domain of 
wild type plasmin 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ValValGlyGlyCysValAlaHisProHisSerTrpProTrpGln 
151015 
ValSerLeuArgThrArgPheGlyMetHisPheCysGlyGlyThr 
202530 
LeuIleSerProGluTrpValLeuThrAlaAlaHisCysLeuGlu 
354045 
LysSerProArgProSerSerTyrLysValIleLeuGlyAlaHis 
505560 
GlnGluValAsnLeuGluProHisGlyGlnGluIleGluValSer 
657075 
ArgLeuPheLeuGluProThrArgLysAspIleAlaLeuLeuLys 
808590 
LeuSerSerProAlaValIleThrAspLysValIleProAlaCys 
95100105 
LeuProSerProAsnTyrValValAlaAspArgThrGluCysPhe 
110115120 
IleThrGlyTrpGlyGluThrGlnGlyThrPheGlyAlaGlyLeu 
125130135 
LeuLysGluAlaGlnLeuProValIleGluAsnLysValCysAsn 
140145150 
ArgTyrGluPheLeuAsnGlyArgValGlnSerThrGluLeuCys 
155160165 
AlaGlyHisLeuAlaGlyGlyThrAspSerCysGlnGlyAspSer 
170175180 
GlyGlyProLeuValCysPheGluLysAspLysTyrIleLeuGln 
185190195 
GlyValThrSerTrpGlyLeuGlyCysAlaArgProAsnLysPro 
200205210 
GlyValTyrValArgValSerArgPheValThrTrpIleGluGly 
215220225 
ValMetArgAsnAsn 
230 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: Misc_feature 
(B) LOCATION: 1..24 
(D) OTHER INFORMATION: mutagenesis oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CTTGGGGACTTCTTCAAGCAGTGG24 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 48 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: misc_feature 
(B) LOCATION: 1..48 
(D) OTHER INFORMATION: mutagenesis oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CACCCCCCTACGATTCTAGGTTTAATTTTAGTTGTACATTTCTTCGGC48 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(D) OTHER INFORMATION: mutagenesis oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GTTCGAGATTCACTTTTTGGTGTGCAC27 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: BB10150 
(B) LOCATION: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
AlaValValProArgValValGlyGlyAla 
1510 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(D) OTHER INFORMATION: mutagenesis oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
CTAGGTACAACCGCTTTCTTCGGCT25 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(D) OTHER INFORMATION: mutagenesis oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
GGTGGGCCACCGCCCCCCCCAC22 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
CysValValProArgValValGlyGlyCys 
1510 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: BB10156 
(B) LOCATION: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
AlaGlyGlnLysThrLeuArgProArgValValGlyGlyAla 
1510 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: BB10158 
(B) LOCATION: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
AlaThrThrLysIleLysProArgIleValGlyGlyAla 
1510 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: BB10170 
(B) LOCATION: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
AlaLeuArgProArgValValGlyGlyAla 
1510 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 39 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(D) OTHER INFORMATION: mutagenesis oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
CAACCCTAGGTCTAAGTGTTTTCTGACCCGCTTTCTTCG39 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(D) OTHER INFORMATION: mutagenesis oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
CAACCCTAGGTTTGATCTTCGTTGTCGCTTTCTTCG36 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(D) OTHER INFORMATION: mutagenesis oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
CACAACCCTAGGTCTAAGCGCTTTCTTCGG30 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: BB10169 
(B) LOCATION: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
CysValGluLeuGlnGlyIleLysProArgIleValGlyGlyCys 
151015 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(D) OTHER INFORMATION: mutagenesis oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
GATTCTAGGTTTAATGCCCTGCAGTTCCACACATTTCTTCGG42 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: BB10171 
(B) LOCATION: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
CysValGluLeuGlnGlyLeuArgProArgValValGlyGlyCys 
151015 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 39 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(D) OTHER INFORMATION: mutagenesis oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
CACCCCCCTACCACTCTGGGTCTCAGGCCCTGCAGTTCC39 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(D) OTHER INFORMATION: mutagenesis oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
CCCTGCGGAGAGTTGGATGGATTCCTGC28 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(D) OTHER INFORMATION: mutagenesis oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
GAAGTGCATTCCTCTCCTCGTACGAAG27 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(D) OTHER INFORMATION: mutagenesis oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
CCACAGAAGTGTCTTCCAAACCTCG25 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(D) OTHER INFORMATION: Plasminogen analogue thrombin 
cleavable site sequence where Xaa at position 1 
represents P4; Xaa at position 2 represents P3; 
Xaa at position 5 represents P1'; and Xaa at 
position 6 represents P2'. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
XaaXaaProArgXaaXaa 
15 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(D) OTHER INFORMATION: alpha- thrombin cleavage site where 
Xaa at position 1 represents P2 and Xaa at position 3 
represents P11. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
XaaArgXaa 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
ValGluLeuGlnGlyValValPro 
15 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(D) OTHER INFORMATION: Plasminogen analogue cleavage site 
where Xaa at position 8 represents P1'and Xaa at 
position 7 represents P2'. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
ThrThrLysIleLysProArgXaaXaa 
15 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
LeuArgProArg 
1 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
CysThrThrLysIleLysProArgIleValGlyGlyCys 
1510 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 810 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
MetGluHisLysGluValValLeuLeuLeuLeuLeuPheLeuLysSer 
19- 15-10-5 
GlyGlnGlyGluProLeuAspAspTyrValAsnThrGlnGlyAlaSer 
1510 
LeuPheSerValThrLysLysGlnLeuGlyAlaGlySerIleGluGlu 
152025 
CysAlaAlaLysCysGluGluAspGluGluPheThrCysArgAlaPhe 
30354045 
GlnTyrHisSerLysGluGlnGlnCysValIleMetAlaGluAsnArg 
505560 
LysSerSerIleIleIleArgMetArgAspValValLeuPheGluLys 
657075 
LysValTyrLeuSerGluCysLysThrGlyAsnGlyLysAsnTyrArg 
808590 
GlyThrMetSerLysThrLysAsnGlyIleThrCysGlnLysTrpSer 
95100105 
SerThrSerProHisArgProArgPheSerProAlaThrHisProSer 
110115120125 
GluGlyLeuGluGluAsnTyrCysArgAsnProAspAsnAspProGln 
130135140 
GlyProTrpCysTyrThrThrAspProGluLysArgTyrAspTyrCys 
145150155 
AspIleLeuGluCysGluGluGluCysMetHisCysSerGlyGluAsn 
160165170 
TyrAspGlyLysIleSerLysThrMetSerGlyLeuGluCysGlnAla 
175180185 
TrpAspSerGlnSerProHisAlaHisGlyTyrIleProSerLysPhe 
190195200205 
ProAsnLysAsnLeuLysLysAsnTyrCysArgAsnProAspArgGlu 
210215220 
LeuArgProTrpCysPheThrThrAspProAsnLysArgTrpGluLeu 
225230235 
CysAspIleProArgCysThrThrProProProSerSerGlyProThr 
240245250 
TyrGlnCysLeuLysGlyThrGlyGluAsnTyrArgGlyAsnValAla 
255260265 
ValThrValSerGlyHisThrCysGlnHisTrpSerAlaGlnThrPro 
270275280285 
HisThrHisAsnArgThrProGluAsnPheProCysLysAsnLeuAsp 
290295300 
GluAsnTyrCysArgAsnProAspGlyLysArgAlaProTrpCysHis 
305310315 
ThrThrAsnSerGlnValArgTrpGluTyrCysLysIleProSerCys 
320325330 
AspSerSerProValSerThrGluGlnLeuAlaProThrAlaProPro 
335340345 
GluLeuThrProValValGlnAspCysTyrHisGlyAspGlyGlnSer 
350355360365 
TyrArgGlyThrSerSerThrThrThrThrGlyLysLysCysGlnSer 
370375380 
TrpSerSerMetThrProHisArgHisGlnLysThrProGluAsnTyr 
385390395 
ProAsnAlaGlyLeuThrMetAsnTyrCysArgAsnProAspAlaAsp 
400405410 
LysGlyProTrpCysPheThrThrAspProSerValArgTrpGluTyr 
415420425 
CysAsnLeuLysLysCysSerGlyThrGluAlaSerValValAlaPro 
430435440445 
ProProValValLeuLeuProAspValGluThrProSerGluGluAsp 
450455460 
CysMetPheGlyAsnGlyLysGlyTyrArgGlyLysArgAlaThrThr 
465470475 
ValThrGlyThrProCysGlnAspTrpAlaAlaGlnGluProHisArg 
480485490 
HisSerIlePheThrProGluThrAsnProArgAlaGlyLeuGluLys 
495500505 
AsnTyrCysArgAsnProAspGlyAspValGlyGlyProTrpCysTyr 
510515520525 
ThrThrAsnProArgLysLeuTyrAspTyrCysAspValProGlnCys 
530535540 
AlaAlaProSerPheAspCysGlyLysProGlnValGluProLysLys 
545550555 
CysProGlyArgValValGlyGlyCysValAlaHisProHisSerTrp 
560565570 
ProTrpGlnValSerLeuArgThrArgPheGlyMetHisPheCysGly 
575580585 
GlyThrLeuIleSerProGluTrpValLeuThrAlaAlaHisCysLeu 
590595600605 
GluLysSerProArgProSerSerTyrLysValIleLeuGlyAlaHis 
610615620 
GlnGluValAsnLeuGluProHisValGlnGluIleGluValSerArg 
625630635 
LeuPheLeuGluProThrArgLysAspIleAlaLeuLeuLysLeuSer 
640645650 
SerProAlaValIleThrAspLysValIleProAlaCysLeuProSer 
655660665 
ProAsnTyrValValAlaAspArgThrGluCysPheIleThrGlyTrp 
670675680685 
GlyGluThrGlnGlyThrPheGlyAlaGlyLeuLeuLysGluAlaGln 
690695700 
LeuProValIleGluAsnLysValCysAsnArgTyrGluPheLeuAsn 
705710715 
GlyArgValGlnSerThrGluLeuCysAlaGlyHisLeuAlaGlyGly 
720725730 
ThrAspSerCysGlnGlyAspSerGlyGlyProLeuValCysPheGlu 
735740745 
LysAspLysTyrIleLeuGlnGlyValThrSerTrpGlyLeuGlyCys 
750755760765 
AlaArgProAsnLysProGlyValTyrValArgValSerArgPheVal 
770775780 
ThrTrpIleGluGlyValMetArgAsnAsn 
785790 
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