WO1992007874A1 - Pharmaceutically active proteins comprising an active protein and an integrinaffinity sequence - Google Patents

Abstract

Proteins such as hirudin analogues have an integrin affinity sequence which is located at the carboxy terminus may have improved plasma lifetimes, may be able to target platelets in a blood clot, and are not degraded by carboxypeptidases on expression. Preferred proteins have the sequence -RGDX (e.g. X = Ser) at the carboxy terminus.

Description

Pharmaceutically active proteins comprising an active protein and an integrinaffinity sequence.

This invention relates to proteins having a pharmacological activity which may be enhanced by the possession of an integrin affinity site. It also relates to nucleic acids coding for such proteins. In preferred embodiments the invention relates to proteins having antithrombotic activity, such as hirudin, that have an integrin affinity sequence located at the carboxy terminus, and to nucleic acids coding for such proteins.

Hirudins are naturally occurring polypeptides of 65 or 66 amino acids in length that are produced by the leech Hirudo medicinalis . Hirudin is an anticoagulating agent which binds to thrombin and prevents blood coagulation by inhibiting thrombin from catalysing the conversion of fibrinogen to fibrin, thus preventing the formation of the protein framework of blood clots. The binding of hirudin also prevents other prothrombic activities of thrombin including activation of factors V, VIII, XIII and platelets. There are three principal variants of hirudin (named HV-1, HV-2 and HV-3) , whose sequences will be described later.

J-Y. Chang, FEBS Letters 164, 307-313 (1983) describes that the C-terminal segment of hirudin is essential for hirudin-thrombin interaction. Removal of the 5 carboxy-ter inal amino acids by protease digestion reduces the thrombin inhibitory activity of hirudin by 92%.

T.J. Rydel et al , Science 249: 277-280 (20th July 1990) describes the crystal structure of the complex between recombinant hirudin and thrombin. It describes that the carboxy terminal segment of hirudin makes numerous electrostatic interactions with an anion-binding exosite of thrombin, which is an extension of the active site cleft dominated by positively charged side chains. The carboxy terminal tail of hirudin adopts a long extended confirmation in the complex and is firmly anchored at the end of the thrombin exosite by hydrophobic contacts of the 3₁₀ helical region (Glu61-Glu65) . Nine of the final 11 carboxy-terminal residues are involved in the exosite interactions (Glu61 and Glu62 are not involved) terminating in a salt bridge between the carboxylate of Glu65 with Lys36 of thrombin. It is stated that the initial step of the process of formation of the complex may centre about the binding of the bulky carboxy terminal 3₁₀ helix in the exosite.

M.G. Grutter et al , The EMBO Journal 9(8): 2361-5 (1990) describe investigations into the crystal structure of the thrombin-hirudin complex. It is stated that hirudin inhibits thrombin by a previously unobserved mechanism. In contrast to other inhibitors of serine proteases, the specificity of hirudin is not due to interaction with the primary specificity pocket of thrombin, but rather through binding at sites both close to and distant from the active site. Binding close to the active site involves the three amino-terminal residues of hirudin. Binding to a site distant from the active site (the exosite) involves the carboxy-terminal residues of hirudin. The carboxyl tail of hirudin (residues 48-65) wraps around thrombin along the putative fibrinogen secondary binding site and makes a number of ionic and hydrophobic interactions with thrombin in this area.

S.J.T. Mao et al, Biochemistry 27, 8150-3 (1988) describe the inhibition of thrombin by C-terminal fragments of hirudin. It is disclosed that a peptide comprising the C-terminal 10 amino acids (S56-Q65) of hirudin inhibits the thrombin-induced polymerisation of fibrinogen but does not inhibit the thrombin catalysed proteolysis of tripeptide chromogenic substrates. This indicates that the C-terminal region of hirudin binds to a non-catalytic site on thrombin that is involved in the interaction with fibrinogen.

J.Y. Clang, Biochemistry 30, 6656-61 (1991), describes that the C-terminal amino acids of C-terminal fragments of hirudin are essential for anticoagulant activity. Deletion of Q65 from Hir^54""65 reduced anticoagulant activity by 46% and removal of both L64 and Q65 reduced activity by more than 96%.

R.O. Hynes, Cell 48, 549-54 (1987) describes integrins as a family of transmembrane glycoproteins which are involved in cell-cell or cell-matrix interactions. Several have been disclosed as binding to extracellular matrix proteins at sites encompassing the Arg Gly Asp (RGD) sequence. These include fibronectin, vitronectin and fibrinogen receptors. Other integrin-ligand binding reactions may not involve RGD sequences. The fibrinogen receptor, αllb B3, (GpIIb/IIIa) binds fibrinogen, fibronectin, vitronectin and von illebrand factor. When platelets are not activated, the platelet membrane glycoprotein GpIIb/IIIa does not bind fibrinogen. However, upon platelet activation, GpIIb/IIIa becomes competent to bind fibrinogen, a process required for platelet aggregation.

R.O. Hynes, Thromb . Haemostas . 66: 40-42 (1991) describes integrins as a family of heterodimeric cell surface proteins, each composed of an α and a β subunit. The o:IIb/B3 integrin, also known as GPIIb/IIIa, binds to fibrinogen, fibronectin, von Willebrand factor and vitronectin. Integrin α5B3 binds to the same ligands and also thro bospondin. Integrin α2Bl (GPIa/IIa) binds to collagen and laminin.

E. Ruoslahti and M.D. Pierschbacher, Science 238, 491-7 (1987) describe that the conformation of the RGD sequence in individual proteins is critical for recognition by integrins. Many proteins contain RGD sequences but most of these are probably not recognised by RGD-directed cell surface receptors. An inactive RGD sequence may not be available at the surface of the molecule containing it, or, if available, its conformation may be so constrained by the secondary and tertiary structure of the protein that it may not fit any of the receptors.

A. Andrieux et al J . Biol . Chem . 264, 9258-65 (1989) describe that the fibrinogen A chain has two RGD sequences; A α 572-5, RGDS, and A α. 95-8, RGDF, but that only the RGDF sequence binds to Gp lib/Ilia. As both the free peptides RGDS and RGDF can bind to Gp Ilb/IIIa it is suggested that the sequences surrounding RGDF, but not RGDS, in fibrinogen may provide the structural constraints necessary for an appropriate orientation of the RGD such that it can bind to Gp Ilb/IIIa.

A. Hautanen et al . J. Biol . Chem . 264, 1437-1442 (1989) describe that the fibronectin receptor binds only to fibronectin and not to the RGD-containing proteins vitronectin, Zn α2-glycoprotein and Tamm-Horsfall protein.

T. Maeda et al, J . Biol . Chem . 264, 15165-8 (1989) describe the insertion of an RGDS containing heptapeptide (SLRGDSA. into a truncated form of Protein A using in vitro mutagenesis. This is achieved by inserting nucleic acid encoding the heptapeptide sequence into one of the Hind III sites in the plasmid expression vector pRIT2T. The resultant Protein A possesses the SLRGDSA sequence in a middle portion of the truncated Protein A protein, and has acquired the property of the RGDS peptide of binding to cellular attachment receptors.

However, K. Sekiguchi et al, Protein Engineering 3:298 (1990) describe that the context in which the RGDS sequence is present in the truncated Protein A has a major effect on the cellular binding activity of the molecule. Protein A variants with the inserted sequences, YAVTGRGDSPASSK and RGDSPASSKPISIN had slightly less cell binding activity than the variant with the SLRGDSA sequence while a variant with the insert YTITVYAVTGRGDS had only weak cell binding activity. The protein A variants possessed an RGDS sequence in a middle portion of the protein A.

R.J. Shebuski et al, Thromb . Haem . 61, 183-8 (1989) , describe that the peptide Ac-RGDS-NH₂ can bind to the platelet GpIIb/IIIa receptor and inhibit platelet aggregation in vitro . The Ac-RGDS-NH₂ peptide can also inhibit platelet-dependent thrombus formation in dog coronary arteries when administered at high dose by direct intracoronary infusion. However, the activity of the peptide is limited by its rapid clearance and thrombus formation occurs rapidly after stopping the infusion.

US-A-4703039 describes a method of prolonging the circulatory lifetime of peptides containing a sequence such as RGDS by chemically conjugating them to carrier macromolecules (e.g. albumin) which have the property of an extended lifetime within the circulatory system.

EP-A-0333356 (Biogen, Inc.) describes peptides that are from 8 to 26 amino acids in length and homologous to at least a portion of the carboxy terminal 26 amino acids of native hirudin. Such analogues may have an Asp₅₃ or Asn₅₃ replaced with an arginine residue so that the peptide contains an Arg₅₃-Gly₅₄-Asp₅₅ (RGD) sequence which may bind to a platelet surface glycoprotein (GpIIb/IIIa) and prevent platelet aggregation independently of the thrombin inhibitory action of the peptide. The RGD sequence may therefore target the hirudin peptide to activated platelets. An example of such a peptide that is prepared is N-acetyl-Arg₅₃ hirudin₅₃_₆₄. However, these short peptides are less active than the full sequence protein (natural hirudin) and have shorter plasma lifetimes.

F.C. Church et al , J. Biol . Chem 266: 11975-11979 (1991) describe an artificial chimeric peptide that contains an RGD sequence coupled to the amino terminus of a carboxy terminal fragment of hirudin (residues 53-64) . The hirudin component was active as the peptide inhibited thrombin and the RGD component was functional as the peptide inhibited integrin-mediated cell adherence. However, it is known that C-terminal peptides of hirudin are less active than the full sequence protein.

EP-A-0332533 (Transgene SA) describes hirudin variants that may have the sequence RGDS introduced into a central portion of the molecule, for example hirudin with the variants Arg₃₃, Asp₃₅ and Ser₃₆. Also disclosed are hirudin variants that have the C-terminus of the molecule modified by the addition of amino acids, for example proline (such as the introduction of ^Pro ₆₆^ ^^{n order} to prevent degradation by carboxypeptidases on expression.

It is thus known that the carboxy-terminal region of hirudin is essential for its interaction with thrombin and thus also its antithrombotic activity. It is also known that the presence of an RGD sequence in a protein may or may not confer on that protein the ability to bind to an integrin: it is currently not possible to predict whether an integrin binding sequence, such as RGD, within a protein will be capable of binding to an integrin. It is also known that the pharmacological effectiveness of hirudin, and many other proteins and their variants is dose-dependent and limited by their often short duration of action caused by rapid clearance from the circulation. This is particularly true of hirudin which has a short plasma half-life due to rapid renal clearance (Markwardt et al , Thrombosis Research 52: 393-400 (1988). Kelly et al, Blood 77: 1006-12 (1991), estimated the half-life of hirudin to be 4-5 minutes in baboons and found that intravenous infusion was necessary to inhibit platelet dependent thro botic processes. Consequently, in order to produce prolonged pharmacological activity, e.g. antithrombotic activity as is generally desirable, the protein must be administered by continuous intravenous infusion or repeated subcutaneous injections. Neither of these modes of administration is desirable.

There is therefore a general need to increase the duration of action of the protein or its variant, which may result in a lower dosage being required. This may be achieved by increasing the residence time (or the plasma lifetime) of the protein in the body.

According to a first aspect of the present invention there is provided a protein comprising a first peptide having pharmacological activity, and a second peptide of no more than fifteen amino acids in length, the second peptide being located at the carboxy terminus of the protein and comprising an integrin affinity sequence, the protein being other than a protein which naturally possesses an integrin affinity sequence at the carboxy terminus.

The Applicants have found that placing an integrin affinity sequence at the carboxy terminus of the protein can surprisingly increase the duration of action of the protein, without significantly affecting the protein's biological activity, which may allow fewer and/or lower doses of the protein to be administered. The increased duration of action achieved may also allow administration of the protein by intravenous bolus rather than infusion.

The Applicants have also found that the provision of the integrin affinity sequence at the carboxy terminus may also prevent degradation of the protein by carboxypeptidases when the protein is expressed in transformed host cells, thus improving on existing preparative techniques. So, for example, if the protein is synthesised by recombinant DNA technology in transformed yeast cells, there is no need to use mutants deficient in carboxypeptidase activity.

These advantages can be realised despite the fact that it is known that the carboxy terminus of hirudin plays a crucial role in the interaction with thrombin (see Chang, Rydel et al, Grutter et al and Mao et al. supra) .

The first peptide may be a naturally occurring protein, or a fragment thereof, for example an antithrombotic protein such as tick anticoagulant peptide, antistasin (a leech protein) and in particular hirudin. Other naturally occurring proteins include a fibrinolytic protein such as tissue plasminogen activator (t-PA) , urokinase or streptokinase; a soluble form of a cell surface receptor used as a receptor by a virus such as soluble CD4; a soluble form of a cell surface receptor such as a cytokine, growth factor, immunoglobulin and/or complement receptor; a growth factor such as epidermal growth factor; a colony stimulating factor (e.g. GM-CSF or G-CSF) ; a cytokine such as interleukin-1; a cytokine inhibitor such as IL-1 inhibitor; a hormone such as growth hormone; an antigen such as an HIV antigen (e.g. HIV p24) ; a soluble form of an adhesin such as ELAM; an enzyme such as asparaginase; an enzyme inhibitor such as alpha-1 antitrypsin; a blood coagulation factor such as factor VII; a neuropeptide such as vasoactive intestinal polypeptide; or a kinin such as bradykinin. It will be appreciated that this list is not exhaustive, and merely exemplary.

Alternatively the first peptide may be a variant of a naturally occurring protein, or a fragment thereof, which may differ structurally from, but have similar biological activity to, a naturally occurring protein such as these listed above. The first peptide also encompasses a non-natural protein constructed by protein engineering whose pharmacological activity may or may not be similar to the natural proteins given.

By the term "fragment", when in relation to a fragment of a peptide, it is meant a portion of that peptide that substantially retains at least one (and preferably all) activity of that peptide. By "variant" is meant a polypeptide having one or more amino acids substituted, added or deleted such that the variant substantially retains at least one and preferably all activity of that peptide. Preferably, the amino acid sequence of the first peptide, or a fragment thereof, is not altered significantly.

By "pharmacological activity" it is meant that the first peptide induces a pharmacological effect on the human or animal to which the protein is administered. Protein sequences which do not induce any biological response or biological or chemical reaction are not included within this definition.

Preferred pharmacological activities include antithrombotic and fibrinolytic activities as well as hormonal, growth factor and antigenic activities.

The invention has particular application in the preparation of antithrombotic proteins with improved plasma lifetimes, and so the first peptide preferably has antithrombotic activity. In this embodiment the protein of the present invention may thus provide a desired antithrombotic effect and as a result of the presence of the integrin affinity sequence may increase the plasma lifetime of the protein, and so also the length of time over which the protein is active and therefore blood clotting inhibited.

The presence of the integrin affinity sequence may provide an additional advantage in that it may serve to target the protein to the site of a blood clot by binding to platelets present at such a clot, for example by binding a receptor such as to the GpIIb/IIIa integrin which is exposed at blood clots on the surface of activated platelets. This binding may thus make such proteins of the present invention more efficient in their antithrombotic activity and may reduce the amount of protein that needs to be administered in order to provide the desired level of inhibition (or even prevention) of blood clotting.

The integrin affinity sequence will generally be capable of binding to an integrin. The term "integrin" includes receptors (such as cell-surface receptors) and other glycoproteins capable of interacting with ligands. Specifically contemplated are cell surface adhesion receptors that may be capable of mediating a cell-cell and/or cell-extracellular matrix interaction.

Suitable receptors include GpIIb/IIIa, vitronectin receptor, fibronectin receptor, fibrinogen receptor, laminin receptor, collagen receptor and von Willebrand receptor. The integrin affinity sequence will generally be capable of binding to an endothelial cell and/or a platelet (for example an activated platelet) .

Also preferred are integrin affinity sequences that comprise the sequence RGD (or other sequences, which will be explained later) and/or are specific for integrins that bind ligands possessing the amino acid sequence RGD or other sequences. For the sequence RGD (or other sequences) examples of such integrins are the fibronectin, fibrinogen, vitronectin and von Willebrand factor receptors. Other sequences that can be used instead of RGD in the integrin affinity sequence and that are within the scope of the present invention include derivatives of the RGD sequence (such as extended RGD sequences) and sequences where amino acids have been substituted or inverted that are capable of binding integrins. Suitable sequences include:

1. HHLGGAKQAGDV (SEQ.ID:7), a fibrinogen gamma chain platelet integrin affinity sequence (400-411) (Kloczewiak et al, Biochemistry 23:1767-74 (1984));

2. GRGDSP (SEQ.ID:8), a fibronectin integrin affinity sequence (A. Hautenen et al, supra) ;

3. KNQDK (SEQ.ID:9), a kappa casein sequence (EP-A-343085) ;

4. SGDR (SEQ.ID:47) an "inverted" sequence, (K.M. Yamada and D.W. Kennedy, J. Cellular Physiology 130: 21-28 (1987));

5. HGDF (SEQ.ID:48) and/or HQAGDV (SEQ. ID: 10) (substituted sequences, see Yamada K.M. and Kennedy D.W. , supra and Tranqui L. et al , J. Cell. Biol. 108: 2519-25 (1989));

6. DGEA (SEQ.ID:20), the minimal recognition sequence from collagen required for binding to the collagen receptor α2Bl (Staatz et al , J. Biol . Chem . 266: 7363-7367 (1991));

7. GREDV (SEQ.ID:45) and REDV (SEQ:21) the latter being an alternative fibronectin integrin affinity sequence (Hubbell et al, Bio /technology 9: 568-572 ( 1991) ) ;

8. KGDW (SEQ.ID:22), a GPIIb-IIIa selective affinity sequence from the snake venom barbourin (Scarborough et al, J. Biol . Chem . 266: 9359-9362 (1991));

9. CKGDWPC (SEQ.ID:23) and/or CRGDWPC (SEQ.ID:24) , which are cyclic peptide sequences in which the two cysteine residues are covalently linked to each other by a disulphide bond. Cyclic RGD peptides bind to both GPIIb-IIIa and vitronectin receptor while cyclic KGD peptides bind to GPIIb-IIIa but not to vitronectin receptors (WO-A-90/15620) ; or

10. GKDGEA (SEQ.ID:46).

However, it should be realised that the integrin affinity sequence may be capable of binding to integrins that are not specific for RGD sequences. An example is the Mac-1 integrin. This integrin is a leukocyte-restricted integrin capable of binding to fibrinogen (in an RGD - independent manner) to a recognition site in fibrinogen not shared with other integrins known to function as fibrinogen receptors (Alteiri D.C. et al , J. Biol . Chem 265: 12119 - 12122 (1990)).

The integrin affinity sequence is preferably suitable for binding to an integrin, such as the fibronectin, collagen or fibrinogen receptor. These receptors are glycoproteins that are found on the surface of endothelial cells and platelets. A preferred integrin affinity sequence comprises the sequence -RGDX where X represents nothing or any amino acid. It is preferred that X represent S, F, W or V; S is the amino acid residue of choice. It will thus be seen that the integrin affinity sequence is preferably at least three (such as RGD) , and often four (such as RGDS, RGDF RGDV, RGDW, SGDR, DGEA, REDV and/or KGDW) , amino acids in length. The full sequence of hirudin types HV-1, HV-2 and HV-3 each with a carboxy terminal -RGDS is given as SEQ.ID:1-3 respectively. However, it will be appreciated that such a sequence may be a 12 amino acid sequence (e.g. HHLGGAKQAGDV , SEQ.ID: 7) , a five amino acid sequence (eg -RGDX-^X² , where X¹ and X² independently represent the same as X) or a six amino acid sequence (eg GRGDSP (SEQ.ID:8) , KQAGDV (SEQ.ID:18), CKGDWPC (SEQ.ID:23), GKDGEA (SEQ.ID: 46) and/or CRGDWPC (SEQ.ID:24)) . Larger integrin affinity sequences, such as HHLGGAKQAGDV (SEQ.ID:7), are, however, contemplated.

It will thus be appreciated that the integrin affinity sequence is preferably from 3 or 4 to 12 or 15 amino acids in length.

In particularly preferred embodiments the second peptide is (i.e. it consists only of) the integrin affinity sequence, although this is not essential. The second peptide is 15 or less, such as 12 or less, and preferably no more than 6, amino acids in length.

Proteins which are not encompassed by the present invention are those which, in their natural form, possess an integrin affinity sequence at their carboxy terminus. However, the invention may also exclude any protein which naturally possesses an integrin affinity sequence (anywhere in the protein) , in some embodiments. Such proteins may be (blood) coagulants, or promote blood clot formation. They may also have the integrin affinity sequence HHLGGAKQAGDV (SEQ.ID:7). An example of this is fibrinogen.

It will be apparent that the protein may have additional peptide sequence(s) placed either at the amino terminus of the first peptide and/or between the first and second peptides.

It should be appreciated that the first and second peptides are separate and do not overlap: that is to say one of the peptides does not encompass the other. Thus preferred proteins of the present application may have the following general formula:

(J^a)_n - Pep¹ - (J^b)_m - Pep² I

wherein:

each of J^a and J" individually represent an amino acid (or an amino acid sequence) which may be the same or different; Pep¹ represents the first peptide having pharmacological, such as antithrombotic, activity; Pep² represents the second peptide, of up to 15 amino acids in length, comprising an integrin affinity sequence; and each of n and m individually represent integers, which may be the same or different, having values of from 0 to 1000. It will be appreciated that the protein of formula I thus encompasses the following three proteins having the general formulae:

(^ja)n ~ ^Pepl ~ ^PeP^{2 τ}- Pep¹ - Pep² III Pep¹ - (J^b)_m " Pep² IV

Proteins of the general formula III may be preferred.

The peptides (J^a)_n arid (J )_m may be any suitable peptide sequences, of any suitable length, from one amino acid upwards provided that they do not substantially interfere with, or inhibit, the integrin affinity of the second peptide (Pep²) . It is also preferred that they do not interfere with, or inhibit, the pharmacological activity of the first peptide (Pep¹) . Thus n and/or m preferably has a value of from 0 up to 10, 50, 100 or even 1000. (J^a)_n and/or (J^b)_m may themselves have antithrombotic activity, and so may be a peptide as defined for the first peptide (Pep¹) .

Alternatively or in addition, (J^a)_n and/or (J )_m may comprise a integrin affinity sequence as defined for the second peptide (Pep²) . Thus in one embodiment ( J^~ ) _Ώ and/or (J )_m may individually represent a naturally occurring polypeptide, or a variant, or a fragment thereof, having an integrin affinity sequence as discussed before, or may represent fibronectin (or a fragment thereof) .

If the protein contains one or more integrin affinity sequences in addition to Pep , then one of these sequences is preferably provided at the N-terminus of either (J^a)_n or Pep¹.

If (J^a)_n is provided with an integrin affinity sequence, then this may be provided at the N-terminus. In this situation, it is preferred that the protein does not contain hirudin (or a variant or fragment thereof) .

The first peptide (Pep¹) can be of any suitable length provided that it possesses pharmacological activity. However, it is preferred that the first peptide is preferably at least 6 (such as at least 8) , amino acids in length.

In this specification the term "antithrombotic activity" in relation to peptides is to be interpreted as that activity possessed by the peptides that are capable of at least partially prohibiting, or reducing, blood clotting; this activity is otherwise known as anticoagulant activity. Preferred peptides have antithrombotic activity. They may therefore be thrombin inhibitors, that is to say they are capable of binding to thrombin so that thrombin does not catalyse the conversion of fibrinogen to fibrin. Such peptides will also usually inhibit, or prevent, the activation of factors V, VIII, XIII and platelets.

The first peptide, if it has antithrombotic activity, is preferably a naturally occurring antithrombotic peptide, or a fragment thereof (that retains antithrombotic activity) . Hirudin is the preferred peptide. The fragment can be of any suitable length provided that the fragment has antithrombotic activity. Such a fragment may be, for example, a fragment of from 12 to 26 amino acids in length. The fragment preferably corresponds to, or is substantially homologous with, at least a portion of the carboxy terminal 26 amino acids of a naturally occurring protein such as the 26 carboxy terminal amino acids of hirudin.

Alternatively the fragment may correspond to, or be substantially homologous with, at least a portion of the amino terminus of the naturally occurring protein; that is to say, some amino acids at the N-terminus may be deleted while the fragment retains antithrombotic activity. However, the native full length sequence of the naturally occurring peptide is preferred. For hirudin, this will be 65 or 66 amino acids in length.

Preferred types of hirudin include HV-1, HV-2 and HV-3. HV-l and HV-2 both contain 65 amino acids, while HV-3 has 66.

In this specification, apart from the sequence listing section, the single letter code will often be used to denote the amino acid, and for the sake of reference the single letter amino acid codes are as follows:

Three-letter Single-letter Amino Acid Abbreviation Symbol

Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Asparagine or aspartic acid Asx B Cysteine Cys C Glutamine Gin Q Glutamic acid Glu E Glutamine or glutamic acid Glx Z Glycine Gly G Histidine His H Isoleucine lie I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

The invention also contemplates a protein comprising a first peptide having pharmacological activity, and an integrin affinity sequence located at the carboxy terminus of the first peptide, other than a protein which naturally possesses an integrin affinity sequence at the carboxy terminus. Proteins in accordance with the first aspect of invention may be prepared by any convenient proce Recombinant DNA technology provides the processes choice.

According to a second aspect of the present inventi there is provided nucleic acid (which may be RNA o preferably, DNA) coding for a protein of the fir aspect. Such nucleic acid is preferably recombina and/or isolated nucleic acid. The nucleic acid may in the form of a vector, such as a plasmid, cosmid virus, and in some embodiments is in an expressib form. Thus a third aspect of the present inventi relates to a vector comprising nucleic acid of t second aspect.

The invention also extends to host cells includi mammalian or other animal cells such as insect cell but preferably unicellular organisms, that contain a preferably express, or are capable of expressing, t nucleic acid of the second aspect. Thus, a four aspect of the present invention relates to a ho transformed with a vector of the third aspec Unicellular hosts may be eukaryotic or prokaryotic a are preferably mammalian or yeast cells (such as of t genus Saccharomyces or Pi chia . A particular preferred host is a yeast of the species Saccharomyc cerevisiae and/or Pichia pastoris .

The host need not be a mutant cell, e.g mutant yea cell with reduced, or without, carboxypeptida activity. Non-mutant yeasts can be used since t integrin affinity sequence can prevent degradation of the protein by any carboxy peptidases present.

The nucleic acid of the second aspect may be prepared by chemical synthesis . For example a large number ( e . g . a n even numb er s uch a s 8 - 14 ) o f oligonucleotides may be prepared , preferably by chemical synthesis , and then joined together (suitably by kinasing pairs of oligonucleotides and ligating the pairs) .

Alternatively the nucleic acid may be prepared using site directed utagenesis. A nucleic acid primer containing the mutation may be annealed to template nucleic acid containing nucleic acid encoding the protein of the first aspect. After preparation of complementary nucleic acid to give double stranded nucleic acid the strand containing the primer can be used to prepare double stranded nucleic acid for insertion into a vector (preferably an expression vector) .

The vector is preferably capable of replication in both E. coli and yeast (e.g. Saccharomyces cerevisiae . Suitably the vector has a selectable marker for maintenance in the yeast host (e.g. Ieu2 gene) and/or an ampicillin resistance locus. The vector also preferably has a promoter, e.g. GAL-10 and/or PGK promoter, for expression. A suitable vector is pSW6 (Accession No. NCIMB 40326) or pJKl. The preferred host is Saccharomyces cerevisiae .

As indicated above, proteins of the invention are useful in medicine. According to a fifth aspect of the present invention, there is provided a protein in accordance with the first aspect for use in human or veterinary medicine, for example particularly as an antithrombotic agent.

According to a sixth aspect of the invention, there is provided a pharmaceutical composition comprising one or more proteins in accordance with the first aspect of the invention and a pharmaceutically or veterinarily acceptable carrier. Such a composition may be adapted for intravenous administration and may therefore be sterile. Examples of compositions in accordance with the invention include preparations of sterile protein(s) of the first aspect in isotonic physiological saline and/or buffer. The composition may include a local anaesthetic to alleviate the pain of injection.

The proteins 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 indicating the quantity of protein in activity units. These units may, for example, be antithrombin units (ATUs) , if the protein has antithrombotic activity. Where the protein of the first aspect is to be administered by infusion, it may be dispensed by means of an infusion bottle containing sterile water for injection or saline. Where it is to be administered by injection, it may be dispensed with an ampoule of water for injection or saline. The infusible or injectible composition may be made up by mixing the ingredients prior to administration. The amount of protein to be administered will depend on the effect required, such as the amount of antithrombosis, the required speed of action, and the seriousness of a condition of a patient (for example in terms of the extent of clotting, both in the number and size of clots) . The precise dose to be administered will, because of the very nature of the condition which the proteins of the invention are intended to treat, will be determined by the physician. As a guide, however, in antithrombotic treatment a patient being treated may generally receive a daily dose of from 500 to 50000 ATUs/kg of body weight, for example about 10000 ATUs/kg, either by injection in for example up to five doses, or by infusion.

The invention may be used in a method for the treatment or prophylaxis of a human or animal, such as thrombotic disease or disorder, the method comprising the administration of an effective non-toxic amount of a protein of the first aspect. Thus, the proteins of the present invention may find use in the treatment of diseases that are caused by either partial or total occlusion of a blood vessel by a blood clot, for example vascular diseases such a myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion and other blood system thromboses.

According to a seventh aspect of the invention, there is therefore provided the use of a protein of the first aspect in the preparation of a medicament such as an antithrombotic agent. According to an eighth aspect of the invention, there is provided a process for the preparation of a protein in accordance with the first aspect, the process comprising coupling successive amino acid residues and/or oligo- and/or polypeptides together. This may achieved by chemical synthesis, but is preferably by translation of a nucleic acid of the second aspect in vivo.

According to a ninth aspect of the present invention there is provided a process for the preparation of a nucleic acid in accordance with the second aspect of the invention, the process comprising coupling successive nucleotides and/or ligating oligonucleotides and/or polynucleotides together. The nucleic acid may be synthesised chemically, although it is preferred to use a nucleic acid-directed polymerase, preferably in vivo. The nucleic acid may be suitably modified using site-directed mutagenesis.

A tenth aspect of the present invention relates to a process for the preparation of a pharmaceutical composition of the sixth aspect, the process comprising admixing a protein of the first aspect with a pharmaceutically or veterinarily acceptable carrier.

An eleventh aspect of the present invention relates to a process for the preparation of a vector of the third aspect, the process comprising coupling successive nucleotides and/or ligating oligonucleotides and/or polynucleotides together. A twelfth aspect of the present invention relates to a process for the preparation of a host of the fourth aspect, the process comprising transforming or transfecting a cell with a vector of the third aspect.

Preferred features and characteristics of one aspect of the present invention are as for another aspect mutatis mutandis .

In the the accompanying drawings:

Figures 1 and 2 are maps of the vectors pSW6 and pJKl respectively used in the Examples and Comparative Examples described later; and

Figure 3 is an assembly diagram of pairs of annealed oligomers that can be kinased to prepare a synthetic hirudin gene.

COMPARATIVE EXAMPLE 1 - Construction of a Hirudin Gene

The techniques of genetic engineering and genetic manipulation used in the manufacture of a synthetic HV-l gene and in its further manipulation for construction of a yeast expression vector are well known to those skilled in art. Descriptions of modern techniques can be found in the laboratory manuals "Current Protocols in Molecular Biology" published by Wiley Interscience and in "Molecular Cloning, A Laboratory Manual" (second edition) edited by Sambrook, Fritsch and Maniatis published by Cold Spring Harbour Laboratories, New York. a. Gene Design

A synthetic hirudin HV-1 gene was designed incorporating useful unique restriction sites to facilitate manipulation (see SEQ. ID: 4) . The selected codons are favoured by either S. cerevisiae or E. coli and are thus suitable for expression in either organism.

b. Gene Construction

The gene sequence was divided into 12 oligodeoxyribonucleotides (see SEQ. ID: 6) . Each oligonucleotide overlapped its adjacent partner by 7 base pairs, thus providing a cohesive end after annealing of complementary pairs of oligonucleotides.

c. Oligonucleotide Synthesis

The oligonucleotides were synthesised by automated phosphoramidite chemistry on an Applied Systems 380B DNA Synthesiser, using cyanoethyl phosphoramidites. The methodology is now widely used and has already been described (Beaucage, S.L. and Caruthers, M.H. Tetrahedron Letters 24, 245 (1981)). The oligonucleotides were de-protected and removed from the CPG support by incubation in concentrated NH₃. Typically, 50 mg of CPG carrying 1 micromole of oligonucleotide was de-protected by incubation for 5 hours at 70°C in 600 μl of concentrated NH₃. The supernatant was transferred to a fresh tube and the oligomer precipitated with 3 volumes of ethanol. Following centrifugation the pellet was dried and resuspended in 1 ml of water. The concentration of crude oligomer was then determined by measuring the absorbance at 260 nm.

For gel purification 10 absorbance units of the crude oligonucleotide was dried down and resuspended in 15 μl of marker dye (90% de-ionised formamide, lOmM Tris, 10 mM borate, ImM EDTA, 0.1% bromophenol blue) . The samples were heated at 90°C for 1 minute and then loaded onto a 1.2 mm thick denaturing polyacrylamide gel with 1.6 mm wide slots. The gel was prepared from a stock of 15% acrylamide, 0.6% bisacrylamide and 7M urea in 1 XTBE (90mM Tris-HCl, pH 8.3, 90mM boric acid, 2.5mM EDTA) and was polymerised with 0.1% ammonium persulphate and 0.025% TEMED. The gel was pre-run for 1 hr. The samples were run at 1500 V for 4 to 5 hours. The bands were visualised by UV shadowing and those corresponding to the full length product cut out and transferred to micro-test tubes. The oligomers were eluted from the gel slice by soaking in AGEB (0.5 M ammonium acetate, 0.01 M magnesium acetate and 0.1% SDS) overnight. The AGEB buffer was then transferred to fresh tubes and the oligomer precipitated with three volumes of ethanol at -70°C for 15 minutes. The precipitate was collected by centrifugion in an Eppendorf microfuge for 10 minutes, the pellet washed in 80% ethanol, the purified oligomer dried, redissolved in 1 ml of water and finally filtered through a 0.45 micron micro-filter. The concentration of purified product was measured by determining its absorbance at 260 nm. d. Gene Assembly

The oligonucleotides were kinased to provide them with a 5' phosphate thus enabling subsequent ligation. (see Figure 3) .

Kinasing of Oligomers

100 pmole of oligomer was dried down and resuspended in 20 μl kinase buffer (70 mM Tris, pH 7.6, 10 mM MgCl₂, 1 mM ATP, 0.2mM spermidine, 0.5 mM dithiothreitol) . 10 μl of T4 polynucleotide kinase was added and the mixture was incubated at 37°C for 30 minutes. The kinase was then inactivated by heating at 70°C for 10 minutes.

Complementary pairs of kinased oligonucleotides were annealed in pairs (90°C, 5 minutes, followed by slow cooling) . The 6 paired oligomers were then mixed together, incubated at 50°C for 5 minutes and allowed to cool. They were then ligated overnight at 16°C with T4 DNA ligase. This is shown diagrammatically in Figure 3 (note P = 5'-phosphate) . Oligomers designated BB2011 and BB2020 were not kinased to prevent multimerisation. The sequences of the oligomers shown in Figure 3 correspond to those given in SEQ.ID:6.

The ligation products were separated on a 2% low gelling temperature agarose gel and the band corresponding to the hirudin HV-l gene was excised and extracted from the gel. The purified fragment was then ligated to Hindlll-EcoRI treated pUC19 plas id DNA. (pUC19, code no.^' 27-4951-01, was purchased from Pharmacia Ltd. , Midsummer Boulevard, Central Milton Keynes, Bucks, MK9 3HP, United Kingdom.) The transformation of E . coli host strains was accomplished using standard procedures. The strain used as a recipient in the cloning using plasmid vectors was HW87 which has the following genotype:

araD139(ara-leu)delta7697 (lacIPOZY)delta74 galU galK hsdR rpsL srl recA56

The recombinant pUC19 HV-1 product was transferred into E. coli host strain HW87 and plated onto L-broth ampicillin plates. Twelve colonies were picked and used to prepare plasmid DNA for sequence analysis. Double stranded dideoxy sequence analysis was used to identify a correct clone using a universal sequencing primer BB22 ( CAGGGTTTTCCCAGTCACG) , ( SEQ . ID : 11 ) complementary to the universal primer region of pUC19. The pUC19 recombinant was used to construct the expression vector.

COMPARATIVE EXAMPLE 2 - Construction of a Hirudin Expression Vector

An expression vector was designed to enable the secretion of hirudin to the extracellular medium after expression in S. cerevisiae. Secretion of hirudin is desirable to facilitate production of protein with an authentic N-terminus, to ease purification, to limit intracellular proteolysis, to reduce potential toxic effects on the yeast host and to allow optimal protein folding via formation of native disulphide bonds. Secretion of hirudin through the yeast membrane was directed by fusion of hirudin to the yeast mating type alpha-factor pre-pro-peptide (a naturally secreted yeast peptide) .

The yeast expression vector pSW6 (Figure 1) is based on the 2 micron circle from S . cerevisiae . (pSW6 was deposited in _7. cerevisiae strain BJ2168 at The National Collections of Industrial and Marine Bacteria Limited, 23 St. Machar Drive, Aberdeen, AB2 1RY, Scotland, United Kingdom on 23rd October 1990 under Accession No. NCIMB 40326.) pSW6 is a shuttle vector capable of replication in both E . coli and S . cerevisiae and contains an origin of replication for both organisms, the leu2 gene (a selectable marker for maintenance in the yeast host) and the ampicillin resistant locus for selection of plasmid maintenance in E. coli . (The DNA sequence of the vector has been determined, the E . coli sequences are derived from the E . coli ColEl-based replicon pAT153.) The full sequence is given as SEQ.ID:19. The ability to passage this vector through E. coli greatly facilitates genetic manipulation on this vector. pSW6 contains an alpha- factor pre-pro peptide fused in-frame to epidermal growth factor (EGF) . The expression of this fusion is under the control of an efficient galactose regulated promoter which contains hybrid DNA sequences from the S . cerevisiae GAL 1-10 promoter and the S . cerevisiae phosphoglycerate kinase (PGK) promoter. Transcription is terminated in this vector by the natural yeast PGK terminator. The EGF gene in pSW6 can be removed by digestion with Hindlll and BamHI. This removes DNA encoding both EGF and 5 amino acids from the C-terminus of the alpha-factor pro-peptide. Genes to be inserted into the pSW6 expression vector must therefore have the general composition: Hindlll site - alpha-factor adapter - gene - Ba HI site.

To rebuild the DNA encoding the 5 amino acids at the C-terminal end of the alpha factor pro peptide and to fuse this to the synthetic hirudin gene, an oligonucleotide adapter (5' AGCTTGGATAAAAGA 3' (top strand, SEQ.ID:12), 5'TCTTTTATCCA 3' (bottom strand, SEQ.ID:13)) containing part of a Hindlll site and codons encoding the Ser, Leu, Asp, Lys and Arg from the C-terminal end of the alpha factor pro peptide was constructed. The alpha-factor adaptor was ligated to the synthetic HV-l gene such that the recombinant gene encoded an in frame alpha-factor propeptide fusion to hirudin. The pUC19 HV-l plasmid was first cleaved with Bspml. the overhanging ends were next filled with DNA polymerase I to create a blunt ended linear DNA fragment. This fragment was separated from uncut plasmid on a 1% low gelling temperature agarose gel, excised and extracted from the agarose gel matrix, then further treated with Hindlll. The fragment was then ligated to the alpha-factor adaptor (synthesised as two complementary oligonucleotides described above) and annealed prior to ligation. The resultant recombinant plasmid was pJC80. The alpha-factor adaptor - hirudin sequence was removed from pJC80 on a Hindlll-BamHI fragment (SEQ.ID:5). The fragment was purified on a 1% low gelling temperature agarose gel and ligated to Hindlll-BamHI treated pSW6 to create pJKl (Figure 2) . This plasmid is the basic vector used for wild-type hirudin HV-l expression. COMPARATIVE EXAMPLE 3 - Expression of Hirudin Synthetic Gene

Plasmid expression vector pJKl was transformed into yeast (S_. cerevisiae) strain BJ2168 (prc-1-407, prbl-1122 pep4-3 leu2 trpl ura3-52 cir+) using the method of Sherman F. et al (Methods in Yeast Genetics , Cold Spring Harbour Laboratory, (1986)). All yeast media was as described by Sherman et al . Using a 500 ml shake flask, a 100 ml culture of yeast containing pJKl was grown in 0.67% synthetic complete medium yeast nitrogen base, with amino acids minus leucine and 1% glucose as a carbon source. After overnight growth at 30°C, the cells were transferred to a 2 litre shake flask containing 1 litre of the same synthetic complete medium except having 1% galactose and 0.2% glucose as the carbon source. This induces expression from the hybrid PGK promoter. Cells were grown in the induction medium for 3 days. After this period the supernatant was harvested and assayed.

COMPARATIVE EXAMPLE 4 - Purification of Hirudin

Hirudin was purified from yeast culture broth. Cells were first removed by centrifugation. The supernatant was then assayed for biological activity (see Pharmacology Examples) . Production levels from shake flask cultures were routinely between 10-15mg/litre of culture. The hirudin was purified by preparative HPLC (DYNAMAX (Trade Mark) C18, 300 Angstroms pore size, 12 μm particle size, 21.4 mm internal diameter, 25cm long) . The column was first equilibrated in 15% acetonitrile, 0.1% trifluoro acetic acid. Then up to 21 of supernatant was loaded onto the column with 15% acetroniltrile at lOml/minute. The protein was eluted using a 15-40% acetonitrile gradient at 10ml/minute.The eluted hirudin peak was diluted with an equal volume of 0.1% TFA before loading onto a 10 mm ID DYNAMAX (Trade mark) C18 column at 3 ml/minute. The protein was eluted with a 23-35% gradient. The purity of the isolated protein was assessed by analytical HPLC (VYDAC (Trade Mark) C18 reverse phase) and N-terminal sequence analysis. Mass spectrometry was used to confirm total sequence.

a. Assessing Purity by Analytical HPLC

Samples were analysed on a VYDAC (Trade Mark) C18 column (15 x 0.46cm, particle size 5 μm) equilibrated with 10% acetonitrile, 0.1% trifluroacetic acid (TFA). Purified protein (20 μg) was loaded in 10% acetonitrile, 0.1% TFA. Protein was eluted at a flow rate of lml/minute using an acetonitrile gradient from 10-40% in 0.1% TFA over 30 minutes. The eluted protein sample was monitored by absorbance at 254 nm.

b. Analysis of Purity by Mono 0 FPLC

Samples were analysed on a Mono Q FPLC column (5 x 0.5cm, Pharmacia) equilibrated in 20 mM Tris HCl (pH 7.5). Approximately 15 μg of lyophilised protein was reconstituted in 1ml 20mM Tris HCl (pH 7.5) and loaded onto the column. Protein was eluted using a gradient of 0-250mM NaCl in 20 mM Tris HCl buffer (pH 7.5) at a flow rate of lml/minute over 30 minutes.

c. N-terminal Sequence Analysis

N-terminal sequence analysis was performed by automated Edman degradation using an Applied Biosystems Protein Sequencer, model 471 A (Applied Biosystems, Foster City, California) .

Purified material that was greater than 95% pure, was dried down in a Speedivac and reconstituted in 0.5ml of 0.9% saline for assay.

EXAMPLE 5 - Construction and Expression of Hirudin RGDS

Hirudin analogues which are altered to include a C-terminal amino acid extension were constructed in order to increase the duration of hirudin antithrombotic activity. Hirudin-RGDS is a hirudin variant in which the amino acid sequence Arg-Gly-Asp-Ser has been added to the C-terminus of hirudin HV-l. The full sequences of hirudin types HV-l, HV-2 and HV-3 with a carboxy terminal -RGDS are given in SEQ. ID.1-3 respectively. The strategy for modification is described below.

Host strains

RZ1032 is a derivative of E. coli that lacks two enzymes of DNA metabolism: (a) dUTPase (dut) which results in a high concentration of intracellular dUTP, and (b) uracil N-glycosylase (ung) which is responsible for removing mis-incorporated uracils from DNA (Kunkel et al, Methods in Enzymol . , 154 , 367-382 ( 1987 ) ) . The principal benef it is that these mutations lead to a higher frequency of mutants in site directed mutagenesis . RZ1032 has the following genotype :

HfrKL16PO/45[ly_sA961-62) , dutl, ungl. thil. recA, Zbd-279: :TnlO, SUPE44

JM103 is a standard recipient strain for manipulations involving M13 based vectors. The genotype of JM103 is JM103 delta (lac-pro) , thi_r supE.strA. endA. sbcB15, hspR4, F' traD36. proAB. lacl^. ZdeltaM15.

Site Directed Mutagenesis

Kinased mutagenesis primer (2.5pmole) was annealed to the single stranded template DNA, which was prepared using RZ1032 as host, (1 μg) in a final reaction mix of 10 μl containing 70 mM Tris, 10 mM MgCl₂. The reaction mixture in a polypropylene micro-testube (Eppendorf) was placed in a beaker containing 250 ml of water at 70°C for 3 minutes followed by 37°C for 30 minutes. The annealed mixture was then placed on ice and the following reagents added: 4 μl of 10 X HM (200 mM HEPES, 100 mM MgCl₂ pH 7.6), 5 μl of a mixture of all 4 deoxyribonucleotide triphosphates each at 5mM, 5 μl of ATP (lOmM) , 5 μl DTT (lOOmM) , 2 μl of T4 DNA ligase (lOOu) , 1.0 μl Klenow fragment of DNA polymerase and water to a final volume of 50 μl. The polymerase reaction mixture was then incubated at 15°C for 4-16 hrs. After the reaction was complete, 150 μl of TE (10 mM Tris, 1 mM EDTA pH 8.0) was added and the mutagenesis mixture stored at -20°C.

For the isolation of mutant clones the mixture was then transformed into the recipient JM103 as follows. A 5 ml overnight culture of JM103 in 2 X YT (1.6% Bactotryptone, 1% Yeast Extract, 1% NaCl) was diluted 1 in a 100 into 50 ml of pre-warmed 2 X YT. The culture was grown at 37°C with aeration until the A600 reached 0.4. The cells were pelleted and resuspended in 0.5 vol of 50 mM CaCl₂ and kept on ice for 15 minutes. The cells were then re-pelleted at 4°C and resuspended in 2.5 ml cold 50 mM CaCl₂. For the transfection, 0.25, 1, 2, 5, 20 and 50 mcl aliquots of the mutagenesis mixture were added to 200 μl of competent cells which were kept on ice for 30 mins. cells were then heated shocked at 42°C for 2 minutes. To each tube was then added 3.5 ml of YT soft agar containing 0.2 ml of a late exponential culture of JM103, the contents were mixed briefly and then poured onto the surface of a pre-warmed plate containing 2 X YT solidified with 1.5% agar. The soft agar layer was allowed to set and the plates then incubated at 37°C overnight.

Single stranded DNA was then prepared from isolated clones as follows: Single plaques were picked into 4 ml of 2 X YT that had been seeded with 10 μl of a fresh overnight culture of JM103 in 2 X YT. The culture was shaken vigorously for 6 hrs. 0.5ml of the culture was then removed and added to 0.5 ml of 50% glycerol to give a reference stock that was stored at -20°C. The remaining culture was centrifuged to remove the cells and 1 ml of supernatant carrying the phage particles was transferred to a fresh Eppendorf tube. 250 μl of 20% PEG6000, 250mM NaCl was then added, mixed and the tubes incubated on ice for 15 minutes. The phage were then pelleted at 10,000 rpm for 10 minutes, the supernatant discarded and the tubes re-centrifuged to collect the final traces of PEG solution which could then be removed and discarded. The phage pellet was thoroughly resuspended in 200 mcl of TEN (10 mM Tris, 1 mM EDTA, 0.3 M NaOAc) . The DNA was isolated by extraction with an equal volume of Tris saturated phenol. The phases were separated by a brief centrifugation and the aqueous phase transferred to a clean tube. The DNA was re-extracted with a mixture of 100 μl of phenol, 100 μl chloroform and the phases again separated by centrifugation. Traces of phenol were removed by three subsequent extractions with chloroform and the DNA finally isolated by precipitation with 2.5 volumes of ethanol at -20°C overnight. The DNA was pelleted at 10,000 rpm for 10 minutes, washed in 70% ethanol, dried and finally resuspended in 50 μl of TE.

A gene encoding hirudin RGDS was constructed by oligonucleotide directed mutagenesis. The hirudin gene of Comparative Example 1 was first transferred into M13 mpl9 on a Hindlll-BamHI DNA fragment. The 42bp base pair oligonucleotide CGGGGATCCCTATTAGCTGTCACCGCGCTGCAGATATTCTTC 3 ' (SEQ.ID:17) was used to direct the mutagenesis. Clones carrying the desired mutation were identified by DNA sequence analysis. The entire clone was sequenced to ensure that no other mutation had inadvertently been introduced. After confirmation of the correct DNA sequence on single stranded templates replicative form DNA of one mutant was prepared. and the DNA encoding the hirudin variants was removed on a Hindlll-BamHI fragment. The fragment was gel purified and ligated to Hindlll-BamHI treated pSW6 thus replacing the EGF gene in pSW6 to create pJK009. pJK009 was transformed first into E. coli host HW87 and characterised by restriction digestion. A 50ml plasmid preparation was prepared and used to transform yeast strain BJ2168.

The procedures of Comparative Examples 3 and 4 were followed for expression and purification of hirudin RGDS. The in vitro and .in vivo characterisation of this variant is described in Comparative Pharmacology Examples A and B.

EXAMPLE 6 - Construction and expression of Hirudin RGDF

Hirudin-RGDF is a hirudin derivative in which the integrin binding amino acid sequence Arg-Gly-Asp-Phe has been attached to the C-terminus of hirudin HV-l. The procedure of Example 5 was used except that the primer used for mutagenesis was the 42bp primer 5' CGGGGATCCCTATTAGAAGTCACCGCGCTGCAGATATTCTTC 3' (SEQ.ID:15) . EXAMPLE 7 - Construction and expression of Hirudin RGDV

Hirudin-RGDV is a hirudin derivative in which the integrin binding amino acid sequence Arg-Gly-Asp- Val has been attached to the C-terminus of native hirudin. The procedure of Example 5 was used except that the oligonucleotide primer comprised a 42bp 5' CGGGGATCCCTATTAAACGTCACCGCGCTGCAGATATTCTTC 3' (SEQ.ID:16).

EXAMPLE 8 - Construction and expression of Hirudin RGDW

Hirudin-RGDW is a hirudin derivative in which the amino acid integrin binding sequence Arg-Gly-Asp-Trp has been attached to the C-terminus of native hirudin. The procedure of Example 5 was used except that the oligonucleotide primer comprised a 42bp 5'CGGGGATCCCTATTACCAGTCACCGCGCTGCAGATATTCTTC 3' (SEQ.ID:14).

EXAMPLE 9 - Mass Spectroscopic Analysis of Hirudin Variants

The C-terminus of recombinant hirudin can be susceptible to proteolysis by carboxypeptidases during production from yeast (R. Bischoff et al, J. Chromatography 476: 245-55, 1989) . As the presence of the C-terminal residues of the hirudin-RGDX variants is important for their activity, we have analysed the purified hirudin variants by mass spectroscopy to determine whether they are of the predicted molecular weight.

Purified samples were analysed by fast atom bombardment mass spectrometry by M-Scan Ltd, Silwood Park, Sunninghill, Ascot, UK. Samples were dissolved in 5% aqueous acetic acid and 2 μl was loaded onto a target previously smeared with 2-4 μl M-nitro benzyl alcohol. Analysis was carried out on a VG Analytical ZAB-2SE high field mass spectrometer operating at Vacc-8kV. A caesium ion gun was used to generate ions for the mass spectra which were recorded using a PDP 11-250J data system. Mass calibration was performed using caesium iodide or caesium iodide glycerol. Spectra were acquired firstly over the mass range m/z 7800-3400 then re-analysed from m/z 8350-7300.

Hirudin-RGDF was found to have a mass of 7439.3 for the protonated molecular ion compared to the calculated molecular weight of 7439. For hirudin-RGDW the centroid of the molecular ion had a mass of 7476.6-7478.5 compared to the calculated value of 7478. The observed and calculated molecular weights of the hirudin variants are essentially the same indicating that for each variant the C-terminus of the molecule is intact. The addition of an RGDX sequence at the C-terminus of hirudin therefore prevents C-terminal proteolysis. EXAMPLES 10 to 25 - Construction and expression of Hirudin-RGDX

Hirudin-RGDX's are hirudin derivatives in which the amino acid integrin binding sequence RGDX has been attached to the C-terminus of recombinant desulphato-hirudin (X can be any amino acid) . Hirudin variants possessing C-terminal extensions of RGDS (Example 5) , RGDF (Example 6) , RGDV (Example 7) and RGDW (Example 8) have already been described (SEQ.ID NOS:25-28) . Hirudin variants possessing the remaining possible C-terminal amino acids were constructed in order to investigate the effect on the duration of action of antithrombotic activity. Table 1 gives the relationship between the Example number and the integrin affinity sequences (including RGDX sequences) employed.

The hirudin-RGDX peptides were constructed and cloned into expression vectors, according to the procedure of Example 5 except that the mutagenesis strategy was complicated by the number of clones sought. The bacteriophage M13 clone containing the hirudin-RGDW DNA sequence of Example 8 was used for the mutagenesis template. A first round of mutagenesis with an oligonucleotide primer BB4395 30bp (5' GGATCCCTATTANNNGTCACCGCGCTGCAG 3') (SEQ.ID NO:49), where N represents an equimolar mixture of the four nucleotides, A, T, G and C, was used to construct four new hirudin variants in which the amino acid sequences Arg-Gly-Asp-Arg (RGDR) Arg-Gly-Asp-Glu (RGDE) and Arg-Gly-Asp-Cys (RGDC) (SEQ. ID:NOS.29-31) have been added to the C-terminus of hirudin HV-l.

Examination of the codon usage of the remaining desired amino acid substitutions at the X position allowed the design of two oligonucleotide primers BB4882 28bp (5' GGATCCCTATTAC(H:C/A/T) (B:T/C/G) GTCACCGCGCTGC 3' ) (SEQ. ID NO.-50) and BB4883 28bp (5' GGATCCCTATTAA (D:G/A/T)NGTCACCGCGCTGC 3'), where N is any one of the four nucleotides A, T, G or C (SEQ. ID NO:51) . Hirudin variants in which the amino acid sequences Arg-Gly-Asp-Lys (RGDK) , Arg-Gly-Asp-Leu (RGDL) , Arg-Gly-Asp-Gly (RGDG) , Arg-Gly-Asp-Met (RGDM) and Arg-Gly-Asp-Gln (RGDQ) (SEQ. ID NOS:32-36) had been added to the C-terminus of hirudin HV-l were identified after mutagenesis with BB4882.

Hirudin variants in which the amino acid sequences Arg-Gly-Asp-Thr (RGDT) , Arg-Gly-Asp-Asp (RGDD) , Arg-Gly-Asp-Asn (RGDN) , Arg-Gly-Asp-Tyr (RGDY) , Arg-Gly-Asp-Pro (RGDP) and Arg-Gly-Asp-Ile (RGDI) (SEQ.ID NOS:37-42) had been added to the C-terminus of hirudin HV-l were identified after mutagenesis with BB4883.

Two specific mutagenic oligonucleotide primers BB5258 28bp (5' GGATCCCTATTAAGCGTCACCGCGCTGC 3') (SEQ.ID NO:52) and BB5259 28bp (5' GGATCCCTATTAGTGGTC- ACCGCGCTGC 3') (SEQ.ID NO:53) were designed and used to construct hirudin variants in which Arg-Gly-Asp-Ala (RGDA) (SEQ.ID NO: 43) and Arg-Gly-Asp-His (RGDH) (SEQ.ID NO:44) respectively, have been added to the C-terminus of hirudin HV-l.

The genes encoding the hirudin RGDX variants described above were transferred into expression vectors according to the procedure of Example 5.

EXAMPLE 26 - Construction and Expression of Hirudin-GREDV

Hirudin-GREDV is a hirudin derivative in which the amino acid sequence Gly-Arg-Glu-Asp-Val (SEQ.ID NO:45) has been attached to the C-terminus of native hirudin HV-l. The procedure of Example 5 was used except that the oligonucleotide primer comprised a 42bp BB6282 (5'GGATCCCTATTAAACGTCTTCGCGACCCTGCAGATATTCTTC 3') (SEQ.ID NO:54) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template. EXAMPLE 27 - Construction and Expression of Hirudin-HHLGGAKQAGDV

Hirudin-HHLGGAKQAGDV is a hirudin derivative in which the amino acid sequence His-His-Leu-Gly-Gly-Ala-Lys- Gln-Ala-Gly-Asp-Val (SEQ.ID NO:7) has been attached to the C-terminus of native hirudin HV-l. The procedure of Example 5 was used except that the oligonucleotide primer comprised a 63bp BB6283 ( 5 ' GGATCCCTATTAAACATCACCTGCCTGTTTTGCACCACCCAGGTGGTGCTGCA- GATATTCTTC 3') (SEQ.ID NO:55) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template.

EXAMPLE 28 - Construction and Expression of Hirudin-CRGDWPC

Hirudin-CRGDWPC is a hirudin derivative in which the amino acid sequence Cys-Arg-Gly-Asp-Trp-Pro-Cys (SEQ.ID NO:24) has been attached to the C-terminus of native hirudin HV-l. The procedure of Example 5 was used except that the oligonucleotide primer comprised a 48bp BB6284 (5' GGATCCCTATTAACATGGCCAGTCACCGCGACACTGCA- GATATTCTTC 3') (SEQ.ID NO:56) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template.

EXAMPLE 29 - Construction and Expression of Hirudin-CKGDWPC

Hirudin-CKGDWPC is a hirudin derivative in which the amino acid sequence Cys-Lys-Gly-Asp-Trp-Pro-Cys (SEQ.ID NO:23) has been attached to the C-terminus of native hirudin HV-l. The procedure of Example 5 was used except that the oligonucleotide primer comprised a 48bp BB6285 (5' GGATCCCTATTAACATGGCCAGTCACCCTTACACTGCA- GATATTCTTC 3') (SEQ.ID NO:57) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template.

EXAMPLE 30 - Construction and Expression of Hirudin-GKDGEA

Hirudin-GKDGEA is a hirudin derivative in which the amino acid sequence Gly-Lys-Asp-Gly-Glu-Ala (SEQ.ID NO:46) has been attached to the C-terminus of native hirudin HV-l. The procedure of Example 5 was used except that the oligonucleotide primer comprised a 45bp BB6281 (5' GAATCCCTATTATGCTTCACCGTCTTTACCCTGCA- GATATTCTTC 3') (SEQ. ID NO: 58) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template. BB6281 contained an undesirable base substitution which was corrected by a further round of mutagenesis with BB6420 22bp (5' GTACCCGGGGATCCCTATTATG 3 ' ) ( SEQ . ID NO : 62 ) .

EXAMPLE 31 - Construction and Expression of IEGR-Hirudin-RGDW

IEGR-Hirudin-RGDW is a hirudin derivative in which the amino acid sequence Ile-Glu-Gly-Arg has been attached to the N-terminus of hirudin-RGDS of Example 5. The procedure of Example 5 was used except that the oligonucleotide primer comprised a 42bp BB4404 (5' GTCGGTGTAAACAACTCTTCCTTCGATTCTTTTATCCAAGCT 3 ' ) (SEQ. ID NO: 59 ) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template .

EXAMPLE 32 - Construction and Expression of Hirudin [II, T2]-RGDW

Hirudin [II, T2]-RGDW is a hirudin derivative in which the N-terminus of hirudin-RGDW of Example 8 (Val-Val) was replaced by the amino acid sequence Ile-Thr. The procedure of Example 5 was used except that the oligonucleotide primer comprised a 34bp BB4401 (5' GTACAGTCGGTGTAGGTGATTCTTTTATCCAAGC 3') (SEQ.ID NO:60) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template.

EXAMPLE 33 - Construction and Expression of Hirudin [K24]-RGDW

Hirudin [K24] -RGDW is a hirudin HV-l derivative in which amino acid Gln24 of hirudin-RGDW of Example 8, has been substituted with Lys. The procedure of Example 5 was used except that the oligonucleotide primer comprised a 29bp BB4402 ( 5 ' GCATTTGTTACCTTTACCACAGACGTTAG 3') (SEQ.ID NO:61) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template. EXAMPLE 34 - Construction and Expression of Hirudin [II, T2, K24]-RGDW

Hirudin [II, T2, K24]-RGDW is a hirudin derivative in which the N-terminus of hirudin-RGDW of Example 8, (Val-Val) , was replaced by the amino acid sequence Ile-Thr and Gln24 of hirudin-RGDW of Example 8, has been substituted with Lys. The procedure of Example 5 was used except that the oligonucleotide primer comprised a 29bp BB4402 (SEQ.ID NO:61) as used in Example 33 and the bacteriophage M13 clone encoding the hirudin [II, T2]-RGDW sequence of Example 32 was used as template.

COMPARATIVE PHARMACOLOGY EXAMPLE A - In vitro inhibition of Thrombin

The ability of hirudin and variants to inhibit thrombin catalysed hydrolysis of the chromogenic substrate tosyl-Gly-Pro-Arg-p-nitroanilide (CHROMOZYM (Trade Mark) TH, Boehringer-Mannheim) was determined. Hirudin samples (50 μl) diluted in 0.1M Tris HCl pH8.5, 0.15M NaCl, 0.1% PEG 6000 were mixed with 50 μl human thrombin (Sigma, 0.8U/ml in the above buffer) and 50 μl CHROMOZYM TH (2.5mM in water) in 96 well plates. The plates were incubated at room temperature for 30 minutes. The reaction was terminated by adding 50 μl 0.5M acetic acid and the absorbance read at 405nm using an automatic plate reader. Quantitation was performed by comparison with a standard hirudin preparation (recombinant [Lys-47]-HV-2 purchased from Sigma: Sigma Chemical Co. Ltd, Fancy Road, Poole, Dorset BH11 7TG, United Kingdom) .

The specific activity of hirudin and the four hirudin-RGD variants is shown in Table 2. The specific activities of hirudin-RGDW, hirudin-RGDS, hirudin-RGDF and hirudin-RGDV determined in this assay were found to be not significantly different from that of unmodified hirudin indicating that the addition of an integrin binding sequence at the C-terminus of hirudin does not impair its activity as a thrombin inhibitor.

TABLE 2

Protein Specific Activity fU/ g)

Hirudin 7693

Hirudin-RGDW 7858

Hirudin-RGDF 7667

Hirudin-RGDV 7275

Hirudin-RGDS 7547

PHARMACOLOGY EXAMPLE B - In Vivo Testing Of

Antithrombotic Activity for Hirudin-RGDS

The antithrombotic activity of the hirudin-RGDS produced using the procedure of Example 5 was measured by determining the time to clot formation using a rat arterio-venous shunt (as described in Markwardt et al Thromb . Haemostasis 47: 226-229 (1982)). Male Sprague-Dawley rats (250 - 400g) were anaesthetised with urethane at a concentration of 1.6 g/kg. Through a mid-line incision in the neck, the trachea was cannulated and the animals breathed spontaneously air enriched with oxygen to maintain arterial blood p0₂ between 100 and 150 mmHg. A carotid artery was cannulated for measurement of blood pressure and heart rate. A jugular vein was cannulated for the infusion of supplementary anaesthetic and the antithrombotic protein to be tested, in this case the hirudin-RGDS protein of Example 5.

A shunt, consisting of two 12.5 cm nylon cannulae, connected by a 2 cm long glass tube 1 mm in diameter, was connected between a carotid artery and a contralateral jugular vein. An artery clip placed on the carotid artery proximal to the shunt prevented blood flow through the shunt until required. The shunt was primed with 0.9% saline. A thermistor bead to measure temperature was placed on the surface of the jugular vein nylon catheter, adjacent to the glass section of the shunt. The output of the thermistor was recorded and an output fall indicated that blood flow had ceased and a clot had formed. The time to clot formation was measured from the time to a fall in temperature had occurred after opening of the shunt by removal of the artery clip.

This time was measured repeatedly in the rats by repeated cannulation of the carotid artery and jugular vein in the same animal. A new shunt was used for each determination of time to clot formation. The time to clot formation was assessed at 30, 60 and 120 minutes following iv adminstration of 10,000 ATU/kg hirudin-RGDS. The values were 8.3+1.9, 6.9+1.1 and 8.5+0.8 minutes, respectively. The value at 120 minutes was significantly different and considerably higher than in the corresponding Comparative Pharmacology Example F indicating an increase in anticoagulant effect and prolonged duration of action up to 120 minutes after administration. The results of in vivo tests including those with hirudin-RGDV and hirudin-RGDF (Pharmacology Examples C and D) , are summarised in Table 3.

PHARMACOLOGY EXAMPLE C - In Vivo Testing of Antithrombotic Activity for Hirudin - RGDF

The procedure of Pharmacology Example B was repeated for the protein hirudin - RGDF, prepared according to the procedure of Example 6. The time to clot formation at 120 minutes following intravenous administration of 10,000 ATU/kg was 5.8+1.4 minutes.

PHARMACOLOGY EXAMPLE D - In Vivo Testing of Antithrombotic Activity for Hirudin - RGDV

The procedure of Pharmacology Example B was repeated for the protein hirudin - RGDV, prepared according to the procedure of Example 7. The time to clot formation at 120 minutes following intravenous administration of 10,000 ATU/kg was 8.1+2.5 minutes. COMPARATIVE PHARMACOLOGY EXAMPLE E - Saline Placebo

The method of in vivo testing previously described in Pharmacology Example B was carried out using vehicle administration only as a (saline) control. The time to clot formation at 30, 60 and 120 minutes following vehicle administration was 4.2+0.6, 3.7+0.5 and 3.3+1.4 minutes respectively.

COMPARATIVE PHARMACOLOGY EXAMPLE F - Hirudin (Applicants')

Comparative Pharmacology Example B was carried out except using hirudin variant HV-l produced using the procedure of Comparative Examples 1 to 4. This was administered at a dose of 10,000 ATU/kg which resulted in significant prolongation of time to clot formation at 30 minutes after administration of the hirudin to 10.9+1.4 minutes and at 60 minutes to 9.0+1.7 minutes. No significant prolongation was observed at 120 miuntes to 4.9+1.3 minutes after hirudin administration when compared with the control in Comparative Pharmacology Example E.

TABLE 3

Time to clot formation (+ minutes)

Time after test protein administration (minutes)

30 60 120

Pharmacology Hirudin-

Example B RGDS 8.3+1.9 6.9+1.1 8.6+0.8

Pharmacology Hirudin-

Example C RGDF 5.8+1.4

Pharmacology Hirudin-

Example D RGDV 8.1+2.5

Comparative Pharmacology

Example E Saline 4.2+0.6 3.7+0.5 3.3+1.4

Comparative

Pharmacology Hirudin

Example F (Applicants') 10.9+1.4 9.0+1.7 4.9+1.3

SEQUENCE LISTINGS

SEQ.ID: 1

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 69 amino acids

SOURCE: experimental

PROPERTIES: antithrombotic

FEATURES: hirudin type HV-l with carboxy terminus - RGDS (66-69)

Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Leu Cys Leu

5 10 15

Cys Glu Gly Ser Asn Val Cys Gly Gin Gly Asn Lys Cys lie Leu

20 25 30

Gly Ser Asp Gly Glu Lys Asn Gin Cys Val Thr Gly Glu Gly Thy

35 40 45

Pro Lys Pro Gin Ser His Asn Asp Gly Asp Phe Glu Glu lie Pro

50 55 60

Glu Glu Tys Leu Gin Arg Gly Asp Ser

65 SEQ.ID: 2

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 69 amino acids

SOURCE: hypothetical

PROPERTIES: antithrombotic

FEATURES: hirudin type HV-2 with carboxy terminus - RGDS (66-69)

lie Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Leu Cys Leu

5 10 15

Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys lie Leu

20 25 30

Gly Ser Asn Gly Lys Gly Asn Gin Cys Val Thr Gly Glu Gly Thr

35 40 45

Pro Asn Pro Glu Ser His Asn Asn Gly Asp Phe Glu Glu lie Pro

50 55 60

Glu Glu Tyr Leu Gin Arg Gly Asp Ser

65

SEQ.ID: 3

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 70 amino acids

SOURCE: hypothetical

PROPERTIES: antithrombotic

FEATURES: hirudin type HV-3 with carboxy terminus -RGDS (67-70)

lie The Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Leu Cys Leu

5 10 15

Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys lie Leu

20 25 30

Gly Ser Gin Gly Lys Asp Asn Gin Cys Val Thr Gly Glu Gly Thr

35 40 45

Pro Lys Pro Gin Ser His Asn Gin Gly Asp Phe Glu Pro lie Pro

50 55 60

Glu Asp Ala Tyr Asp Glu Arg Gly Asp Ser

65 70

SEQ.ID: 4

SEQUENCE TYPE: nucleotide with corresponding protein

SEQUENCE LENGTH: 201 base pairs

STRANDEDNESS: double

TOPOLOGY: linear

MOLECULE TYPE: synthetic DNA

SOURCE: synthetic

FEATURES: hirudin type HV-l gene from 195 to 201 bp non-translated stop codons

GTT GTT TAC ACC GAC TGT ACT GAA TCC GGA CAA AAC CTG TGT TTG 45 CAA CAA ATG TGG CTG ACA TGA CTT AGG CCT GTT TTG GAC ACA AAC Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Leu Cys Leu

5 10 15

TGT GAG GGT TCT AAC GTC TGT GGT CAG GGT AAC AAA TGC ATC CTG 90 ACA CTC CCA AGA TTG CAG ACA CCA GTC CCA TTG TTT ACG TAG GAC Cys Glu Gly Ser Asn Val Cys Gly Gin Gly Asn Lys Cys lie Leu

20 25 30

GGT TCC GAC GGT GAA AAG AAC CAA TGT GTC ACT GGT GAA GGT ACC 135 CCA AGG CTG CCA CTT TTC TTG GTT ACA CAG TGA CCA CTT CCA TGG Gly Ser Asp Gly Glu Lys Asn Gin Cys Val Thr Gly Glu Gly Thr

35 40 45

CCA AAG CCG CAG TCC CAC AAC GAT GGA GAT TTC GAA GAA ATC CCA 180 GGT TTC GGC GTC AGG GTG TTG CTA CCT CTA AAG CTT CTT TAG GGT Pro Lys Pro Gin Ser His Asn Asp Gly Asp Phe Glu Glu lie Pro

50 55 60

GAA GAA TAT CTG CAG TAATAG 201 CTT CTT ATA GAC GTC ATTATC Glu Glu Tyr Leu Gin

65 SEQ.ID:

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 223 base pairs STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA SOURCE: synthetic FEATURES: hirudin type HV-l gene with 5 amino acid adaptor (corresponding to C- terminus of factor) at amino terminus; from 1 to 6 bp (AAGCTT) is Hindlll site from 118 to 123 bp (GGATCC) is BamI site.

AAGCTTGGAT AAAAGAGTTG TTTACACCGA CTGTACTGAA TCCGGACAAA 50

ACCTGTGTTT GTGTGAGGGT TCTAACGTCT GTGGTCAGGG TAACAAATGC 100

ATCCTGGGTT CCGACGGTGA AAAGAACCAA TGTGTCACTG GTGAAGGTAC 150

CCCAAAGCCG CAGTCCCACA ACGATGGAGA TTTCGAAGAA ATCCCAGAAG 200

AATATCTGCA GTAATAGGGA TCC 223 SEQ.ID: 6

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 223 base pairs

STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: synthetic DNA SOURCE: synthetic FEATURES: oligomers designed for construction of synthetic type HV-l gene.

BB2011 BB2013

AGCTTACCTG CCATGGTTGT TTACACCGAC TGTACTGAAT C CGGACAAAA 50 I I I M I I I I I I I I

ATGGAC GGTACCAACA AATGTGGCTG ACATGACTTA GGCCTGTT TT BB2012

BB2015 CCTGTGTTTG TGTGAGGGTT CTAACGTC TG TGGTCAGGGT AACAAATGCA 100 Mill

GGACACAAAC ACACTCCCAA GATTGCAGACACC AG TCCCA TTGTTTACGT

BB2014

BB2017 TCCTGGGTTC CGACGGTG AA AAGAACCAAT GTGTCACTGG TGAAGGTACC 150

AGGACCCAAG GCTGCCACTTTTCT T GGTTA CACAGTGACC ACTTCCATGG BB2016 BB2018 BB2019 CCA AAGCCGC AGTCCCACAA CGATGGAGAT TTCGAAGAAA TC CCAGAAGA

III II I

GGTTTCGGCG TCAGGGTGTT GCTACCTCTA AAGCTTCTTT AGGGTCTTC T

BB2020

BB2021 ATATCTGCAG TAATAGGGAT CCG III

TATAGACGTC ATTATCCCTA GGCTTAA BB2022

SEQ.ID: 7

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 12 amino acids

FEATURES: residues 400-411 of gamma chain of fibrinogen PROPERTIES: platelet binding site (HHLGGAKQAGDV)

His His Leu Gly Gly Ala Lys Gin Ala Gly Asp Val

5 10

SEQ.ID: 8

SEQUENCE TYPE: protein SEQUENCE LENGTH: 6 amino acids PROPERTIES: integrin affinity sequence in fibronectin

Gly Arg Gly Asp Ser Pro

5

SEQ.ID: 9

SEQUENCE TYPE: protein SEQUENCE LENGTH: 5 amino acids

PROPERTIES: integrin affinity sequence in kappa chain of casein

Lys Asn Gin Asp Lys

5

SEQ.ID: 10

SEQUENCE TYPE: protein SEQUENCE LENGTH: 6 amino acids

His Glu Arg Gly Asp Val

5 SEQ.ID: 11

SEQUENCE TYPE: nucleotide

SEQUENCE LENGTH: 19 base pairs

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: synthetic DNA

FEATURES: primer complementary to region of pUC19

CAGGGTTTTC CCAGTCACG 19

SEQ.ID: 12

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 15 base pairs

STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA FEATURES: encodes 5 amino acids from carboxy terminus of alpha factor

AGCTTGGATA AAAGA 15

SEQ.ID: 13

SEQUENCE TYPE: nucleotide

SEQUENCE LENGTH: 11 base pairs

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: synthetic DNA

FEATURES: complementary to part of SEQ.ID: 12

TCTTTTATCC A 11

SEQ.ID: 14

SEQUENCE TYPE: nucleotide

SEQUENCE LENGTH: 42 base pairs

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: synthetic DNA

PROPERTIES: mutagenesis primer used for hirudin-RGDW

CGGGGATCCC TATTACCAGT CACCGCGCTG CAGATATTCT TC 42

SEQ.ID: 15

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 42 base pairs STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA PROPERTIES: mutagenesis primer used for hirudin-RGDF

CGGGGATCCC TATTAGAAGT CACCGCGCTG CAGATATTCT TC 42

SEQ.ID: 16

SEQUENCE TYPE: nucleotide

SEQUENCE LENGTH: 42 base pairs

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: synthetic DNA

PROPERTIES: mutagenesis primer used for hirudin-RGDV

CGGGGATCCC TATTAAACGT CACCGCGCTG CAGATATTCT TC 42 SEQ.ID: 17

SEQUENCE TYPE: nucleotide

SEQUENCE LENGTH: 42 base pairs

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: synthetic DNA

PROPERTIES: mutagenesis primer used for hirudin-RGDS

CGGGGATCCC TATTAGCTGT CACCGCGCTG CAGATATTCT TC 42

SEQ.ID: 18

SEQUENCE TYPE: protein SEQUENCE LENGTH: 6 amino acids

Lys Gin Ala Gly Asp Val

5

SEQ.ID: 19

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 7984 base pairs STRANDEDNESS: single TOPOLOGY: circular SOURCE: experimental

1 TTCCCATGTC TCTACTGGTG GTGGTGCTTC TTTGGAATTA TTGGAAGGTA 5

51 AGGAATTGCC AGGTGTTGCT TTCTTATCCG AAAAGAAATA AATTGAATTG 1

101 AATTGAAATC GATAGATCAA TTTTTTTCTT TTCTCTTTCC CCATCCTTTA 1

151 CGCTAAAATA ATAGTTTATT TTATTTTTTG AATATTTTTT ATTTATATAC 2

201 GTATATATAG ACTATTATTT ACTTTTAATA GATTATTAAG ATTTTTATTA 2

251 AAAAAAAATT CGTCCCTCTT TTTAATGCCT TTTATGCAGT TTTTTTTTCC 3

301 CATTCGATAT TTCTATGTTC GGGTTTCAGC GTATTTTAAG TTTAATAACT 35

351 CGAAAATTCT GCGTTTCGAA AAAGCTCTGC ATTAATGAAT CGGCCAACGC 40

401 GCGGGGAGAG GCGGTTTGCG TATTGGGCGC TCTTCCGCTT CCTCGCTCAC 45

451 TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT 50

501 CAAAGGCGGT AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG 55

551 AACATGTGAG CAAAAGGCCA GCAAAAGGCC AGGAACCGTA AAAAGGCCGC 60

601 GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA 65

651 ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC 70

701 CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT 75

751 GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC 80

801 TTTCTCATAG CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC 85

851 TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC 90

901 CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT 95

951 CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA 10

1001 GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG 10

1051 AAGGACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA 11

1101 AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT 11

1151 GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA 12

1201 AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA 12 1251 ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC 130

1301 TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA 135

1351 TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA 14

1401 TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC 14

1451 GTGTAGATAA CTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC 15

1501 AATGATACCG CGAGACCCAC GCTCACCGGC TCCAGATTTA TCAGCAATAA 155

1551 ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC AACTTTATCC 16

1601 GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC 16

1651 GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTACA GGCATCGTGG 17

1701 TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA 17

1751 TCAAGGCGAG TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC 18

1801 CTTCGGTCCT CCGATCGTTG TCAGAAGTAA GTTGGCCGCA GTGTTATCAC 18

1851 TCATGGTTAT GGCAGCACTG CATAATTCTC TTACTGTCAT GCCATCCGTA 19

1901 AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA 19

1951 GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAACA CGGGATAATA 20

2001 CCGCGCCACA TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT 20

2051 TCGGGGCGAA AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT 21

2101 GTAACCCACT CGTGCACCCA ACTGATCTTC AGCATCTTTT ACTTTCACCA 21

2151 GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AAAATGCCGC AAAAAAGGGA 22

2201 ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA 22

2251 TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG 23

2301 AATGTATTTA GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA 23

2351 AAAGTGCCAC CTGACGTCTA AGAAACCATT ATTATCATGA CATTAACCTA 24

2401 TAAAAATAGG CGTATCACGA GGCCCTTTCG TCTTCAAGAA TTCTGAACCA 24

2451 GTCCTAAAAC GAGTAAATAG GACCGGCAAT TCTTCAAGCA ATAAACAGGA 25

2501 ATACCAATTA TTAAAAGATA ACTTAGTCAG ATCGTACAAT AAagctAGCT 25

2551 TTGAAGAAAA ATGCGCCTTA TTCAATCTTT GCTATAAAAA ATGGCCCAAA 26

2601 ATCTCACATT GGAAGACATT TGATGACCTC ATTTCTTTCA ATGAAGGGCC 26

2651 TAACGGAGTT GACTAATGTT GTGGGAAATT GGAGCGATAA GCGTGCTTCT 27

2701 GCCGTGGCCA GGACAACGTA TACTCATCAG ATAACAGCAA TACCTGATCA 27

2751 CTACTTCGCA CTAGTTTCTC GGTACTATGC ATATGATCCA ATATCAAAGG 28

2801 AAATGATAGC ATTGAAGGAT GAGACTAATC CAATTGAGGA GTGGCAGCAT 28

2851 ATAGAACAGC TAAAGGGTAG TGCTGAAGGA AGCATACGAT ACCCCGCATG 29

2901 GAATGGGATA ATATCACAGG AGGTACTAGA CTACCTTTCA TCCTACATAA 29 2951 ATAGACGCAT ATAAGTACGC ATTTAAGCAT AAACACGCAC TATGCCGTTC

3001 TTCTCATGTA TATATATATA CAGGCAACAC GCAGATATAG GTGCGACGTG

3051 AACAGTGAGC TGTATGTGCG CAGCTCGCGT TGCATTTTCG GAAGCGCTCG

3101 TTTTCGGAAA CGCTTTGAAG TTCCTATTCC GAAGTTCCTA TTCTCTAGAA

3151 AGTATAGGAA CTTCAGAGCG CTTTTGAAAA CCAAAAGCGC TCTGAAGACG

3201 CACTTTCAAA AAACCAAAAA CGCACCGGAC TGTAACGAGC TACTAAAATA

3251 TTGCGAATAC CGCTTCCACA AACATTGCTC AAAAGTATCT CTTTGCTATA

3301 TATCTCTGTG CTATATCCCT ATATAACCTA CCCATCCACC TTTCGCTCCT

3351 TGAACTTGCA TCTAAACTCG ACCTCTACAT TTTTTATGTT TATCTCTAGT

3401 ATTACTCTTT AGACAAAAAA ATTGTAGTAA GAACTATTCA TAGAGTGAAT

3451 CGAAAACAAT ACGAAAATGT AAACATTTCC TATACGTAGT ATATAGAGAC

3501 AAAATAGAAG AAACCGTTCA TAATTTTCTG ACCAATGAAG AATCATCAAC

3551 GCTATCACTT TCTGTTCACA AAGTATGCGC AATCCACATC GGTATAGAAT

3601 ATAATCGGGG ATGCCTTTAT CTTGAAAAAA TGCACCCGCA GCTTCGCTAG

3651 TAATCAGTAA ACGCGGGAAG TGGAGTCAGG CTTTTTTTAT GGAAGAGAAA

3701 ATAGACACCA AAGTAGCCTT CTTCTAACCT TAACGGACCT ACAGTGCAAA

3751 AAGTTATCAA GAGACTGCAT TATAGAGCGC ACAAAGGAGA AAAAAAGTAA

3801 TCTAAGATGC TTTGTTAGAA AAATAGCGCT CTCGGGATGC ATTTTTGTAG

3851 AACAAAAAAG AAGTATAGAT TCTTTGTTGG TAAAATAGCG CTCTCGCGTT

3901 GCATTTCTGT TCTGTAAAAA TGCAGCTCAG ATTCTTTGTT TGAAAAATTA

3951 GCGCTCTCGC GTTGCATTTT TGTTTTACAA AAATGAAGCA CAGATTCTTC

4001 GTTGGTAAAA TAGCGCTTTC GCGTTGCATT TCTGTTCTGT AAAAATGCAG

4051 CTCAGATTCT TTGTTTGAAA AATTAGCGCT CTCGCGTTGC ATTTTTGTTC

4101 TACAAAATGA AGCACAGATG CTTCGTTAAC AAAGATATGC TATTGAAGTG

4151 CAAGATGGAA ACGCAGAAAA TGAACCGGGG ATGCGACGTG CAAGATTACC

4201 TATGCAATAG ATGCAATAGT TTCTCCAGGA ACCGAAATAC ATACATTGTC

4251 TTCCGTAAAG CGCTAGACTA TATATTATTA TACAGGTTCA AATATACTAT

4301 CTGTTTCAGG GAAAACTCCC AGGTTCGGAT GTTCAAAATT CAATGATGGG

4351 TAACAAGTAC GATCGTAAAT CTGTAAAACA GTTTGTCGGA TATTAGGCTG

4401 TATCTCCTCA AAGCGTATTC GAATATCATT GAGAAGCTGC ATTTTTTTTT

4451 TTTTTTATAT ATATTTCAAG GATATACCAT TGTAATGCCT GCCCCTAAGA

4501 AGATCGTCGT TTTGCCAGGT GACCACGTTG GTCAAGAAAT CACAGCCGAA

4551 GCCATTAAGG TTCTTAAAGC TATTTCTGAT GTTCGTTCCA ATGTCAAGTT

4601 CGATTTCGAA AATCATTTAA TTGGTGGTGC TGCTATCGAT GCTACAGGTG

6351 AAAACTGTTT GCATTCAAAA ATAGGTAGCA TACAATTAAA ACATGGCGGG 6401 CATGTATCAT TGCCCTTATC TTGTGCAGTT AGACGCGAAT TTTTCGAAGA 6451 AGTACCTTCA AAGAATGGGG TCTTATCTTG TTTTGCAAGT ACCACTGAGC 6501 AGGATAATAA TAGAAATGAT AATATACTAT AGTAGAGATA ACGTCGATGA 6551 CTTCCCATAC TGTAATTGCT TTTAGTTGTG TATTTTTAGT GTGCAAGTTT 6601 CTGTAAATCG ATTAATTTTT TTTTCTTTCC TCTTTTTATT AACCTTAATT 6651 TTTATTTTAG ATTCCTGACT TCAACTCAAG ACGCACAGAT ATTATAACAT 6701 CTGCATAATA GGCATTTGCA AGAATTACTC GTGAGTAAGG AAAGAGTGAG 6751 GAACTATCGC ATACCTGCAT TTAAAGATGC CGATTTGGGC GCGAATCCTT 6801 TATTTTGGCT TCACCCTCAT ACTATTATCA GGGCCAGAAA AAGGAAGTGT 6851 TTCCCTCCTT CTTGAATTGA TGTTACCCTC ATAAAGCACG TGGCCTCTTA 6901 TCGAGAAAGA AATTACCGTC GCTCGTGATT TGTTTGCAAA AAGAACAAAA 6951 CTGAAAAAAC CCCGGATCTT TTGAATTCCC ACGGATTAGA AGCCGCCGAG 7001 CGGGTGACAG CCCTCCGAAG GAAGACTCTC CTCCGTGCGT CCTCGTCTTC 7051 ACCGGTCGCG TTCCTGAAAC GCAGATGTGC CTCGCGCCGC ACTGCTCCGA 7101 ACAATAAAGA TTCTACAATA CTAGGGGGAT CGGTCGTCAC ACAACAAGGT 7151 CCTAGCGACG GCTCACAGGT TTTGTAACAA GCAATCGAAG GTTCTGGAAT

7201 GGCGGGGAAA GGGTTTAGTA CCACATGCTA TGATGCCCAC TGTGATCTCC 7251 AGAGCAAAGT TCGTTCGATC GTACTGTACT CTCTCTCTTT CAAACAGAAT

7301 TGTCCGAATC GTGTGACAAC AACAGCCTGT TCTCACACAC TCTTTTCTTC 7351 TAACCAAGGG GGTGGTTTAG TTTAGTAGAA CCTCGTGAAA CTTACATTTA 7401 CATATATATA AACTTGCATA AATTGGTCAA TGGAAGAAAT ACATATTTGG

7451 TCTTTTCTAA TTCGTAGTTT TTCAAGTTCT TAGATGCTTT CTTTTTCTCT 7501 TTTTTACAGA TCATCAAGGA AGTAATTATC TACTTTTTAC AACAAATACA 7551 AAAGATCTAT GAGATTTCCT TCAATTTTTA CTGCAGTTTT ATTCGCAGCA 7601 TCCTCCGCAT TAGCTGCTCC AGTCAACACT ACAACAGAAG ATGAAACGGC 7651 ACAAATTCCG GCTGAAGCTG TCATCGGTTA CTTAGATTTA GAAGGGGATT 7701 TCGATGTTGC TGTTTTGCCA TTTTCCAACA GCACAAATAA CGGGTTATTG 7751 TTTATAAATA CTACTATTGC CAGCATTGCT GCTAAAGAAG AAGGGGTAAG 7801 CTTGGATAAA AGAAACAGCG ACTCTGAATG CCCGCTGAGC CATGATGGCT 7851 ACTGCCTGCA CGACGGTGTA TGCATGTATA TCGAAGCTCT GGACAAATAC 7901 GCATGCAACT GCGTAGTTGG TTACATCGGC GAACGTTGCC AGTACCGCGA 7951 CCTGAAATGG TGGGAGCTCC GTTAATAAGG ATCC 7984 SEQ.ID: 20

SEQUENCE TYPE: protein SEQUENCE LENGTH: 4 amino acids PROPERTIES: integrin affinity sequence in collagen (DGEA)

Asp Gly Glu Ala

SEQ.ID: 21

SEQUENCE TYPE: protein SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence in fibronectin (REDV)

Arg Glu Asp Val

SEQ.ID: 22

SEQUENCE TYPE: protein SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence in snake venom barbourin (KGDW)

Lys Gly Asp Trp SEQ.ID: 23

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 7 amino acids

PROPERTIES: integrin affinity sequence selective for GPIIb-IIIa and vitronectin receptor

(CKGDWPC)

Cys Lys Gly Asp Trp Pro Cys

5

SEQ.ID: 24

SEQUENCE TYPE: protein SEQUENCE LENGTH: 7 amino acids

PROPERTIES: integrin affinity sequence selective for GPIIb-IIa .(CRGDWPC)

Cys Arg Gly Asp Trp Pro Cys

5

SEQ.ID: 25

SEQUENCE TYPE: protein SEQUENCE LENGTH: 4 amino acids PROPERTIES: integrin affinity sequence (RGDS)

Arg Gly Asp Trp

SEQ.ID: 26

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDF)

Arg Gly Asp Phe

SEQ.ID: 27

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDV)

Arg Gly Asp Val SEQ.ID: 28

SEQUENCE TYPE: protein SEQUENCE LENGTH: 4 amino acids PROPERTIES: integrin affinity sequence (RGDW)

Arg Gly Asp Trp

SEQ.ID: 29

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDR)

Arg Gly Asp Arg

SEQ.ID: 30

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDE)

Arg Gly Asp Glu SEQ.ID: 31

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDC)

Arg Gly Asp Cys

SEQ.ID: 32

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDK)

Arg Gly Asp Lys

SEQ.ID: 33

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDL)

Arg Gly Asp Leu SEQ.ID: 34

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDG)

Arg Gly Asp Gly

SEQ.ID: 35

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDM)

Arg Gly Asp Met

SEQ.ID: 36

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDQ)

Arg Gly Asp Gin SEQ.ID: 37

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDT)

Arg Gly Asp Thr

SEQ.ID: 38

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDD)

Arg Gly Asp Asp

SEQ.ID: 39

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDN)

Arg Gly Asp Asn SEQ.ID: 40

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDY)

Arg Gly Asp Thr

SEQ.ID: 41

SEQUENCE TYPE: protein SEQUENCE LENGTH: 4 amino acids PROPERTIES: integrin affinity sequence (RGDP)

Arg Gly Asp Pro

SEQ.ID: 42

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDI)

Arg Gly Asp lie SEQ.ID: 43

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDA)

Arg Gly Asp Ala

SEQ.ID: 44

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (RGDH)

Arg Gly Asp His

SEQ.ID: 45

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 5 amino acids

PROPERTIES: integrin affinity sequence (GREDV)

Gly Arg Glu Asp Val

5 SEQ.ID: 46

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 6 amino acids

PROPERTIES: integrin affinity sequence (GKDGEA)

Gly Lys Asp Gly Glu Ala

5

SEQ.ID: 47

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (SGDR)

Ser Gly Asp Arg

SEQ.ID: 48

SEQUENCE TYPE: protein

SEQUENCE LENGTH: 4 amino acids

PROPERTIES: integrin affinity sequence (HGDF)

His Gly Asp Phe SEQ.ID: 49

SEQUENCE TYPE: nucleotide

SEQUENCE LENGTH: 30 base pairs

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: synthetic DNA : BB4395

PROPERTIES: mutagenesis primer used for hirudin-RGDR/E/C

GGATCCCTAT TANNNGTCAC CGCGCTGCAG 30

SEQ.ID: 50

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 28 base pairs

STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA : BB4882 PROPERTIES: mutagenesis primer

H=C, A or T

B=T, C or G

GGATCCCTAT TACHBGTCAC CGCGCTGC 28 SEQ.ID: 51

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 28 base pairs

STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA : BB4883 PROPERTIES: mutagenesis primer

N = G, T, A or C

D = G, A or T

GGATCCCTAT TAADNGTCAC CGCGCTGC 28

SEQ.ID: 52

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 28 base pairs STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA : BB5258 PROPERTIES: mutagenesis primer used for hirudin-RGDA GGATCCCTAT TAAGCGTCAC CGCGCTGC 28

SEQ.ID: 53

SEQUENCE TYPE: nucleotide

SEQUENCE LENGTH: 28 base pairs

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: synthetic DNA : BB5259

PROPERTIES: mutagenesis primer used for hirudin-RGDH

GGATCCCTAT TAGTGGTCAC CGCGCTGC 28

SEQ.ID: 54

SEQUENCE TYPE: nucleotide

SEQUENCE LENGTH: 42 base pairs

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: synthetic DNA : BB6282

PROPERTIES: mutagenesis primer used for hirudin-GREDV GGATCCCTAT TAAACGTCTT CGCGACCCTG CAGATATTCT TC 42

SEQ.ID: 55

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 63 base pairs

STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA : BB6283 PROPERTIES: mutagenesis primer used for hirudin-

HHLGGAKQAGDV

GGATCCCTAT TAAACATCAC CTGCCTGTTT TGCACCACCC AGGTGGTGCT 50 GCAGATATTC TTC 63

SEQ.ID: 56

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 48 base pairs

STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA : BB6284 PROPERTIES: mutagenesis primer used for hirudin-

CRGDWPC

GGATCCCTAT TAACATGGCC AGTCACCGCG ACACTGCAGA TATTCTTC 48 SEQ.ID: 57

SEQUENCE TYPE: nucleotide

SEQUENCE LENGTH: 48 base pairs

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: synthetic DNA : BB6285

PROPERTIES: mutagenesis primer used for hirudin-

CKGDWPC

GGATCCCTAT TAACATGGCC AGTCACCCTT ACACTGCAGA TATTCTTC 48

SEQ.ID: 58

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 45 base pairs

STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA : BB6281 PROPERTIES: mutagenesis primer used for hirudin-

GKDGEA

GAATCCCTAT TATGCTTCAC CGTCTTTACC CTGCAGATAT TCTTC 45 SEQ.ID: 59

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 42 base pairs STRANDEDNESS: single TOPOLOGY: linear

MOLECULE TYPE: synthetic DNA : BB4404

PROPERTIES: mutagenesis primer used for IEGR hirudin- RGDW

GTCGGTGTAA ACAACTCTTC CTTCGATTCT TTTATCCAAG CT 42

SEQ.ID: 60

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 34 base pairs

STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA : BB4401 PROPERTIES: mutagenesis primer used for hirudin [I1,T1]~

RGDW

GTACAGTCGG TGTAGGTGAT TCTTTTATCC AAGC 34 SEQ.ID: 61

SEQUENCE TYPE: nucleotide

SEQUENCE LENGTH: 29 base pairs

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: synthetic DNA : BB4402

PROPERTIES: mutagenesis primer used for hirudin[K24]-

RGDW

GCATTTGTTA CCTTTACCAC AGACGTTAG 29

SEQ.ID: 62

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 22 base pairs STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA : BB6420 PROPERTIES: mutagenesis primer used for hirudin-

GKDGEA

GTACCCGGGG ATCCCTATTA TG 22

Claims

1. A protein comprising a first peptide having pharmacological activity, and a second peptide of no more than fifteen amino acids in length, the second peptide being located at the carboxy terminus of the protein and comprising an integrin affinity sequence, the protein being other than a protein which naturally possesses an integrin affinity sequence at its carboxy terminus.

2. A protein as claimed in claim 1 wherein the pharmacological activity is antithrombotic activity.

3. A protein as claimed in claim 1 or 2 which is capable of inhibiting the conversion of fibrinogen to fibrin by thrombin.

4. A protein as claimed in claim 1 , 2 or 3 wherein the first peptide is hirudin, or a hirudin fragment, or a variant thereof.

5. A protein as claimed in any of claims 1 to 4 wherein the first peptide is hirudin of type HV-l, HV-2 or HV-3.

6. A protein as claimed in any of claims 1 to 5 wherein the integrin affinity sequence comprises the sequence -RGDX where X represents nothing or any amino acid.

7. A protein as claimed in claim 6 wherein X represents S, V, W or F.

8. A protein as claimed in claims 6 or 7 wherein X represents S.

9. Nucleic acid coding for a protein, the protein comprising a first peptide having pharmacological activity, and a second peptide of no more than fifteen amino acids in length, the second peptide being located at the carboxy terminus of the protein and comprising an integrin binding sequence, the protein being other than a protein which naturally possesses an integrin affinity sequence at its carboxy terminus.

10. Nucleic acid as claimed in claim 9 coding for a protein as claimed in any of claims 2 to 8.

11. A vector comprising nucleic acid as claimed in claim 9 or 10.

12. A host transformed with a vector as claimed in claim 11.

13. A host as claimed in claim 12 which is Saccharomyces cerevisiae or Pichia pastoris .

14. A protein comprising a first peptide having pharmacological activity, and a second peptide of no more than fifteen amino acids in length, the second peptide being located at the carboxy terminus of the protein and comprising an integrin affinity sequence, the protein being other than a protein which naturally possesses an integrin affinity sequence at its carboxy terminus; for use in medicine.

15. A protein as claimed in claim 14 which is a protein according to any of claims 2 to 8.

16. A pharmaceutical composition comprising one or more proteins, at least one protein comprising a first peptide having pharmacological activity, and a second peptide of no more than fifteen amino acids in length, the second peptide being located at the carboxy terminus of the protein and comprising an integrin affinity sequence, the protein being other than a protein which naturally possesses an integrin affinity sequence at its carboxy terminus; and a pharmaceutically or veterinarily acceptable carrier.

17. A pharmaceutical composition as claimed in claim 16 wherein the protein is a protein according to any of claims 2 to 8.

18. The use of a protein comprising a first peptide having pharmacological activity, and a second peptide of no more than fifteen amino acids in length, the second peptide being located at the carboxy terminus of the protein and comprising an integrin affinity sequence, the protein being other than a protein which naturally possesses an integrin affinity sequence at its carboxy terminus; in the preparation of a medicament.

19. The use as claimed in claim 18 wherein the medicament is an antithrombotic agent.

20. A method of treatment of a human or animal, the method comprising the administration of an effective non-toxic amount of a protein comprising a first peptide having pharmacological activity, and a second peptide of no more than fifteen amino acids in length, the second peptide being located at the carboxy terminus of the protein and comprising an integrin affinity sequence, the protein being other than a protein which naturally possesses an integrin affinity sequence at its carboxy terminus.

21. A method as claimed in claim 20 for the treatment or prophylaxis of a thrombotic disease or disorder such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis or peripheral arterial occlusion.