MXPA99008525A - Bi- or multifunctional molecules based on a dendroaspin scaffold - Google Patents

Abstract

Dendroaspin, a polypeptide neurotoxin analogue is modified by recombinant DNA techniques, particularly"loop grafting"to provide a modified polypeptide. The modified polypeptide is constructed so as to retain dendroaspin activity e.g. platelet adhesion to fibrinogen in addition to possessing one or more further biological or biochemical activities not native to dendroaspin, e.g. platelet derived growth factor (PDGF) activity or hirudin activity.

Description

MOLECULES BL- OR MULT1- FUNCTIONAL BASED ON A SCAFFOLDING OF DENDROASP1NA DESCRIPTION OF THE INVENTION The present invention relates to dendroaspine-based chimeric molecules, which have anticoagulant, antiplatelet and other activities. The invention also relates to nucleic acid molecules that communicate these chimeric dendroaspine molecules, cloning and expression vectors comprising said nucleic acids and host cells transformed with expression vectors, in order to provide recombinant chimeric multifunctional dendroaspin. The invention further relates to pharmaceutical compositions comprising chimeric dendroaspin for use in the prevention or treatment of diseases associated with thrombus formation or platelet aggregation. The invention furthermore also relates to the use of a dendroaspine scaffold in the design and generation of chimeric dendroaspine derivatives having inhibitory activity against the platelet binding activity of the platelet plus some other functionality such as an anticoagulant or antithrombotic action. The role of the blood formulation is to provide an insoluble fibrin matrix for the consolidation and stabilization of a hemostatic plug. The formation of an interlaced fibrin module results from a series of biochemical interactions involving a plasma protein scale. Acute vascular diseases, such as myocardial infarction, shock, pulmonary embolism, deep vein thrombosis and peripheral arterial occlusion are caused either by a partial or total occlusion of the blood passage through a module. The formation of a clot with a blood vessel is called thrombosis and depends on the aggregation of platelets. In the context of blood vessel damage (such as may occur in surgical procedures), the interaction of blood platelets with the endothelial surface of damaged blood vessels and with other platelets is an important factor in the course of clot development. or thrombi. Currently, several agents are available to prevent the formation of clots, such as aspirin, dipyridamole, and filopidine. These products generally inhibit the activation and aggregation of platelets, or delay the blood clotting process, but have the potential side effect of causing prolonged bleeding. In addition, the effect of such products can only be invested by new platelets being formed or provided. Platelet aggregation depends on the binding of fibrinogen and other serum proteins to the glycoprotein llb / llla receptor complex on the platelet plasma membrane. GP llb / llla is a member of a large family of cell adhesion receptors known as integrins, many of which are known to recognize a tripeptide recognition sequence Arg-Gly-Asp (RGD). Heparin and low molecular weight heparins have been widely used to treat conditions such as venous thromboembolism, where thrombin activity is responsible for the development or expansion of a thrombus. Although effective, heparin produces many unwanted side effects, including hemorrhage and thrombocytopenia. A more specific and less toxic anticoagulant, therefore, is required. Direct thrombin inhibitors are available and their examples are hirudin, hirugen and hirulogo, (the last two being derivatives of synthetic hirudin), PPACK (a synthetic tripeptide) and argatroban (an arginine derivative). The actions of these inhibitors were reviewed by Lefkovits J and Topol E J (1994), Circulation 90: 1522-1536. Although in theory, the risk of bleeding with direct thrombin inhibitors is lower than other antithrombotic drugs, due to its specific mono-objective nature, absence of direct platelet effects and short half-life, bleeding remains the strongest adverse effect. . There is a variety of other thrombin inhibitors, which have been developed (listed in Table 1 of Lefkovits J and Topol E J above), but these have been considered too toxic for clinical use. Localized narrowing of an artery caused by atherosclerosis is a condition that can usually be remedied surgically through the balloon angioplasty technique. The procedure is invasive and causes some tissue damage to the arterial wall, which can result in the formation of a thrombus. Extracellular proteins such as fibronectin in the arterial wall are exposed to bleeding in the artery. Platelets bind to the RGD motif of fibronectin through integrin receptors, which in turn leads to platelet aggregation and the initiation of the cascade of coagulation reactions. An agent that specifically inhibits platelet aggregation at damage sites and that also inhibits coagulation at these sites is required. The agent must be non-toxic and free of unwanted side effects such as a risk of generalized bleeding. Integrins are a family of cell surface receptors that mediate the adhesion of cells to each other or to the extracellular matrix (Kieffer N & amp; amp;; Philips D R (1990) Annu Rev Cell Biol 6: 329-357; Hynes R O (1992) Cell 69_: 11-25; McEver R P (1992) Curr Opin Cell Biol 4: 840-849; Smyth S S et al. (1993) Blood 8 _: 2827-2843; Giancotti F G and Mainiero F (1994) Biochim Biophys Acta 1198: 47-64). They are composed of transmembrane and ß non-covalently associated subunits. There are 16 different subunits a and 8 different β subunits that heterodimerize to produce about 20 different types of receptors (Clark E A &Brugge J S (1995) Science 268: 233-239). Among the integrins, the ß3 platelet membrane integrin is one of the best characterized. After cell activation, the integrin ap_ß3 binds several glycoproteins, predominantly through the tripeptide sequence Arg-Gly-Asp (RGD) (Pierschbacher MD and Ruoslahti E (1984) supra; Plow EF and others (1987) Blood 70 .: 110-115; Pytela R et al. (1986) Science 231: 1559-1562), present in fibrinogen (Nachman RL and Nachman LLK (1992) J Clin Invest 69: 263-269), fibronectin (Gardner JM and Hynes RO (1985) Cell 42: 439-448), von Willebrand factor and (Ruggeri Z et al. (1983) J Clin Invest 72: 1-12), vitronectin (Pytela RM et al. (1985) Proc Nati Acad Sci USA 8_2: 5766-5770), and thrombospondin (Karczewski J et al. (1989) J Biol Chem 264: 21322-21326). The nature of the interactions between these glycoprotein ligands and their integrin receptors is known to be complex, and conformational changes occur both in the receptor (Sims PJ et al. (1991) J Biol Chem 266: 7345-7352) and in the ligand (Ugarova T et al. (1995) Thromb Haemostasis 7_4: 253-257). Recently, many proteins have been identified from a variety of snake venoms as potent inhibitors of platelet aggregation and integrin-independent cell adhesion. Most of these proteins belonging to the so-called "disintegrin" family share a high level of sequence homology, are small (4-8 kDa), rich in cysteine and contain the sequence RGD (Gould RJ and others (1990 Proc Soc Exp Biol Med 195: 168-171) or KGD (Scarborough RM et al. (1991) J Biol Chem 266: 9359-9362). In addition to the disintegrin family, a number of non-disintegrin RGD proteins of similar inhibitory potency, high degree of bisulfide binding and small size have been isolated from both snake venom Elapidae family (McDowell RS et al. (1992) Biochemistry 31: 4766-4772; Williams JA and others (1992) Biochem Soc Trans 21_: 73S) as well as leech homogenates (Knapp A et al. (1992) J Biol Chem 267: 24230-24234). All these proteins are inhibitors of approximately 1000 times more potent interactions of glycoprotein ligands with integrin receptors than simple linear RGD peptides; a characteristic that is attributed to an optimally favorable conformation of the RGD motif maintained within the protein scaffold. The NMR structures of several inhibitors including quistrin (Adier M et al. (1991) Science 253: 445-448; Adier M and Magner G (1992) Biochemistry 3 _: 1031-1039; Adier M et al. (1993) Biochemistry 32 .: 282-289), flavoridina (Senn H and Klaus W (1993) J Mol Biol 234: 907-925), equistatine (Saudek V et al. (1991) Biochemistry 30: 7369-7372; Saudek V et al. (1991) Eur J Biochem 202; 329-328; Cooke RM et al. (1991) Eur J Biochem 202: 323-328; Cooke RM et al. (1992) Protein Eng 5: 473-477), Albolabrine (Jaseja M et al. (1993) Eur J Biochem 218: 853-860), decorsin (Krezel AM et al. (1994) Science 264: 1944-1947), and dendroaspin (Jaseja M et al. (1994) Eur J Biochem 226: 861-868; Sutcliffe MJ and others ( 1994) Nature Struct Biol 1_: 802-807), have been reported, and the only common structural aspect is the placement of the RGD motif at the end of a loop exposed to solvent, a feature of primary importance for its inhibitory action. Recent studies have implicated a role for amino acids around RGD of tripeptide to regulate the specific character of ligand binding shown by snake venom proteins. Scarborough R M et al. (1993) J Biol Chem 268: 1058-1065 examined a scale of disintegrins and observed that those containing RGDW were very effective in inhibiting the interactions of fibrinogen to purified an_ß3, but not to vitronectin and fibronectin to avß3 and a5ß? purified, respectively, while the inverse ones were true for the disintegrins containing the RGDNP of sequences. Other regions of amino acid sequence divergence also contributed (Scarborough et al. (1993) supra). Dendroaspine, a short chain neurotoxin analog containing the RGD sequence, and disintegrin quistrin, which exhibit small total sequence homology but have similar amino acids flanking the RGD sequence (PRGDMP), both are potent platelet adhesion inhibitors. fibrinogens, but poor antagonists of platelet binding to immobilized fibronectin (Lu X et al. (1994) Biochem J 304: 929-936). In contrast, elegantin, which has a homology sequence of 65% to the quistrin but markedly different amino acids around RGD (ARGDNP), preferentially inhibited the adhesion of platelets to fibronectin opposite fibrinogen and binds to an alloestherically distinct site in the anbß3 complex. Smith JW et al. (1995) Journal of Biological Chemistry 270: 30486-30490 performed protein "loop graft" experiments to construct a tissue-type plasminogen activator variant (t-PA), which bound the platelet integrin to , nbß3. The amino acids in a surface loop of the epidermal growth factor (EGF) domain of t-PA were replaced with residues from a region of complementarity determination (CDR) forming a CDR of monoclonal antibody reagent against the adhesive integrin receptor an ß3. The resulting variant of t-PA (t-PA grafted in a loop) was bound to anbß3 with monomolecular affinity and had a complete activity for both synthetic and natural substrates. The effects and applicability of the loop graft are jointly not predictable and doubtful. The present invention has now discovered that the dendroaspine scaffold itself leads to modification. When dendroaspine (including the RGD motif) is modified to incorporate additional functional amino acid sequences, for example, active portions or agonist, antagonist or factor inhibitor motifs in the coagulation cascade, the resulting molecules are particularly useful as anticoagulants and do not present the disadvantages associated with existing anticoagulants. In a first aspect, the present invention provides a hybrid polypeptide comprising a first amino acid sequence, including the RGD motif and conferring dendroaspine activity and an additional amino acid sequence conferring activity to a different one than that of the dendroaspine activity. The invention also provides a hybrid polypeptide having integrin binding activity comprising a dendroaspine scaffold and an additional non-dendroaspine amino acid sequence, preferably of different activity. Advantageously, the molecules of the invention have an integrin binding activity, which when administered in vivo results in the binding of the molecules to platelets thus inhibiting the aggregation of the platelets, at sites of damage. In addition, the non-wild type dendroaspine domain provides optional, additional, secondary functionality, for example, antithrombotic action, inhibiting cell migration and proliferation and regulation of signal transduction. The molecules of the invention are, therefore, bi- or multifunctional in their anti-coagulation activities, particularly thrombus formation and arterial / venous wall thinning at the sites of damage. The polypeptides of the invention may have activities against leukocyte recruitment, immune system activation, tissue fibrosis and tumorigenesis. The polypeptide of the invention may comprise at least two of these additional amino acid sequences, preferably the two sequences are equal. The additional amino acid sequence may comprise two or more amino acid sequence portions separated by at least one amino acid residue of dendroaspin. The two or more sequence portions can be transposed with respect to each other and in the linear order of amino acids in the additional natural amino acid sequence. In other words, the natural order of the two or more portions of amino acid sequence is altered, although the actual sequence of each portion can not necessarily be altered. Said additional sequence can be selected from platelet-derived growth factor (PDGF), glycoprotein (GP) IBa, hirudin, thrombin, thrombomodulin (particularly its fifth EGF-like domain), vascular epidermal growth factor (VEGF), factor-beta1 of transformation growth (TGFßl), basic fibroblast growth factor (bFGF), angiotensin II (Ang II), factor VIII and von Willebrand factor (vWF). In this way, the molecules of the invention can be made multifunctional, so that they are active against more aggregation of platelets, for example, another component in the coagulation cascade (e.g., thrombin activity), or the intercellular signaling cascade ( for example, growth factor). The modified dendroaspins of the invention can be engineered such that the additional amino acid sequence has an integrin binding activity, thus providing a dendroaspine-based molecule with enhanced integrin binding activity.

The polypeptide of the invention preferably comprises an amino acid sequence as shown in Figure 3. Prior to the inclusion of said additional amino acid sequence, the dendroaspine scaffold of the invention includes homologous molecules, which may share approximately 50% the homology of amino acid sequence, preferably about 65%, preferably about 75% and most preferably about 85% homology with dendroaspin. By excluding the amino acid sequence encoding said additional amino acid sequence, the nucleic acid sequences encoding the polypeptide of the invention can share about 50% of the nucleotide homology, preferably about 65%, preferably about 75%, and most preferably about 85% homology with a dendroaspine nucleotide sequence. The polypeptides of the invention may comprise a greater or lesser number of amino acid residues compared to the 59 amino acids of dendroaspin. For example, the molecules of the invention may comprise a number of amino acid residues on the scale of 45 to 159, preferably about 49 to 85, preferably about 53 to 69, and most preferably about 57 to 61. The sequence of additional amino acid is preferably incorporated into, (a) loop I and / or loop II; (b) loop I and / or loop lll; (c) loop II and / or loop lll; or (d) loop I, loop II and loop III the dendroaspine scaffold. The loop I comprises the amino acid residues 2-16, the loop II the residues 23-36 and the loop III the residues 40-50. However, additional amino acids being incorporated can be extended to or replace regions outside the loops, ie residues 1-3, 17-22 and 37-39, so that the residues from the no-loop regions are increased or replaced. by those of the additional amino acid sequence or sequences that are being inserted. Additional amino acid residues are preferably incorporated either into loop I or loop II. In this way, the link III containing RGD is unaltered and in this way the integrin binding function of dendroaspin is retenin. An additional RGD motif can be introduced to the dendroaspine scaffold, preferably in loop I or loop II, thereby increasing the activity of dendroaspine. A preferred site for the inserted additional sequence is at a site on the dendroaspine scaffold between the amino acid residues: 4-16, 18-21, 23-36, or 52-59. Each additional amino acid sequence or portion inserted from an additional amino acid sequence is preferably an amino acid sequence in the range of 3-40 amino acid residues, preferably 3-16, and most preferably 3-14 amino acid residues in length. The start of the additional amino acid sequence inserted can be in any of the amino acid residues 1-57 of the dendroaspine scaffold. The term of the additional amino acid sequence inserted can be in any of the amino acid residues 3-59 of the dendroaspine scaffold. When two additional amino acid sequences are inserted into the dendroaspine scaffold then the linear distance between them is preferably in the range of 1-35 amino acids, most preferably 1-14 amino acids. When more than two additional amino acid sequences are inserted, then there is preferably at least one natural dendroaspin amino acid residue separating each additional amino acid sequence. The loop containing RGD can be modified through insertion, deletion or substitution of one of more amino acid residues, preferably a maximum of 8 or a minimum of one amino acid can be modified within the lll loop of dendroaspin. The RGD loop preferably has an amino acid sequence as shown in Figure 3B. An advantage of modifying the RGD loop region is that the integrin binding activity can be improved and becomes more specific for certain glycoprotein ligands. Also, if one or more of the additional "extraneous" amino acid sequences grafted to the dendroaspine scaffold has steric effects on the RGD motif, then the lll loop around the RGD site can be modified to overcome any steric hindrance by restoring as such, or such time improving the RGD functionality.

The loop I and / or loop II can be modified through insertion, elimination or substitution of one or more amino acid residues. Any suitable number of amino acids can be inserted into the dendroaspine scaffold to give the desired bi-or multifunctional activity, although a number of residues on the scale of 14 to 36 is preferred for insertion or one or more sites on the dendroaspine scaffold . The modification of the loops may be necessary if an additional "extraneous" amino acid sequence grafted to the dendroaspine scaffold has a steric hindrance effect either in another grafted domain or in the loop containing RGD. The computer-aided molecular model using the Insight II software (Molecular Simulations Inc) can be used to predict the structure of the "grafted-in-the-loop" dendroaspins of this invention. In cases where the steric effects between the loops may serve to cause the loss of functionality, these effects may be "designed" by modifying the appropriate parts of the dendroaspine molecule in an appropriate manner. Sometimes, this may involve the insertion of a number of suitable amino acid residues to extend one or more of the loop structures. The preferred modification includes the insertion of polygon in the loop or loops of the dendroaspin scaffold in order to extend them. Other modifications comprising repeat units of an amino acid residue or residue numbers may be used. Computer modeling studies can be used to design the necessary loop modifications in order to extend dendroaspine bonds. In the design of a bi-functional or multifunctional molecule according to the invention, the "fine tuning" of the desired activity, stability or other biological or biochemical characteristic can be achieved by altering the individual selected amino acid residues through substitution or elimination. Modification through an insertion of an amino acid residue or residues at a selected site is also within the scope of this "fine tuning" aspect of the invention. The site-directed mutagenesis techniques available to alter the amino acid sequence at a particular site in the molecule will be well known to those skilled in the art. In a second aspect, the invention provides a nucleic acid molecule that encodes a peptide as defined above. The nucleic acid can be operably linked to a promoter and optionally to a nucleic acid sequence encoding a heterologous protein or peptide thus encoding a fusion product. The promoter is preferably inducible β-D-isopropyl-thiogalactopyranoside (IPTG) and a heterologous protein or peptide can be glutathione S-transferase (GST). This aspect of the invention also includes a plasmid comprising a nucleic acid as described above. The plasmid is preferably PGEX-3X. In a third aspect, the invention provides a host cell transformed with a plasmid as defined above, preferably said host cell is E. coli. Therefore, the invention also provides a cell culture comprising transformed host cells as defined above. In a fourth aspect, the invention provides a method for producing a polypeptide as defined above which comprises culturing a host cell as defined above in order to express said polypeptide, extract the polypeptide from the culture and purify it. In a fifth aspect, the invention provides a method for producing a multifunctional anticoagulant comprising the steps of: a) constructing an expression vector comprising a nucleic acid sequence encoding a dendroaspine scaffold operably linked to a promoter and optionally linked to a nucleic acid that encodes a heterologous protein for coexpression with the same; b) modifying at least a portion of the nucleic acid sequence of the vector encoding the dendroaspine scaffold, excluding the RGD motif, through one or more of insertion, deletion or substitution of nucleic acid residues, so that the Dendroaspine scaffold expression comprises an additional amino acid sequence of activity other than that of dendroaspine activity; c) transforming a host cell with the vector and causing the host cell to express the dendroaspine sequence. The method preferably further comprises the steps of: d) extracting the modified dendroaspin from a host cell culture; e) purifying modified dendroaspin from the cell culture extract, optionally including the step of separating dendroaspin from a co-expressed heterologous protein. The heterologous protein is preferably GST and the purification preferably involves affinity chromatography using Sepharose 4B of glutathione contained in the GST purification modules followed by factor Xa cleavage of dendroaspins modified from GST. Therefore, the invention provides a polypeptide as defined above that is obtained through the method of producing a multifunctional anticoagulant as previously defined. In a sixth aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide as defined above, optionally further comprising a pharmaceutically acceptable excipient or carrier. A multitude of polypeptides of the invention of different functionalities can be combined together in a pharmaceutically acceptable form in order to provide a desired treatment. A multitude of polypeptides of the invention of different functionalities can be combined together in a pharmaceutically acceptable form in order to provide a desirable treatment. The polypeptide of the invention is preferably formulated for intravenous injection or intravenous infusion, although other methods of administration are possible, eg, subcutaneous or intramuscular if it is desired to provide a slow release into the circulatory system of an individual. It is also possible to formulate the polypeptide for use with implanted controlled release devices, such as those used to administer, for example, growth hormone. A formulation may comprise extravasated blood combined with a polypeptide of the invention at a concentration on the scale of 1nM-60μM. This blood can be stored ready for use in a form and provides an immediate and convenient supply of blood for transfusion in case when coagulation should be avoided, such as during or immediately after surgical procedures. In a seventh aspect, the invention provides a polypeptide as described above for use as a pharmaceutical product. In an eighth aspect, the invention provides the use of a polypeptide as defined above for the manufacture of a medicament for the treatment of disease associated with thrombosis; more particularly thrombosis, myocardial infarction, retinal neovascularization, endothelial damage, deregulated apoptosis, abnormal cell migration, leukocyte recruitment, activation of the immune system, tissue fibrosis and tumorigenesis. The invention also provides methods for the treatment of disease associated with thrombosis; more particularly thrombosis, myocardial infarction, retinal neovascularization, endothelial damage, deregulated apoptosis, abnormal cell migration, leukocyte recruitment, activation of the immune system, tissue fibrosis and tumorigenesis. The methods comprise administering a therapeutically effective amount of a polypeptide as defined above. Preferred embodiments of the invention will now be described with reference to the following examples and drawings, in which: Figure 1 shows the basic three-dimensional structure of a dendroaspine molecule. N denotes the term -NH and -C denotes the term -COOH. The amino acid residues are listed. Figure 2A shows the corresponding nucleotide and amino acid sequence of dendroaspin. The individual synthetic oligonucleotides are indicated by the numbers 1-10. Figure 2B is a partial restriction map of the PGEX-DEG plasmid.

Figure 3A comprises alignments of modified dendroaspins, wherein the inserted amino acid sequences are listed below the amino acid sequence of dendroaspin. Figure 3B is similar to Figure 3A and presents other alignments of the amino acid sequences (one letter code) of several modified dendroaspins. Figure 3C is similar to Figure 3A and shows modified molecules of the invention. Figure 3D is a tab showing various activities of the modified molecules of the invention. Figure 4 shows the PGEX-3X expression plasmid used to clone and express the modified dendroaspine fusion protein. Figure 5A shows a 12.5% SDS-PAGE gel comparing the total cell lysates of either induced (lane 1) or uninduced (lane 2) bacteria transformed with the recombinant plasmid PGEX-DEG harboring the modified dendroaspine gene. Figure 5B shows a 10% SDS-PAGE gel comparing the total cell lysate (lane 1) of induced bacteria transformed with PGEX-DEG and an affinity purified extract of said lysate (lane 2). Figure 5C shows a 20% SDS-PAGE gel of dendroaspine fusion protein purified by affinity and modified by GST digested with the Xa factor. Lane 1 is dendroaspin, lane 2 is GST-Den and lane 3 is GST-Den digested with Xa factor. Figure 6 is a purified recombinant dendroaspine reverse phase HPLC elution profile purified on a Vydac C 8 8 column grown with an acetonitrile gradient (the retention time of recombinant dendroaspin at 44 minutes is indicated as an arrow). Figure 6B is a reverse phase HPLC elution profile of the peak fraction eluting at 44 minutes in Figure 6A worked under similar conditions. Figure 7 shows the immunoprecipitation of dendroaspin containing the loop I PDGF, using the antibodies R38 and R65 arising in loop I and R51 arising from both loop I and loop III.

A 32 kDa protein was observed, which corresponds to the mutant dendroaspin of GST. Lane 1 is DEN-PFGF probed with R38, lane 2 is probed with R65 and lane 3 probed with R51. Figure 8 is a bar graph showing the results of an experiment, where PDGF-dendroaspine was used to inhibit PDGF-induced proliferation of human fibroblast cells. "Peptide 1" corresponds to the linear amino acid sequence of loop I of PDGF. Dendroaspina is a homologue of the short chain neurotoxin of snake venom of Elapidae, which lacks neurotixity.

The neurotoxin difference contains an Arg-Gly-Asp- (RGD) motif and functions as an inhibitor of platelet aggregation and platelet adhesion with power comparable to the disintegrins of Viperidae poisons. The structure of dendroaspine in solution has been determined using NMR spectroscopy (Sutcliffe M J et al. (1994) Nature Structure Biol 1: 802-807). The structure contains a nucleus similar to that of short-chain neurotoxins, but with a novel arrangement of loops and a RGD motif exposed to solvents. Dendroaspin in this way is an integrin antagonist with a well-defined fold different from that of disintegrins, based on the neurotoxin scaffold. The structure of dendroaspine consists of a core region from which three loops, denoted as I, II and III (residues 4-16, 23-36 and 40-50) extend outward (Figure 1). The core contains the four disulfide bonds, which are spatially close to each other and hold the bonds together. The amino acid sequence of dendroaspine is shown in Figure 2. In the following examples, the materials used include: Restriction enzymes, T4 polynucleotide kinase, T4 DNA ligase, IPTG (isopropyl-β-D-thio-galactopyranoside) and DH5a competent cells purchased from Life Technologies Ltd (UK) or Promega Ltd (Southampton, UK). The DNA polymerase Vent (exo-) was supplied by New England Biolabs Ltd (Hitchin, UK). Oligonucleotides were made either at King's College School of Medicine &; Dentistry (London, UK), or by Cruachem Ltd (Glasgow, UK), and in addition to purified by PAGE denaturation in a 15% acrylamide gel / 8 M urea. Deoxynucleotide triphosphates (dNPT's), triphosphates of deoxynucleotide (ddNTP's) and plasmid pGEX-3X, a vector expressing a gene cloned as a fusion protein linked to glutathione S-transferase (GST) and glutathione-Sepharose CL-4B, were purchased from Pharmacia Biotech Ltd (Herts , UK). The "Geneclean" equipment and the plasmid maxi equipment were purchased at Bio 101 (La Jolla CA, USA) and Qiagen Ltd (Surrey, UK), respectively. Sequenase 2.0 sequencing was obtained from Cambridge Bioscience (Cambridge, UK). [35S] dATP [aS] and 125l (15.3 mCi / mg iodide) were supplied by NEN Dupont (Herts, UK) and Amersham International Pie (Amersham, Bucks, England), respectively.

EXAMPLE 1 Construction of Expression Vectors Encoding Variants of Dendroaspina The wild type dendroaspine gene was inserted into a PGEX-3X plasmid (Figure 2) successfully, and expressed according to the method of Lu et al. (1996) J Biol Chem 271: 289-295. Starting with the wild type gene for dendroaspine, dendroaspine gene variants were engineered using recombinant DNA technology. For the longer insertion variants, oligonucleotides encoding non-dendroaspine or heterologous amino acids were simply inserted directly into the wild-type dendroaspina gene digested by restriction, and then ligated. For minor changes such as modification of some amino acid residues including insertion, substitution or elimination, the site-directed mutagenesis equipment was used at the Transformer ™ site of Clonetech Laboratories, according to the manufacturers' instructions. Figure 2A shows the nucleotide sequence of the synthetic dendroaspine gene (Den). The gene was designed based on the known amino acid sequence (Williams JA et al. ((1992)) Biochem Soc Trans 21: 73S), and the codons for each amino acid were adopted from those that were highly expressed in E. coli (Fiers W ((1982)) Gene 1_8_: 199-209). Ten synthetic oligonucleotides are shown in parentheses and individually listed from 1 to 10 either above the coding structure or below the non-coding structure. The stop codon is indicated with an asterisk. The three-letter amino acid code was used and the total of 59 amino acids of Den were only listed as 1 for the N-terminal arginine residue and 59 for the C-terminal leucine. Figure 2B is a partial restriction map of pGEX-DEG.

Only the dendroaspine gene (Den) and its relevant upstream region are shown. Figure 3A shows the amino acid sequences of various modified dendroaspine molecules, which will be described in detail later. In each case, the modified inserted residues are fixed below the dendroaspine amino acid sequence.

EXAMPLE 2 Modified Dendroaspin Containing a Factor Domain Platelet Derived Growth (PDGF) Platelet-derived growth factor (PDGF) is a 30 kDa polypeptide and a major serum mitogen and chemotactic factor for mesenchymal cells. Originally purified from human platelets, PDGF has subsequently been found to be produced through other cell types, such as smooth muscle cells, placental cytotrophoblast cells, fibroblasts and connective tissue. It has been shown that PDGF promotes cell migration and proliferation and these are key events in natural processes such as embryogenesis and wound healing. PDGF has also been implicated in a number of disease states, such as atherosclerosis, fibrosis and rheumatoid arthritis (Heldin CH and Westermark B (1990) Cell Regul 1_: 555-556 and Engstrom U and others (1991) J Biol Chem 267: 16581 -16587). PDGF is a dimer of two similar chains called A and B chains. The amino acid sequences derived from chain B PDGF are inserted into the loop II of dendroaspin as shown in Figures 3A and 3C, maintaining the adhesive property of the RGD sequence of the loop lll within dendroaspina. The modified dendroaspins are then examined for competitive inhibition of PDGF activity by analyzing hemolymph and fibroblast cell proliferation and integrin antagonist activity. Figure 3D summarizes the results obtained with the example given in Figure 3C. Modified dendroaspine molecules inhibit platelet aggregation induced by ADP and the proliferation of fibroblast cells induced by PDGF.

Assembly and cloning of the dendroaspine gene containing the I PDGF loop. The dendroaspine gene containing the PDGF I loop was assembled from the fragments (77 mer, 76 mer, 42 mer and 44 mer) of the wild-type gene, after digestion with Bam Hl, EcoR I, Hinf I and Hpa II, and a pair of oligos of complementary phosphorylated mutagenesis 81 mer and 80 mer). A mixture of a total of 6 fragments was heated at 85 ° C for 5 minutes and collected by cooling slowly below room temperature (RT). The ligation was performed at 16 ° C for 16 hours in a total volume of 50 μl containing about 1 nM of each fragment, 50mM Tris-HCl (pH7.6), 10mM MgCl2, 1mM DTT, 1mM ATP and 5% PEG 8000 , and 5 units of T4 ligase. After ligation, a 1 μl ligation mixture was used as a template together with two 5'-pendant oligonucleotides as primers and 2 units of Vent polymerase for PCR. The only product obtained was the expected size verified through 2% agarose gel. The ligation gel was then cloned into the PGEX-3X vector to produce the recomnt plasmid PGEX-3X comprising the dendroaspin gene modified with PDGF.

Transformation and isolation of plasmid DNA. The PGEX-3X vector comprising the PDGF-modified dendroaspine gene (approximately 5ng) was used to transform 50 μl of E. coli from strain DH5a. One vial controls were established containing only competent cells, a vial containing unbound plasmid DNA and a vial containing wild-type plasmid DNA. The incubation media used for the bacteria were the SOC medium (containing 20 g / l of bacto-tryptone, 5 g / l of bacto-yeast extract, 0.5 g NaCl, 2.5 mM KCl, 20 μM MgCl 2 and 20 mM glucose). The transformation step was followed by a recovery period, where the bacterial culture was incubated without ampicillin for one hour. After the transformation, a sufficient quantity of each of the bacterial cultures was plated. The SOC agar medium containing ampicillin was used to grow the bacteria. The PGEX-3X ampicillin selection marker confers resistance to ampicillin in any transformed bacteria allowing it to develop while eliminating any untransformed bacteria. The control plate remained clear after an overnight incubation. Individual colonies were picked from the plates, each representing a modified dendroaspine gene. These were grown in SOC medium containing ampicillin for a few hours. A small amount of each was observed and transferred to a new SOC medium containing ampicillin. Sterile glycerol was added to the original bacterial culture to allow storage at -70 ° C as a supply supply. The remaining cultures were incubated overnight. Isolation of the plasmid DNA from the transformed bacterial culture was performed through a plasmid minipreparation (for rapid test) or maxipreparation (for DNA sequencing) following the manufacturer's instructions (QIAGEN). A plasmid minipreparation was performed on each to isolate the plasmid DNA all at once; the majority of which comprised the modified dendroaspine gene. DNA sequencing was performed on the region of the plasmid containing the dendroaspine gene fragment to verify that the modification was present and corrected. The complete DNA sequencing of the inserted fragments was performed using a dideoxy chain termination method of Sanger et al. (1977) Proc Nati Acad Sci 7_4: 5463-5467.

Protein expression. E. coli from strain DH5a was transformed with modified plasmid PGEX-3X, using standard methods particularly as those described in Sambrook et al. (1989) Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY. The transformed cultures were used for protein expression. The media were inoculated with a seed culture overnight (1%, v / v) and developed in a medium of LB / ampicillin (100μg / ml) and shaken at 37 ° C until an A6oo of 0.7 was obtained. Then IPTG was added to a final concentration of 0.1mM for induction. The cells were grown for a further 4 hours at a lower temperature of 30 ° C and harvested by centrifugation.

Purification of fusion protein. The recombinant dendroaspins were purified as follows, suspending the cell pellets in pH regulator (pH 7.4) from pH regulated saline (PBS) containing 1% Triton X-100 and the protease inhibitors methylphenylsulfonyl fluoride (1nM) pepstatin (1μM), aprotinin (2μg / ml), soybean trypsin inhibitor (1μg / ml), 1mM EDTA, and sound was applied on ice. The mixture with sound was centrifuged at 7,800 x g at 4 ° C for 10 minutes to form pellets of cell waste and insoluble material. The recombinant GST-dendroaspina and dendroaspinas modified with GST of supernatants, were purified through affinity chromatography in columns of glutationa-Sepharose CL-4B by absorption in PBS containing 150 mM of NaCl and with an elution with 50 mM Tris-HCl containing 10 mM of reduced glutathione (pH 8.0). The elution of material absorbed with glutathione (pH 8.0) resulted in the appearance of a larger band migrating at 31 kDa (GST-dendroaspine fusion protein) in polyacrylamide gels (Figure 5A and 5B). Appropriate fractions comprising the 32 kDa fusion proteins were then digested in the presence of 150 mM NaCl, 1 mM CaCl2, and Factor Xa (1: 100 p / p, Factor Xa: fusion protein) at 4 ° C for 24 hours. The treatment of the GST-proteins purified with factor Xa delibered recombinant proteins migrating as 7-kDa bands (Figure 5C), approaching the size of dendroaspine, and free GST appearing as an intensification of a band of 28-kDa in gel electrophoresis of SDS-psiacrylamide. The digested mixture was loaded onto an analytical reverse phase HPLC column Vydac C18 (TP104) and eluted with a linear gradient of 0-26% acetonitrile (1.78% per minute) containing 0.1% trifluoroacetic acid, followed by 26- 36% acetonitrile in 0.1% trifluoroacetic acid (0.25% per minute). When necessary, additional analytical columns were operated under the same conditions. Figures 6A and 6B show the elution profile of reverse phase HPLC operations.

Western staining Electrophoresis was applied to fusion proteins (10μg) on a SDS-PAGE gel (12%) and transferred through semi-dry staining to nitrocellulose membranes using a current density of 0.8mA / cm2. The nitrocellulose membranes were blocked for 2 minutes at room temperature with 5% dry milk in TBS / Tween pH buffer (20mM Tris, 50mM NaCl, 0.1% Tween) after which they were probed with a corresponding primary antibody during the overnight and then with either a second anti-mouse or anti-rabbit antibody linked to horseradish peroxidase for 2 hours. The stains were then developed with aminoethylcarbasol (Figure 7).

Production of the R51 antibody. Peptide 1 (linear amino acid sequence of the PDGF loop) was conjugated to bovine thyroglobulin (Tg) through rabbit serum albumin (RSA), through the thiol group, using hetero-bifunctional crosslinkers, 4- (N -maleimidomethyl) cyclohexan-1-carboxylic acid sulfosuccinimidyl (Sulfo-SMCC) and gamma-maleimidobutyrate succinimide (GMBS), respectively. The manufacturer's own procedure was used. The conjugates were made in 1mg / ml in PBS and stored at -20 ° C. New Zealand white rabbits were immunized with Tg-peptide 1 (100 μg each per rabbit injected subcutaneously into 1 ml of 1: 1 PBS: Freund's Complete Assist). Rabbits were boosted after 6 and 12 weeks using the same dose but in Freund's incomplete adjuvant. Final blood samples were taken 10 days after the last boost injection. The antisera were formed in aliquots and stored at -20 ° C.

Production of antibodies R38 and R65. IgC (R38 and R65) was purified from the aforementioned antisera using protein-G affinity chromatography (H1-TRAP Protein-G, Pharmacia, Upsala, Sweden), as described by the manufacturers. The purity was purified by SDS-PAGE. The IgC solutions in PBS were filtered in sterile form, formed in aliquots and stored at -20 ° C.

EXAMPLE 3 Identification of Function RGD (Dendroaspine Activity) Aggregation of platelets. Platelet aggregation was measured through the increase in light transmission as described by Lu et al. (1994) Biochem J 304: 929-936, and was used to identify the function of RGD. Briefly, a platelet-rich plasma (PRP) was prepared from human blood coded from healthy individuals by centrifugation at 200 g for 15 minutes. The platelets were prepared from PRP through centrifugation at 1200 g for 15 minutes. Pellets were washed and resuspended in adhesion / aggregation buffer (145mM NaCl, 5mM KCl, 1mM MgCl 2, 2mM CaCl 2, 10mM glucose, 3.5mg / ml BSA and 10mM HEPES, pH 7.35), and adjusted to an account of 3 x 108 / ml. Platelet aggregation (320 μl incubations) was induced with 10 μM ADP in the presence of 1.67 mg / ml fibrinogen and was measured using a Payton Dual-Aggregometer meter linked to a plot chart.

Platelet adhesion. Platelet adhesion was also measured as described to further identify the function of RGD as described by Lu et al. (1994) supra. Briefly, 96-well plates were coated overnight at 4 ° C with either human fibrinogen or reconstituted fibronection in PBS (pH 7.4) at appropriate concentrations (2-10μg / ml, 100μl volumes). The platelets were treated with antagonists at appropriate concentrations for 3 minutes before the addition of 90μl of the mixtures to the microtiter plates, which were reloaded with 10μl of 500μM ADP giving a final concentration of 50μM and the number of adherent platelets by measuring the endogenous acid phosphatase using 130μl / developing pH regulator cavity (sodium acetate, 10mM p-nitrophenyl phosphate, 0.1% triton X-100, pH 5.5) and read at 410 / 630nm in a automatic plate reader.

Iodization and ligand studies and ligand binding assays. Iodization of all dendroaspine variants was performed using Enzymobead Enzyme Radioiodination Reagent Radioiodination Reagent (Biorad Laboratories) according to the manufacturer's instructions. The binding of 125 I-labeled disintegrins, dendroaspine and dendroaspins modified to washed platelets was performed under equilibrium conditions essentially as previously described by Lu et al. (1994) supra. The incubation mixture contained 300 μl of washed platelets (3 x 108 / ml), 10 μl of agonist (1.75 mm ADP giving a final concentration of 50 μm), 10 μl of samples of 125 μl-labeled protein, 5-20 μl of Resuspension pH regulator and a final volume of 250 μl was developed. In antibody inhibition studies, platelet suspensions were treated with antibody for 30 minutes before exposure to ADPA and then the 125l protein samples were added and the mixture was incubated at room temperature for an additional 60 minutes. Incubations were terminated by loading the mixture in 25% sucrose (w / v), 1% BSA pad and centrifugation at 12,000g for 10 minutes. Both the platelet pellets and the supernatants were contracted to determine the levels of binding and free ligand. Previous binding levels were determined in the presence of a 50-fold excess of samples of cold protein or disintegrin or 10mm EDTA.

Identification of grafted ties. The identification of the functions of a grafted loop depends on the function of the proteins of origin of the grafted loop.

EXAMPLE 4 Tests for the effects of the I PDGF in dendroaspine ELISAS of competition (CELIA). The relevant amount of Den-PDGF, p PDGF as tablets was added simultaneously with appropriately diluted rabbit anti-PDGF antiserum and incubated for 2 hours. Each anti-rabbit antibody was used at a dilution within the linear part of the standard direct ELISA curve and gave an OD of 1.2 to 1.5 to 405 nm, after a 30 minute incubation with substrate, when analyzed against the conjugate appropriate for RSA-peptide.

Cell culture and [3H] -thymidine incorporation assay. Fibroblasts from the dermal foreskin (used between passage number 7-10) were maintained in complete DMEM containing 10% FBS. The sub-confluent monolayers of 75cm2 tissue culture flasks were triptimized, counted and seeded on 24-well flat bottom tissue culture plates (Costar) or 48 cavities (Falcon) at cell densities of 10,000 cells / cavity , and developed until approximately 65% or confluent (2 days). The cells were made immobile by replacing the medium with complete DMEM containing 0.2% or 0.5% FBS for 48-72 hours. All cell cultures were performed at 37 ° C in a humidified atmosphere containing 8% CO2 in air. The inhibition of cell proliferation induced by growth factor was directed by replacing the medium, at time zero, with complete DMEM containing varying concentrations of Den-PDGF and peptide P1 (linear sequence of the PDGF domain), respectively. The concentration of PDGF for induction was used on a scale of 10-20ng / ml. [3H] -thymidine (0.3 mCi / 1 OOμl / well) was added after 20 hours-22 hours and incubated until 27.5-28 hours. The medium was aspirated, the cells were washed twice with cold PBS and fixed through the addition of 500μl of 10% TCA and incubated for 0 minutes at 4 ° C. The cells were then washed once with 0.5 ml of 70% ethanol and stored at -20 ° C. The cells were then solubilized through the addition of 500μl of 0.1 N NaOH to each well for 30 minutes at room temperature. The cellular incorporated [3H] -thymidine was quantified by counting by scintillation to a 400μl sample of each cavity. The percentage of inhibition of cell proliferation was worked as follows: ([3 H] -thymidine incorporated with PDGF) - [3 H] -thymidine incorporated with PDGF + M * or Pl) x100 [3 H] -thymidine incorporated with PDGF) - ([3 H] -thymidine incorporated with 0.2% FCS) M *: denotes modified dendroaspins Figure 8 shows that the results of the tests where PDGF-dendroaspine inhibits PDGF-induced proliferation at 10-34% (there was no inhibition that was found when wild-type dendroaspine was used as control) on the 6.5-60μM scale.

EXAMPLE 5 Modified dendroaspin containing a sequence derived from the fifth thrombomodulin EGF domain Thrombomodulin serves as a thrombin receptor. Thrombin is a trypsin-like serine protease that fills a central role in both hemostasis and thrombosis. In the coagulation cascade, thrombin is the final key enzyme, proteolytically separating fibrinogen to release fibrinopeptides A and B and generate fibrin monomers, which can then be polymerized to form a hemostatic drum. In addition to the cleavage of fibrinogen, thrombin exerts a positive feedback in its own production generating Va and Villa coagulation factors, which act as co-factors of thrombin activation. Factor XIII is also activated by thrombin and is crosslinked and stabilized by the fibrin polymer. The natural anticoagulant mechanisms limit these processes through inhibition by serpin, antithrombin III and through the activation of protein C by the thrombin / thrombomodulin complex. Thrombomodulin is a cell receptor located on the endothelial cell surface and binds and alters the specific molecular character of thrombin, reducing its ability to catalyze clot formation, while converting thrombin to a potent activator of protein C. Activated protein C destroys the factors Va and Villa ending the coagulation cascade. In this way, the rest between the pro- and anti-coagulant mechanism maintains normal physiological conditions and allows the local generation of thrombin, while preventing it from becoming a systemic or potentially dangerous process. In addition, thrombin also activates platelets and endothelial cells. After activation of platelets by thrombin, the platelets undergo a shape change, aggregation and release their storage granule contents (e.g., platelet factor-4, ADP, 5-hydroxytryptamine). Thrombin also increases the synthesis and secretion of thromboxane A2 and the platelet activation factor. The interaction of thrombin with endothelial cells also results in the secretion of several agents (eg, activated tissue plasminogen, PDGF, endothelin), as well as the acceleration of the activation of protein C that binds to thrombomodulin, which initiates the anticoagulant pathway of protein C. As examples, a sequence derived from the fifth epidermal growth factor (EGF) as a thrombomodulin domain was grafted into dendroaspin. The thrombin binding affinity of the novel protein was determined based on the inhibition of coagulation by fibrinogen. An appropriate plasmid expression vector was made according to the procedures of Example 1, except that a thrombomodulin domain was engrafted into dendroaspin as shown in Figure 3A, where the sequence grafted into loop II of dendroaspin is shown. The expression, isolation and purification of dendroaspin modified with TM were also analyzed according to Example 1, and the RGD function of the modified proteins was tested as described in Example 2. Human thrombomodulin has Mr of approximately 10 kDa and consists of an N-terminal domain that is homologous to the C-type lectin family, six epidermal growth factor (EGF) type domains repeated in a group, a Ser / Thr-rich domain, a transmembrane domain, and a short cytoplasmic tail. In a particular example shown in Figure 3C, a sequence derived from the fifth EGF-like domain of thrombomodulin in dendroaspine has been inserted and additional modifications of appropriate parts of dendroaspine were made to avoid steric understanding that causes the loss of its biological functions . As indicated in Figure 3D, the modified dendroaspine molecules have platelet aggregation activities induced by ADP and thrombin and also prolongs the coagulation time of thrombin.

EXAMPLE 6 Assays for effects of thrombomodulin domain in dendroaspine Coagulation time of fibrinogen induced by thrombin. The coagulation time of fibrinogen induced by thrombin was determined using an Emelung KC-10 instrument as follows. 50μl of thrombin (3.3μg / ml) was mixed in 50mM Tris-HCl, pH 7.5, containing 150mM NaCl and 5mM CaCI2 with 10 10 μl of various concentrations of both dendroaspina modified (Den-TM) and wild-type (as a control). for comparison). After a 2 minute incubation at 37 ° C, 10 μl of fibrinogen 160 μg / ml was added in the same pH buffer to determine the clotting time.

Activation of Protein C. To verify whether Den-TM has any influence on the activation of protein C, a two-step assay was used as described by Tsiang et al. (1990 = Biochemistry 29: 10602-10612.In the first stage, thrombin and protein C were added to appropriate final concentrations in a reaction volume of 110 μl, with or without wild-type dendroaspins or mutants with either 2mM CaCI2 or 1mM Na2 EDTA, reaction mixtures were incubated for 30 minutes and stopped at Through the addition of antithrombin III and heparin, the active protein C generated was analyzed through hydrolysis of the substrate S-2366.

Aggregation of platelets induced by thrombin. The aggregation of thrombin-induced platelets in washed platelets for both wild-type dendroaspins and mutants was determined as described in Example 3 above, except that thrombin was used as an agonist.

Thrombomodulin binding. The thrombomodulin binding was performed as described by Tsiang et al. (1990) supra.

EXAMPLE 7 Modified Dendroaspin Containing a Sequence Derivated from QC-Protein (GP) IB Glycoprotein (GP) IBa was required for the expression of the high-affinity α-thrombin binding site site in platelets (Marco et al. (1994) J Biol Chem 269: 6478-6484). This function may be of crucial importance in the initiation of hemostasis and thrombosis and may play an important role in the development of pathological vascular occlusion. A modified dendroaspin, which contains a sequence derived from GP IB, was engineered to create both thrombin and integrin agonist activities.

An appropriate plasmid expression vector was made as described in Example 1, except that a glycoprotein IBa domain was inserted into the I loop of the dendroaspine molecule as shown in Figures 3A and 3C, where the residues of amino acid of the grafted domain were aligned with the complete amino acid sequence of dendroaspin. As summarized in Figure 3D, the modified dendroaspine molecules inhibit thrombin activity and platelet aggregation.

Assemble cloning of the glycoprotein IB domain gene (GP IB) containing dendroaspin. The dendroaspine gene containing the GP IB domain was assembled from the fragments (81 mer, 82 mer, 42 mer and 44 mer) of the wild-type gene, after digestion with Bam Hl, EcoR I, Hinf I and Hpa II, and a pair of phosphorylated mutagenesis oligos (83 mer and 82 mer). The experimental procedures of recoside, ligation, PCR and assembly were similar to those previously described for Den-PDGF. The expression, isolation and purification of dendroaspin modified with GP IBa was performed as described in Example 1 and the RGD function of the molecule was analyzed according to Example 3.

EXAMPLE 8 Assays for the effects of the GP IB domain in dendroaspine Measurement of thrombin binding to platelets. The binding of thrombin to washed platelets was measured in a pH regulator of calcium-free aggregation / adhesion (see measurement of platelet aggregation in Example 3). Washed platelets were equilibrated at 37 ° C for 10 minutes before assay and then incubated with 125 I-thrombin for 10 minutes at 25 ° C. The binding as a function of ligand concentration was determined with a constant concentration (0.1nM) of 125i-a-thrombin mixed with increasing concentrations (between 1 and 200nM) of unlabelled a-thrombin. The binding was initiated through platelets to the thrombin mixture. The platelets and the bound thrombin were separated from the unbound thrombin after 10 minutes of incubation through centrifugation by a 20% sucrose layer at 12 minutes., 000 g for 4 minutes (Lu et al. (1994) supra). The effect of dendroaspin containing the GP IB domain on the binding of thrombin to platelets was evaluated by mixing varying concentrations of the mutants with the washed platelets and adding a constant concentration of 125 I-thrombin. The results were analyzed as previously described (Lu et al. (1996) supra).

Measurement of platelet aggregation and secretion. The release of ATP from dense platelet granules was measured by the luciferin-luciferase assay. Washed platelets were resuspended in 0.4 ml of calcium-free aggregation / adhesion pH regulator at a count of 2.5 x 108 / ml, 0.4 ml aliquots were measured using a lumiagregometer (Chrono-Log Corp). Then 50 μl of the luciferin-luciferase reagent was added followed by α-thrombin to the final concentration of 0.25 nM; the resulting release of ATP was determined by registering the luminescence change, using a lumiagregometer (Chrono-Log Corp). Platelet aggregation was measured as described in Example 3 above. To test the inhibitory effect of mutant dendroaspina containing the GP IB domain on the release of ATP and platelet aggregation, mutant proteins were added and mixed with platelets for 5 minutes at 37 ° C before the addition of luciferin-luciferase and thrombin.

Amidolytic activity of thrombin and fibrinogen coagulation.

The amidolytic activity of thrombin and fibrinogen coagulation was determined as described in Example 10 below.

EXAMPLE 9 Modified dendroaspine containing a hirudin domain Hirudin, a potent thrombin inhibitor of the blood-sucking species Hirudo medicinalis, is an individual polypeptide chain protein containing 65 amino acid residues (Maraganore et al. (1989) J Biol Chem 264: 8692-8698). A modified dendroaspin was produced, which includes amino acid residues Asn52-Leu64, or Phe45-Gln65 from hirudin. The new construction contains both antithrombin binding domains and platelet anti-adhesives. The expression vector of the plasmid was made as described in Example 7, except that the additional mutation site was made for a minor change corresponding to the amino acid residues PRP in loop II of dendroaspin. For minor changes, the mutagenesis engineered equipment was used on the Transformer ™ site of Clonetech Laboratories. The experimental procedures were analyzed according to the manufacturer's instructions, except that the hirudin domain was inserted into dendroaspine loops I and II. The modified molecules are shown in Figures 3A and 3C, where the amino acid residues derived from hirudin were aligned with the original dendroaspin sequence. Figure 3D indicates how the molecule of Figure 3C delays the coagulation time of thrombin and inhibits ADP or platelet aggregation induced by thrombin. The expression, isolation and purification of Den-HR was essentially as described in Example 1. The RDG function of the molecule was stabilized according to the test methods of Example 3.

EXAMPLE 10 Assays for the effects of the hirudin domain in dendroaspine Amidolytic activity of thrombin and fibrinogen coagulation. The a-thrombin amidolytic activity was performed using the chromogenic substrate S-2238 (Chromogenix) and α-thrombin at a final concentration of 0.06 units / ml. Thrombin releases p-n? Troaniline from the substrate and the speed of this reaction was checked in 405 nm microtiter plates using an automatic spectrophotometer (Autoreader III, Ortho Diagnostic System). The inhibitory effect of Den-HR on the cleavage of thrombin from the chromogenic substrate was measured at a final concentration between nM-mM. Thrombin and Den-HR were premixed and reactions were initiated through the addition of substrate. To evaluate the activation of coagulation by fibrinogen, 200μl of α-thrombin (final concentration, 1nM) in 0.05 M sodium phosphate, pH 6.5 (BSA, final concentration, 1%) was incubated for 5 minutes at room temperature, and then they added 200μl of normal plasma containing 0.011 M trisodium citrate, as an anticoagulant. The time to observe the start of fibrinogen coagulation (thrombin time) was measured using an automatic clot measurer at 37 ° C. The effect of the mutants on coagulation with fibrinogen through α-thrombin was determined by replacing the pH regulator HEPES in the first mixture with mutants at a concentration scale.

EXAMPLE 11 Dendroaspin modified containing a thrombin-based peptide that blocks the procoating activity of thrombin The thrombomodulin binding site within human thrombin has been located in a region in the B chain of thrombin. Its sequence was introduced to dendroaspin as shown in Figure 3B to generate a bifunctional molecule, which blocks the procoagulant activities of thrombin and also inhibits the inhibition of platelet aggregation induced by ADP / thrombin. The functional characterization of this mutant includes the measurement of fibrinogen coagulation time induced by thrombin as described in Example 6 and the platelet aggregation measurement induced by ADP / thrombin as described in Examples 3 and 6 above. Figure 3D summarizes the properties of the bifunctional molecule as being capable of delaying coagulation of thrombin and inhibits platelet aggregation induced either by ADP or thrombin. In the case of the molecules of Figure 3C, the modification of the loops became necessary due to a steric effect caused by the introduction of a sequence (foreign), for example, when a sequence derived from PDGF introduced to dendroaspin, generated a anti-PDGF activity, but antiplatelet activity was lost. This steric effect was designed through the introduction of a similar RGD loop as the lll loop of dendroaspin in loop I. The sequences of engineered molecules based on the dendroaspine scaffold and their functions are summarized in the following Table I and Figure I, respectively.

EXAMPLE 12 Site-directed mutagenesis of modified dendroaspins Each of the modified dendroaspins described above can be modified around the RGD loop or any other portion of the molecule as desired through site-directed mutagenesis. The methods used are as described by Lu et al. (1996) supra. Possible modifications of flanking region RGD are shown in Figure 3B, together with the results of dendroaspine activity assays. Some modifications increase the activity, while other modifications deny the activity.

EXAMPLE 13 Antithrombotic activity of dendroaspins modified in a guinea pig arterial thrombosis model of india The four modified dendroaspins produced in the above examples can be tested for antithrombotic activity in vivo in an Indian guinea pig model for arterial thrombosis as described in detail by Carteaux J P et al. (1995) Circulation 9: 1568-1574. A dose scale of each dendroaspine modified on the scale of 0.1 mg / kg of body weight to 1 mg / kg can be administered separately to animals through intravenous infusion. Control tests of 50-150 IU / kg of heparin and placebo were performed in parallel.

EXAMPLE 14 Activation of modified dendroaspins in cell proliferation after arterial damage in a rabbit atherosclerosis model Den-PDGF, Den-TM, Den-GP and Den-HR in the rabbit can be tested in an in vivo model system as described by Ragosla M et al. (1996) Circulation 93: 1194-1200. After the induction of atherosclerosis, the rabbits were infused intravenously with a dose of 0.1 mg-0.5mg / kg of these modified dendroaspins. A first group of control animals was treated intra-arterially with an individual heparin bolus (150 IU / kg). A second animal control group can be set to receive saline instead of modified dendroaspins. Balloon angioplasty was then performed in the test and control animals and followed by quantitative angiography, measurement of activated partial thromboesplastin time (aPTT), incorporation of 3H-thymidine into the damaged artery, and studies of luminal narrowing followed.

EXAMPLE 15 Thrombolytic activity of modified dendroaspins tested in a pig system model in vivo The system model of Mruk J S et al. (1995) Circulation 93: 762-799, was employed. Pigs with occlusive thrombi were administered intravenously 0.1mg-1mg / kg of the modified dendroaspines mentioned in Example 12 above. A dose of heparin or hirudin (bolus of 100 IU / kg followed by infusion of 20 IU / kg) can be administered to the control animals. Animals that only received saline served as controls.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Thrombosis Research Institute (B) STREET: Emmanuel Kave Building, Manresa Road (C) CITY: Chelesa (D) STATE: London (E) COUNTRY: United Kingdom ( F) POSTAL CODE (ZONE): SW3 6LR (A) NAME: Lu Xinje (B) STREET: 78 Lambourn Cióse (C) CITY: Hanwell (D) STATE: London (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZONE): W7 2LN (ii) TITLE THE INVENTION: Multi-Functional Bi- O Molecules Based on a Dendroaspine Scaffold (iii) NUMBER OF SEQUENCES: 45 (iv) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Reléase # 1.0, Version # 1.30 (EPO) (v) CURRENT REQUEST DATE: NO. OF APPLICATION: 9800848 (vi) DATE OF PREVIOUS APPLICATION: (A) NO. OF APPLICATION: GB 9705787.1 (B) DATE OF SUBMISSION: March 20, 1997 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 217 base pairs (B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: peptide (ix) ASPECT: (A) NAME / KEY: CDS (B) LOCATION: 2 ... 217 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: T GGG ATC CAT ATC GAA GGT CGT CGT ATC TGC TAC AAC CAT CTT GGT 46 Gly lie His lie Glu Gly Arg Arg lie Cys Tyr Asn His Leu Gly 1 5 10 15 ACT AAA CCG CCG ACT ACT GAA ACT TGC CAG GAA GAC TCT TGC TAC AAA 94 Thr Lys Pro Pro Thr Thr Glu Thr Cys Gln Glu Asp Ser Cys Tyr Lys 20 25 30 AAC ATC TGG ACT TTC GAC AAC ATC ATC CGT CGT GGT TGC GGT TGC TTC 142 Asn lie Trp Thr Phe Asp Asn lie lie Arg Arg Gly Cys Gly Cys Phe 35 40 45 ACT CCG CGT GGT GAC ATG CCG GGT CCG TAC TGC TGC GAA TCT GAC AAA 190 Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Cys Cys Glu As Asp Lys 50 55 60 TGC AAC CTT TGA GAA TTC TCG TGA TGA 217 Cys Asn Leu * 65 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Gly lie His lie Glu Gly Arg Arg lie Cys Tyr Asn His Leu Gly Thr 1 5 10 15 Lys Pro Pro Thr Thr Glu Thr Cys Gln Glu Asp Ser Cys Tyr Lys Asn 20 25 30 lie Trp Thr Phe Asp Asn lie Arg Arg Gly Cys Gly Cys Phe Thr 35 40 45 Pro Arg Gly Asp Met Pro Gly Pro Tyr Cys Cys Glu As Asp Lys Cys 50 55 60 Asn Leu * 65 (2) INFORMATION FOR SEC ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Arg lie Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn lie Trp Thr Phe Asp Asn lie 20 25 30 lie Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly 35 40 45 Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Arg lie Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Pro Gly 1 5 10 15 Pro Cys Gln Glu Asp Ser Cys Tyr Lys Asn lie Trp Thr Phe Asp Asn 20 25 30 lie lie Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 60 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Arg lie Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Pro Gly 1 5 10 15 Cys Gln Glu Asp Ser Cys He Ser Arg Arg Leu He Asp Arg Thr Asn 20 25 30 Wing Asn Phe Leu Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly 35 40 45 Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Arg lie Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Pro Gly 1 5 10 15 Pro Cys Gln Glu Asp Ser Cys He Ser Arg Arg Leu lie Asp Arg Thr 20 25 30 Asn Wing Asn Phe Leu Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 60 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 62 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Arg He Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Pro Gly 1 5 10 15 Cys Gln Glu Asp Ser Cys He Ser Arg Arg Leu He Asp Arg Thr Asn 20 25 30 Wing Asn Phe Leu Pro Gly Pro Cys Gly Cys Phe Thr Pro Arg Gly Asp 35 40 45 Met Pro Gly Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 60 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Arg He Cys He Ser Arg Arg Leu He Asp Arg Thr Asn Wing Asn Phe 1 5 10 15 Leu Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn 20 25 30 He He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 60 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Arg He Cys He Ser Arg Arg Leu He Asp Arg Thr Asn Wing Asn Phe 1 5 10 15 Leu Cys Gln Glu Asp Ser Cys Arg Lys He Glu He Val Arg Lys Lys 20 25 30 He He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 60 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Arg He Cys Gly Asp Thr Asp Leu Tyr Asp Tyr Tyr Pro Glu Glu Asp 1 5 10 15 Thr Glu Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp 20 25 30 Asn He He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met 35 40 45 Pro Gly Pro Tyr Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 60 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Arg He Cys Gly Asp Gly Asp Phe Glu Glu He Pro Glu Glu Tyr Leu 1 5 10 15 Cys Gln Glu Asp Ser Cys Arg Lys He Glu He Val Arg Pro Arg Pro 20 25 30 He Arg Gly Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly 35 40 45 Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF SEQUENCE: (A) LENGTH: 60 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Arg He Cys Gly Asp Gly Asp Phe Glu Glu He Pro Arg Pro Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Gly Asp Gly Asp Phe Glu Glu He Pro Glu 20 25 30 Glu Tyr Pro Gly Pro Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 60 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Pro Glu Gly Arg He Leu Asp Asp Gly Phe 20 25 30 He Thr Asp He Asp Glu Cys Gly Cys Phe Thr Pro Arg Gly Asp Met 35 40 45 Pro Gly Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 60 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Gly Asp Thr Asp Leu Tyr Asp Tyr Tyr Pro 20 25 30 Glu Glu Asp Thr Glu Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 60 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 63 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Gly Asp Thr Asp Leu Tyr Asp Tyr Tyr Pro 20 25 30 Glu Glu Asp Thr Glu Pro Gly Pro Cys Gly Cys Phe Thr Pro Arg Gly 35 40 45 Asp Met Pro Gly Pro Tys Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 60 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 73 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Arg He Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Pro Gly 1 5 10 15 Pro Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn 20 25 30 He lie Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Phe Pro Arg Pro Gln Ser His Asn Asp Gly Asp Phe 50 55 60 Glu Glu He Pro Glu Glu Tyr Leu Gln 65 70 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 69 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro 20 25 30 Tyr Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Cys 35 40 45 Phe Pro Arg Pro Gln Ser His Asn Asp Gly Asp Phe Glu Glu He Pro 50 55 60 Glu Glu Tyr Leu Gln 65 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 72 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly 35 40 45 Pro Tyr Cys Phe Pro Arg Pro Gln Ser His Asn Asp Gly Asp Phe Glu 50 55 60 Glu He Pro Glu Glu Tyr Leu Gln 65 70 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 74 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Arg He Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Pro Gly 1 5 10 15 Pro Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn 20 25 30 He He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Pro Gly Pro Glu Cys Pro Glu Cys Tyr He Leu Asp 50 55 60 Asp Gly Phe He Cys Thr Asp He Asp Glu 65 70 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 70 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro 20 25 30 Tyr Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Cys 35 40 45 Pro Gly Pro Glu Cys Pro Glu Cys Tyr He Leu Asp Asp Gly Phe He 50 55 60 Cys Thr Asp He Asp Glu 65 70 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 73 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21 Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly 35 40 45 Pro Tyr Cys Pro Gly Pro Glu Cys Pro Glu Cys Tyr He Leu Asp Asp 50 55 60 Gly Phe He Cys Thr Asp He Asp Glu 65 70 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Arg He Pro Arg Gly Asp Met Pro Asp 35 40 45 Asp Arg Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Arg Arg Ala Arg Gly Asp Asn Pro Asp 35 40 45 Asp Arg Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Asp 35 40 45 Asp Arg Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Asn Pro Gly 35 40 45 Pro Tyr Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Wing Arg Gly Asp Asn Pro Gly 35 40 45 Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Wing Arg Gly Asp Asn Wing Gly 35 40 45 Wing Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Ala Arg Gly Asp Asn Ala Gly 35 40 45 Pro Tyr Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly 35 40 45 Wing Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Ala Pro Arg Gly Asp Met Pro Gly 35 40 45 Pro Tyr Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Phe Pro Gly 35 40 45 Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Trp Pro Gly 35 40 45 Pro Tyr Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Ser Pro Gly 35 40 45 Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Asp Pro Gly 35 40 45 Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly 35 40 45 Pro Arg Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Gly 35 40 45 Pro Thr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xí) SEQUENCE DESCRIPTION: SEQ ID NO: 37: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Leu Thr 35 40 45 Pro Tyr Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro Asp 35 40 45 Asp Tyr Cys Cys Glu As Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 39: (i) CHARACTERISTICS OF SEQUENCE: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Lys Gly Asp Met Pro Gly 35 40 45 Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: Arg He Cys Tyr Asn His Leu Gly Thr Lys Pro Pro Thr Thr Glu Thr 1 5 10 15 Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn He 20 25 30 He Arg Arg Gly Cys Gly Cys Phe Thr Pro Lys Gly Asp Trp Pro Gly 35 40 45 Pro Tyr Cys Cys Glu Ser Asp Lys Cys Asn Leu 50 55 (2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 74 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: Arg He Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Pro Gly 1 5 10 15 Pro Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn 20 25 30 He He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Pro Gly Pro Glu Cys Pro Glu Gly Tyr He Leu Asp 50 55 60 Asp Gly Phe He Cys Thr Asp He Asp Glu 65 70 (2) INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 67 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: Arg He Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Pro Gly 1 5 10 15 Pro Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn 20 25 30 He He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Gly Asp Thr Asp Leu Tyr Asp Tyr Tyr Pro Glu Glu 50 55 60 Asp Thr Glu 65 (2) INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 64 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: Arg He Cys Phe Thr Pro Arg Gly Asp Met Pro Gly Pro Tyr Pro Gly 1 5 10 15 Pro Cys Gln Glu Asp Ser Cys Tyr Lys Asn He Trp Thr Phe Asp Asn 20 25 30 He He Arg Arg Gly Cys Gly Cys Phe Thr Pro Arg Gly Asp Met Pro 35 40 45 Gly Pro Tyr Cys Thr Trp Thr Wing Asn Val Gly Lys Gly Gln Pro Ser 50 55 60 (2) INFORMATION FOR SEQ ID NO: 44 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: other nucleic acid ( ix) ASPECT: (A) NAME / KEY: CDS (B) LOCATION: < 1 ... 33 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: ATC GAA GGT CGT GGG ATC CCC GGG AAT TCA TCG TGACTGACTG AC 45 He Glu Gly Arg Gly He Pro Gly Asn Ser Ser 75 80 (2) INFORMATION FOR SEQ ID NO: 45: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 45: He Glu Gly Arg Gly He Pro Gly Asn Ser Ser 1 5 10

Claims (32)

1. A hybrid dendroaspine-based peptide comprising a first amino acid sequence including the RGD motif and conferring platelet binding activity and an additional amino acid sequence conferring activity different from the platelet binding activity or glutathione S-transferase activity . 2. A hybrid polypeptide, comprising a dendroaspine scaffold and which has integrin binding activity in a domain and a non-dendroaspine amino acid sequence, which confers a second functionality different from the activity of

S-transferase of glutathione in another domain.

3. A polypeptide according to claim 1 or claim 2, comprising at least two of said additional amino acid sequences, optionally, said additional sequences are the same.

4. A polypeptide according to any of claims 1 to 3, wherein the additional amino acid sequence comprises two or more portions of amino acid sequence separated by at least one amino acid residue of dendroaspine.

5. A polypeptide according to any of claims 1 to 4, wherein said additional sequence is selected from: platelet-derived growth factor (PDGF), glycoprotein IBa, hirudin, thrombomodulin, vascular epidermal growth factor, factor- ß1 transforming growth, basic fibroblast growth factor, angiotensin II, factor VIII and von Willebrand factor.

6. A peptide according to any of the preceding claims, characterized in that it comprises an amino acid sequence as shown in Figure 3A or 3C.

7. A polypeptide according to any of the preceding claims, wherein said additional sequence is incorporated into: (a) loop I and / or loop II; (b) loop I and / or loop lll; (c) loop II and / or loop lll; or (d) loop I, loop II and loop lll of the dendroaspin scaffold.

8. A polypeptide according to claim 7, wherein the additional sequence is incorporated into either loop I or loop II.

9. A polypeptide according to any of the preceding claims, wherein the loop containing RGD is modified through the insertion, deletion or substitution of one or more amino acid residues, preferably a maximum of 8 and a minimum of 1 amino acid can be modified within the lll loop of dendroaspin.

10. A polypeptide according to claim 9, wherein the RDG loop has an amino acid sequence as shown in Figure 3B.

11. A polypeptide according to any of the preceding claims, wherein the loop I and / or loop II is modified through insertion, elimination or substitution of one or more amino acid residues, preferably a number of amino acid residues in the scale from 14 to 36.

12. A polypeptide according to any of the preceding claims, wherein said additional sequence is inserted into the dendroaspine scaffold between the selected amino acid residues of one or more than 2-16, 21- 36, 21-31, 28-32, 9-13, 21-33, or at the end of the dendroaspine scaffold after residue 50.

13. A nucleic acid molecule encoding a polypeptide of any of claims 1 to 12

14. A nucleic acid according to claim 13, operably linked to a promoter and optionally to a nucleic acid sequence encoding a heterologous protein or peptide to thereby encode a fusion product.

15. A nucleic acid according to claim 14, wherein the promoter is inducible IPTG and optionally the heterologous protein or peptide is glutathione S-transferase.

16. A plasmid comprising a nucleic acid according to any of claims 13 to 15.

17. A plasmid pGEX-3X comprising the nucleic acid of claim 13.

18. A host cell transformed with a plasmid of according to claim 16 or 17.

19. - A host cell according to claim 18 which is E. coli.

20. A cell culture comprising host cells according to claims 18 or 19.

21. A method for producing a polypeptide as defined in any of claims 1 to 12, which comprises culturing a host cell of claim 18. or claim 19 in order to express said polypeptide, extract the polypeptide from the culture and purify it.

22. A method for producing a multifunctional anticoagulant comprising: a) constructing an expression vector comprising a nucleic acid sequence encoding a dendroaspine scaffold operably linked to a promoter and optionally aligned with a nucleic acid encoding a heterologous protein for the coexpression with it; b) modifying at least a portion of the nucleic acid sequence of the vector encoding the dendroaspine scaffold, excluding the RGD motif of platelet binding, through one or more of insertion, deletion or substitution of the nucleic acid residue, so that the expression of the dendroaspine scaffold comprises an additional amino acid sequence different from the glutathione S-transferase activity, which confers a second functionality in addition to the platelet binding in the expressed scaffold; c) transforming a host cell with the vector and causing the host cell to express the modified dendroaspine nucleic acid sequence.

23. A method according to claim 22, further comprising the steps of: d) extracting the modified dendroaspin from a host cell culture, e) purifying the modified dendroaspin from the cell culture extract, further including the step of Separate dendroaspin from a co-expressed heterologous protein.

24. A method according to claim 23, wherein the heterologous protein is glutathione S-transferase (GST) and the purification involves affinity chromatography of GST followed by dendroaspin cleavage modified from GST.

25. A method according to any of claims 21 to 24, which comprises the use of site-directed mutagenesis to alter one or more selected amino acid residues through substitution or elimination.

26. A method according to any of claims 21 to 25, further comprising formulating the multifunctional anticoagulant expressed to a pharmaceutical formulation.

27. A polypeptide according to any of claims 1 to 12 or obtained by the method according to any of claims 21 to 25.

28. - A pharmaceutical composition comprising a therapeutically effective amount of a polypeptide of any of claims 1 to 12 or 27. 29.- A composition according to claim 28, further comprising a pharmaceutically acceptable excipient or carrier. 30. A polypeptide according to any of claims 1 to 12 or 27 for use as a pharmaceutical. 31. The use of a polypeptide according to any of claims 1 to 12 or 27 for the manufacture of a medicament for the treatment or prophylaxis of diseases associated with thrombosis. 32. The use according to claim 31, wherein said disease is one or more of thrombosis, myocardial infarction, retinal neovascularization, endothelial damage, deregulated apoptosis, abnormal cell migration, leukocyte recruitment, activation of the immune system, tissue fibrosis and tumorigenesis.