WO2009009399A2 - Ixostatins and their use - Google Patents

Abstract

The present invention relates to the discovery of the Ixostatin gene and Ixostatin protein, a molecule that interacts with vitronectin and enhances fibrinolysis and inhibits endothelial cell adhesion to vitronectin. Novel biological tools, prophylactics, therapeutics, diagnostics, and methods of use of the foregoing are also disclosed.

Description

IXOSTATINS AND THEIR USE

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional Patent Application No.

60/948,388, filed July 6, 2007, the disclosure of which is incorporated in its entirety herein by reference.

FIELD OF THE INVENTION

Aspects of the present invention concern the discovery of a gene that encodes Ixostatin, a protein that interacts with vitronectin, enhances fibrinolysis, and inhibits endothelial cell adhesion to vitronectin. Novel biological tools, prophylactics, therapeutics, diagnostics, and methods of use of the foregoing are embodiments.

BACKGROUND OF THE INVENTION

The interaction between vitronectin (VN) with its physiological ligands PAI-I, uPAR, and integrin αvβ3 plays an important role in physiological processes such as clot dissolution (fibrinolysis) and cell migration (Delias and Loskutoff, Thromb. Haemost., 93, 631-640 (2005). The plasminogen activation system is also involved in pathological processes such metabolic syndrome (Alessi and Juhan- Vague, Arterioscler. Thromb. Vase. Biol. 26:2200-2207 (2006), cardiovascular and obstetric diseases (Estelles et al., Thromb. Haemost., 79: 500-508 (1998); Kohler and Grant, N. Engl. J. Med., 342: 1792- 1801 (2000); Thogersen et al., Circulation, 98:2241-2247 (1998)), and has a clear role in vascular remodeling, tumor growth and metastasis (Stafansson et al., Curr. Pharm.

Des., 9:1545-1564 (2003)). Accordingly, elevated levels of active PAI-I (Keijer et al., J. Biol. Chem., 266:10700-10707 (1991)) are associated with several thrombotic diseases such as myocardial infarction and deep venous thrombosis, and also indicate a poor prognosis for survival in several metastatic human cancers (de Bock and Wang, Med. Res. Rev., 24: 13-39 (2004); Foekens et al., Cancer Res., 60:636-643 (2000)). Not surprisingly, there has been much interest in the development of agents to block this system (Zhou et al., Nat. Struct. Biol, 10:541-544 (2003). Some investigators have attempted to neutralize this system using PAI-I inhibitors (Biemond et al., Circulation, 91:1175-1181 (1995); Gils and Declerck, Thromb. Haemost., 91:425-437 (2004); Hennan et al., J. Pharmakol. Exp. Ther., 314:710-716 (2005), integrin αvβ3 and αvβ 5 antagonists (Trikha et al., Int. J. Cancer, 110:326-335 (2004), anti-VN antibodies (Trikha et al., (2004), approaches in gene therapy that target uPAR (de Bock and Wang, (2004); Pillay et al., Trends Biotechnol, 25: 33-39 (2007)).

The salivary glands from blood-sucking arthropods are an important source of molecules that affect vascular biology and hemostasis (Ribeiro and Francischetti, Annu. Rev. Entomol., 48:73-88 (2003). Specific inhibitors targeting the blood coagulation cascade, such as thrombin (Francischetti et al., Biochemistry, 38:16678-16685 (1999), FXa (Cappello et al., Proc. Natl. Acad. Sci USA, 92:6152-6156 (1995); Waxman et al.,

Science, 248:593-596 (1990)) and FVIIa/TF (Francischetti et al., Blood, 99:3602-3612 (2002); Stassens et al., Proc. Natl. Acad. Sci USA, 93:2149-2154 (1996)) have been cloned and expressed and the mechanism of action of these molecules have been studied in detail. Other molecules that affect platelet activation through integrin receptor antagonisms (Mans et al, J. Biol. Chem., 277:21371-21378 (2002)), or removal of pro- aggregatory amines (e.g ADP, serotonin) that induce platelet aggregation (Andersen et al., J. Biol. Chem., 278:461 1-4617 (2003); Calvo et al., J. Biol Chem., 281 :1935-1942 (2006); Francischetti et al., J Biol. Chem., 275:12639-12650 (2000)), or enzymatic degradation of platelet stimulating molecules, such as ADP and PAF (Champagne et al., Proc. Natl. Acad. Sci USA, 92:694-698 (1995); Ribeiro and Francischetti, J. Exp. Biol,

204:3887-3894 (2001)) have been investigated. Molecules that target the tonus of the vessel wall, including NO-releasing proteins (Ribeiro et al., Science, 260:539-541 (1993)), vasodilating peptides, such as maxadilan, which activates the PACAP receptors (Champagne and Ribeiro, Proc. Natl. Acad. Sci USA, 91 :138-142 (1994)), and catechol oxidases, which degrade epinephrine (Ribeiro and Valenzuela, J. Exp. Biol., 202:809-

816 (1999) have also been analyzed. Some of these molecules or their prototypes have been introduced into human clinical trials to address the ability of these compounds to treatt thrombotic disorders, for example: bivalirudin (Angiomax) - a thrombin inhibitor isolated from the leach (Stone et al., N. Engl. J. Med., 355:2203-2216 (2006)); epitifibatide (Integrilin) - an integrin αllbβ3 antagonist isolated from snake venom

(Bhatt and Topol, JAMA, 284:1549-1558 (2000)); and rNAPc2 - a Tissue Factor Pathway Inhibitor (TFPI) isolated from hookworm (Moons et al., J. Am. Coll. Cardiol., 41 : 2147-2153 (2003)).

For all of the above reasons, it is important to identify antithrombogenic molecules, which block the binding of PAI-I to VN.

SUMMARY OF THE INVENTION

A new family of genes that encode a novel protein containing a cysteine rich domain (CRD) have been discovered (see SEQ ID NOs: 1-4). The first member of this family has been named Ixostatin-1. A second member of the family, which bears 45% total identity and 65% total homology to Ixostatin-1 but greater homology within regions of the molecule, is named Ixostatin-2.

Embodiments described herein include a purified or isolated nucleic acid encoding an Ixostatin-like polypeptide having a cysteine rich domain. Nucleic acids encoding Ixostatins, Ixostatin polypeptides, and fragments of these molecules (e.g., fragments that are less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides or 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, or 122 consecutive amino acids) are also aspects of the invention. Some embodiments also include SEQ. ID. NO: 1, SEQ. ID. NO: 3, SEQ. ID NO: 13, SEQ. ID. NO: 14, SEQ. ID NO: 15, or SEQ. ID.

NO: 16, a sequence complementary thereto, or a fragment thereof (e.g., fragments that are less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides).

Nucleic acids that hybridize to said nucleic acids having the nucleotide sequence selected from the group consisting of: SEQ. ID. NO: 1, SEQ. ID. NO: 3, SEQ. ID NO:

13, SEQ. ID. NO: 14, SEQ. ID NO: 15, and SEQ. ID. NO: 16 or fragments thereof (e.g., fragments that are less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides) under high stringency conditions (e.g., wash conditions with IX SSC and 0.1% SDS at 60 degrees Centigrade) or medium stringency conditions (e.g., wash conditions with IX SSC and 0.1% SDS at 50 degrees Centigrade) are also aspects of the invention. Still further, nucleic acids that share identity or homology to SEQ. ID. NO: 1, SEQ. ID. NO: 3, SEQ. ID NO: 13, SEQ. ID. NO: 14, SEQ. ID NO: 15, or SEQ. ID. NO: 16 or fragments thereof (e.g., fragments that are less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides) such as nucleic acids having greater than or equal to 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% homology or identity to a nucleotide sequence selected from the group consisting of: SEQ. ID. NO: 1, SEQ. ID. NO: 3, SEQ. ID NO: 13, SEQ. ID. NO: 14, SEQ. ID NO: 15, and SEQ. ID. NO: 16 or fragments thereof (e.g., fragments that are less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides) are aspects of the invention.

Other embodiments include Ixostatin-like purified or isolated polypeptides having a cysteine rich domain. Ixostatins, Ixostatin polypeptides, and fragments of these molecules (e.g., fragments that are less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, or 122 consecutive amino acids) are embodiments. Some embodied polypeptides also have an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4 and fragments thereof (e.g., fragments that are less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, or 122 consecutive amino acids).

Antibodies to Ixostatins and fragments of Ixostatin-like polypeptides are also embodiments. These antibodies can be monoclonal or polyclonal. Antibodies capable of specifically binding to a protein comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO: 4 or a fragment thereof (e.g., fragments that are less than, greater than, or equal to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 115, or 122 consecutive amino acids) are embodiments. Desirable antibodies are capable of specifically bind to Ixostatin- 1 protein, but not to Ixostatin-2 protein or vice versa (i.e., a purified or isolated antibody capable of specifically binding Ixostatin-2 protein, but does not specifically bind Ixostatin- 1 protein).

Methods of identifying a binding partner that interacts with Ixostatin- 1 or Ixostatin-2 are also embodiments. By one approach, a support comprising Ixostatin- 1, Ixostatin-2 or a representative fragment thereof (e.g., fragments that are less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 115, or 122 consecutive amino acids) is provided; the support is contacted with a candidate binding partner; and a biological complex comprising Ixostatin-1 or Ixostatin-2; and the candidate binding partner, is detected. The detection of such a complex indicates that said candidate binding partner interacts with Ixostatin-1 or Ixostatin-2. In certain aspects, the support is a microarray substrate, a bead, or a membrane. A computerized system for identifying an agent that interacts with Ixostatin-1 or

Ixostatin-2 is also an embodiment. One embodiment, for example, includes a first data base comprising a protein model of the amino acid sequences set forth in SEQ ID NO. 2 or 4 or a fragment thereof (e.g., fragments that are less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, or 122 consecutive amino acids); a second data base comprising the composition of a plurality of candidate binding partners; a search program that compares the protein model of the first data base with the compositions of the candidate agents of the second database; and a retrieval program that identifies a candidate binding partner that interacts with the protein model of the first database. In some embodiments, the candidate binding partners are selected from the group consisting of: a peptide, a peptidomimetic, and a chemical. Another related embodiment concerns a computer-based system for identifying a target sequence having homology to an Ixostatin molecule. This system includes a database comprising one of the sequences of SEQ ID NOS: 1-4 or a representative fragment thereof (e.g., polypeptide fragments that are less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, or 122 consecutive amino acids or nucleic acid fragments that are less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides); a search program that compares a target sequence to sequences in the database to identify homologous sequence(s), and a retrieval program that obtains said homologous sequence(s). Another way to identify an agent that modulates Ixostatin-mediated activity involves providing a support having an Ixostatin protein or a representative fragment thereof (e.g., fragments that are less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, or 122 consecutive amino acids); contacting the support with a binding partner that binds to the Ixostatin protein or representative fragment thereof (e.g., fragments that are less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100,

1 15, or 122 consecutive amino acids); contacting the support with a candidate agent; and detecting the presence or absence of binding of the binding partner to the Ixostatin protein and thereby identifying the agent as one that modulates Ixostatin-mediated activity. In certain aspects, the support can be, for example, a microarray substrate, a bead, a membrane and the like.

Another embodiment concerns a transgenic animal (e.g., mouse), wherein the Ixostatin-1 or Ixostatin-2 gene or fragment thereof (e.g., fragments that are less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides) is inserted into the genome of the animal. Furthermore, a method for producing a genetically altered animal (e.g., mouse) that exhibits improved fibrinolysis is contemplated. This method is practiced by providing an Ixostatin gene targeting construct comprising an Ixostatin gene; introducing said Ixostatin gene and a selectable marker sequence into an animal's embryonic stem cell (e.g., a mouse); introducing said embryonic stem cell into an animal embryo; transplanting said embryo into a pseudopregnant animal; allowing said embryo to develop to term; identifying a genetically altered animal whose genome comprises an Ixostatin gene in one or both alleles; and breeding the genetically altered animal to obtain a genetically altered animal whose genome comprises an Ixostatin gene, wherein said insertion in said animal exhibits improved fibrinolysis. Preferred Ixostatin nucleic acids for use in these methods include SEQ. ID. NO: 1, SEQ. ID. NO: 3, SEQ. ID NO: 13, SEQ. ID. NO: 14, SEQ. ID NO: 15, or SEQ. ID. NO: 16 or a fragment thereof (e.g., a nucleic acid fragment that is less than, greater than, or equal to

20, 30, 40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides).

Also provided herein are methods of treating or inhibiting progression of a malignant tumor in an animal by selecting or identifying an animal in need of a molecule that treats or inhibts progression of a malignant tumor and providing said animal with a therapeutically effective amount of an Ixostatin polypeptide or fragment thereof or a nucleic acid encoding said molecule (e.g., an Ixostatin polypeptide fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 115, or 122 consecutive amino acids or a nucleic acid fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300,

345, 366, 400, 463, 500, or 523 consectutive nucleotides), or an antibody capable of specifically binding to an Ixostatin protein. Preferred Ixostatin nucleic acids for use in these methods include SEQ. ID. NO: 1, SEQ. ID. NO: 3, SEQ. ID NO: 13, SEQ. ID. NO: 14, SEQ. ID NO: 15, or SEQ. ID. NO: 16 or a fragment thereof (e.g., a nucleic acid fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides) and preferred Ixostatin polypeptides for use in these methods include SEQ ID NOS. 2 and 4 or a fragment thereof (e.g., an Ixostatin polypeptide fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 115, or 122 consecutive amino acids). In certain embodiments, the animal is human. In certain embodiments, the Ixostatin polypeptide is Ixostatin- 1 or Ixostatin-2. In certain embodiments, the malignant tumor is selected from the group consisting of: melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular (liver) carcinoma, thyroid tumor, gastric (stomach) cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer, esophageal carcinoma, head and neck cancers, mesothelioma, sarcomas, biliary

(cholangiocarcinoma), small bowel adenocarcinoma, pediatric malignancies and epidermoid carcinoma. In some aspects of this embodiment, the treatment or inhibition of progression of the malignant tumor is measured, monitored, or analyzed after contact with the Ixostatin polypeptide, fragment thereof or a nucleic acid encoding one or more of said molecules. Such measurements, monitoring, and analysis can be conducted by clinical examination by qualified medical personnel or by diagnostic approaches conventional in the field or as described herein.

Also provided herein are methods of reducing clot formation comprising identifying a subject in need of a reduction in clot formation and providing to said subject a therapeutically effective amount of an Ixostatin or fragment thereof or nucleic acid encoding one or more of these molecules (e.g., an Ixostatin polypeptide fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, or 122 consecutive amino acids or a nucleic acid fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides), or an antibody capable of specifically binding to an Ixostatin protein. Preferred Ixostatin nucleic acids for use in these methods include SEQ. ID. NO: 1, SEQ. ID. NO: 3, SEQ. ID NO: 13, SEQ. ID. NO: 14, SEQ. ID NO: 15, or SEQ. ID. NO: 16 or a fragment thereof (e.g., a nucleic acid fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides) and preferred Ixostatin polypeptides for use in these methods include SEQ ID NOS. 2 and 4 or a fragment thereof (e.g., an Ixostatin polypeptide fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, or 122 consecutive amino acids).

In certain embodiments, the animal is human. In certain embodiments, the Ixostatin polypeptide is Ixostatin- 1, Ixostatin-2, fragments or mutants thereof. In certain embodiments, the method of reducing clot formation can be part of a treatment regimen where an antithrombogenic compound would be used. Nonlimiting examples include coronary thrombosis, pulmonary embolism, myocardial infarction, deep vein thrombosis, cerebral thrombosis, postoperative fibrinolytic shutdown, or rapid thrombogenic actions, which can occur following implantation of a medical device. In some aspects of this embodiment, the reduction in clot formation is measured, monitored, or analyzed after contact with the Ixostatin polypeptide, fragment thereof or a nucleic acid encoding one or more of said molecules. Such measurements, monitoring, and analysis can be conducted by clinical examination by qualified medical personnel or by diagnostic approaches conventional in the field or as described herein. Other embodiments include antithrombogenic medical devices. For example, medical devices, such as stents and catheters, which may include a therapeutically effective amount of an Ixostatin or fragment thereof or nucleic acid encoding one or more of these molecules (e.g., an Ixostatin polypeptide fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, or 122 consecutive amino acids or a nucleic acid fragment that is less than, greater than, or equal to 20, 30,

40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides). Preferred Ixostatin nucleic acids for use in these methods include SEQ. ID. NO: 1, SEQ. ID. NO: 3, SEQ. ID NO: 13, SEQ. ID. NO: 14, SEQ. ID NO: 15, or SEQ. ID. NO: 16 or a fragment thereof (e.g., a nucleic acid fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, or

125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides) and preferred Ixostatin polypeptides for use in these methods include SEQ ID NOS. 2 and 4 or a fragment thereof (e.g., an Ixostatin polypeptide fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, or 122 consecutive amino acids).

Other embodiments include a vector comprising the purified or isolated nucleic acid encoding an Ixostatin or fragment thereof, (e.g., an Ixostatin polypeptide fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, or 122 consecutive amino acids or a nucleic acid fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides). Preferred Ixostatin nucleic acids for use in such vectors include SEQ. ID. NO: 1, SEQ. ID. NO: 3, SEQ. ID NO: 13, SEQ.

ID. NO: 14, SEQ. ID NO: 15, or SEQ. ID. NO: 16 or a fragment thereof (e.g., a nucleic acid fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125, 150, 175, 200, 250, 300, 345, 366, 400, 463, 500, or 523 consectutive nucleotides). In certain embodiments, a cultured cell comprises the vector. Also provided herein are therapeutic anticoagulant formulations comprising an

Ixostatin polypeptide or fragment thereof in combination with a pharmaceutically acceptable carrier. Preferred Ixostatin polypeptides for use in these methods include SEQ ID NOS. 2 and 4 or a fragment thereof (e.g., an Ixostatin polypeptide fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 115, or 122 consecutive amino acids). The therapeutic formulations can further comprise a second antithrombogenic agent, including any of heparin, hirudin, albumin, phospholipids, streptokinase, tissue plasminogen activator (tPA), urokinase (uPA), hydrophilic polymers such as hyaluronic acid, chitosan, methyl cellulose, poly(ethylene oxide), poly(vinyl pyrrolidone), growth factors such as endothelial cell growth factor, epithelial growth factor, osteoblast growth factor, fibroblast growth factor, platelet derived growth factor (PDGF), or an angiogenic growth factor.

Also provided herein is a kit for determining Ixostatin protein expression which includes a probe indicative of Ixostatin protein expression in cells.

Other embodiments include a vaccine for the treatment of animals, comprising an Ixostatin polypeptide or fragment thereof in combination with a pharmaceutically acceptable carrier. Preferred Ixostatin polypeptides for use in these methods include SEQ ID NOS. 2 and 4 or a fragment thereof (e.g., an Ixostatin polypeptide fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 115, or 122 consecutive amino acids).

Other embodiments include use of an Ixostatin polypeptide or fragment thereof in the treatment of a thrombogenic disease. Preferred Ixostatin polypeptides for use in these methods include SEQ ID NOS. 2 and 4 or a fragment thereof (e.g., an Ixostatin polypeptide fragment that is less than, greater than, or equal to 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, or 122 consecutive amino acids). Nonlimiting examples of thrombogenic disease include coronary thrombosis, pulmonary embolism, myocardial infarction, deep vein thrombosis, cerebral thrombosis, postoperative fibrinolytic shutdown, or a rapid thrombogenic action, which can occur following implantation of a medical device. In certain aspects the Ixostatin polypeptide is expressed from a nucleic acid encoding an Ixostatin polypeptide or fragment thereof. In certain aspects the Ixostatin polypeptide is SEQ ID NO: 2 or SEQ ID NO: 4 or a polypeptide encoded by SEQ ID NOs: 1, 3, 13, 14, 15, or 16 or a codon-optimized version thereof. Also provided herein is a method of preventing angiogenesis comprising: identifying a subject in need of angiogenesis prevention and providing to the subject a therapeutically effective amount of a molecule selected from the group consisting of: an Ixostatin polypeptide or fragment thereof, a nucleic acid encoding an Ixostatin polypeptide or fragment thereof, and an antibody capable of specifically binding to an Ixostatin protein. In certain aspects the molecule is an Ixostatin polypeptide or fragment thereof. In certain aspects the molecule is a nucleic acid encoding an Ixostatin polypeptide or fragment thereof. In certain aspects the molecule is SEQ ID NO: 2 or SEQ ID NO: 4 or a polypeptide encoded by SEQ ID NOs: 1, 3, 13, 14, 15, or 16 or a codon-optimized version thereof. Also provided herein is a method of preventing metastasis comprising: identifying a subject in need of a molecule that prevents matastasis and providing said subject with a therapeutically effective amount said molecule, wherein said molecule is selected from the group consisting of: an Ixostatin polypeptide or fragment thereof, a nucleic acid encoding an Ixostatin polypeptide or fragment thereof, and an antibody capable of specifically binding to an Ixostatin protein. In certain aspects the molecule is an Ixostatin polypeptide or fragment thereof. In certain aspects the molecule is a nucleic acid encoding an Ixostatin polypeptide or fragment thereof. In certain aspects the molecule is SEQ ID NO: 2 or SEQ ID NO: 4 or a polypeptide encoded by SEQ ID NOs: 1, 3, 13, 14, 15, or 16 or a codon-optimized version thereof.

Also provided herein is a method for identifying proteins that bind to vitronectin, comprising: providing a homogenate from an arthropod, contacting said homogenate with vitronectin or a fragment thereof and identifying a binding complex indicative of a protein binding to vitronectin. In certain aspects the vitronectin or fragment thereof comprises the somatomedin B (SMTB) domain of vitronectin.

Also provided herein is a purified or isolated polypeptide comprising a cysteine rich domain wherein the cysteine rich domain comprises the conserved pattern CX_IOCX_{I I}CX_I4CX₃CX₂₄CX_SCX₄C, wherein X is any amino acid residue, wherein Xi₀ comprises 1 to 10 residues between the first and second cysteines, Xn comprises 1 to 11 residues between the second and third cysteines, Xi₄ comprises 1 to 14 residues between the third and fourth cysteines, X₃ comprises 1 to 3 residues between the fourth and fifth cysteines, X₂₄ comprises 1 to 24 residues between the fifth and sixth cysteines, X₅ comprises 1 to 5 residues between the sixth and seventh cysteines and X₄ comprises

1 to 4 residues between the seventh and eighth cysteines.

BRIEF DESCRIPTION OF THE DRAWINGS Figures IA-D show the amino acid sequence of Ixostatins and their homology to other proteins. Figure IA shows the sequence homology between Ixostatin-1

(gi22164273) and Ixostatin-2 (gi22164272) with ixostatin-like proteins from tick salivary gland. Figure IB shows the sequence homology between Ixostatin-1 (gi22164273) and Ixostatin-2 (gi22164272) with the cysteine-rich domain of reprolysin from Aedes sp. (ReproAedes, gi 108883454), Anopheles sp. (ReproAnoph, gil l 8779268) reprolysin and human ADAMTS-4 (ADAMTS4, prot no. 075173). In

Figure 1C the unrooted cladogram for the sequences depicted in IA is shown. Figure ID is a diagram showing the similarities between Ixostatin and the cysteine-rich domain of ADAMTS-4 (aggregacanase-1).

Figures 2A-C show the purification and identification of Ixostatin-1 -His. Figure 2A shows the Ixostatin-1 -His containing fraction after purification by reverse phase HPLC. Figure 2B shows a coomassie blue stained gel with a single band corresponding to purified Ixostatin-1 -His. The Edman sequence (SEQ ID NO: 12) of the N-terminus of Ixostatin-1-His is shown. Figure 2C shows the mobility by Western blot of Ixostatin-1-His under non-reducing (NR) and reducing (R) conditions.

Figures 3A-B are graphs showing the kinetics of Ixostatin-1 with various matrix proteins, as exhibited by surface Plasmon resonance. Figure 3A shows kinetics of Ixostatin-1 binding to vitronectin. Response of immobilized vitronectin (in relative units (RU) to increasing concentrations of Ixostatin-1 is shown as a function over time. Ixostatin-1 concentrations are (from top to bottom): 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.1 nM, 1.5 nM, 0.75 nM. Figure 3B shows Plasmon resonance experiments of Ixostatin-1 (50 nM) with immobilized collagen, fibronectin or von Willebrandt factor.

Figure 4 is a graph demonstrating that Ixostatin modulates in vitro fibrinolysis. Calcium was added to plasma in the presence of tPA (2nM) in the absence or presence of Ixostatin-1 (100 nM). Fibrinolysis did not take place with plasma alone or plasma in the presence of Ixostatin. Figure 5 is a plot showing that Ixostatin inhibits MVEC adhesion to vitronectin.

Ixostatin-1 (10 and 100 nM) was incubated with vitronectin for 30 minutes, followed by addition of calcein-labeleed MVEC. Fluorescence of adherent cells is shown as a function of Ixostatin concentration.

Figure 6 shows the molecular characterization of Ixostatin-2 and identification in the /. scapulaήs saliva. (A) Purification of recombinant Ixostatin-2 after vitronectin- affinity column and reverse-phase columns as described in Methods section. The shaded bar indicates the salivary fractions that are positive for Ixostatin-2 binding to vitronectin, by SPR. (B) NU-PAGE of purified Ixostatin-2 under denaturating and reducing conditions. (C) Plasmon resonance experiments: Ixostatin-2 (15 nM) interacts with monomeric (or multimeric vitronectin, not shown) but not with collagen, laminin, fibronectin, von Willebrand Factor, and fibrinogen. (D) Reverse phase of saliva (solid line) and identification of the active fraction that binds to vitronectin (circle). The green bar indicates the salivary fractions that are positive for Ixostatin-2 according to an ELISA. The elution pattern of recombinant Ixostatin-2 activity (measured by SPR) chromatographed under identical conditions as saliva, is superimposed (square). (E) and

(F), saliva respectively interacts with both monomeric and multimeric vitronectin at different dilutions: a, 1:50; b, 1:100; c, 1:200; d, 1 :400; e, 1:1,000. Figure 7 shows kinetics of Ixostatin-2 interaction with vitronectin. Typical sensorgrams of Ixostatin-2 interaction with (A) monomeric human, and (B) multimeric human vitronectin. Different concentrations of recombinant Ixostatin-2 (in nM: a, 20; b, 10; c, 5; d, 2.5; and e, 1.25) were injected over immobilized vitronectin for 180 s. Dissociation of Ixostatin-2-vitronectin complex was monitored for 2000 s, and a 1 : 1 binding model was used to calculate kinetic parameters. Sensorgrams are representative experiments, and the results are summarized in Table 1.

Figure 8 shows Ixostatin-2 binds to the Somatomedin (SMTB) domain. (A) SMTB was chemically synthesized and purified by reverse-phase chromatography. The inset shows the mass spectrometry for the synthetic peptide. (B) PAI-I was injected at different concentrations (in nM: a, 1.8; b, 0.9; c 0.45; d, 0.225; e, 0.11) to the SMTB domain for 180 sec, followed by 2000 sec dissociation time. (C) Ixostatin-2 was injected to the SMTB domain as above at the following concentrations (in nM: a, 20; b, 10; c, 5; d, 2.5; and e, 1.25) (D) Saliva displays SMTB-binding properties. Saliva was injected as above at the following dilutions: a, 1 :50; b, 1 :100; c, 1 :200; d, 1 :400; and e,

1:1,000.

Figure 9 shows the purification and kinetics of bacterial Ixostatin-2 interaction with vitronectin. (A) Ixostatin-2 was purified in a reverse phase column and the active fraction submitted for mass spectrometry. The ml wt obtained for the folded peptide was similar to the theoretical mass for folded Ixostatin-2 containing an extra methionine. Ixostatin-2 was loaded in a 4-12% NU-PAGE gel (inset), transferred to PVDF and the Edman degradation confirmed the identity of the peptide. T ypical sensorgrams of Ixostatin-2 interaction with (B) multimeric human vitronectin, or (C) Somatomedin B (SMTB). Different concentrations of recombinant Ixostatin-2 (in nM: a, 25; b, 12.5; c, 6.25; and d, 3.1) were injected over immobilized collagen for 180 s.

Dissociation of Ixostatin-2-vitronectin complex was monitored for 2000 s. Sensorgrams are representative experiments.

Figure 10 sh ows that Ixostatin-2 prevents cell adhesion to vitronectin, but display negligible effects on fibrinolysis. (A) integrin αvβ3-expressing endothelial cell or (B) u-PAR bearing U937 cells were incubated with vitronectin for 90 and 120 min, respectively, in presence of Ixostatin-2 at the indicated concentrations. In controls, wells were coated with BSA only. One hundred % adhesion was estimated in the absence of Ixostatin-2, and 0% adhesion was obtained in the presence of (A) 1 mM cyclic RGDS or (B) 1 μM PAI-I. (C) Ixostatin-2 does not affect fibrinolysis. Ixostatin-2 (tracings b-d, 0-100 nM) was incubated with plasma for 3 hours followed by activation of the coagulation cascade with 10 mM CaCl₂, in the presence of dc-tPA (0.6 nM). Tracing a, no dc-TPA was added (n=3). Typical experiments are shown in = 3).

Figure 11 is a schematic diagram of the Ixostatin-2 interaction with the SMTB domain of vitronectin. Through interaction with SMTB, Ixostatin-2 prevents integrin αvβ3 and uPAR-dependent cell adhesion to vitronectin, potentially inhibiting angiogenesis and related events such as wound healing and tissue repair.

DETAILED DESCRIPTION OF THE INVENTION

A new family of structurally distinctive proteins, designated Ixostatins, has been discovered. Two members of this family have been identified, cloned, sequenced, and characterized. These proteins can be identified by their cysteine rich domain (CRD). Several embodiments of the invention have biotechnological, diagnostic, and therapeutic use. For example, the nucleic acids of the invention and/or proteins of the invention can be used as probes to isolate more Ixostatins, detect the presence of wild type or mutant Ixostatins in various tissues, and can be incorporated into constructs for preparing recombinant Ixostatin proteins or can be expressed from such constructs. The sequences of the nucleic acids of the invention and/or proteins of the invention can also be incorporated into computer systems, used with modeling software so as to enable some forms of rational drug design. The nucleic acids of the invention and/or proteins of the invention, as well as, the binding partners of the invention, can be incorporated into pharmaceuticals and used for the treatment of cell adhesion fibrinolytic disorders. The nucleic acid embodiments of the invention include nucleotides encoding

Ixostatin molecules and fragments thereof and variants such as spliced variants, allelic variants, synonomous sequences, and homologous or orthologous molecules. Some embodiments for example, include genomic DNA, RNA, and cDNA encoding Ixostatins. The nucleic acid embodiments of the invention also include partial or complete DNA sequences shown in the sequence listing (SEQ. ID. NOS.: 1, 3, 13, 14, 15, and 16), nucleotide sequences encoding the amino acid sequences shown in the sequence listing (SEQ. ID. NOS.: 2 and 4) and complements thereof. Nucleic acid sequences encoding Ixostatins from other organisms are also embodiments, as are methods for obtaining such sequences. The nucleic acid embodiments can be altered, mutated, or changed such that the alteration, mutation, or change results in a conservative amino acid replacement. The nucleic acid embodiments can also be altered, mutated, or changed such that the alteration, mutation, or change results in a non-conservative amino acid replacement. Some embodiments of the invention, for example, include nucleic acids encoding Ixostatin molecules that have one or more of the Ixostatin domains deleted or combined in a novel fashion so as to create an "Ixostatin-like hybrid" molecule. Further, some embodiments relate to nucleic acids encoding Ixostatin-like hybrids having multimerized domains, synthetic domains, and domains from other proteins.

Some polypeptide embodiments include partial or complete amino acid sequences shown in the sequence listing (SEQ. ID. NOS.: 2 and 4) and functional equivalents to such molecules including, but not limited to, the polypeptides of SEQ. ID. NOS. 2 and 4 having non-conservative amino acid substitutions and peptidomimetics that resemble these molecules. Additional polypeptide embodiments include mutant Ixostatins having nonconservative amino acid replacements, in particular mutants that result in gain or loss of Ixostatin function. Further, the polypeptide embodiments include Ixostatin-like hybrids having one or more of the Ixostatin domains deleted or combined in a novel fashion or multimerized domains, synthetic domains, and domains from other proteins. Polypeptides that are homologous to Ixostatin- 1 and/or Ixostatin-2 are also embodiments and methods of obtaining such molecules are provided. Additionally, methods of preparing the polypeptide embodiments by chemical synthesis and recombinant techniques are disclosed. Approaches to creating genetically altered organisms that express either a wild-type or mutant Ixostatin transgene (i.e. Ixostatin transgenic or knockout animals) are also provided.

Several embodiments also include antibodies that recognize wild-type and mutant Ixostatins. Approaches to manufacture monoclonal and polyclonal antibodies are disclosed.

Approaches to rational drug design are also provided, and these methods can be used to isolate new Ixostatin family members and to identify molecules that interact with the Ixostatins, referred to as "binding partners". Several computer-based methodologies are discussed, which involve three-dimensional modeling of the Ixostatin nucleic acid and/or protein sequences and the nucleic acid and protein sequences encoding known or suspected binding partners (e.g., antibodies and vitronectin or vitronectin somatomedin domains).

Ixostatin characterization assays are also described. These assays test the functionality of an Ixostatin molecule and identify binding partners that interact with the

Ixostatins. Some functional assays involve the use of multimeric Ixostatins and/or binding partners, which are Ixostatins, hybrids, or binding partners disposed on a support, such as a resin, bead, lipid vesicle or cell membrane. These multimeric agents are contacted with candidate binding partners and the association of the binding partner with the multimeric agent is determined. Successful binding agents can be further analyzed for their effect on

Ixostatin function by using cell based assays. One such assay evaluates the effect of Ixostatins, hybrids, and binding partners on the activation of mitogen activated kinase, RAS, or the phosphorylation of myelin basic protein. Other Ixostatin characterization assays involve molecular biology techniques designed to identify protein-protein interactions (e.g., two-hybrid systems).

Several pharmaceutical embodiments described herein include medicaments that contain Ixostatins, Ixostatin-like hybrids, and binding partners, which interact with Ixostatins. These medicaments can be prepared in accordance with conventional methods of galenic pharmacy for administration to organisms in need of treatment. A therapeutically effective amount of an Ixostatin molecule, Ixostatin-like hybrid molecule, or a binding partner of Ixostatin can be incorporated into a pharmaceutical composition with or without a carrier. Routes of administration of the pharmaceuticals of the invention include, but are not limited to, topical, transdermal, parenteral, gastrointestinal, transbronchial, and transalveolar. These pharmaceuticals can be provided to organisms in need of treatment for cell adhesion and fibrinolytic disorders.

The section below describes several of the nucleic acid embodiments of the invention.

Nucleic acids encoding Ixostatins and Ixostatin-like hybrids

A new family of structurally distinctive molecules, designated Ixostatins, has been discovered. These molecules can be identified by their cysteine rich domain

(CRD). Several nucleic acid embodiments described herein include nucleotides encoding

Ixostatin molecules and fragments thereof and variants, such as spliced variants, allelic variants, synonomous sequences, and homologous or orthologous molecules. Some embodiments for example, include genomic DNA, RNA, and cDNA encoding Ixostatins. Ixostatins can be present in many different organisms including but not limited to plants, insects, animals, and mammals. Further, molecules that resemble Ixostatins by the organization of their structure (e.g., a molecule having a CRD) and hybrid molecules having one or more of the aforementioned domains are embodiments of the invention.

The discovery of Ixostatin-1 and Ixostatin-2 was made while examining sequences of clones generated from a cDNA library from /. scapularis (tick) saliva. {See Example 1). The coding sequence of Ixostatin-1 cDNA and Ixostatin-1 protein are provided in the Sequence Listing (SEQ. ID. NOS. 1 and 2), respectively. A BLAST search using Ixostatin-1 did yield a variety of tick proteins and putative proteins related to Ixostatin-1.

The BLAST using tick Ixostatin-1 also revealed another Ixostatin family member, designated Ixostatin-2. (FIGURE 1). The coding sequence of Ixostatin-2 cDNA and Ixostatin-2 protein are provided in the Sequence Listing (SEQ. ID. NOS.: 3 and 4), respectively. Ixostatin-1 and Ixostatin-2 display approximately 45% amino acid identity overall, however, conservation is appreciably higher in specific sub-regions of the gene.

Data presented, infra, demonstrate that: Ixostatin-1 is a low molecular weight, approximately 9kDa protein that can exist as a dimer (Example 2). In addition,

Ixostatin-1 and Ixostatin-2 display remarkably tight interaction to the somatomedin B (STMB) domain of vitronectin (Example 3). It was also found that Ixostatin-1 accelerates clot dissolution initiated by tPA (Example 4) and can inhibit endothelial cell adhesion to vitronectin (Examples 5 and 14). The Ixostatin nucleotide sequences also include: (a) the DNA sequences shown in the sequence listing (SEQ. ID. NOS.: 1, 3, 13, 14, 15, and 16); (b) nucleotide sequences encoding the amino acid sequences shown in the sequence listing (SEQ. ID. NOS.: 2 and 4); (c) any nucleotide sequence that hybridizes to the complement of the DNA sequences shown in the sequence listing (SEQ. ID. NOS.: 1, 3, 13, 14, 15, and 16) under stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄,

7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 50°C and washing in 0.2 X SSC/0.2% SDS at 50°C; and (d) any nucleotide sequence that hybridizes to the complement of the DNA sequences that encode an amino acid sequence provided in the sequence listing (SEQ. ID. NOS.: 2 AND 4) under less stringent conditions (e.g., hybridization in 0.5 M NaHPO₄, 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 37°C and washing in 0.2X SSC/0.2% SDS at 37°C. Embodiments also include Ixostatins that are isolated from other organisms

(e.g., plants, molds, yeast, insects, animals, and mammals) and mutant Ixostatins, whether naturally occurring or engineered. Approaches to isolate Ixostatin homologs in other species are provided infra. Embodiments also include fragments, modifications, derivatives, and variants of the sequences described above. Desired embodiments, for example, include nucleic acids having at least 9 consecutive nucleotides of an Ixostatin or a sequence complementary thereto and preferred fragments of the invention include at least 9 consecutive nucleotides of Ixostatin- 1 or Ixostatin-2 or a sequence complementary thereto. In this regard, the nucleic acid embodiments of the invention can have from 9 to approximately 523 consecutive nucleotides for Ixostatin- 1 and from 9 to approximately 463 nucleotides for Ixostatin-2. Some DNA fragments of the invention, for example, comprise, consist, or consist essentially of a nucleic acid with less than or equal to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,

100, 125, 150, 175, 200, 250, 300, 350, 400, 500, and 523 consecutive nucleotides of an Ixostatin gene (e.g., a sequence of SEQ. ID. NOS.: 1, 3, 13, 14, 15, and 16 or a complement thereof). Preferably, the nucleic acid embodiments, however, comprise at least 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleotides of a sequence of SEQ. ID. NOS.: 1, 3, 13, 14, 15, and 16 or complement thereof. More preferably, the nucleic acid embodiments comprise at least 20-30 consecutive nucleotides or complement thereof.

The nucleic acid embodiments can also be altered by mutation such as substitutions, additions, or deletions that provide for sequences encoding functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same Ixostatin amino acid sequence as depicted in SEQ. ID. NOS.: 2 and 4 can be used in some embodiments of the present invention. These include, but are not limited to, nucleic acid sequences comprising all or portions of Ixostatin-1 or Ixostatin-2 or nucleic acids that complement all or part of Ixostatin-1 or Ixostatin-2 that have been altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change, or a functionally non-equivalent amino acid residue within the sequence, thus producing a detectable change.

The mutant Ixostatin nucleic acids also include nucleic acids encoding Ixostatin polypeptides or peptides having a non-conservative change that affects the functionality of the molecule (e.g. modulates fibrinolysis or cell adhesion). Additional mutant Ixostatins include nucleic acids encoding molecules in the Ixostatin CRD domain is deleted. Further, some Ixostatin mutant nucleic acids encode one or more Ixostatin domains combined in a novel fashion so as to create an "Ixostatin-like hybrid" molecule, also referred to as a "hybrid". These hybrids can be used to modulate (i.e., inhibit or enhance) fibrinolysis or cell adhesion, for example. Some nucleic acids also encode multimerized Ixostatins or hybrids, which are characterized by a structure having at least two of the same domain (e.g., a hybrid having two CRD domains).

Several assays can be employed to evaluate these molecules for their ability to modulate fibrinolysis or cell adhesion and many are discussed in detail infra. The Ixostatin-like hybrids that are identified for their ability to modulate fibrinolysis or cell adhesion can be used in biotechnological assays and can be formulated in pharmaceuticals for the treatment of diseases and abnormalities in various organisms.

Some mutant Ixostatin nucleic acid embodiments include nucleic acids encoding Ixostatin-like hybrids, wherein one or more regions of the protein are swapped with synthetic polypeptides. For example, nucleic acids encoding the Ixostatin-1 CRD can be joined to a nucleic acid encoding a synthetic hydrophobic domain (e.g., poly-leucine) so as to create a reagent that better associates with a membrane. Similarly, the nucleic acids encoding the various domains of Ixostatin-1 or Ixostatin-2 can be swapped with nucleic acids encoding domains from other proteins (besides Ixostatins) involved in fibrinolysis or cell adhesion. In this manner, many different nucleic acids encoding designer peptides can be created and these molecules can be used to modulate specific cellular events, for example. The nucleic acid sequences described above also have biotechnological and diagnostic use, e.g., in nucleic acid hybridization assays, Southern and Northern Blot analysis, etc. By using the Ixostatin nucleic acid sequences disclosed herein (e.g., SEQ. ID. NOS.: 1, 3, 13, 14, 15, and 16), probes that complement Ixostatin- 1 or Ixostatin-2 can be designed and manufactured by oligonucleotide synthesis. Desirable probes comprise a nucleic acid sequence of (SEQ. ID. NOS.: 1, 3, 13, 14, 15, and 16) that is unique to Ixostatins with preferred probes comprising a nucleic acid fragment of (SEQ. ID. NOS.:

1, 3, 13, 14, 15, and 16) that is unique to Ixostatin- 1 or Ixostatin-2. These probes can be used to screen cDNA or genomic libraries from various organisms (e.g., plants, molds, fungi, yeast, insects, animals, and mammals) so as to isolate natural sources of the nucleic acid embodiments described herein. Screening can be by filter hybridization, for example, using duplicate filters. The labeled probe preferably contains at least 15-

30 base pairs of a nucleic acid sequence of (SEQ. ID. NOS.: 1, 3, 13, 14, 15, and 16) that are unique to Ixostatin- 1 or Ixostatin-2. The hybridization washing conditions used are preferably of a lower stringency when the cDNA library is derived from an organism different from the type of organism from which the labeled sequence is originated.

With respect to the cloning of an Ixostatin- 1 or Ixostatin-2 homolog, using murine Ixostatin- 1 or Ixostatin-2 probes, for example, hybridization can be performed in 0.5M NaHPO₄, 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 37°C overnight and washing can be performed in 0.2X SSC/0.2% SDS at 37°C. Various stringency conditions are well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N. Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.

Further, sequences from nucleic acids complementing Ixostatin- 1 or Ixostatin-2, or portions thereof, can be used to make oligonucleotide primers by conventional oligonucleotide synthesis for use in isolation and diagnostic procedures that employ the Polymerase Chain Reaction (PCR) or other enzyme-mediated nucleic acid amplification techniques. An Ixostatin gene homolog can be isolated from a nucleic acid of the organism of interest by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the Ixostatin gene products disclosed herein. The template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from, for example, cells or tissue of an organism known or believed to express an Ixostatin. A variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see Molecular Cloning to Genetic Engineering White, B.A. Ed. in Methods in Molecular Biology 67: Humana

Press, Totowa (1997), the disclosure of which is incorporated herein by reference in its entirety and the publication entitled "PCR Methods and Applications" (1991, Cold Spring Harbor Laboratory Press), the disclosure of which is incorporated herein by reference in its entirety. For amplification of mRNAs, it is within the scope of the invention to reverse transcribe mRNA into cDNA followed by PCR (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Patent No. 5,322,770, the disclosure of which is incorporated herein by reference in its entirety. Another technique involves the use of Reverse Transcriptase Asymmetric Gap Ligase Chain Reaction (RT-AGLCR), as described by Marshall R.L. et al. (PCR Methods and Applications 4:80-84, 1994), the disclosure of which is incorporated herein by reference in its entirety. Briefly, RNA is isolated, following standard procedures, from an appropriate cellular or tissue source. A reverse transcription reaction is performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment as a primer of first strand synthesis. The resulting RNA/DNA hybrid is then "tailed" with guanines using a standard terminal transferase reaction. The hybrid is then digested with RNAse H, and second strand synthesis is primed with a poly-C primer. Thus, cDNA sequences upstream of the amplified fragment are easily isolated. For a review of cloning strategies which can be used, see e.g., Sambrook et al., 1989, supra. In each of these amplification procedures, primers on either side of the sequence to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase, such as Taq polymerase, PfU polymerase, or Vent polymerase. The nucleic acid in the sample is denatured and the primers are specifically hybridized to complementary nucleic acid sequences in the sample. The hybridized primers are then extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites. PCR has further been described in several patents including US Patents 4,683,195, 4,683,202 and 4,965,188, the disclosure of which is incorporated herein by reference in their entirety.

The primers are selected to be substantially complementary to a portion of the nucleic acid sequence of (SEQ. ID. NOS.: 1, 3, 13, 14, 15, and 16) that is unique to Ixostatin-1 or Ixostatin-2, thereby allowing the sequences between the primers to be amplified. Preferably, primers are 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 nucleotides in length. The formation of stable hybrids depends on the melting temperature (Tm) of the DNA. The Tm depends on the length of the primer, the ionic strength of the solution and the G+C content. The higher the G+C content of the primer, the higher is the melting temperature because G:C pairs are held by three H bonds whereas A:T pairs have only two. The G+C content of the amplification primers of the present invention preferably ranges between 10 and 75 %, more preferably between 35 and 60 %, and most preferably between 40 and 55 %. The appropriate length for primers under a particular set of assay conditions can be empirically determined by one of skill in the art.

The spacing of the primers relates to the length of the segment to be amplified. In the context of the present invention, amplified segments carrying nucleic acid sequence encoding fragments of Ixostatin-1 or Ixostatin-2 can range in size from at least about 25 bp to 35 kb. Amplification fragments from 25-1000 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp are highly preferred. It will be appreciated that amplification primers can be of any sequence that allows for specific amplification of a region of an Ixostatin and can, for example, include modifications such as restriction sites to facilitate cloning.

The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of an Ixostatin gene. The PCR fragment can then be used to isolate a full length cDNA clone by a variety of methods. For example, the amplified fragment can be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library. Alternatively, the labeled fragment can be used to isolate genomic clones via the screening of a genomic library. The identification and characterization of genomic clones from many different organisms (particularly humans) is helpful for designing diagnostic tests and clinical protocols for treating and preventing aberrations or diseases involving defects in fibrinolysis or cell adhesion. For example, sequences derived from regions adjacent to the intron/exon boundaries of human Ixostatin genes can be used to design primers for use in amplification assays to detect mutations within the exons, introns, splice sites (e.g. splice acceptor and/or donor sites), etc., that can be used in diagnostics. The Ixostatin gene sequences can additionally be used to isolate mutant Ixostatin gene alleles. Such mutant alleles can be isolated from organisms either known or proposed to have a genotype that contributes to a disorder involving aberrant fibrinolysis or cell adhesion. Mutant alleles and mutant allele products can then be utilized in the therapeutic and diagnostic systems described below. Additionally, such Ixostatin gene sequences can be used to detect Ixostatin gene regulatory (e.g., promoter or promotor/enhancer) defects that can affect fibrinolysis or cell adhesion.

A cDNA of a mutant Ixostatin gene can be isolated, for example, by using PCR. In this case, the first cDNA strand can be synthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolated from tissue known or suspected to be expressed in an individual putatively carrying the mutant Ixostatin allele, and by extending the new strand with reverse transcriptase. The second strand of the cDNA is then synthesized using an oligonucleotide that hybridizes specifically to the 5' end of the normal gene. Using these two primers, the product is then amplified via PCR, cloned into a suitable vector, and organismed to DNA sequence analysis through methods well known to those of skill in the art. By comparing the DNA sequence of the mutant Ixostatin allele to that of the normal Ixostatin allele, the mutation(s) responsible for the loss or alteration of function of the mutant Ixostatin gene product can be ascertained.

Alternatively, a genomic library can be constructed using DNA obtained from an organism suspected of or known to carry the mutant Ixostatin allele, or a cDNA library can be constructed using RNA from a tissue known, or suspected, to express the mutant Ixostatin allele. The normal Ixostatin gene or any suitable fragment thereof can then be labeled and used as a probe to identify the corresponding mutant Ixostatin allele in such libraries. Clones containing the mutant Ixostatin gene sequences can then be purified and subjected to sequence analysis according to methods well known to those of skill in the art.

Additionally, an expression library can be constructed utilizing cDNA synthesized from, for example, RNA isolated from a tissue known, or suspected, to express a mutant Ixostatin allele in an organism suspected of, or known to carry, such a mutant allele. In this manner, gene products made by the putatively mutant tissue can be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against the normal Ixostatin gene product. (For screening techniques, see, for example, Harlow, E. and Lane, eds., 1988, Antibodies: A

Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor.) For instance, the anti-Ixostatin-1 antibody described in Example 5 was found to cross-react with amphibian Ixostatin- 1. By using conventional antibody screening techniques and the anti-Ixostatin-1 antibody described in Example 5, one can isolate Ixostatin- 1 from expression libraries of various organisms. In cases where an Ixostatin mutation results in an expressed gene product with altered function (e.g., as a result of a missense or a frameshift mutation), a polyclonal set of antibodies against Ixostatin are likely to cross- react with the mutant Ixostatin gene product. Library clones detected via their reaction with such labeled antibodies can be purified and subjected to sequence analysis according to methods well known to those of skill in the art.

The invention also encompasses (a) DNA vectors that contain any of the foregoing Ixostatin coding sequences and/or their complements (i.e., antisense or RNAi vectors); (b) DNA expression vectors that contain any of the foregoing Ixostatin coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences; and (c) genetically engineered host cells that contain any of the foregoing Ixostatin coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell. These recombinant constructs are capable of replicating autonomously in a host cell. Alternatively, the recombinant constructs can become integrated into the chromosomal DNA of a host cell. Such recombinant polynucleotides typically comprise an Ixostatin genomic or cDNA polynucleotide of semi-synthetic or synthetic origin by virtue of human manipulation. Therefore, recombinant nucleic acids comprising Ixostatin sequences and complements thereof that are not naturally occurring are provided by embodiments of this invention. Although nucleic acids encoding an Ixostatin or nucleic acids having sequences that complement an Ixostatin gene as they appear in nature can be employed, they will often be altered, e.g., by deletion, substitution, or insertion and can be accompanied by sequence not present in humans. As used herein, regulatory elements include, but are not limited to, inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression. Such regulatory elements include, but are not limited to, the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3- phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast α-mating factors. In addition, recombinant Ixostatin-encoding nucleic acid sequences and their complementary sequences can be engineered so as to modify processing or expression of the Ixostatin. For example, and not by way of limitation, the Ixostatin-1 or Ixostatin- 2 gene can be combined with a promoter sequence and/or ribosome binding site, or a signal sequence can be inserted upstream of Ixostatin-encoding sequences to permit secretion of the Ixostatin and thereby facilitate harvesting or bioavailability.

Additionally, a given Ixostatin-1 or Ixostatin-2 nucleic acid can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction sites or destroy preexisting ones, or to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site- directed mutagenesis. (Hutchinson et al., J. Biol. Chem., 253:6551 (1978), herein incorporated by reference).

Further, nucleic acids encoding other proteins or domains of other proteins can be joined to nucleic acids encoding an Ixostatin so as to create a fusion protein. Nucleotides encoding fusion proteins can include, but are not limited to, a full length

Ixostatin, a truncated Ixostatin or a peptide fragment of an Ixostatin fused to an unrelated protein or peptide, such as for example, a transmembrane sequence, which better anchors the Ixostatin peptide fragment to the cell membrane; an Ig Fc domain which increases the stability and half life of the resulting fusion protein (e.g., Ixostatin- Ig); or an enzyme, fluorescent protein, luminescent protein which can be used as a marker (e.g., an Ixostatin-Green Fluorescent Protein ("Ixostatin-GFP") fusion protein). The fusion proteins are useful as biotechnological tools or pharmaceuticals or both, as wall be discussed infra. The section below describes several of the polypeptides of the invention and methods of making these molecules.

Ixostatin polypeptides Ixostatins, Ixostatin polypeptides, fragments of these molecules, and chemicals that resemble these molecules including, but not limited to peptidomimetics, modified Ixostatins, and derivatives or variants of Ixostatins are also embodiments. Ixostatin polypeptides can be present either naturally or through genetic engineering in a number of organisms (e.g., plants, insects, amphibians, reptiles, birds, other animals, cats, dogs, rodents, primates, humans, and other mammals). The Ixostatin family members have a novel structure that contains a CRD.

Both Ixostatin- 1 or Ixostatin-2 has a cleaved, N-terminal signal peptide that allows for insertion into membranes via a conventional ER-to-Golgi routing (Figure IA, underlined). Ixostatin-1 and Ixostatin-2 also have all of the critically conserved amino acid motifs that are diagnostic of CRDs and exhibit the correct predicted spacing of the conserved features of CRD secondary structure (Figure 1C).

The nucleic acids encoding an Ixostatin or fragments thereof, described in the previous section, can be manipulated using conventional techniques in molecular biology so as to create recombinant constructs that express Ixostatin protein or fragments of Ixostatin protein. The Ixostatin polypeptides or derivatives thereof, include but are not limited to, those containing as a primary amino acid sequence all of the amino acid sequence substantially as depicted in the Sequence Listing (SEQ. ID. NOS.: 2 and 4) and fragments of SEQ. ID. NOS.: 2 and 4 at least three amino acids in length including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. Preferred fragments of a sequence of SEQ. ID. NOS.: 2 and 4 are at least three amino acids and comprise amino acid sequence unique to Ixostatins including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. The Ixostatin peptide fragments can comprise, consist, or consist essentially of peptides that are less than or equal to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,

19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 1OQ 115, or 121 amino acids in length.

Embodiments encompass proteins that are functionally equivalent to the Ixostatins encoded by the nucleotide sequences described in SEQ. ID. NOS.: 2 and 4, as judged by any of a number of criteria, including but not limited to the ability to bind vitronectin, the binding affinity for a particular matrix protein, the resulting biological effect of Ixostatin interaction, e.g., change in fibrinolysis, cell adhesion or change in PAI-I levels. Such functionally equivalent Ixostatins include, but are not limited to, additions or substitutions of amino acid residues within the amino acid sequence encoded by the Ixostatin nucleotide sequences described above but, which result in a silent change, thus producing a functionally equivalent gene product. For example, embodiments include Ixostatins that have one or more amino acid residues within the Ixostatin polypeptide of SEQ. ID. NOS.: 2 and 4 and fragments of SEQ. ID. NOS.: 2 and 4 that are substituted by another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. The aromatic amino acids include phenylalanine, tryptophan, and tyrosine.

Additional embodiments include mutant Ixostatins (e.g., Ixostatin-1 and Ixostatin-2), wherein one or more amino acid residues within the Ixostatin polypeptide of SEQ. ID. NOS.: 2 and 4 and fragments of SEQ. ID. NOS.: 2 and 4 are substituted by another amino acid resulting in a non-conservative change. While random mutations can be made to Ixostatin-1 or Ixostatin-2 DNA (using random mutagenesis techniques well known to those skilled in the art) and the resulting mutant Ixostatins tested for activity, site-directed mutations of the Ixostatin-1 or Ixostatin-2 coding sequence can be engineered (using site-directed mutagenesis techniques well known to those skilled in the art) to generate mutant Ixostatins with increased function, e.g., higher binding affinity for a specific matrix protein, and/or greater fibrinolysis promotion capability; or decreased function, e.g., lower binding affinity for a particular matrix protein, and/or decreased fibrinolysis promotion capability.

For example, a comparison of the Ixostatin cysteine rich domain (CRD) to the most closely related CRDs in public databases is shown in Figures IB and C and the amino acid identities are indicated in black and the conservative substitutions are indicated in gray. Mutant Ixostatins can be engineered so that regions of amino acid identity and conservation (indicated in black and gray in Figures IB and C) are maintained, whereas the variable residues are altered, e.g., by deletion or insertion of an amino acid residue(s) or by substitution of one or more different amino acid residues.

Non-conservative changes can be engineered at these variable positions to alter function, e.g., vitronectin binding affinity or fibrinolysis promotion capability, or both. Alternatively, where alteration of function is desired, deletion or non-conservative alterations of the conserved regions (indicated in black and gray in Figure 2) can be engineered. For example, deletion or non-conservative alterations (substitutions or insertions) of amino acid residues in regions of the polypeptide can be engineered to produce a mutant Ixostatin that binds vitronectin but prevents PAI-I dependent fibrinolysis.

Other embodiments include polypeptides that have homology to an Ixostatin and function as a membrane bound modulator of fibrinolysis or cell adhesion. The term

"homology to Ixostatin" is meant to include nucleic acid or protein sequence homology or three-dimensional homology. Several techniques exist to determine nucleic acid or protein sequence homology and/or three-dimensional homology of proteins. These methods are routinely employed to discover the extent of homology that one sequence, domain, or model has to a target sequence, domain, or model. Because the region of

Ixostatin (e.g., a region within a CRD) that modulates fibrinolysis can be quite small (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 22, 25, 30 amino acids in length), embodiments of the invention can exhibit a vast degree of homology to full-length Ixostatin. For example, a fusion protein having a small region of Ixostatin can exhibit a low degree of overall homology to Ixostatin yet retain the ability to function as a modulator of fibrinolysis or cell adhesion equivalent to Ixostatin. Thus, some embodiments can have from 1% homology to 100% homology to full-length Ixostatin. That is, embodiments can comprise, consist, or consist essentially of 1.0%, 2.0%, 3.0%, 4.0%,. 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 21.0%, 22.0%, 23.0%, 24.0%, 25.0%, 26.0%, 27.0%, 28.0%, 29.0%, 30.0%, 31.0%, 32.0%, 33.0%, 34.0%, 35.0%, 36.0%, 37.0%, 38.0%, 39.0%, 40.0%, 41.0%, 42.0%, 43.0%, 44.0%, 45.0%, 46.0%, 47.0%, 48.0%,

49.0%, 50.0%, 51.0%, 52.0%, 53.0%, 54.0%,. 55.0%, 56.0%, 57.0%, 58.0%, 59.0%, 60.0%, 61.0%, 62.0%, 63.0%, 64.0%, 65.0%, 66.0%, 67.0%, 68.0%, 69.0%, 70.0%, 71.0%, 72.0%, 73.0%, 74.0%, 75.0%, 76.0%, 77.0%, 78.0%, 79.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, and 100.0% homology to a full- length Ixostatin (e.g., Ixostatin-1 or Ixostatin-2).

Therefore, embodiments include polypeptides varying in size from 3 amino acids up to and including the full-length Ixostatin protein that have 1% - 100% homology to an Ixostatin and exhibit the ability to function as a membrane-bound modulator of fibrinolysis or cell adhesion. Several homology searching programs based on nucleic acid or amino acid sequence are known in the art and can be used to identify molecules that are homologous to Ixostatin-1 and/or Ixostatin-2. Some approaches to identify molecules homologous to Ixostatins are provided infra.

The Ixostatins and Ixostatin-like hybrids can be prepared by chemical synthesis methods (such as solid phase peptide synthesis) using techniques known in the art such as those set forth by Merrifield et al., J. Am. Chem. Soc. 85:2149 (1964), Houghten et al., Proc. Natl. Acad. Sci. USA, 82:51 :32 (1985), Stewart and Young (Solid phase peptide synthesis. Pierce Chem Co., Rockford, IL (1984), and Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., N. Y. herein incorporated by reference. Such polypeptides can be synthesized with or without a methionine on the amino terminus. Chemically synthesized Ixostatin and fragments of Ixostatin can be oxidized using methods set forth in these references to form disulfide bridges. Ixostatins and fragments of Ixostatin can be employed as biologically active or immunological substitutes for natural, purified Ixostatin and fragments of Ixostatin. While the Ixostatins and hybrids can be chemically synthesized, it can be more effective to produce these polypeptides by recombinant DNA technology using techniques well known in the art. Such methods can be used to construct expression vectors containing the Ixostatin-1 or Ixostatin-2 nucleotide sequences, for example, and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Alternatively, RNA capable of encoding an Ixostatin nucleotide sequences can be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in Oligonucleotide Synthesis, 1984, Gait, M. J. ed., IRL Press, Oxford, which is incorporated by reference herein in its entirety.

In several embodiments, Ixostatins, fragments of Ixostatins, and Ixostatin-like hybrids are expressed in a cell line. For example, some cells are made to express the Ixostatin polypeptide of SEQ. ID. NOS.: 2 and 4 or fragments of SEQ. ID. NOS.: 2 and

4. The sequences, constructs, vectors, clones, and other materials comprising the present invention can advantageously be in enriched or isolated form. As used herein, "enriched" means that the concentration of the material is at least about 2, 5, 10, 100, or 1000 times its natural concentration (for example), advantageously 0.01%, by weight, preferably at least about 0.1% by weight. Enriched preparations from about 0.5%, 1%,

5%, 10%, and 20% by weight are also contemplated. The term "isolated" requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated. It is also advantageous that the sequences be in purified form. The term "purified" does not require absolute purity; rather, it is intended as a relative definition. Isolated proteins have been conventionally purified to electrophoretic homogeneity by Coomassie staining, for example. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.

A variety of host-expression vector systems can be utilized to express the Ixostatins of the invention. Where the Ixostatin or hybrid is a soluble derivative (e.g., hybrids having a truncated or deleted hydrophobic domain) it can be recovered from the culture, i.e., from the host cell in cases where the peptide or polypeptide is not secreted, and from the culture media in cases where the peptide or polypeptide is secreted by the cells. However, the expression systems also encompass engineered host cells that express the Ixostatin or functional equivalents in situ, i.e., anchored in the cell membrane. Purification or enrichment of the Ixostatin from such expression systems can be accomplished using appropriate detergents and lipid micelles and methods well known to those skilled in the art. However, such engineered host cells themselves can be used in situations where it is important not only to retain the structural and functional characteristics of the Ixostatin, but to assess biological activity, e.g., in drug screening assays.

The expression systems that can be used for purposes of the invention include, but are not limited to, microorganisms such as bacteria (e.g., E. coli or B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing Ixostatin nucleotide sequences; yeast (e.g., Saccharomyces, Pi chid) transformed with recombinant yeast expression vectors containing the Ixostatin nucleotide sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the Ixostatin sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing Ixostatin nucleotide sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metal lothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the Ixostatin gene product being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of Ixostatin protein or for raising antibodies to the Ixostatin protein, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J., 2:1791 (1983), in which the Ixostatin coding sequence can be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res., 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem., 264:5503-5509 (1989)); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa calif ornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The Ixostatin gene coding sequence can be cloned individually into non-essential regions (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedrin promoter). Successful insertion of Ixostatin gene coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus, (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed.

(E.g., see Smith et al., J. Virol 46: 584 (1983); and Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the Ixostatin nucleotide sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the Ixostatin gene product in infected hosts. (E.g., See Logan & Shenk, Proc. Natl. Acad. ScL USA 81 :3655-3659 (1984)). Specific initiation signals can also be required for efficient translation of inserted Ixostatin nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire Ixostatin gene or cDNA, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals are needed. However, in cases where only a portion of the Ixostatin coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, should be provided. Furthermore, the initiation codon should be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bittner et al., Methods in Enzymol, 153:516-544 (1987)).

In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products are important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the Ixostatin sequences described above can be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells are allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn are cloned and expanded into cell lines. This method is advantageously used to engineer cell lines which express the Ixostatin gene product. Such engineered cell lines are particularly useful in screening and evaluation of compounds that affect the endogenous activity of the Ixostatin gene product.

A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., Cell 1 1 :223 (1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:2026 (1962), and adenine phosphoribosyltransferase (Lowy, et al., Cell 22:817 (1980) genes can be employed in tk^~, hgprf or aprf cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al., Proc. Natl. Acad.

Sci. USA 77:3567 (1980); O'Hare, et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981); neo, which confers resistance to the aminoglycoside G- 418 (Colberre-Garapin, et al., J. MoI. Biol. 150: 1 (1981); and hygro, which confers resistance to hygromycin (Santerre, et al., Gene 30: 147 (1984)).

Alternatively, any fusion protein can be readily purified by utilizing an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines. (Janknecht, et al., Proc. Natl. Acad. Sci. USA 88: 8972- 8976 (1991)). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. Example 2 provides a more detailed description of methods to express the proteins encoded by the nucleic acids of the invention.

In addition to the naturally occurring Ixostatins or peptide-based hybrids, embodiments include derivative or modified molecules that produce a more desirable cellular response. For example, a derivative Ixostatin can include a polypeptide that has been engineered to have one or more cystine residues incorporated into the protein so as to promote the formation of a more stable derivative through disulfide bond formation.

(See e.g., US Pat. No. 4,908,773). In the past, investigators have employed computers and computer graphics programs to aid in assessing the appropriateness of potential cystine linkage sites. (Perry, L. J., & Wetzel, R., Science, 226:555-557 (1984); Pabo, C.

O., et al., Biochemistry, 25:5987-5991 (1986); Bott, R., et al., European Patent Application Ser. No. 130,756; Perry, L. J., & Wetzel, R., Biochemistry, 25:733-739 (1986); Wetzel, R. B., European Patent Application Ser. No. 155,832). The introduction of a cystine residue in a polypeptide can be accomplished using conventional molecular biology techniques. Additional Ixostatin and hybrid derivatives include peptidomimetics that resemble a polypeptide of interest. The naturally occurring amino acids employed in the biological production of peptides all have the L-configuration. Synthetic peptides can be prepared employing conventional synthetic methods, utilizing L-amino acids, D- amino acids, or various combinations of amino acids of the two different configurations. Synthetic compounds that mimic the conformation and desirable features of a particular peptide, e.g., an oligopeptide, once such peptide has been found, but that avoids the undesirable features, e.g., flexibility (loss of conformation) and bond breakdown are known as a "peptidomimetics". {See, e.g., Spatola, A. F. Chemistry and Biochemistry of Amino Acids. Peptides, and Proteins (Weistein, B, Ed.), Vol. 7, pp. 267-357, Marcel Dekker, New York (1983), which describes the use of the methylenethio bioisostere

[CH₂ S] as an amide replacement in enkephalin analogues; and Szelke et al., In peptides: Structure and Function, Proceedings of the Eighth American Peptide Symposium, (Hruby and Rich, Eds.); pp. 579-582, Pierce Chemical Co., Rockford, 111. (1983), which describes renin inhibitors having both the methyleneamino [CH₂NH] and hydroxy ethylene [CHOHCH₂ ] bioisosteres at the Leu- VaI amide bond in the 6-13 octapeptide derived from angiotensinogen).

In general, the design and synthesis of a peptidomimetic involves starting with the amino acid sequence of the peptide and conformational data (e.g., geometry data, such as bond lengths and angles) of a desired peptide (e.g., the most probable simulated peptide). That data is then used to determine the geometries that should be designed into the peptidomimetic. Numerous methods and techniques are known in the art for performing this step, any of which could be used. {See, e.g., Farmer, P. S., Drug Design, (Aliens, E. J. ed.), Vol. 10, pp. 1 19-143 (Academic Press, New York, London, Toronto, Sydney and San Francisco) (1980); Farmer, et al., in TIPS, 9/82, pp. 362-365; Verber et al., in TINS, 9/85, pp. 392-396; Kaltenbronn et al., in J. Med. Chem. 33: 838-

845 (1990); and Spatola, A. F., in Chemistry and Biochemistry of Amino Acids. Peptides, and Proteins, Vol. 7, pp. 267-357, Chapter 5, "Peptide Backbone Modifications: A Structure-Activity Analysis of Peptides Containing Amide Bond Surrogates. Conformational Constraints, and Relations" (B. Weisten, ed.; Marcell Dekker: New York, pub.) (1983); Kemp, D. S., "Peptidomimetics and the Template Approach to Nucleation of beta-sheets and alpha-helices in Peptides," Tibech, Vol. 8, pp. 249-255 (1990). Additional teachings can be found in U.S. Patent Nos. 5,288,707;

5,552,534; 5,81 1,515; 5,817,626; 5,817,879; 5,821,231; and 5,874,529. The section below describes antibodies of the invention and methods of making these molecules.

Anti-Ixostatin antibodies Following synthesis or expression and isolation or purification of the Ixostatin protein or a portion thereof, the isolated or purified protein can be used to generate antibodies and tools for identifying agents that interact with Ixostatin and fragments of Ixostatin. Depending on the context, the term "antibodies" can encompass polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Antibodies that recognize Ixostatin and fragments of Ixostatin have many uses including, but not limited to, biotechnological applications, therapeutic/prophylactic applications, and diagnostic applications.

For the production of antibodies, various hosts including goats, rabbits, rats, mice, etc. can be immunized by injection with Ixostatin or any portion, fragment or oligopeptide that retains immunogenic properties. Depending on the host species, various adjuvants can be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG {Bacillus Calmette-Gueriή) and Corγnebacterium parvum are also potentially useful adjuvants.

Peptides used to induce specific antibodies can have an amino acid sequence consisting of at least three amino acids, and preferably at least 10 to 15 amino acids. Preferably, short stretches of amino acids encoding fragments of Ixostatin are fused with those of another protein such as keyhole limpet hemocyanin such that an antibody is produced against the chimeric molecule. While antibodies capable of specifically recognizing Ixostatin can be generated by injecting synthetic 3-mer, 10-mer, and 15-mer peptides that correspond to a protein sequence of Ixostatin into mice, a more diverse set of antibodies can be generated by using recombinant Ixostatin, purified Ixostatin, or fragments of Ixostatin.

To generate antibodies to Ixostatin and fragments of Ixostatin, substantially pure Ixostatin or a fragment of Ixostatin is isolated from a transfected or transformed cell. The concentration of the polypeptide in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml. Monoclonal or polyclonal antibody to the polypeptide of interest can then be prepared as follows:

Monoclonal antibodies to Ixostatin or a fragment of Ixostatin can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein {Nature 256:495-497 (1975), the human B-cell hybridoma technique (Kosbor et al. Immunol Today 4:72 (1983); Cote et al Proc Natl Acad Sci 80:2026-2030 (1983), and the EB V-hybridoma technique Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc, New York N. Y., pp 77-96 (1985). In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used. (Morrison et al. Proc Natl Acad Sci 81 :6851-6855 (1984); Neuberger et al. Nature 312:604-608(1984); Takeda et al. Nature 314:452-454(1985). Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce Ixostatin-specific single chain antibodies. Antibodies can also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al., Proc Natl Acad Sci 86: 3833-3837 (1989), and Winter G. and

Milstein C; Nature 349:293-299 (1991).

Antibody fragments that contain specific binding sites for Ixostatin can also be generated. For example, such fragments include, but are not limited to, the F(ab')₂ fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (Huse W. D. et al. Science 256: 1275-1281 (1989)).

By one approach, monoclonal antibodies to Ixostatin or fragments thereof are made as follows. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein or peptides derived therefrom over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused in the presence of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.

Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall, E., Meth. Enzymol. 70:419 (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al.

Basic Methods in Molecular Biology Elsevier, New York. Section 21-2.

Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein or peptides derived therefrom described above, which can be unmodified or modified to enhance immunogenicity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and can require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971).

Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. (See, for example, Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12μM). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980)). Antibody preparations prepared according to either protocol are useful in quantitative immunoassays that determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively (e.g., in diagnostic embodiments that identify the presence of Ixostatin in biological samples). In the discussion that follows, several methods of molecular modeling and rational drug design are described. These techniques can be applied to identify additional Ixostatin family members, compounds that resemble an Ixostatin or fragment or derivative thereof, and molecules that interact with Ixostatins and, thereby modulate their function.

Rational Drug Design Rational drug design involving polypeptides requires identifying and defining a first peptide with which the designed drug is to interact, and using the first target peptide to define the requirements for a second peptide. With such requirements defined, one can find or prepare an appropriate peptide or non-peptide that meets all or substantially all of the defined requirements. Thus, one goal of rational drug design is to produce structural or functional analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, null compounds) in order to fashion drugs that are, for example, more or less potent forms of the ligand. (See, e.g., Hodgson, Bio. Technology 9:19-21 (1991)). An example of rational drug design is shown in Erickson et al., Science 249:527-533 (1990). Combinatorial chemistry is the science of synthesizing and testing compounds for bioactivity en masse, instead of one by one, the aim being to discover drugs and materials more quickly and inexpensively than was formerly possible. Rational drug design and combinatorial chemistry have become more intimately related in recent years due to the development of approaches in computer-aided protein modeling and drug discovery. {See e.g., US Pat. No. 4,908,773; 5,884,230; 5,873,052; 5,331 ,573; and 5,888,738).

The use of molecular modeling as a tool for rational drug design and combinatorial chemistry has dramatically increased due to the advent of computer graphics. Not only is it possible to view molecules on computer screens in three dimensions but it is also possible to examine the interactions of macromolecules such as enzymes and receptors and rationally design derivative molecules to test. (See Boorman, Chem. Eng. News 70:18-26 (1992). A vast amount of user-friendly software and hardware is now available and virtually all pharmaceutical companies have computer modeling groups devoted to rational drug design. Molecular Simulations Inc., for example, sells several sophisticated programs that allow a user to start from an amino acid sequence, build a two or three-dimensional model of the protein or polypeptide, compare it to other two and three-dimensional models, and analyze the interactions of compounds, drugs, and peptides with a three dimensional model in real time. Accordingly, in some embodiments of the invention, software is used to compare regions of Ixostatins (e.g., Ixostatin-1 and Ixostatin-2) and molecules that interact with Ixostatins (collectively referred to as "binding partners" — e.g., anti-Ixostatin antibodies, vitronectin, and vitronectin somatomedin domain), and fragments or derivatives of these molecules with other molecules, such as peptides, peptidomimetics, and chemicals, so that therapeutic interactions can be predicted and designed. (See Schneider, Genetic Engineering News December: page 20 (1998), Tempczyk et al., Molecular Simulations Inc. Solutions April (1997) and Butenhof, Molecular Simulations Inc. Case Notes (August 1998) for a discussion of molecular modeling). For example, the protein sequence of an Ixostatin or binding partner, or domains of these molecules (or nucleic acid sequence encoding these polypeptides or both), can be entered onto a computer readable medium for recording and manipulation. It will be appreciated by those skilled in the art that a computer readable medium having these sequences can interface with software that converts or manipulates the sequences to obtain structural and functional information, such as protein models. That is, the functionality of a software program that converts or manipulates these sequences includes the ability to compare these sequences to other sequences or structures of molecules that are present on publicly and commercially available databases so as to conduct rational drug design.

The Ixostatin or binding partner polypeptide or nucleic acid sequence or both can be stored, recorded, and manipulated on any medium that can be read and accessed by a computer. As used herein, the words "recorded" and "stored" refer to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently known methods for recording information on a computer readable medium to generate manufactures comprising the nucleotide or polypeptide sequence information of this embodiment. A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide or polypeptide sequence. The choice of the data storage structure will generally be based on the component chosen to access the stored information. Computer readable media include magnetically readable media, optically readable media, or electronically readable media. For example, the computer readable media can be a hard disc, a floppy disc, a magnetic tape, zip disk, CD-ROM, DVD-ROM, RAM, or ROM as well as other types of other media known to those skilled in the art. The computer readable media on which the sequence information is stored can be in a personal computer, a network, a server or other computer systems known to those skilled in the art.

Embodiments of the invention utilize computer-based systems that contain the sequence information described herein and convert this information into other types of usable information (e.g., protein models for rational drug design). The term "a computer- based system" refers to the hardware, software, and any database used to analyze an Ixostatin or a binding partner nucleic acid or polypeptide sequence or both, or fragments of these biomolecules so as to construct models or to conduct rational drug design. The computer-based system preferably includes the storage media described above, and a processor for accessing and manipulating the sequence data. The hardware of the computer-based systems of this embodiment comprise a central processing unit (CPU) and a database. A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable.

In one particular embodiment, the computer system includes a processor connected to a bus that is connected to a main memory (preferably implemented as RAM) and a variety of secondary storage devices, such as a hard drive and removable medium storage device. The removable medium storage device can represent, for example, a floppy disk drive, a DVD drive, an optical disk drive, a compact disk drive, a magnetic tape drive, etc. A removable storage medium, such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded therein can be inserted into the removable storage device. The computer system includes appropriate software for reading the control logic and/or the data from the removable medium storage device once inserted in the removable medium storage device. The Ixostatin or binding partner nucleic acid or polypeptide sequence or both can be stored in a well known manner in the main memory, any of the secondary storage devices, and/or a removable storage medium. Software for accessing and processing these sequences (such as search tools, compare tools, and modeling tools etc.) reside in main memory during execution.

As used herein, "a database" refers to memory that can store an Ixostatin or binding partner nucleotide or polypeptide sequence information, protein model information, information on other peptides, chemicals, peptidomimetics, and other agents that interact with Ixostatin proteins, and values or results from functional assays. Additionally, a "database" refers to a memory access component that can access manufactures having recorded thereon Ixostatin or binding partner nucleotide or polypeptide sequence information, protein model information, information on other peptides, chemicals, peptidomimetics, and other agents that interact with Ixostatins, and values or results from functional assays. In other embodiments, a database stores an " Ixostatin functional profile" comprising the values and results (e.g., ability to associate with vitronectin or modulate fibrinolysis) from one or more " Ixostatin functional assays", as described herein or known in the art, and relationships between these values or results. The sequence data and values or results from Ixostatin functional assays can be stored and manipulated in a variety of data processor programs in a variety of formats. For example, the sequence data can be stored as text in a word processing file, an ASCII file, a html file, or a pdf file in a variety of database programs familiar to those of skill in the art.

A "search program" refers to one or more programs that are implemented on the computer-based system to compare an Ixostatin or binding partner nucleotide or polypeptide sequence with other nucleotide or polypeptide sequences and agents including but not limited to peptides, peptidomimetics, and chemicals stored within a database. A search program also refers to one or more programs that compare one or more protein models to several protein models that exist in a database and one or more protein models to several peptides, peptidomimetics, and chemicals that exist in a database. A search program is used, for example, to compare one Ixostatin functional profile to one or more Ixostatin functional profiles that are present in a database. Still further, a search program can be used to compare values or results from Ixostatin functional assays and agents that modulate Ixostatin-mediated fibrinolysis or cell adhesion.

A "retrieval program" refers to one or more programs that can be implemented on the computer-based system to identify a homologous nucleic acid sequence, a homologous protein sequence, or a homologous protein model. A retrieval program can also used to identify peptides, peptidomimetics, and chemicals that interact with an Ixostatin protein sequence, or an Ixostatin protein model stored in a database. Further, a retrieval program is used to identify a specific agent that modulates Ixostatin-mediated fibrinolysis or cell adhesion to a desired set of values, results, or profile. That is, a retrieval program can also be used to obtain "a binding partner profile" that is composed of a chemical structure, nucleic acid sequence, or polypeptide sequence or model of an agent that interacts with an Ixostatin and, thereby modulates (inhibits or enhances) fibrinolysis or cell adhesion. Further, a binding partner profile can have one or more symbols that represent these molecules and/or models, an identifier that represents one or more agents including, but not limited to peptides and peptidomimetics (referred to collectively as "peptide agents") and chemicals, and a value or result from a functional assay.

As a starting point to rational drug design, a two or three dimensional model of a polypeptide of interest is created (e.g., Ixostatin- 1, Ixostatin-2, or a binding partner, such as a vitronectin somatomedin domain or an antibody). In the past, the three- dimensional structure of proteins has been determined in a number of ways. Perhaps the best known way of determining protein structure involves the use of x-ray crystallography. A general review of this technique can be found in Van Holde, K.E. Physical Biochemistry, Prentice-Hall, NJ. pp. 221-239 (1971). Using this technique, it is possible to elucidate three-dimensional structure with good precision. Additionally, protein structure can be determined through the use of techniques of neutron diffraction, or by nuclear magnetic resonance (NMR). (See, e.g., Moore, WJ., Physical Chemistry, 4^th Edition, Prentice-Hall, NJ. (1972)).

Alternatively, protein models of a polypeptide of interest can be constructed using computer-based protein modeling techniques. By one approach, the protein folding problem is solved by finding target sequences that are most compatible with profiles representing the structural environments of the residues in known three- dimensional protein structures. (See, e.g., U.S. Patent No. 5,436,850). In another technique, the known three-dimensional structures of proteins in a given family are superimposed to define the structurally conserved regions in that family. This protein modeling technique also uses the known three-dimensional structure of a homologous protein to approximate the structure of a polypeptide of interest. (See e.g., U.S. Patent

Nos. 5,557,535; 5,884,230; and 5,873,052). Conventional homology modeling techniques have been used routinely to build models of proteases and antibodies. (Sowdhamini et al., Protein Engineering 10:207, 215 (1997)). Comparative approaches can also be used to develop three-dimensional protein models when the protein of interest has poor sequence identity to template proteins. In some cases, proteins fold into similar three-dimensional structures despite having very weak sequence identities. For example, the three-dimensional structures of a number of helical cytokines fold in similar three-dimensional topology in spite of weak sequence homology.

The recent development of threading methods and "fuzzy" approaches now enables the identification of likely folding patterns and functional protein domains in a number of situations where the structural relatedness between target and template(s) is not detectable at the sequence level. By one method, fold recognition is performed using Multiple Sequence Threading (MST) and structural equivalences are deduced from the threading output using a distance geometry program that constructs a low resolution model. A full-atom representation is then constructed using a molecular modeling package.

According to this 3-step approach, candidate templates are first identified by using the novel fold recognition algorithm MST, which is capable of performing simultaneous threading of multiple aligned sequences onto one or more 3-D structures. In a second step, the structural equivalences obtained from the MST output are converted into interresidue distance restraints and fed into the distance geometry program, together with auxiliary information obtained from secondary structure predictions. The program combines the restraints in an unbiased manner and rapidly generates a large number of low resolution model confirmations. In a third step, these low resolution model confirmations are converted into full-atom models and organismed to energy minimization using the molecular modeling package. (See e.g., Aszόdi et al., Proteins: Structure, Function, and Genetics, Supplement 1:38-42 (1997)). In a preferred approach, a commercially available program (Molecular Simulations Inc.) and accompanying modules are used to create a two and/or three dimensional model of a polypeptide of interest from an amino acid sequence. A three-dimensional graphics program that can interface with several modules that perform numerous structural analysis and enable real-time rational drug design and combinatorial chemistry is commercially available. Modules allow one to rapidly create a two dimensional or three dimensional model of a polypeptide, carbohydrate, nucleic acid, chemical or combinations of the foregoing from their sequence or structure. The modeling tools associated with these programs support many different data file formats including Brookhaven and Cambridge databases; AMPAC/MOPAC and QCPE programs; Molecular Design Limited Molfile and

SD files, Sybel Mol2 files, VRML, and Pict files.

Additionally, the techniques described above can be supplemented with techniques in molecular biology to design models of the protein of interest. For example, a polypeptide of interest can be analyzed by an alanine scan (Wells, Methods in Enzymol. 202:390-41 1 (1991)) or other types of site-directed mutagenesis analysis. In alanine scan, each amino acid residue of the polypeptide of interest is sequentially replaced by alanine in a step-wise fashion (i.e., only one alanine point mutation is incorporated per molecule starting at position #1 and proceeding through the entire molecule), and the effect of the mutation on the peptide's activity in a functional assay is determined. Each of the amino acid residues of the peptide is analyzed in this manner and the regions important for the modulation of fibrinolysis or cell adhesion, for example, are identified. These functionally important regions can be recorded on a computer readable medium, stored in a database in a computer system, and a search program can be employed to generate a protein model of the functionally important regions. Once a model of the polypeptide of interest is created, it can be compared to other models so as to identify new members of the Ixostatin family and binding partners. By starting with the amino acid sequence or protein model of Ixostatin- 1 or Ixostatin-2 or a binding partner, for example, molecules having two-dimensional and/or three- dimensional homology can be rapidly identified. In one approach, a percent sequence identity can be determined by standard methods that are commonly used to compare the similarity and position of the amino acid of two polypeptides. Using a computer program such as BLAST or FASTA, two polypeptides can be aligned for optimal matching of their respective amino acids (either along the full length of one or both sequences, or along a predetermined portion of one or both sequences). Such programs provide "default" opening penalty and a "default" gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in: Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3 (1978)) can be used in conjunction with the computer program. The percent identity can then be calculated as:

total number of identical matches X 100

[length of the longer sequence within the matched span + number of gaps introduced into the longer sequence in order to align the two sequences]

Accordingly, the protein sequence corresponding to an Ixostatin or a binding partner or a fragment or derivative of these molecules can be compared to known sequences on a protein basis. Protein sequences corresponding to an Ixostatin, or a binding partner or a fragment or derivative of these molecules are compared, for example, to known amino acid sequences found in Swissprot release 35, PIR release 53 and Genpept release 108 public databases using BLASTP with the parameter W=8 and allowing a maximum of 10 matches. In addition, the protein sequences are compared to publicly known amino acid sequences of Swissprot using BLASTX with the parameter

E=0.001. The molecules identified as members of the family of Ixostatins or candidate binding partners desirably have at least 35% homology and preferably have 40%, 45%, 50% or 55% or greater homology to Ixostatin- 1 or Ixostatin-2 The Ixostatin family members and candidate binding partners that interact with an Ixostatin can have the following degrees of homology or identity to Ixostatin- 1 or Ixostatin-2 or both, for example: 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The Ixostatin family members and candidate binding partners having greater than or equal to 35% homology are identified and are subsequently examined using an Ixostatin functional assay. In another embodiment, computer modeling and the sequence-to-structure-to- function paradigm is exploited to identify more members of the Ixostatin family candidate binding partners. By this approach, first the structure of an Ixostatin (e.g., Ixostatin- 1 or Ixostatin-2) or a candidate binding partner (e.g., somatomedin domain or antibody) having a known response in a characterization assay is determined from its sequence using a threading algorithm, which aligns the sequence to the best matching structure in a structural database. Next, the protein's active site (i.e., the site important for a desired response in the characterization assay) is identified and a "fuzzy functional form" (FFF) ~ a three-dimensional descriptor of the active site of a protein ~ is created. (See e.g., Fetrow et al., J. MoI. Biol. 282:703-71 1 (1998) and Fetrow and Skolnick, J.

MoI. Biol. 281 : 949-968 (1998).

The FFFs are built by iteratively superimposing the protein geometries from a series of functionally related proteins with known structures. The FFFs are not overly specific, however, and the degree to which the descriptors can be relaxed is explored. In essence, conserved and functionally important residues for a desired response are identified and a set of geometric and conformational constraints for a specific function are defined in the form of a computer algorithm. The program then searches experimentally determined protein structures from a protein structural database for sets of residues that satisfy the specified constraints. In this manner, homologous three- dimensional structures can be compared and degrees (e.g., percentages of three- dimensional homology) can be ascertained. The ability to search three-dimensional structure databases for structural similarity to a protein of interest can also be accomplished by employing commercially available modules.

By using this computational protocol, genome sequence data bases such as maintained by various organizations can be rapidly screened for specific protein active sites and for identification of the residues at those active sites that resemble a desired molecule. Several other groups have developed databases of short sequence patterns or motifs designed to identify a given function or activity of a protein. Many of these databases can use short stretches of sequence information to identify sequence patterns that are specific for a given function; thus they avoid the problems arising from the necessity of matching entire sequences. By a similar approach, a candidate binding partner can be identified and manufactured as follows. First, a molecular model of one or more molecules that are known to interact with an Ixostatin or portions of these molecules that interact with an Ixostatin are created using one of the techniques discussed above or as known in the art. Next, chemical libraries and databases are searched for molecules similar in structure to the known molecule. That is, a search can be made of a three dimensional data base for non-peptide (organic) structures (e.g., non-peptide analogs, and/or dipeptide analogs) having three dimensional similarity to the known structure of the target compound. (See, e.g., the Cambridge Crystal Structure Data Base, Crystallographic Data Center, Lensfield Road, Cambridge, CB2 IEW, England; and Allen, F. H., et al., Acta

Crystallogr., B35: 2331-2339 (1979).) The identified candidate binding partners that interact with Ixostatins can then be analyzed in a functional assay (e.g., a fibrinolysis assay or cell adhesion assay or both) and new molecules can be modeled after the candidate binding partners that produce a desirable response. By cycling in this fashion, libraries of molecules that interact with Ixostatins and produce a desirable or optimal response in a functional assay can be selected.

It is noted that search algorithms for three dimensional data base comparisons are available in the literature. (See, e.g., Cooper, et al., J. Comput. -Aided MoI. Design, 3: 253-259 (1989) and references cited therein; Brent, et al., J. Comput.-Aided MoI. Design, 2: 31 1-310 (1988) and references cited therein.) Commercial software for such searches is also available from vendors such as Day Light Information Systems, Inc., Irvine, Calif. 92714, and Molecular Design Limited, 2132 Faralton Drive, San Leandro, Calif. 94577. The searching is done in a systematic fashion by simulating or synthesizing analogs having a substitute moiety at every residue level. Preferably, care is taken that replacement of portions of the backbone does not disturb the tertiary structure and that the side chain substitutions are compatible to retain the receptor substrate interactions.

By another approach, protein models of binding partners that interact with an Ixostatin (e.g., a vitronectin somatomedin domain or antibody) can be made by the methods described above and these models can be used to predict the interaction of new molecules. Once a model of a binding partner is identified, the active sites or regions of interaction can be identified. Such active sites might typically be ligand binding sites. The active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the Ixostatin with a ligand, such as a vitronectin somatomedin domain or specific matrix proteins. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the

Ixostatin the complexed ligand is found (e.g. CRD). Next, the three dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular structure. On the other hand, solid or liquid phase NMR can be used to determine certain intra-molecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures. The geometric structures can be measured with a complexed ligand, natural or artificial, which may increase the accuracy of the active site structure determined.

If an incomplete or insufficiently accurate structure is determined, the methods of computer based numerical modeling can be used to complete the structure or improve its accuracy. Any recognized modeling method can be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models. For most types of models, standard molecular force fields, representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry. The incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods. Finally, having determined the structure of the active site of the known binding partner, either experimentally, by modeling, or by a combination, candidate binding partners can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. Alternatively, these methods can be used to identify improved binding partners from an already known binding partner. The composition of the known binding partner can be modified and the structural effects of modification can be determined using the experimental and computer modeling methods described above applied to the new composition. The altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity. A number of articles review computer modeling of drugs interactive with specific-proteins, such as Rotivinen, et al., 1988, Acta Pharmaceutical Fennica 97:159- 166; Ripka, New Scientist 54-57 (Jun. 16, 1988); McKinaly and Rossmann, 1989, Annu. Rev. Pharmacol. Toxiciol. 29:1 1 1-122; Perry and Davies, OSAR: Quantitative Structure- Activity Relationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R. Soc. Lond. 236:125-140 and 141-162; and, with respect to a model receptor for nucleic acid components, Askew, et al., 1989, J. Am. Chem. Soc. 111:1082-1090. Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc. (Pasadena, Calif), Allelix, Inc. (Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario). Although these are primarily designed for application to drugs specific to particular proteins, they can be adapted to design of drugs specific for the modulation of Ixostatin-mediated cell adhesion, fibrinolysis and other Ixostatin functions.

Many more computer programs and databases can be used with embodiments of the invention to identify new members of the Ixostatin family and binding partners that modulate Ixostatin function. The following list is intended not to limit the invention but to provide guidance to programs and databases that are useful with the approaches discussed above. The programs and databases that can be used include, but are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX

(Altschul et al, J. MoI. Biol. 215: 403 (1990), herein incorporated by reference), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444 (1988), herein incorporated by reference), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (Molecular Simulations Inc.), Cerius².DBAccess (Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II, (Molecular Simulations Inc.), Discover (Molecular Simulations Inc.), CHARMm (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.),

Homology (Molecular Simulations Inc.), Modeler (Molecular Simulations Inc.), Modeller 4 (SaIi and Blundell J. MoI. Biol. 234:217-241 (1997)), ISIS (Molecular Simulations Inc.), Quanta/Protein Design (Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), Biopendium (Inpharmatica),

SBdBase (Structural Bioinformatics), the EMBL/Swissprotein database, the MDL Available Chemicals Directory database, the MDL Drug Data Report data base, the Comprehensive Medicinal Chemistry database, Derwents's World Drug Index database, and the BioByteMasterFile database. Many other programs and data bases would be apparent to one of skill in the art given the present disclosure.

Once candidate binding partners have been identified, desirably, they are analyzed in a functional assay. Further cycles of modeling and functional assays can be employed to more narrowly define the parameters needed in a binding partner. Each binding partner and its response in a functional assay can be recorded on a computer readable media and a database or library of binding partners and respective responses in a functional assay can be generated. These databases or libraries can be used by researchers to identify important differences between active and inactive molecules so that compound libraries are enriched for binding partners that have favorable characteristics. The section below describes several Ixostatin functional assays that can be used to characterize new Ixostatin family members and candidate binding partners.

Ixostatin characterization assays

The term "Ixostatin characterization assay" or "Ixostatin functional assay" or

"functional assay" the results of which can be recorded as a value in a "Ixostatin functional profile", include assays that directly or indirectly evaluate the presence of an Ixostatin nucleic acid or protein in a cell and the ability of an Ixostatin to modulate fibrinolysis or cell adhesion. Examples 4 and 5 teach assays that are considered for the purposes of this disclosure to be Ixostatin functional assays. Many more are provided in the discussion below.

Some functional assays involve binding assays that utilize multimeric agents. One form of multimeric agent concerns a manufacture comprising an Ixostatin, hybrid, binding partner, or fragment thereof disposed on a support. These multimeric agents provide the Ixostatin, hybrid, binding partner, or fragment thereof in such a form or in such a way that a sufficient affinity is achieved. A multimeric agent having an Ixostatin, hybrid, or binding partner or fragment thereof is obtained by joining the desired polypeptide to a macromolecular support. A "support" can be a termed a carrier, a protein, a resin, a cell membrane, or any macromolecular structure used to join or immobilize such molecules. Solid supports include, but are not limited to, the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, animal cells, Duracyte®, artificial cells, and others. An Ixostatin, hybrid, or binding partner or fragment thereof can also be joined to inorganic carriers, such as silicon oxide material (e.g., silica gel, zeolite, diatomaceous earth or aminated glass) by, for example, a covalent linkage through a hydroxy, carboxy or amino group and a reactive group on the carrier.

In several multimeric agents, the macromolecular support has a hydrophobic surface that interacts with a portion of the Ixostatin, hybrid, or binding partner or fragment thereof by a hydrophobic non-covalent interaction. In some cases, the hydrophobic surface of the support is a polymer such as plastic or any other polymer in which hydrophobic groups have been linked such as polystyrene, polyethylene or polyvinyl. Additionally, an Ixostatin, hybrid, or binding partner or fragment thereof can be covalently bound to carriers including proteins and oligo/polysaccarides (e.g. cellulose, starch, glycogen, chitosane or aminated sepharose). In these later multimeric agents, a reactive group on the molecule, such as a hydroxy or an amino group, is used to join to a reactive group on the carrier so as to create the covalent bond. Additional multimeric agents comprise a support that has other reactive groups that are chemically activated so as to attach the Ixostatin, hybrid, or binding partner or fragment thereof. For example, cyanogen bromide activated matrices, epoxy activated matrices, thio and thiopropyl gels, nitrophenyl chloroformate and N-hydroxy succinimide chlorformate linkages, or oxirane acrylic supports are used. (Sigma). Additionally, a cell based approach can be used characterize new Ixostatin family members or Ixostatin hybrids or to rapidly identify binding partners that interact with an Ixostatin and, thereby, modulate fibrinolysis. Preferably, molecules identified in the support-bound Ixostatin assay described above are used in the cell based approach, however, randomly generated compounds can also be used.

Other Ixostatin characterization assays take advantage of techniques in molecular biology that are employed to discover protein:protein interactions. One method that detects protein-protein interactions in vivo, the two-hybrid system, is described in detail for illustration only and not by way of limitation. Other similar assays that can be adapted to identify binding partners include:

(1) the two-hybrid systems (Field & Song, Nature 340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA 88:9578-9582 (1991); and Young KH, Biol. Reprod. 58:302-31 1 (1998), all references herein expressly incorporated by reference);

(2) reverse two-hybrid system (Leanna & Hannink, Nucl. Acid Res. 24:3341- 3347 (1996), herein incorporated by reference);

(3) repressed transactivator system (Sadowski et al., U.S. Pat. No. 5,885,779), herein incorporated by reference); (4) phage display (Lowman HB, Annu. Rev. Biophys. Biomol. Struct.

26:401-424 (1997), herein incorporated by reference); and (5) GST/HIS pull down assays, mutant operators (Granger et al., WO

98/01879) and the like (See also Mathis G., Clin. Chem. 41 :139-147

(1995); Lam K.S. Anticancer Drug Res., 12:145-167 (1997); and Phizicky et al., Microbiol. Rev. 59:94-123 (1995), all references herein expressly incorporated by reference).

An adaptation of the system described by Chien et al., 1991, Proc. Natl. Acad.

Sci. USA, 88:9578-9582, herein incorporated by reference), which is commercially available from Clontech (Palo Alto, Calif.) is as follows. Plasmids are constructed that encode two hybrid proteins: one plasmid consists of nucleotides encoding the DNA- binding domain of a transcription activator protein fused to a nucleotide sequence encoding an Ixostatin or fragment thereof, and the other plasmid consists of nucleotides encoding the transcription activator protein's activation domain fused to a cDNA encoding an unknown protein that has been recombined into this plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., HBS or lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene: the DNA-binding domain hybrid cannot because it does not provide activation function and the activation domain hybrid cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product.

The two-hybrid system or related methodology can be used to screen activation domain libraries for proteins that interact with the "bait" gene product. By way of example, and not by way of limitation, Ixostatins can be used as the bait gene product.

Total genomic or cDNA sequences are fused to the DNA encoding an activation domain. This library and a plasmid encoding a hybrid of a bait gene encoding the Ixostatin product (Ixostatin- 1) fused to the DNA-binding domain are cotransformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene. For example, and not by way of limitation, a bait gene sequence encoding an Ixostatin can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library plasmids.

A cDNA library of the cell line from which proteins that interact with bait Ixostatin are to be detected can be made using methods routinely practiced in the art. According to the particular system described herein, for example, the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcriptional activation domain of GAL4. This library can be co-transformed along with the bait Ixostatin gene-GAL4 fusion plasmid into a yeast strain which contains a lacZ gene driven by a promoter which contains GAL4 activation sequence. A cDNA encoded protein, fused to GAL4 transcriptional activation domain, which interacts with bait Ixostatin gene product will reconstitute an active GAL4 protein and thereby drive expression of the lacZ gene. Colonies that express lacZ can be detected and the cDNA can then be purified from these strains, and used to produce and isolate the binding partner by techniques routinely practiced in the art. In the disclosure below, several diagnostic embodiments of the invention are described.

Pharmaceutical preparations and methods of administration

The Ixostatins, hybrids, binding agents, and fragments thereof are suitable for incorporation into pharmaceuticals that treat organisms in need of a compound that modulates fibrinolysis or cell adhesion. These pharmacologically active compounds can be processed in accordance with conventional methods of galenic pharmacy to produce medicinal agents for administration to organisms, e.g., plants, insects, mold, yeast, animals, and mammals including humans. The active ingredients can be incorporated into a pharmaceutical product with and without modification. Further, the manufacture of pharmaceuticals or therapeutic agents that deliver the pharmacologically active compounds of this invention by several routes are aspects of the invention. For example, and not by way of limitation, DNA, RNA, and viral vectors having sequence encoding the Ixostatins, hybrids, binding partners, or fragments thereof are used with embodiments. Nucleic acids encoding Ixostatins, hybrids, binding partners, or fragments thereof can be administered alone or in combination with other active ingredients.

The compounds described herein can be employed in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application that do not deleteriously react with the pharmacologically active ingredients of this invention.

Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyetylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc.

Many more suitable vehicles are described in Remmington's Pharmaceutical Sciences, 15th Edition, EastonrMack Publishing Company, pages 1405-1412 and 1461- 1487(1975) and The National Formulary XIV, 14th Edition, Washington, American Pharmaceutical Association (1975), herein incorporated by reference. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like that do not deleteriously react with the active compounds.

The effective dose and method of administration of a particular pharmaceutical formulation having Ixostatins, hybrids, binding partners, or fragments thereof can vary based on the individual needs of the patient and the treatment or preventative measure sought. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population). For example, the Ixostatins, hybrids, binding partners, or fragments thereof discussed above, can be administered to the knockout mice of the invention and the effect on fibrinolysis or cell adhesion can be determined. The data obtained from these assays is then used in formulating a range of dosage for use with other organisms, including humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with no toxicity. The dosage varies within this range depending upon type of Ixostatin, hybrid, binding partner, or fragment thereof, the dosage form employed, sensitivity of the organism, and the route of administration.

Normal dosage amounts of various Ixostatins, hybrids, binding partners, or fragments thereof can vary from approximately 1 to 100,000 micrograms, up to a total dose of about 10 grams, depending upon the route of administration. Desirable dosages include 250μg, 500μg, lmg, 50mg, lOOmg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg,

900mg, Ig, l. lg, 1.2g, 1.3g, 1.4g, 1.5g, 1.6g, 1.7g, 1.8g, 1.9g, 2g, 3g, 4g, 5, 6g, 7g, 8g, 9g, and 1Og.

In some embodiments, the dose of Ixostatins, hybrids, binding partners, or fragments thereof preferably produces a tissue or blood concentration or both from approximately 0.1 μM to 50OmM. Desirable doses produce a tissue or blood concentration or both of about 1 to 800μM. Preferable doses produce a tissue or blood concentration of greater than about lOμM to about 500μM. Preferable doses are, for example, the amount of Ixostatins, hybrids, binding partners, or fragments thereof required to achieve a tissue or blood concentration or both of lOμM, 15μM, 20μM, 25μM, 30μM, 35μM, 40μM, 45μM, 50μM, 55μM, 60μM, 65μM, 70μM, 75μM, 80μM, 85μM, 90μM, 95μM, lOOμM, HOμM, 120μM, 130μM, 140μM, 145μM, 150μM, 160μM, 170μM, 180μM, 190μM, 200μM, 220μM, 240μM, 250μM, 260μM, 280μM,

300μM, 320μM, 340μM, 360μM, 380μM, 400μM, 420μM, 440μM, 460μM, 480μM, and 500μM. Although doses that produce a tissue concentration of greater than 800μM are not preferred, they can be used with some embodiments of the invention. A constant infusion of the Ixostatins, hybrids, binding partners, or fragments thereof can also be provided so as to maintain a stable concentration in the tissues as measured by blood levels.

The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that can be taken into account include the severity of the disease, age of the organism, and weight or size of the organism; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Short acting pharmaceutical compositions are administered daily whereas long acting pharmaceutical compositions are administered every 2, 3 to 4 days, every week, or once every two weeks. Depending on half-life and clearance rate of the particular formulation, the pharmaceutical compositions of the invention are administered once, twice, three, four, five, six, seven, eight, nine, ten or more times per day.

Routes of administration of the pharmaceuticals of the invention include, but are not limited to, topical, transdermal, parenteral, gastrointestinal, transbronchial, and transalveolar. Transdermal administration is accomplished by application of a cream, rinse, gel, etc. capable of allowing the pharmacologically active compounds to penetrate the skin. Parenteral routes of administration include, but are not limited to, electrical or direct injection such as direct injection into a central venous line, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. Gastrointestinal routes of administration include, but are not limited to, ingestion and rectal.

Transbronchial and transalveolar routes of administration include, but are not limited to, inhalation, either via the mouth or intranasally. Compositions having the pharmacologically active compounds of this invention that are suitable for transdermal or topical administration include, but are not limited to, pharmaceutically acceptable suspensions, oils, creams, and ointments applied directly to the skin or incorporated into a protective carrier such as a transdermal device ("transdermal patch"). Examples of suitable creams, ointments, etc. can be found, for instance, in the Physician's Desk Reference. Examples of suitable transdermal devices are described, for instance, in U.S. Patent No. 4,818,540 issued April 4, 1989 to Chinen, et al., herein incorporated by reference.

Compositions having the pharmacologically active compounds of this invention that are suitable for parenteral administration include, but are not limited to, pharmaceutically acceptable sterile isotonic solutions. Such solutions include, but are not limited to, saline and phosphate buffered saline for injection into a central venous line, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection.

Compositions having the pharmacologically active compounds of this invention that are suitable for transbronchial and transalveolar administration include, but not limited to, various types of aerosols for inhalation. Devices suitable for transbronchial and transalveolar administration of these are also embodiments. Such devices include, but are not limited to, atomizers and vaporizers. Many forms of currently available atomizers and vaporizers can be readily adapted to deliver compositions having the pharmacologically active compounds of the invention.

Compositions having the pharmacologically active compounds of this invention that are suitable for gastrointestinal administration include, but not limited to, pharmaceutically acceptable powders, pills or liquids for ingestion and suppositories for rectal administration. Due to the ease of use, gastrointestinal administration, particularly oral, is a preferred embodiment. Once the pharmaceutical comprising the

Ixostatin, hybrid, binding partner, or fragment thereof has been obtained, it can be administered to a organism in need to treat or prevent a defect in fibrinolysis or cell adhesion.

Vaccine compositions

Various nucleic acid-based vaccines therapeutics are known and it is contemplated that these compositions and approaches to immunotherapy can be used in a number of animals. By one approach, for example, a gene encoding one of the Ixostatin proteins can be optimized for expression in a particular animal (e.g., domestic animals, such as dogs, cats, or horses, or humans (see Example 7). By one approach, SEQ. ID. NO.: 13 is cloned into an expression vector capable of expressing the polypeptide when introduced into a subject. The expression construct is introduced into the subject in a mixture of an adjuvant. For example, the adjuvant is administered shortly after the expression construct at the same site. Alternatively, RNA encoding the Ixostatin polypeptide antigen of interest is provided to the subject in a mixture with ribavirin or in conjunction with an adjuvant. Where the antigen is to be DNA (e.g., preparation of a DNA vaccine composition), suitable promoters include Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine and human metalothionein can be used. Examples of polyadenylation signals useful with some embodiments, especially in the production of a genetic vaccine for humans, include but are not limited to, SV40 polyadenylation signals and LTR polyadenylation signals. In particular, the SV40 polyadenylation signal, which is in pCEP4 plasmid (Invitrogen, San Diego

Calif.), referred to as the SV40 polyadenylation signal, is used.

In addition to the regulatory elements required for gene expression, other elements may also be included in a gene construct. Such additional elements include enhancers. The enhancer may be selected from the group including but not limited to: human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV. Gene constructs can be provided with mammalian origin of replication in order to maintain the construct extrachromosomally and produce multiple copies of the construct in the cell. Plasmids pCEP4 and pREP4 from Invitrogen (San Diego, CA) contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-I coding region, which produces high copy episomal replication without integration. All forms of DNA, whether replicating or non-replicating, which do not become integrated into the genome, and which are expressible, can be used. Preferably, the genetic vaccines comprise an adjuvant and a nucleic acid encoding Ixostatin-1, Ixostatin-2, or a fragment or mutant thereof (SEQ. ID. NOs.: 2 and 4). Example 8 below describes the preparation of a genetic vaccine suitable for use in humans.

Treatment of Vitronectin-Related Conditions

Provided herein are methods of treating or inhibiting vitronectin-related conditions other than cancer by selecting or identifying an animal in need of treatment or inhibition of a such a condition and providing to the animal a therapeutically effective dose of a Ixostatin or fragment thereof or nucleic acid encoding one of these molecules. In certain embodiments, the animal is human. In certain embodiments, the Ixostatin polypeptide is Ixostatin-1 or Ixostatin-2.

Blockade of vitronectin by Ixostatin is a potentially useful strategy to prevent cell attachment to integrin αvβ3 and uPAR without the need to target multiple receptors involved in adhesion. This is a relevant finding because vitronectin has been found to deposit for example in liver cirrhosis, glomerulonephritis diabetic nephopathy, and in the brain of patients with multiple sclerosis (see Koukoulis GK, et al. Hum Pathol.

2001;32:1356-1362; Huang Y, et al. J Clin Invest. 2003;l 12:379-388; and Han MH, et al. Nature. 2008 ;451 : 1076- 1081. each of which is hereby incorporated by reference in its entirety).

Therefore, Ixostatin may be regarded as a new inhibitor of vitronectin-dependent conditions that prevents or ameliorate liver cirrhosis, glomerulonephritis, diabetic nephopathy, angiogenesis and multiple sclerosis by a mechanism related to inhibition of cell deposition to vitronectin.

Treatment of Cancer

Provided herein are methods of treating or inhibiting a malignant tumor in an animal by selecting or identifying an animal in need of treatment or inhibition of a malignant tumor and providing to the animal a therapeutically effective dose of a Ixostatin or fragment thereof or nucleic acid encoding one of these molecules. In certain embodiments, the animal is human. In certain embodiments, the Ixostatin polypeptide is Ixostatin-1 or Ixostatin-2. Blockade of vitronectin by Ixostatin is a potentially useful strategy to prevent cell attachment to integrin αvβ3 and uPAR without the need to target multiple receptors involved in adhesion. This is a relevant finding because vitronectin has been found to deposit in tissues containing malignant cells such as breast, ovarian, and hepatocellular cancer (see Aaboe M, et al. Biochim Biophys Acta. 2003;1638:72-82; Kenny HA, et al.

J Clin Invest. 2008; Jaskiewicz K, et al. Anticancer Res. 1993; 13:2229-2237. each of which is hereby incorporated by reference in its entirety).

Thus, in certain embodiments, the malignant tumor can be selected from the group consisting of: melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular (liver) carcinoma, thyroid tumor, gastric (stomach) cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, ovarian cancer, hepatocellular cancer, pancreatic cancer, esophageal carcinoma, head and neck cancers, mesothelioma, sarcomas, biliary (cholangiocarcinoma), small bowel adenocarcinoma, pediatric malignancies and epidermoid carcinoma.

Without being bound to any particular theory, Ixostatins may negatively affect tumor growth and metastasis because cell adhesion and migration are modulated by PAI-I (Delias and Loskutoff, Thromb. Haemost., 93, 631-640 (2005). It is also plausible to suggest that decrease of PAI-I functionality by Ixostatin may be associated with an anti -angiogenic phenotype in vivo.

Herein are provided data that show that Ixostatin affects angiogenesis by a mechanism unrelated to interference with PAI-I function. As provided in Example 5 herein, Ixostatin- 1 effectively prevents adhesion of uPAR-bearing cells (endothelial cells) to VN-containing matrices. Therefore, blockade of VN by Ixostatin is a potentially useful strategy to prevent cell attachment without the need to target multiple receptors involved in adhesion. Therefore, Ixostatin can prevent tumor growth and metastasis by a dual mechanism: inhibition of malignant cell deposition to VN in one hand, and shift of the patient hemostatic status to a pro-fibrinolytic tonus, on the other.

The anti-tumor treatment defined herein may be applied as a sole therapy or may involve, in addition to the Ixostatin-based compounds described herein, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include one or more of the following compounds within the listed categories of anti tumor agents: (i) other antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example, cis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan, temozolamide and nitrosoureas); antimetabolites (for example, gemcitabine and antifolates such as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, and hydroxyurea); antitumor antibiotics (for example, anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example, vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol and taxotere and polokinase inhibitors); and topoisomerase inhibitors (for example, epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin);

(ii) cytostatic agents such as antioestrogens (for example, tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene), antiandrogens

(for example, bicalutamide, fiutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example, goserelin, leuprorelin and buserelin), progestogens (for example, megestrol acetate), aromatase inhibitors (for example, as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5α-reductase, such as finasteride;

(iii) anti-invasion agents (for example, c-Src kinase family inhibitors like 4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-l- yl)ethoxy]-5-tetrahydropyran-4-yloxyquinazoline (AZD0530; International Patent Application WO 01/94341) and N-(2-chloro-6-methylphenyl)-2-{6-[4-(2- hydroxyethyl)piperazin-l-yl]-2-methylpyrimidin-4-ylamino}thiazole-5- carboxamide (dasatinib, BMS-354825; J. Med. Chem.. 2004, 47, 6658-6661), and metalloproteinase inhibitors like marimastat, other inhibitors of urokinase plasminogen activator receptor function or, inhibitors of cathepsins, inhibitors of serine proteases, for example, matriptase, hepsin, urokinase, and inhibitors of heparanase);

(iv) inhibitors of growth factor function: for example, growth factor antibodies and growth factor receptor antibodies (for example, the anti-erbB2 antibody trastuzumab [Herceptin™], the anti-EGFR antibody panitumumab, the anti-erbB 1 antibody cetuximab [Erbitux, C225] and any growth factor or growth factor receptor antibodies disclosed by Stern et al. Critical reviews in oncology /haematology, 2005, Vol. 54, pp 11-29); such inhibitors also include tyrosine kinase inhibitors, for example, inhibitors of the epidermal growth factor family (for example, EGFR family tyrosine kinase inhibitors such as N-(3- chloro-4-fluorophenyl)-7-methoxy-6-(3-moφholinopropoxy)quinazolin-4-amine (gefitinib, ZDl 839), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin- 4-amine (erlotinib, OSI-774) and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7- (3-morpholinopropoxy)-quinazolin-4-amine (CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib, inhibitors of the hepatocyte growth factor family, inhibitors of the platelet-derived growth factor family, such as imatinib, inhibitors of serine/threonine kinases (for example, Ras/Raf signalling inhibitors such as farnesyl transferase inhibitors, for example, sorafenib (BAY 43-9006)), inhibitors of cell signalling through MEK and/or AKT kinases, inhibitors of the hepatocyte growth factor family, c-kit inhibitors, abl kinase inhibitors, IGF receptor (insulin-like growth factor) kinase inhibitors; aurora kinase inhibitors (for example, AZDl 152, PH739358, VX-680, MLΝ8054, R763, MP235, MP529, VX-528 AND AX39459) and cyclin dependent kinase inhibitors, such as CDK2 and/or CDK4 inhibitors;

(v) antiangiogenic agents such as, those which inhibit the effects of vascular endothelial growth factor, [for example, the anti-vascular endothelial cell growth factor antibody bevacizumab (Avastin™) and VEGF receptor tyrosine kinase inhibitors such as 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(l- methylpiperidin-4-ylmethoxy)quinazoline (ZD6474; Example 2 within WO

01/32651), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-l- ylpropoxy)quinazoline (AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO 98/35985) and SUl 1248 (sunitinib; WO 01/60814), compounds such as, those disclosed in International Patent Applications WO97/22596, WO 97/30035, WO 97/32856 and WO 98/13354 and compounds that work by other mechanisms (for example, linomide, inhibitors of integrin αvβ3 function and angiostatin)]; (vi) vascular damaging agents such as, Combretastatin A4 and compounds disclosed in International Patent Applications WO 99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213;

(vii) antisense therapies, for example, those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense;

(viii) gene therapy approaches, including for example, approaches to replace aberrant genes such as aberrant p53 or aberrant BRCAl or BRC A2, GDEPT (gene-directed enzyme pro-drug therapy) approaches such as, those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy; and

(ix) immunotherapy approaches, including for example, ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumor cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease

T-cell anergy, approaches using transfected immune cells such as, cytokine-transfected dendritic cells, approaches using cytokine-transfected tumor cell lines and approaches using anti-idiotypic antibodies.

In one embodiment the anti-tumor treatment defined herein may involve, in addition to the Ixostatin-based compounds described herein, treatment with other antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example, cis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan, temozolamide and nitrosoureas); antimetabolites (for example, gemcitabine and antifolates such as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, and hydroxyurea); antitumor antibiotics (for example, anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example, vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol and taxotere and polokinase inhibitors); and topoisomerase inhibitors (for example, epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin).

In one embodiment the anti-tumor treatment defined herein may involve, in addition to the Ixostatin-based compounds described herein, treatment with gemcitabine.

Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ the compounds of this invention, or pharmaceutically acceptable salts thereof, within the dosage range described hereinbefore and the other pharmaceutically active agent within its approved dosage range.

Methods of reducing clot formation

Several embodiments also concern methods of reducing clot formation comprising providing to an animal a therapeutically effective dose of an Ixostatin disclosed herein. In certain embodiments, the animal is human. In certain embodiments, the Ixostatin polypeptide is Ixostatin- 1, Ixostatin-2, fragments or mutants thereof.

In certain embodiments, the method of reducing clot formation can be part of a treatment regimen where an antithrombogenic would be used. Nonlimiting examples include: coronary thrombosis, pulmonary embolism, myocardial infarction, deep vein thrombosis, cerebral thrombosis, postoperative fibrinolytic shutdown, or rapid thrombogenic actions, which can occur following implantation of a medical device.

The compositions provided herein may be used in combination with a variety of compositions that have been reported for use in reducing clot formation, including antithrombogenic agents. Antithrombogenic, as this term is used herein, is intended to encompass essentially any composition or medical device with the ability to inhibit thrombin-catalyzed fibrin clot formation, its ability to inhibit the amidolytic activity of thrombin, or by its ability to cause a substantial reduction in other known measures of the thrombogenic response when compared with a medical device that has not been so treated.

Antithrombogenic agents are well known and readily available to the individual skilled in this art. Examples of antithrombogenic or nonthrombogenic agents and materials suitable for use in combination, mixed with, or co-administered with an Ixostain -like polypeptide, as described herein, may include or be at least partly comprised of heparin, hirudin, albumin, phospholipids, streptokinase, tissue plasminogen activator (tPA), urokinase (uPA), hydrophilic polymers such as hyaluronic acid, chitosan, methyl cellulose, poly(ethylene oxide), poly(vinyl pyrrolidone), growth factors such as endothelial cell growth factor, epithelial growth factor, osteoblast growth factor, fibroblast growth factor, platelet derived growth factor (PDGF), and angiogenic growth factor, other like compounds, or functionally equivalent variants and/or derivatives thereof. The section below describes several medical devices that incorporate one or moer of the embodied molecules described herein.

Medical devices

Medical devices, such as stents, catheters and the like may be treated with Ixostatin alone or in combination with another antithrombogenic agent. The approach by which an antithrombogenic agent is incorporated into or onto some or all of a medical device is not limiting, and may be selected from any of a number of methods available in the art, some illustrative examples of which are described in U.S. Patent No. 6,528,107, the entirety of which is expressly incoporated by reference hererin.

For example, U.S. Pat. No. 5,679,659, assigned to Medtronic Inc., the disclosure of which is incorporated herein by reference, describes a method for making a heparinized medical device. In this method, heparin is reacted with a periodate compound and this mixture is reacted and then applied to immobilized amine groups on a medical device surface. The application to the immobilized amine groups causes a reaction between the aldehyde groups on the heparin and the immobilized amine groups to form a Schiff base. A mild reducing agent is used to stabilize the Schiff base into a secondary amine.

Other methods for providing antithrombogenic surfaces, for example, as described in U.S. Pat. Nos. 5,512,329 and 5,741,551, the disclosures of which are incorporated herein by reference, and other related patents assigned to BSI Corporation, relate to methods for modifying substrate surfaces by bonding molecules, e.g., protein molecules, to substrates through external activation of latent reactive groups carried on the molecules. The latent reactive groups are groups which respond to specific applied external stimuli to undergo active specie generation with resultant covalent bonding to an adjacent support surface. Latent reactive groups are those groups of atoms in a molecule which retain their covalent bonds unchanged under conditions of storage but which, upon activation, form covalent bonds with other molecules. The latent reactive groups generate active species such as free radicals, nitrenes, carbenes, and excited states of ketones upon absorption of external electromagnetic or kinetic (thermal) energy. Latent reactive groups are generally well known and may be chosen to be responsive to various portions of the electromagnetic spectrum.

In addition to the examples described above, many other antithrombogenic treatment methods are similarly known and available to the skilled individual in the art for use in conjunction with the compositions of this invention, including, but not limited to, methods for providing substrate surfaces with agents such as heparin, e.g., U.S. Pat. Nos. 3,51 1,684, 3,585,647, 4,254,180, 4,331,697, 4,676,974, 4,526,714, 4,634,762, 4,678,660, 4,678,671 and 5,877,263, phospholipids, e.g., U.S. Pat. No. 5,556,632, chitosan, e.g., U.S. Pat. No. 4,326,532, antithrombogenic polymers, e.g., U.S. Pat. Nos.

4,521,564, 4,600,652 and 4,642,242, and others, e.g., U.S. Pat. Nos. 4,973,493, 4,979,959, 5,263,992, 5,414,075, 5,512,329 and 5,741,551, the disclosures of which are incorporated herein by reference.

EXAMPLE 1

IDENTIFICATION AND CLONING OF IXOSTATIN

Tick saliva was obtained by inducing partially engorged adult female / scapularis to salivate (3-4 days post attachment to a rabbit) into capillary tubes using the modified pilocarpine induction method (Tatchell, J. Parasitol. 53:1106-1 107 (1967). Saliva and salivary gland were store frozen at - 80⁰C until needed.

Salivary gland cDNA construction was done as detailed before (Valenzuela et al., J. Exp Biol. 205:2843-2864 (2002)). Briefly, the Micro-FastTrack messenger RNA (mRNA) isolation kit (Invitrogen, San Diego, CA) was used to isolate the mRNA. The / scapularis salivary gland mRNA (200 ng) was reverse transcribed to cDNA followed by double-strand synthesis and ligated into a Lambda Triplex2 vector; the resulting ligation reaction was packed using Gigapack gold III from Stratagene/Biocrest (Cedar Creek, TN), according to manufacturer's instructions. The library obtained was plated by infecting log-phase XLl -blue cells. Randomly picked clones from this library were sequenced exactly as described before (Valenzuela et al., J. Exp Biol. 205:2843-2864 (2002)). After identifying a cDNA with high similarity to tissue factor pathway inhibitor (TFPI) following the Basic Local Alignment Search Tool X (BLASTX) program of the cDNA against the National Center for Biotechnological Information

(NCBI) nonredundant database, an aliquot (~100 ng) of Ixostatin-1 and -2 polymerase chain reaction (PCR) sample was re-amplified, and the entire cDNA was fully sequenced using custom primers.

Sequence analysis

Sequence similarity searches were performed using the BLAST program (NCBI) (Altschul et al, J. Mol. Biol. 215: 403 (1990), herein incorporated by reference). Cleavage site predictions of the mature proteins used the SignalP program (Bendtsen et al. J. MoI. Biol., 340:783-795, (2004)). Alignments of protein sequences were done with the ClustalW program version 1.7 (Thompson et al., Nucleic Acids Res. 22:4673-4680

(1994)). Dendrograms were obtained using Clustal X version 1.8 (Thompson et al., Nucleic Acids Res. 24:4876-4882 (1997)), and the output was used to generate filogenetic trees using Tree View softare (Page et al., Computer Applications in the Biosciences 12: 357-358 (1996)). The molar extinction coefficient (ε_{280 nm})of mature Ixostatin-1 and Ixostatin-2 at 280 nm was obtained at the website molbio.info.nih.gov/molbio/gcglite, yielding for mature Ixostatin-1 (gi 22164273) a value of ε_{28O n}m⁼ 3040 MT¹XnT¹; A_28Onm/_cm (1 mg/mL) = 0.262, mol wt. 1 1,592.34 (103 aa) and pi 6.95; mature Ixostatin-2 (gi 22164272) ε_{280 nm} = 8730 M^-1Xm^"1; A_{280 nm/cm} (1 mg/mL) = 0.853, mol wt 10,231.90 (91 aa), p/ 7.53. No O- or ΛMinked glycosylation sites were detected .

SEQ ID NOs: 2 and 4 set forth the predicted amino acid sequence for Ixostatin-1 and Ixostatin-2 identified in I. scapularis salivary gland. Figure IA shows the Clustal alignment for Ixostatins 1 and 2 (SEQ ID NOs: 2 and 4) and several cysteine-rich proteins from tick saliva (SEQ ID NOs: 17-29). The alignment of Ixostatins with the cysteine-rich domain from putative metalloproteases from Aedes, Anopheles or humans aggrecanase (SEQ ID NOs: 30-34) (e.g.: ADAMTS-4) is shown in Figure IB. Ixostatin-1-HIS and Ixostatin-2-HIS constructs

In order to identify the function of Ixostatin-1 and -2, the corresponding cDNA was PCR amplified with primers designed to add a sequence coding for 6xHIS tag in the 3' end. For expression of Ixostatin-1, and Ixostatin-2, the full-length cDNA (~ 100 ng) was used as a template to amplify the cDNA that begins at the initial methionine (underlined in the following primer sequences) and ends at the first stop codon. Forward primers were designed to introduce a Kozak consensus sequence (ANNATGG (SEQ ID NO: 5)) and the reverse primers were designed to introduce a 6xHIS Tag (italicized in the following primer sequences) before the stop codon. The forward primer for Ixostatin-1 was: 5'-AAA ATG CAA CTG GCC CTC TTC CTG GTT GTG- 3' (SEQ ID NO: 6) and the reverse primer was: 5'-TTA ATG ATG ATG ATG ATG ATG TAC GAA GGT CAT TTG GCA TGT GTC-3¹ (SEQ ID NO: 7). For Ixostatin-2, the forward primer was: 5'-AAA ATG CAA CTG GCC CTC TTC ATG ATT ATG - 3' (SEQ ID NO: 8) and the reverse primer was: 5 '-TTA ATG ATG ATG ATG ATG ATG

TAC GAA GGT CAT TTG GCA TGT GTC-3' (SEQ ID NO: 9). PCR was performed using High Fidelity Taq polymerase (0.5 U/ml) and condition were: 1 cycle of 75^°C for 2 mins; 1 cycle of 94^°C for 3 min; 25 cycles of 94^°C for 1 min, 55^°C for 30 sec, and 72^°C for 1 min. Then, 0.25U/ml of platinum taq polymerase was added for 10 min, at 72^°C to add overhanging A in both 3' ends of the PCR product to allow for efficient cloning in the TOPO vector (Invitrogen, CA). A single band of- 350 bp was visualized on 1.2 % agarose gel with ethidium bromide for Ixostatin-1 and Ixostatin-2 PCR products. An aliquot was taken, and the PCR product was cloned into the vector pIB/V5-His TOPO (Invitrogen, San Diego, CA) following manufacturer's specifications (Invitrogen, CA). The vector was used to transform TOPlO chemically competent E.coli cells by heat shock procedure and selection was performed using LB-ampicilin plates. Because blue and white selections were not possible, 16 colonies were randomly selected for PCR analysis using primers that flank Ixostatin-1, and -2 inserts: OpIE2 forward: 5'-CGC AAC GAT CTG GTA AAC AC-3' (SEQ ID NO: 10) and OpIE2 reverse: GAC AAT ACA AAC TAA GAT TTA GTC AG-3' (SEQ ID NO: 1 1). The resulting PCR products were sequenced and analyzed as described (Francischetti et al., Blood 99:3602-3612 (2002)). Expression vectors containing clones with the correct sequence and right orientation were selected for plasmid preparation and subsequent transfection of High Five insect cells.

Ixostatin-1-His and Ixostatin-2-His plasmid preparation TOP 10 cells containing Ixostatin-1, and Ixostatin-2 plasmids were grown in 1 liter of LB/ampicilin (100 μg/ml) overnight. Plasmid purification was carried using Mega-prep kit, following the manufacturer instructions (Sigma Chemical, Co., USA). The plasmid was eluted in 15 ml water and centrifuged at 4,000 rpm for 30 min. The supernatant was concentrated by centrifugation (2,500 rpm, 25 min) to approximately 2 ml using a 50 kDa cut-off Amicon-Ultra (Millipore Co, MA), and filtered in a GV filter

(0.22 μm). Plasmid concentration was estimated at 260 nm, using a Nanodrop spectrophotometer (Nanodrop Technologies Inc., CA). The plasmid concentration was adjusted to approximately lmg/ml with DNAse, RNAse-free water (Invirtogen, CA).

The following example describes expression and purification of Ixostatin for further use in studies to characterize its function.

EXAMPLE 2 EXPRESSION AND PURIFICATION OF IXOSTATIN After cloning Ixostatin-1 and Ixostatin-2 in a pIB/HIS-5 vector as described in the previous example, High-Five cells were transfected and the supernatant used for protein purification in a Ni-agarose column. The eluate was further purified in a HPLC C- 18 column. Ixostatin-1-His and Ixostatin-2-His expression in High Five Insect Cells High Five™ cells (BTI-Tn-4, Invitrogen, San Diego, CA) were cultivated in serum free medium supplemented with gentamycin (10 μg/ml) and GlutaMAX (2mM, Invitrogen, San Diego, CA), and were grown to 50-60% confluency in T-175 flasks. The cells were the transfected with Ixostatin-1-His or Ixostatin-2-His constructs (75 μg plasmid/200 μl cellfectin/20 ml medium without antibiotics/T-175 flask) for 4 hours at 28 C. Then, 30 ml of serum free media (supplemented with 15 μg/ml gentamycin) was added and after 3 days, the supernatant (50 mL) was collected, centrifuged (8,000 rpm, 30 min) and stored frozen for further analysis. An aliquot was used to confirm by Western blot that Ixostatin-1 and Ixostatin-2 were expressed, using anti-His monoclonal antibodies (as described below).

Purification of Recombinant Ixostatin-1 -His and Ixostatin-2-His

Five milliliters of Ni²⁺-Agarose gel (Invitrogen, CA) was added to 500 ml of supernatant containing Ixostatin-1 or Ixostatin-2, and the volume divided in 10x50 ml Falcon tubes. The tubes were allowed to rotate for 2 hours at 4 C in a lymphocyte mixer and centrifuged (2500 rpm for 15 min). The agarose gel containing Ixostatin-1 - His and Ixostatin-2-His was then washed once with 50 ml TBS pH 7.4, Ix 50 ml imidazol 10 μM, and incubated for 30 min with 5 ml of 300 mM imidazol, and centrifuged (2500 rpm for 15 min). The supernatant enriched with Ixostain-1-His, and Ixostatin-2-His (confirmed by Western blot as described below) was diluted in water and applied to a 10 x 250 mm Vydac 218TP510 octadecyl-silica reverse phase column (The Separation Group, Inc. Hesperia, CA) eluted at 1.5 ml/min with 60-min gradient from 10-80% acetonitrile in water containing 0.1% TFA.

Western Blot and ELISA assays to detect Ixostatin-1 -His and Ixostatin-2-His

To detect HIS-tag Ixostatin-1 by ELISA, an aliquot (5 μl) of each fraction eluted from the reverse phase column (above) were transferred to a 96-well plate and freeze dried. Wells of the 96-well plate were blocked with 200 μl TBS-BSA (BSA 2%, v/v, in TBS) for 2 hours and then incubated for 1 hour with anti-tetra His monoclonal antibody (0.5 μg/ml)(Gibco) or anti-6xhis monoclonal antibody (0.5 μg/ml)(Covance, Emeryville, CA) in TBS-BSA-Tween (BSA 5%, v/v, Tween, 0.05 % v/v in TBS). After washing four times with TBS-BSA-Tween, the wells were incubated with a 1 :2,000 dilution of anti-mouse alkaline-phosphatase coupled monoclonal antibody for 1 hour, and washed four times with TBS-BSA-Tween. One hundred μl of substrate (p- Nitrophenyl Phosphate Liquid Substrate System, Sigma Chemical Co.) was added and reactions were followed for 10 min and stopped with 100 μl of NaOH (0.375 N, final concentration). Absorbance was then detected at A405 nm. For Western blot analysis, His-tag-containing recombinant proteins were incubated with LDS buffer (denaturaing conditions) or LDS-dithiothreitol (DTT; denaturing and reducing conditions), warmed at 7O C for 10 min, loaded and run on a 4-12% NU-P AGE™ gel (MES buffer, Invitrogen, CA), and then electroblotted to PVDF membranes (0.22 um) using Transfer Buffer (Invitrogen, CA). Membranes were blocked with 1% BSA (in TBS), and incubated with anti-tetra His monoclonal antibody at a concentration of 0.5 μg/ml in TBS-BSA-Tween ("TBT," BSA 1%, v/v, Tween, 0.05 % v/v in TBS). Membranes were washed in TBT and incubated with a 1 :5,000 dilution of anti-mouse alkaline-phosphatase coupled monoclonal antibody for 1 hour. After washing, 10 ml of Western Blue stabilized substrate for alkaline phosphatase

(Promega, WI) was added, and color development followed for 10-20 min. Reactions were stopped by water.

PAGE of recombinant Ixostatin A sample of purified recombinant Ixostatin- 1 (2 μg) was incubated with LDS- dithiothreitol (DTT; denaturing and reducing conditions), warmed at 70 C for 10 min, and loaded into a 4-12% NUPAGE™ gel using MES buffer. For Ixostatin-2 analysis, a sample of purified recombinant Ixostatin-2 was added to LDS buffer in the presence of 5% B-mercaptoethanol, subsequently boiled for 10 min, and loaded into a 4-12% NU- PAGE using MES buffer.

Electrophoresis was performed using a PowerEasy™ 500 power supply (Invitrogen, CA). Gels were stained with Coomassie Brilliant Blue R-250 solution for 1 hour followed by destaining with Ix Destain Solution (Bio-Rad, Hercules, CA). Molecular weight markers were myosin (188 kDa), BSA (62 kDa), Glutamic Dehydrogenase (49 kDa.), Alcohol Dehydrogenase (38 kDa.), Carbonic Anhydrase (28 kDa.), Myoglobin (18 kDa), Lysozyme (14 kDa.), Aprotinin (6 kDa.), and Insulin, B chain (3 kDa.).

As shown in Figure 2A, purification by reverse phase HPLC resulted in elution of a single peak corresponding to Ixostatin- 1. Figure 2B shows a coomassie blue staining of the Ixostatin- 1 fraction under denaturing and reducing conditions, and demonstrates the purified nature of the expressed protein. The N-terminus of purified Ixostatin- 1 was sequenced by Edman degradation, and had the following sequence: EKSESGLVIYKEFESLQEG (SEQ ID NO: 12). This sequence was identical to the sequence predicted by the cDNA for the first 19 amino acids. As shown in Figure 2C, Western blot analysis of the purified Ixostatin-1 using anti-His antibody indicated that the protein behaves as a dimer (mol.wt. 18 kDa) under denaturating condition, and as monomer under reducing conditions (mol. wt. 9 kDa).

With purified Ixostatin proteins, functional assays were perfomed, as set forth in the following examples.

EXAMPLE 3 KINETIC ANALYSIS OF VITRONECTIN BINDING

Because Ixostatin-1 sequence has similarity to the cysteine-rich domain of Aggrecanase, the following experiments were performed using plasmon resonance technology to determine whether Ixostatins interact with matrix proteins. Binding Analysis with Surface Plasmon Resonance Surface plasmon resonance (SPR) studies were performed using a BIAcore X biosensor system (BIAcore AB, Sweden). Vitronectin (VN) at 50 μg/ml was immobilized (1500 RU) onto the surface of a sensor chip CM5 in 1O mM sodium acetate, pH 4.5, using the amine-coupling kit supplied by the manufacturer. To subtract the nonspecific component from the apparent binding response, the blank flow cell was prepared by the same immobilizing procedure but without VN. Binding analyses of

Ixostatin-1 to VN were performed at 25°C in running buffer (1O mM HEPES, pH 7.4, 15O mM NaCl cand 0.005% Tween 20). Forty microliters of varying concentrations of Ixostatin-1 (0-100 nM), was injected at a flow rate of 20 μl/min, and association was monitored. After return to buffer flow, dissociation was followed during 2 min. The sensor chip surface was regenerated by a pulse injection of 10 mM

HCl after each experiment. Kinetic binding constants were obtained using BIAevaluation 3.0 software (BIAcore).

The results show that Ixostatins interact with VN. As set forth in Figure 3A, kinetic analysis of the Ixostatin-1 -VN interaction yielded a Kd in the nanomolar range. Similar experiments also demonstrated similarly high affinity interaction of Ixostatin-2 with VN (see Example 14). Additional SPR experiments were performed to assess whether Ixostatins interact with other matrix proteins. Figure 3B shows that Ixostatin-1 does not interact with collagen type I, fibronectin or von Willebrandt factor. In addition, binding was not detectable for Collagen types H-VI, fibrinogen or laminin.

EXAMPLE 4

/N VITRO FIBRINOLYSIS ASSAY

In order to assess the effect of Ixostatin on plasma clotting, fibrinolysis and fibrin interaction, the following in vitro experiment was performed. Microplate-based clotting assays were performed at 37°C in flat-bottom, polystyrene 96-well plates (Corning) by monitoring turbidity changes at 605 run using a VERSAmax microplate reader (Molecular Devices) as described (Smith et al., Proc. Natl. Acad. Sci. USA 103:903-908 (2006)). Briefly, 50 μL human pooled normal plasma (diluted 1 :2 in TBS) was incubated with double-chain tissue plasminogen activator (dc- TPA) (2 nM) in the presence or in the absence of Ixostatin-1 (100 nM), followed by addition Of CaCl₂ (10 mM, final concentration). As a control, wells were not incubated with dc-TPA, or were incubated with Ixostatin-1 (100 nM), in the absence of dc-TPA. Clotting did not occur in the absence OfCaCl₂.

For Ixostatin 2, eight μl of human plasma (diluted 1 :2 in TBS-BSA 0.3%) was incubated with 0, 10 and 100 nM Ixostatin-2 for 3 hours at 37⁰C. Then, 10 μl CaCl₂ (10 mM, final concentration) was added to start reactions in the presence of 0.6 nM dc- TPA. In some wells, no dc-TPA was added. Clotting and fibrinolysis were followed turbidimetrically at 650 nm using Versamax plate reader (Molecular Devices, CA).

The results of the assay are set forth in Figure 4 and in Example 14. The results show that coagulation activation when triggered by Ca2+ alone ("plasma") was not followed by fibrinolysis. Similar results were obtained when Ixostatin-1 was added to plasma ("plasma + Ixostatin"). When dc-TPA was added to the wells, fibrinolysis started at ~ 8 min and continued until fibrin was completely digested at 53 min ("plasma

+ tPA"). In the presence of Ixostatin-1, fibrinolysis also started at ~ 8 min, but was complete at 48 min ("plasma + tPA + Ixostatin")(n=3). These results show that

Ixostatin-1 accelerates clot dissolution initiated by tPA. EXAMPLE 5 ADHESION ASSAY

In order to assess the function of Ixostatin in VN-dependent cell adhesion, the following adhesion assay was performed using human umbilical vein endothelial cells (MVECs).

Human umbilical vein endothelial cells (MVECs) were purchased from Clonetics (San Diego, CA). After trypsinization, cells were grown to confluence in 96- well plates in 200 μL endothelial cell basal medium-2 (EBM-2-PLUS) supplemented with endothelial cell growth medium (EBM-2) in a humidified incubator at 37⁰C with 5% CO2. On the day of the experiment, MVEC were trypsinized and re-suspended in 5 ml EBM-2 -BSA (EBM-2 medium containing 0.3% BSA, w/v) and incubated with 2 μM Calcein-AM for 30 min, at RT. Cells were washed 2x with and re-suspended in EBM-2- BSA at a concentration to 500,000 cells/ml.

Vitronectin (0.2 μg/ml in PBS, 100 μl/well, in quadruplicate) was added to Immunobind 96 well plates (Nunc) and immobilized overnight at 4⁰C. Wells were washed three times with PBS, and blocked with BSA (2% w/v) for two hours. 50 μl of

Ixostatin-2 (0-100 nM, in EBM-2-BSA) were added to the wells and incubated for 1 hour, followed by addition of 50 μl of MVEC (25,000 cells/well). Calcein-labeled

MVEC were incubated with victronectin-coated plates for 90 min. Labeled U937 cells were incubated with vitronectin-coated plates for 120 min. After incubation, wells were inverted and washed 5 times with EBM-2-BSA and fluorescence was measured with an excitation wavelength of 490 nm, and an emission wavelength of 530 nm using a

Microfluor plate reader (Molecular Devices, CA). Fluorescence produced by cells attached to VN, in the absence of Ixostatin was set as 100% adhesion; adhesion to wells coated with BSA only (or in the presence of RGDS or PAI-I) was set as 0% adhesion.

The adhesion assay results are shown in Figure 5. Figure 5 shows that Ixostatin- 1 dose-dependently inhibits MVEC adhesion to VN. At 10 nM Ixostatin- 1, 10% inhibition was observed, while complete inhibition was attained at 100 nM of Ixostatin- 1 (n=3). EXAMPLE 6

ANTIBODIES TO IXOSTATINS

Polyclonal antibodies specific for Ixostatin-1 or Ixostatin-2 are prepared, affinity purified, and used to detect Ixostatin-1 or Ixostatin-2 in a Western blot. Briefly, for Ixostain-1 antibodies, rabbit antiserum is raised against a synthetic peptide derived from the Cysteine-Rich domain (residues 89 to 107 of Ixostatin-1) using standard procedures.

(Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Lab. Press,

Cold Spring Harbor (1988), herein incorporated by reference). A GST-Ixostatin-1 fusion protein containing residues 1 to 11 1 of Ixostatin-1 was prepared and used to affinity purify the Ixostatin-1 antiserum (Quickpure kit from Sterogene).

The antibodies are used to detect naturally-occuring and recombinant Ixostatins and Ixostatin-like proteins and Ixostatin fragments in Western blot assays.

EXAMPLE 7 CODON OPTIMIZATION FOR EXPRESSION IN HUMAN CELLS

The nucleotide sequences encoding the full-length Ixostatin-1 and Ixostatin-2 proteins were modified to generate sequences optimized for expression in human cells without altering the encoded polypeptide sequences, according to Sharp et al. (1988) Nucleic Acids Res. 16:8207-1 1, hereby incorporated by reference. The optimized sequence for Ixostatin-1 (SEQ ID NO: 13) bears 75% identity to the original /. scapularis sequence. The optimized sequence for Ixostatin-2 (SEQ ID NO: 14) bears 78% identity to the original /. scapularis sequence.

EXAMPLE 8 IXOSTATIN DNA VACCINES AND THERAPEUTICS

An expression plasmid is designed to express Ixostatin-1 (SEQ. ID. NO.: 2). The Ixostatin-1 coding sequence (SEQ ID NO: 13) is inserted into plasmid A so that it is under the transcriptional control of the CMV promoter and the RSV enhancer element. (See U.S. Pat. No. 6,235,888 to Pachuk , et al., herein expressly incorporated by reference in its entirety). Plasmid Plasmid backbone A is 3969 base pairs in length; it contains a PBR origin of replication for replicating in E. coli and a kanamycin resistance gene. Inserts such as Ixostatin-1, are cloned into a polylinker region, which places the insert between and operably linked to the promoter and polyadenylation signal. Transcription of the cloned inserts is under the control of the CMV promoter and the RSV enhancer elements. A polyadenylation signal is provided by the presence of an SV40 poly A signal situated just 3' of the cloning site. An Ixostatin-1 containing vaccine composition is then made by mixing 500μg of the Ixostatin-1 construct with

1 mg of ribavirin.

Said vaccine composition can be used to raise antibodies in a mammal (e.g., mice or rabbits) or can be injected intramuscularly into a human so as to to raise antibodies. The recipient preferably receives three immunization boosts of the mixture at 4-week intervals, as well. By the third boost, the titer of antibody specific for

Ixostatin-1 will be significantly increased.

EXAMPLE 9 INHIBITION OF TUMOR CELL GROWTH IN HUMAN PATIENTS A group of human cancer patients diagnosed with pancreatic cancer is randomized into treatment groups. Each patient group is treated with weekly intravenous injections of full-length Ixostatin-1 or Ixostatin-2 described herein in addition to the standard chemotherapeutic regimen. Each patient is dosed with an effective amount of the protein (ranging from 1-100 mg/hr for 6 hr. C. I., daily x 5 days and repeated every two weeks) duing a period of two months. A control group is given only the standard chemotherapeutic regimen.

At periodic times during and after the treatment regimen, tumor burden is assessed by magnetic resonance imaging (MRI). It is found that the patients who have received bi-weekly treatment with Ixostatin-1 or Ixostatin-2 show significant reductions in tumor size, time delay to progression or prolonged survival compared to patients that do not receive antibody treatment. In some treated patients, the tumors are no longer detectable. In contrast, tumor size increases or remains substantially the same in the control group. EXAMPLE 10

INHIBITION OF TUMOR METASTASIS IN HUMAN PATIENTS A group of human cancer patients diagnosed with non-small cell lung cancer is randomized into treatment groups. In addition to a standard chemotherapeutic regimen, each patient group is treated with weekly intravenous injections of Ixostatin-1 or

Ixostatin-2 described herein. Each patient is dosed with an effective amount of the (ranging from 1-100 mg/hr for 6 hr. C.I., daily x 5 days and repeated every two weeks) during a period of two months. A control group is given only the standard chemotherapeutic regimen. At periodic times during and after the treatment regimen, metastatic lesions are assessed by positron emission tomography (PET). It is found that the patients who have received bi-weekly treatment with Ixostatin-1 or Ixostatin-2 show significant reductions in the number and size of metastatic lesions, compared to patients that do not receive Ixostatin treatment. EXAMPLE I l

/N VIVO CLOT LYSIΝG ACTIVITY

An in vivo experiment is performed in rabbits to demonstrate the dose response of Ixostatin-1 or Ixostatin-2 either alone or in combination with t-PA. Thrombolytic activities are determined in rabbits using a extracorporeal shunt which contains a thrombus labeled with 1-125 fibrinogen. Lysis is measured by the disappearance of radioactivity, measured by an external sodium iodine crystal. Wild type t-PA is given as a 10% bolus with the remainder of the dose infused over the following 90 min.

Ixostatin-1 and Ixostatin-2 are tested at single dose of 0.1 mg/kg using a 0.05 mg/kg bolus followed by an infusion of 0.05 mg/kg for 90 min. All lysis is determined at the end of the 90 min. infusion.

The results show that Ixostatin has a surprisingly high clot lysing activity, and enhances the clot lysing activity of tPA.

EXAMPLE 12 TREATMENT OF THROMBOSIS IN A HUMAN PATIENT

Ixostatins may be parenterally administered to subjects suffering from cardiovascular diseases or conditions, including acute thrombosis. Dosage or dose rate may parallel that currently in use in clinical investigations of other cardiovascular, thrombolytic agents, e.g. about 1-2 mg/kg body weight as an intravenous or intraarterial dose over 1.5-12 hours in patients suffering from conditions, such as myocardial infarction and pulmonary embolism.

EXAMPLE 13 TREATMENT OF A HUMAN PATIENT USING AN IXOST ATIN-IMPREGNATED

STENT

A human patient is diagnosed with cardiovascular disease. During an angioplasty procedure, a drug-eluting mesh stent impregnated with Ixostatin-1 and tPA is placed in an occluded artery. The treated artery is monitored periodically after the procedure, and the patient will experience improved cardiovascular health.

EXAMPLE 14 CHARACTERIZATION OF IXOSTATIN-2

Cloning, expression and purification of Ixostatin, and its identification as a specific vitronectin-binding protein

Ixostatin is cysteine-rich peptide found in the salivary gland of the ticks /. scapularis and /. pacificus. Figures IA and IB show that the position of the cysteines in Ixostatin is highly conserved among several members of this family and characteristically display the pattern CXlOCXl ICXl 4CX3CX24CX5CX4C. In addition, most Ixostatins present the sequence NLKDGTPCG (SEQ ID NO: 35), which appears to be a signature for this family of proteins that share a commom ancestor (Figure 1C). The DGTPC signature is also found in mosquitoes reprolysins (Figure IB) and notably, in the cysteine-rich domain of ADAMTS-4 (aggrecanase-1) as diagrammatical Iy depicted in Figure ID.

In an attempt to identify a biological function for Ixostatins, the cDNA coding for Ixostatin-2 was PCR amplified and a nucleotide sequence coding for 6xHIS tag was added to the T end of the cDNA. After cloning the insert in a PIB/HIS-5 vector, High Five cells were transfected with the appropriate expression vector as described in

Material and Methods. After 3 days, the supernatant for Ixostatin-2 was purified in a Ni 2+ agarose column. Ixostatin-2 was eluted with 300 mM imidazol and chromatographed in a reversed-phase column (RP-HPLC) (Figure 6A). A single active peak (estimated by SPR) corresponding to Ixostatin-2 was found at ~ 16 ml of retention volume. Western blot analysis using anti-His antibody indicate that Ixostatin-2 consistently migrates as a doublet of ~ 10 kDa protein under reducing condition, likely a result of cysteine re-oxidation or glycosylation (Figure 6B). The N-terminus of purified

Ixostatin-2 was sequenced by Edman degradation and the aminoacids DIFGVMKYL (SEQ ID NO: 36) were identified which are identical to the sequence predicted by the cDNA. Similar results were obtained for Ixostatin-1 (see Example 2).

Polyclonal Antibody anti-Ixostatin Production in Mice

Ten micrograms of recombinant Ixostatin-2 produced in E. coli was injected in both ears of Webster mice. Additional injections were performed two, four and six weeks later to boost the immune response. Previous to injection of Ixostatin-2, pre- immune serum was obtained from all mice. Antibody titer was estimated by ELISA using bacterial Ixostatin-2 (1 μg/well) immobilized overnight in PBS.

Cell Culture

U937 (myelomonocytic cells) were obtained from American Type Culture Collection (Rockville, MD) and were cultured in RPMI- 1640 medium containing fetal calf serum (FCS) 10% (v/v), penicillin (100 U/ml) and streptomycin (100 μg/ml) in a humidified incubator at 37⁰C with 5% CO₂. U937 cells were differentiated with PMA (16 nM) overnight. Differentiated U937 (suspension cultures) were trypsinezed, washed and resuspended in RPMI 1640 with 0.3% (w/v) BSA and loaded with calcein AM for 30 min at rt (2 μM), and washed. Cells were re-suspended in RPMI-BSA and used for cell adhesion assays as below. Human microvascular endothelial cells were purchased from Clonetics (San Diego, CA). Cells were grown to 80% confluency in T-25 flasks in 10 ml endothelial cell basal medium-2 (EBM-2) containing 2% fetal bovine serum, and human Fibroblast Growth Factor, Vascular Endothelial Growth Factor, R³-Insulin- like Growth Factor, ascorbic acid, human Epidermal Growth Factor, and gentamicin- anfotericin B. On the day of the experiment, cells were trypsinezed and re-suspended in

EBM-2 media containing 0.3% BSA (EBM-BSA media), without growth factors. Cells (400-500,000/ml) were incubated with 2 μM calcium AM for 30 min at room temperature, centrifuged and re-suspended in EBM-BSA media.

Ixostatin is a Vitronectin binding protein Due to its similarity to aggrecanase-1, an enzyme that interacts with matrix proteins and involved in cartilage remodeling, a screening assay was designed to test whether Ixostatin-2 recognizes any matrix protein, using SPR as follows.

SPR experiments were carried out in a TlOO instrument (Biacore Inc., Uppsala, Sweden) following the manufacturer's instructions. This instrument features an integrated degasser, allowing problem-free kinetic measurements at temperatures up to

45oC, as well as a temperature-controlled flow cell and sample compartment. The Biacore TlOO evaluation software was utilized for kinetic and thermodynamic evaluation. Sensor CM5, amine coupling reagents, and buffers were also purchased from Biacore Inc (Piscataway, NJ). HBS-P (10 mM Hepes, pH 7.4, 150 mM NaCl, and 0.005% (v/v) P20 surfactant) was used as the running buffer for all SPR experiments.

All SPR experiments were carried out three times. Immobilization and kinetic analysis. Monomeric or multimeric human, vitronectin (30 μg/ml) in acetate buffer pH 4.5 was immobilized over a CM5 sensor via amine coupling resulting in a final immobilization of 1,671.8 and 1,81 1.6 RU, respectively. Mouse and rat multimeric vitronectin were immobilized as above, yielding a final response of 2,435.5 and 1,416.3 RU, respectively. Somatomedin B 1-44 domain (50 μg/ml) in acetate buffer pH 4.0 was immobilized for 1800 sec, at a flow of 5 μl/min and yielded 523.2 RU. Blank flow cells were used to subtract the buffer effect on sensorgrams. Kinetic experiments were carried out with a contact time of 180 s at a flow rate of 30 μl/min at 25⁰C. Ixostatin- vitronectin complex dissociation was monitored for 2000 s, and the sensor surface was regenerated by a pulse of 60 s of 20 mM HCl at 30 μl/minute. Sensorgrams were fitted using the 1 :1 model interaction model as follows: ka A+B<→AB kd

Model parameters are: ka, association rate constant for analyte binding; kd, dissociation rate constant for analyte from the complex. It was found that Ixostatin-2 specifically interacts with vitronectin (Figure 6C), but no binding could be detected for collagen, fibronectin, laminin, von Willebrand Factor and fibrinogen. Similar results were obtained for Ixostatin-1 (see Example 3).

Ixostatin is found in the saliva

Next, experiments were performed to identify whether native Ixostatin was found in the saliva. Figure 6D shows the chromatogram of 10 μl saliva applied to a high resolution RP-HPLC column, performed as described hereinabove.

Saliva fractions were tested for binding to immobilized vitronectin, using SPR, and activity was found in two peaks at ~ 7 and ~9 ml of retention volume. Both peaks were positive for Ixostatin-2 according to ELISA assays, and notably, the active peak corresponding to Ixostatin-2 display the same retention time of the second peak obtained with saliva. One explanation is that two peaks have been found because different salivary Ixostatins behave distinctly in the RP-HPLC. Figure 6E and 6F respectively show that saliva at dilutions < 1 :400 dose-dependently display monomeric and multimeric vitronectin-binding properties. The concentration of Ixostatin in the saliva is estimated to be in nM range.

Ixostatin exhibits high-affinity binding to vitronectin To investigate binding kinetics of Ixostatin-2 interaction with vitronectin, SPR experiments were performed as described in Methods. Typical sensorgrams obtained for Ixostatin-2 interaction with monomeric and multimeric vitronectin are shown in Figs 3A and 3B, respectively. In both cases, the best fit was attained using a one state reaction model and a KD of ~ 0.5 nM for both monomeric and multimeric vitronectin was determined.

The results for the experiment depicted in Figure 7 are shown in Table 1. Responses were obtained by injecting Ixostatin-2 over immobilized vitronectin for 180 sec at a flow rate of 30 μl/min. Dissociation was followed for 2000 sec. Data was fitted according to 1 :1 model. Ixostatin also binds to multimeric mouse and rat vitronectin (not shown).

Table 1. Kinetics of Ixostatin-2 interaction with human vitronectin estimated by SPR. Ka Kd KD

(M-¹S ¹) (S-¹) (nM) χ²

Monomeric vitronectin 6.698x10° 0.000291 0.43 0. 58 Multimeric vitronectin 1.171x10⁵ 0.000089 0.76 1.55

SMTB domain is a high-affinity binding site for Ixostatin

Somatomedin B (SMTB) domain of vitronectin that comprises the first 44 amino acid of the protein mediates its interaction with PAI-I, uPAR and integrin αvβ3. In an attempt to test whether Ixostatin binds to SMTB domain, the peptide was synthesized, refolded, and purified to homogeneity as shown in Figure 8A.

SMTB domain (theoretical mol wt 5,003.54) DQESCKGRCTEGFN VDKKCQCDELCSYYQSCCTDYTAECKPQVT (SEQ ID NO: 37) synthesis was carried out on an Applied Biosystems' model 433A peptide synthesizer using solid- phase, Fmoc (9-fluorenylmethoxycarbonyl) peptide synthesis methodology with HBTU

2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium haxafluorophosphate); NMP, 1- methylpyrolidin-2-one activation. All cysteine residues were trityl-protected. The Fmoc groups were removed from the N-terminal amino group of the resin-bound peptide with 20% of 4-methylpiperidine in NMP. Deprotection/cleavage from the resin was carried out with TFA/thioanisole/EDT (92:5:3, v/v/v) mixture at room temperature for 2.5 hrs.

The linear SMTB was purified by RP-HPLC on Phenomenex Gemini Cl 8 (21.2mm x 250mm) column utilizing water/acetonitrile gradient. The peptide was refolded according to (Okumura Y, et al. J Biol Chem. 2002;277:9395-9404). Refolded material was purified by RP-HPLC utilizing conditions used for the linear form. The final, folded peptide showed the correct molecular mass of 5,003.00.

Mass spectrometry analysis of the fraction containing SMTB domain indicates a molecular weight of 5,003.00 da, which is in agreement with the theoretical mass of 5,003.5 for the peptide with 8 oxidized cysteines (Figure 8A, inset). The peptide was immobilized in a CM5 chip, and PAI-I was used as a control to demonstrate that SMTB was functionally folded. Figure 8B shows that PAI-I binds to SMTB with a KD in the pM range as previously reported for PAI-I -vitronectin interaction (Okumura Y, et al. J Biol Chem. 2002;277:9395-9404; Arroyo De Prada N, et al. Eur J Biochem. 2002;269:184-192.), validating the use of synthetic SMTB for additional experimentation. Figure 8C shows sensorgram for Ixostatin-2 interaction with SMTB, which yield a KD of -0.5 nM (Table 2) while Figure 8D shows that saliva at dilution < 1:400 display SMTB binding properties. Notably, the fractions from saliva that bind to vitronectin also bind to immobilized SMTB domain (not shown). As a control, binding was not observed when other arthropod recombinant salivary protein (250 nM each) were used as analyte, including cystatin, yellow protein, nitrophorin, ixolaris and aegyptin.

Experiments were performed as indicated in Table 1. Data were fitted according to 1 :1 model for Ixostatin-2 and PAI-I-SMTB interactions.

Table 2. Kinetics of Ixostatin-2 and PAI-I interaction with SMTB estimated bv SPR. ka kd KD (M-¹S-¹) (S-¹) (nM) χ² lxostatin-2 1.942x10^b 0 .00078 0.40 0 .483 PAI-1 5.898x10⁵ 0.00040 0.08 0.341

Bacterial Ixostatin binds to vitronectin

Because of relatively low levels of expression of Ixostatin-2 in High Five cells, Ixostatin-2 was produced in E. coli. Synthetic cDNA for Ixostatin-2 was produced by Biobasics (Ontario, CA). The sequence displays and N-terminal Ndel and a C-terminal Xhol restriction site. The Ndel site added a 5'-methionine codon to all sequences that acts as start codon in the bacterial expression system, whereas the Xhol site was incorporated after the stop codon. pET 17b constructs were confirmed before transformation of Escherichia coli strain BL21 cells. F or recombinant protein production, 30 ml of Luria Bertani broth (with added chloramphenicol and ampicilin) was inoculated and grown overnight (maximum of 16 h). Luria Bertani broth (1 liter, with added chloramphenicol and ampicilin) was inoculated with 10 ml of the overnight culture and grown at 37 °C with shaking at 250 rpm until an optical density of 0.6-0.8 (A600 nm) was reached (~ 3h) before isopropyl- 1 -thio-b-D-galactopyranoside (1 ΠTM final concentration) was added to induce expression. The flask was shaken for 3 h under the same conditions; cells were harvested by centrifugation and washed once in 20 ΠIM Tris-HCl, pH 8.0, before the cell pellet was frozen and stored until use. The frozen cell pellet was resuspended in 200 ml of 20 niM Tris-HCl, pH 8.0, and cells disrupted using a probe sonicator before collecting the inclusion bodies by centrifugation. Inclusion bodies were washed with 20 ΠTM Tris- HCl, pH 8.0, and 1% Triton X-100. The remaining pellet was washed three times with 20 ITiM Tris-HCl, pH 8.0, before solubilization in 20 ml of 20 HIM Tris-HCl, pH 8.0, 6 M guanidinium hydrochloride, 10 HIM dithiothreitol. The solubilized material was diluted into 4 liter of 20 ITIM Tris-HCl, pH 8.5, 0.2 M arginine monohydrochloride, 1 mM EDTA,

0.2 mM GSSG, and 1 mM GSH for 48 hrs at 4⁰C, under agitation. The samples were concentrated by ultrafiltration and then purified by RP-HPLC as above.

Figure 9A depicts the mass spectrometry for the recombinant protein. The estimated molecular mass was compatible with the predicted calculated mass of the inhibitor with an extra methione. Figures 9B and 9C confirm that Ixostatin-2 binds to multimeric vitronectin and to the SMTB 1-44 with comparable affinities of KD 0.29 nM and 0.46 nM, respectively.

Ixostatin-2 (250 nM, final concentration) was preincubated for 15 minutes at 37⁰C with the enzymes listed, followed by addition of the appropriate fiuorogenic or chromogenic substrate (250 μM). Reactions were followed for 30 minutes at 37⁰C.

Substrate hydrolysis rate was followed in a Spectramax Gemini XPS 96 well plate fluorescence reader (Molecular Devices, Sunnyvale, CA) using 365 nm excitation and 450nm emission wavelength with a cutoff at 435 nm. The effect of Ixostatin-2 was estimated by setting the initial velocity obtained in the presence of enzyme alone (without inhibitor) as 100%. Table 3 shows that Ixostatin-2 is devoid of inhibitory activity toward proteases.

Table 3. Specificity of Ixostatin-2.

Enzymes (nM) Residual

Activity

(%)

Thrombin (0.01 nM) 99.2+2.7

Factor Xa (0.8 nM) 106.7+2

Kallikrein (1.2 nM) 93.1+3.2

Trypsin (0.24 nM) 97.1+1.4

Chymotrypsin (0.06 nM) 99.7+1.8

Elastase (0.008 nM) 126.8+3.8

Granzyme B (15 nM) 101.1+1.2

Cathepsin G (1O nM) 103.8+3.1 uPA (0.7 nM) 86.8+2.8 tPA (5 nM) 99.8+1 Plasmin (0.25 nM) 106.3+5.2

Ixostatin prevents the interaction of αvβ3- and uPAR interaction with vitronectin, but displays negligible effects on fibrinolysis. The SMTB domain mediates integrin αvβ3-, uPAR- and PAI-I -mediated binding to vitronectin. Figures 1OA and 1OB show that Ixostatin-2 dose-dependently inhibits αvβ3 -expressing MVEC and uPAR-bearing U937 cell adhesion to vitronectin with IC50 ~ of 30 and 100 nM, respectively. As a control for both adhesion experiments, complete blockade of cell adhesion was attained with cyclic RGDS (1 mM) and PAI-I (1 μM), respectively (not shown). On the other hand, Ixostatin-2 (100 nM) did not interfere with fibrinolysis mediated by dc-TPA in vitro (Figure 10C). Finally, Figure 11 summarizes the putative binding site for Ixostatin in the SMTB domain of vitronectin and the mechanism by which it negatively modulates cell adhesion, migration and angiogenesis.

Although the invention has been described with reference to embodiments and examples, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All references cited herein are hereby expressly incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A purified or isolated nucleic acid encoding an Ixostatin polypeptide comprising a cysteine rich domain.

2. The nucleic acid of Claim 1, wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of: SEQ. ID. NO: 1 , SEQ. ID.

NO: 3, SEQ. ID NO: 13, SEQ. ID. NO: 14, SEQ. ID. NO: 15, and SEQ. ID. NO. 16 or a sequence complementary thereto.

3. A purified or isolated nucleic acid sequence encoding a polypeptide copmprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO: 4.

4. The nucleic acid sequence of Claim 3, wherein said sequence is selected from the group consisting of: SEQ. ID. NO: 1, SEQ. ID. NO: 3, SEQ. ID NO: 13, SEQ. ID. NO: 14, SEQ. ID. NO: 15, and SEQ. ID. NO. 16.

5. A purified or isolated Ixostatin polypeptide comprising a cysteine rich domain.

6. The polypeptide of Claim 5, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4.

7. An antibody capable of specifically binding to a protein comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2 and SEQ ID

NO: 4.

8. The antibody of Claim 7, wherein said antibody specifically binds to a polypeptide comprising at least 10 consecutive amino acids of said protein.

9. The antibody of Claim 7 or 8, wherein the antibody is a monoclonal antibody.

10. A purified or isolated antibody, which specifically binds Ixostatin- 1 protein, but does not specifically bind to Ixostatin-2 protein.

1 1. A purified or isolated antibody which specifically binds Ixostatin-2 protein, but does not specifically bind Ixostatin- 1 protein.

12. A method of identifying a binding partner that interacts with Ixostatin- 1 or Ixostatin-2 comprising: providing a support comprising Ixostatin-1, Ixostatin-2 or a representative fragment thereof; contacting the support with a candidate binding partner; and detecting a biological complex comprising Ixostatin-1 or Ixostatin-2 and the candidate binding partner, wherein detection of such complex indicates that said candidate binding partner interacts with Ixostatin-1 or Ixostatin-2.

13. The method of Claim 12, wherein said support is a microarray substrate.

14. The method of Claim 12, wherein said support is a bead.

15. The method of Claim 12, wherein said support is a membrane.

16. The method of Claim 12, wherein said Ixostatin-1 or Ixostatin-2 is SEQ

ID NO: 2 or SEQ ID NO: 4 or a polypeptide encoded by SEQ ID NOs: 1, 3, 13, 14, 15, or 16 or a codon-optimized version thereof.

17. A method of treating or inhibiting progression of a malignant tumor in a subject comprising: identifying a subject in need of a molecule that treats or inhibts progression of a malignant tumor and providing said animal with a therapeutically effective amount said molecule, wherein said molecule is selected from the group consisting of: an Ixostatin polypeptide or fragment thereof, a nucleic acid encoding an Ixostatin polypeptide or fragment thereof, and an antibody capable of specifically binding to an Ixostatin protein.

18. The method of Claim 18, wherein said molecule is an Ixostatin polypeptide or fragment thereof.

19. The method of Claim 18, wherein said molecule is a nucleic acid encoding an Ixostatin polypeptide or fragment thereof.

20. The method of Claim 18, wherein said molecule is SEQ ID NO: 2 or SEQ ID NO: 4 or a polypeptide encoded by SEQ ID NOs: 1, 3, 13, 14, 15, or 16 or a codon-optimized version thereof.

21. The method of Claim 18, wherein said molecule is an antibody capable of specifically binding to an Ixostatin protein.

22. The method of Claim 18, wherein said animal is human.

23. A method of reducing clot formation comprising: identifying a subject in need of a reduction in clot formation and providing to said subject a therapeutically effective amount of a molecule selected from the group consisting of: an Ixostatin polypeptide or fragment thereof, a nucleic acid encoding an Ixostatin polypeptide or fragment thereof, and an antibody capable of specifically binding to an Ixostatin protein.

24. The method of Claim 24, wherein said subject in need of a reduction in clot formation suffers from a thrombosis-related disease.

25. The method of Claim 25, wherein said thrombosis-related disease is any one of: coronary thrombosis, pulmonary embolism, myocardial infarction, deep vein thrombosis, cerebral thrombosis, postoperative fibrinolytic shutdown, or a rapid thrombogenic action which can occur following implantation of a medical device.

26. The method of Claim 24, wherein said molecule is SEQ ID NO: 2 or

SEQ ID NO: 4 or a polypeptide encoded by SEQ ID NOs: 1, 3, 13, 14, 15, or 16 or a codon-optimized version thereof.

27. A method of treating a thrombogenic disease comprising: identifying a subject suffering from a thrombogenic diease and providing to said subject a therapeutically effective amount of a molecule selected from the group consisting of: an

Ixostatin polypeptide or fragment thereof, a nucleic acid encoding an Ixostatin polypeptide or fragment thereof, and an antibody capable of specifically binding to an Ixostatin protein.

28. The method of Claim 28, wherein said thrombogenic disease is any one of: coronary thrombosis, pulmonary embolism, myocardial infarction, deep vein thrombosis, cerebral thrombosis, postoperative fibrinolytic shutdown, or a rapid thrombogenic action which can occur following implantation of a medical device.

29. The method of Claim 28, wherein said molecule is SEQ ID NO: 2 or SEQ ID NO: 4 or a polypeptide encoded by SEQ ID NOs: 1, 3, 13, 14, 15, or 16 or a codon-optimized version thereof.

30. An antithrombogenic medical device, wherein said device comprises a therapeutically effective amount of an Ixostatin or fragment thereof.

31. The antithrombogenic medical device of Claim 31, wherein said Ixostatin is SEQ ID NO: 2 or SEQ ID NO: 4 or a polypeptide encoded by SEQ ID NOs: 1, 3, 13, 14, 15, or 16 or a codon-optimized version thereof.

32. A vector comprising the purified or isolated nucleic acid of Claim 1.

33. A cultured cell line comprising the vector of Claim 33.

34. A therapeutic anticoagulant formulation comprising: an Ixostatin polypeptide or fragment thereof in combination with a pharmaceutically acceptable carrier.

35. The therapeutic anticoagulant formulation of Claim 35, wherein said Ixostatin polypeptide is SEQ ID NO: 2 or SEQ ID NO: 4 or a polypeptide encoded by

SEQ ID NOs: 1, 3, 13, 14, 15, or 16 or a codon-optimized version thereof.

36. The therapeutic formulation of Claim 35, further comprising a second antithrombogenic agent.

37. The therapeutic formulation of Claim 37, wherein said second antithrombogenic agent is any one of: heparin, hirudin, albumin, phospholipids, streptokinase, tissue plasminogen activator (tPA), urokinase (uPA), an hydrophilic polymers, hyaluronic acid, chitosan, methyl cellulose, poly(ethylene oxide), polyvinyl pyrrolidone), a growth factor, endothelial cell growth factor, epithelial growth factor, osteoblast growth factor, fibroblast growth factor, platelet derived growth factor (PDGF), or an angiogenic growth factor.

38. A kit for determining Ixostatin protein expression comprising: a probe indicative of Ixostatin protein expression in cells.

39. A vaccine for the treatment of animals, comprising an Ixostatin polypeptide or fragment thereof, or a nucleic acid encoding an Ixostatin polypeptide or fragment thereof.

40. Use of an Ixostatin polypeptide or fragment thereof for the treatment of a thrombogenic disease.

41. The use of Claim 41, wherein said thrombogenic disease is any one of: coronary thrombosis, pulmonary embolism, myocardial infarction, deep vein thrombosis, cerebral thrombosis, postoperative fibrinolytic shutdown, or a rapid thrombogenic action which can occur following implantation of a medical device.

42. A method of preventing angiogenesis comprising: identifying a subject in need of angiogenesis prevention and providing to said subject a therapeutically effective amount of a molecule selected from the group consisting of: an Ixostatin polypeptide or fragment thereof, a nucleic acid encoding an Ixostatin polypeptide or fragment thereof, and an antibody capable of specifically binding to an Ixostatin protein.

43. The method of Claim 42, wherein said molecule is an Ixostatin polypeptide or fragment thereof.

44. The method of Claim 42, wherein said molecule is a nucleic acid encoding an Ixostatin polypeptide or fragment thereof.

45. The method of Claim 42, wherein said molecule is SEQ ID NO: 2 or

SEQ ID NO: 4 or a polypeptide encoded by SEQ ID NOs: 1, 3, 13, 14, 15, or 16 or a codon-optimized version thereof.

46. A method of preventing metastasis comprising: identifying a subject in need of a molecule that prevents matastasis and providing said subject with a therapeutically effective amount said molecule, wherein said molecule is selected from the group consisting of: an Ixostatin polypeptide or fragment thereof, a nucleic acid encoding an Ixostatin polypeptide or fragment thereof, and an antibody capable of specifically binding to an Ixostatin protein.

47. The method of Claim 46, wherein said molecule is an Ixostatin polypeptide or fragment thereof.

48. The method of Claim 46, wherein said molecule is a nucleic acid encoding an Ixostatin polypeptide or fragment thereof.

49. The method of Claim 46, wherein said molecule is SEQ ID NO: 2 or SEQ ID NO: 4 or a polypeptide encoded by SEQ ID NOs: 1, 3, 13, 14, 15, or 16 or a codon-optimized version thereof.

50. A method for identifying proteins that bind to vitronectin comprising: providing a homogenate from an arthropod, contacting said homogenate with vitronectin or a fragment thereof and identifying a binding complex indicative of a protein binding to vitronectin.

51. The method of Claim 50, wherein said vitronectin or fragment thereof comprises the somatomedin B (SMTB) domain of vitronectin.

52. The polypeptide of Claim 5, wherein said cysteine rich domain comprises the conserved pattern CX_IOCX_{I I}CX_I4CX₃CX₂₄CX₅CX₄C, wherein X is any amino acid residue, wherein Xio comprises 1 to 10 residues between the first and second cysteines, Xn comprises 1 to 1 1 residues between the second and third cysteines, XH comprises 1 to 14 residues between the third and fourth cysteines, X₃ comprises 1 to 3 residues between the fourth and fifth cysteines, X₂₄ comprises 1 to 24 residues between the fifth and sixth cysteines, X₅ comprises 1 to 5 residues between the sixth and seventh cysteines and X₄ comprises 1 to 4 residues between the seventh and eighth cysteines.