Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2002-01-29
2004-11-09
Chan, Christina (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S389100, C530S391300, C530S391900
Reexamination Certificate
active
06815534
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel human gene encoding a polypeptide which is a homolog of tissue-type plasminogen activator (t-PA). More specifically, isolated nucleic acid molecules are provided encoding a human polypeptide named tissue-plasminogen activator-like protease, hereinafter referred to as “t-PALP”. t-PALP polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic methods for detecting disorders related to the circulatory system and therapeutic methods for treating such disorders. The invention further relates to screening methods for identifying agonists and antagonists of t-PALP activity.
BACKGROUND OF THE INVENTION
The plasmin coagulation system is activated in response to vascular injury. Within a few minutes of the injury, prothrombin is activated through the coagulation cascade to give rise to thrombin. Thrombin then converts fibrinogen to insoluble fibrin, which then interdigitates with and strengthens the primary platelet. Abnormal blood clotting can lead to many vascular diseases, such as stroke, deep-vein thrombosis, peripheral arterial occlusion, pulmonary embolism, and myocardiothrombosis, each of which constitutes a major health risk. Such diseases are primarily caused by partial or total occlusion of a blood vessel by a blood clot. Such clots consist essentially of a mass of fibrin and platelets. The prevention of clot formation and the dissolution of existing clots are two major therapeutic avenues frequently used for the treatment of disease states related to blood clots. Prevention of clot formation is primarily achieved through the inhibition of thrombin activity, whereas the dissolution of existing clots is frequently achieved by the activation of plasminogen which dissolves the existing blood clot (thereby affecting the fibrinolysis pathway).
The fibrinolytic system is activated by the deposition of fibrin. The conversion of fibrinogen to fibrin results in the exposure of many lysine residues on the surface of the molecule. A factor released from endothelial cells, termed tissue-type plasminogen activator (t-PA), activates plasminogen. Only upon activation can plasminogen bind to exposed lysine residues on the surface of fibrin, resulting in the degradation of fibrin, and, ultimately, the degradation of the blood clot itself.
In man and other animals, t-PA plays an essential role in the dissolution of fibrin clots (see, e.g., Verstraete and Collen, (1986)
Blood
67:1425). t-PA is composed of several domains which share sequence homology with other proteins. These are the fibronectin finger-like domain, the epidermal growth factor-like domain, the kringle domain (of which t-PA has two), and the protease domain (Pennica, D., et al., (1983) Nature 301:214-221; Banyai, L., et al., (1983)
FEBS Lett.
163:37-41). Only the function of the protease domain (residues 276-527) has been unambiguously defined. This finding was first based on the observed sequence homology with other known serine proteases. More recently, limited reduction of the two-chain form of t-PA has allowed the direct isolation and functional characterization of the protease region (Rijken and Groeneveld, (1986)
J. Biol. Chem.,
261:3098).
In addition to the role played by human t-PA and related protease-like molecules in the fibrinolytic system, this same family of molecules also play important roles in carcinogenesis. On the one hand, numerous studies have implicated the plasminogen activator and/or protease activity of t-PA and related molecules in promoting progression of carcinogenesis and metastasis (for example see: Alizadeh, H., (1995) Curr. Eye Res. 14:449; Yamashita, J., (1993) Br. J. Cancer 68:524; Yamashita, J., (1992) Int. J. Clin. Lab Res. 21:227; Koller, A., (1984) Eur. Urol. 10:389). As such, inhibitors of the plasminogen activator and/or protease activity of t-PA and related molecules may provide useful therapeutics in combating cancer.
On the other hand, there is also now a large body of evidence which shows that specific domains from proteins such as t-PA can actually inhibit tumorigenesis and metastasis by inhibiting endothelial cell-mediated vascularization (i.e. angiogenesis) which is required for tumor growth. The specific domains mediating such anti-angiogenic activity have been identified as “kringle” domains. Kringle domains are triple-looped, disulfide cross-linked domains occurring with varying copy numbers in some serine proteases and plasma proteins. The kringle domain has been found in proteins such as: Apolipoprotein A (38 copies); Blood coagulation factor XII (Hageman factor) (1 copy); Hepatocyte growth factor (HGF) (4 copies); Hepatocyte growth factor-like protein (4 copies); Hepatocyte growth factor activator (1 copy); Plasminogen (5 copies); Thrombin (2 copies); Urokinase-type plasminogen activator (1 copy); and Tissue plasminogen activator (TPA) (2 copies). The signature pattern of a kringle domain is [F/Y]-C-R-N-P-[D/N/R] (SEQ ID NO:28), where C (cysteine) is involved in disulfide bond formation.
Kringle domains appear to be effective inhibitors of endothelial cell angiogenesis, and thus, effective inhibitors of tumorigenesis and metastasis. It has been demonstrated, for example, that a four-kringle domain containing protein called HGF/NK4 inhibits invasion of multiple tumorigenic cell types in both in vitro and in vivo assays (Date, K., et al. (1998) Oncogene 17:3045). Similarly, angiostatin (a fragment of plasminogen containing four kringle domains) has also been shown to inhibit tumor vascularization, growth, and metastasis (O'Reilly M. S., et al., (1994) Cell 79:315; O'Reilly, M. S., et al., (1996) Nat. Med. 2:689). Furthermore, a fragment of plasminogen containing just three kringle domains has been demonstrated to markedly reduce growth of malignant brain tumors in mice (Joe, J. Y., et al., (1999) Int. J. Cancer 82:694). Finally, it has also been demonstrated that a single kringle domain of angiostatin is sufficient to significantly inhibit endothelial cell angiogenesis (Cao, Y., et al., (1996) J. Biol. Chem. 271:29461). Therefore, t-PALP polynucleotides and/or polypeptides of the invention may provide particularly good therapeutic molecules for use in treating cancer and/or tumorigenesis, as well as in therapeutically modulating angiogenesis.
There is a clear need, therefore, for identification and characterization for such enzymes that influence the fibrinolytic system, both normally and in disease states. In particular, there is a need to isolate and characterize additional human tissue plasminogen activator and related protease-like molecules which possess such functions as the activation of plasminogen and may be employed, therefore, for preventing, ameliorating or correcting dysfunctions or disease states or, alternatively, augmenting the positive, natural actions of such enzymes.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding at least a portion of the t-PALP polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 or the complete amino acid sequence encoded by the cDNA clone deposited as plasmid DNA HMSIB42 (ATCC Deposit Number 209023) on May 8, 1997. The nucleotide sequence determined by sequencing the deposited t-PALP clone, which is shown in
FIGS. 1A
,
1
B, and
1
C (SEQ ID NO:1), contains an open reading frame encoding a complete polypeptide of 263 amino acid residues, including an initiation codon encoding an N-terminal methionine at nucleotide positions 124-126, and a predicted molecular weight of about 28.2 kDa. Nucleic acid molecules of the invention include those encoding the complete amino acid sequence excepting the N-terminal methionine shown in SEQ ID NO:2, or the complete amino acid sequence excepting the N-terminal methionine encoded by the cDNA clone in ATCC Deposit Number 209023, which molecules also can encode additional amino acids fused to the N-terminus of the t-PALP amino acid seque
Ebner Reinhard
Moore Paul A.
Ruben Steven M.
Chan Christina
Haddad Maher
Human Genome Sciences Inc.
Human Genome Sciences Inc.
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