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
2000-12-12
2004-06-15
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...
Reexamination Certificate
active
06750323
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to platelet activation proteins.
The normal hemostatic system regulates bleeding and thrombosis through a series of complex interactions between components of the blood vessel wall, circulating blood platelets, and plasma proteins.
Because vascular injury causes a rapid loss of the protein, fluid, and cellular components of the blood, animals have developed rapid responses to patch the vessel and initiate its repair. These rapid responses are initiated by the platelet, a highly specialized cell that reacts to vascular injury. Normally, platelets circulate in the blood as quiescent and nonadherent cells, monitoring the integrity of the blood vessel. In response to vascular injury, platelets adhere to de-endothelialized areas and activate. Platelet activation induces profound morphologic and functional changes in the cell. Platelets change shape, aggregate with other platelets, and adhere to other cells. With full activation, platelets secrete the contents of their lysosomal, alpha, and dense granules, thereby expressing adhesion molecules, growth factors, coagulation enzymes, and other specialized molecules. Molecules expressed by activated platelets execute many of the complex cellular and biochemical processes that staunch the loss of blood and begin the process of vascular repair.
The cellular and biochemical processes initiated by platelets in response to vascular injury can be lifesaving, but in the absence of such injury these same processes can be deleterious. For example, unregulated arterial platelet thrombosis can occlude the blood supply to organs and lead to strokes, heart attacks, and limb necrosis.
SUMMARY OF THE INVENTION
A novel polypeptide, designated activated platelet protein-2 (APP-2), which is preferentially expressed on activated human platelets but not resting platelets, has now been discovered.
The invention includes a substantially pure DNA encoding a platelet activation polypeptide having a molecular weight of approximately 25 kilodaltons (kDa). Under non-reducing conditions, it can be naturally found in covalent association with two other proteins in a 145 kDa complex. The 25 kDa polypeptide as expressed in human platelets contains at least 2 putative phosphorylation sites. The protein of the invention can be characterized as containing an epitope which binds to the monoclonal antibody (MAb) 3B2.
Preferably, the encoded polypeptide is human APP-2, which includes at least 95% of the amino acid sequence of SEQ ID NO:4 (e.g. the protein encoded by SEQ ID NO:1). A preferred example of such a DNA would contain the nucleotide sequence of SEQ ID NO:3 or any degenerate variant of SEQ ID NO:3.
Most preferably, the DNA includes the nucleotide sequence of SEQ ID NO:2, or any degenerate variant of SEQ ID NO:2.
A substantially pure DNA containing a strand of at least 12 nucleotides, e.g., a hybridization probe of at least 20 nucleotides, 50 nucleotides, 100 nucleotides or more, which hybridizes at high stringency to a DNA having the sequence of SEQ ID NO:2, or the complement thereof, is also within the invention. Expression of APP-2 in a cell can be detected by (a) contacting mRNA obtained from the cell with a labeled hybridization probe comprising, for example, a single-stranded segment of isolated DNA encoding a fragment of APP-2; and (b) detecting hybridization of the probe with the MRNA.
By “high stringency” is meant the following DNA hybridization and wash conditions: hybridization at 42° C. in the presence of 50% formamide; a first wash at 65° C. with 2×SSC containing 1% SDS; followed by a second wash at 65° C. with 0.1×SSC.
By “substantially pure DNA” is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the DNA sequence of interest. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which (a) is part of a hybrid gene encoding additional polypeptide sequence, e.g., a fusion protein, or (b) has a sequence that is not a naturally-occurring nucleotide sequence (e.g., a degenerate variant of a natural sequence, or a sequence containing mutations which do not occur naturally). Also included is a recombinant DNA which includes a portion of SEQ ID NO:2 and which encodes an alternative splice variant of APP-2, e.g., a polypeptide, the amino terminus of which differs from the amino terminus of SEQ ID NO:1.
The DNA should have at least about 50% identity to the coding sequence of SEQ ID NO:1 or 3, and preferably at least 70% (e.g., 80t, 90% or 95%). The identity between two nucleic acid or polypeptide sequences is a direct function of the number of matching or identical positions. For example, when a subunit position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by an adenine, then they are identical at that position. For example, if half, e.g., 5 positions in a sequence 10 nucleotides in length, are identical, then the sequences have 50% sequence identity. The length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). For purposes of calculating % sequence identity, gaps are considered to be mismatches.
The invention also includes a vector containing a DNA encoding a polypeptide which includes the amino acid sequence of SEQ ID NO:1, e.g., a construct in which the coding sequence is operably linked to a promoter or other regulatory sequences for expression of the polypeptide, and a cell containing such a vector. The cell may be procaryotic or eukaryotic (e.g., a mammalian cell such an a human cell) and preferably expresses the recombinant polypeptide encoded by SEQ ID NO:2.
The invention also includes a substantially pure platelet activation polypeptide as described above. By “platelet activation polypeptide” is meant a polypeptide having the amino acid sequence of a protein that is naturally preferentially expressed by activated platelets compared to resting platelets of an animal. Preferably, the animal is a vertebrate, e.g. a mammal such as a primate, including a human; alternatively the mammal is a rat, mouse, rabbit, guinea pig, hamster, cow, pig, horse, goat, sheep, dog, or cat.
Preferably, the polypeptide contains the amino acid sequence of human APP-2 (SEQ ID NO:1), e.g., in the form of a Flag-APP-2 fusion protein. By “polypeptide” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). The amino acid sequence of the polypeptide differs solely from SEQ ID NO:1 by conservative amino acid substitutions, e.g., substitution of one amino a acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the protein (e.g., its binding to Mab 3B2 or its covalent association in the 145 kDa complex). Preferably, the amino acid sequence of the platelet activation polypeptide is at least 50%, more preferably 70%, even more preferably 85% or 90%, and most preferably 95% identical to SEQ ID NO:1.
By a “substantially pure polypeptide” is meant a polypeptide which has been separated from components which naturally accompany it. Typically,
Clement Christophe Y.
Reed Guy
Beattie Ingrid A.
Chan Christina
Haddad Maher
Mintz Levin Cohn Ferris Glovsky and Popeo P.C.
President and Fellows of Harvard College
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