Guinea pig proteinase-activated receptor 4 and its activating peptide
AssigneeOrtho-McNeil Pharmaceutical, Inc.
InventorsAndrew Darrow, Claudia Derian, Michael Addo, Patricia Andrade-Gordon
Life of this patent
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Abstract
This invention relates to nucleic acid encoding guinea pig PAR 4 and to the protein encoded thereby. The guinea pig DNA and protein are useful for the development of models of human platelet aggregation. The invention further relates to an animal model to assess the role of PAR antagonists in thrombosis.
Claims34
An isolated and purified nucleic acid molecule encoding guinea pig PAR-4 protein, said molecule selected from the group consisting of:
- (a) a nucleic acid molecule encoding a protein having at least a 75% identity to a polypeptide comprising amino acids 1 to 388 of SEQ ID NO:3;
- (b) a nucleic acid molecule which is complementary to the polynucleotide of (a);
- (c) a nucleic acid molecule comprising at least 15 sequential bases of the polynucleotide of (a) or (b);
- and
- (d) a nucleic acid molecule that hybridizes under stringent conditions to the polynucleotide molecule of (a).
An isolated and purified nucleic acid encoding a PAR-4 fusion protein comprising at least amino acids 219 to 243 of guinea pig PAR4.
An expression vector for expression of a guinea pig PAR-4 protein in a recombinant host, wherein said vector comprises a nucleic acid molecule that hybridizes under stringent conditions to the polynucleotide molecule of SEQ ID NO:2.
A substantially pure protein comprising a sequence of least 15 consecutive amino acids corresponding to amino acids 219 through 243 of SEQ ID NO:3.
A peptide consisting of at least 15 consecutive amino acids from SEQ ID NO:3.
A monospecific antibody immunologically reactive with guinea pig PAR-4 protein.
A Protease Activated Receptor (PAR) activating peptide comprising the sequence SFPGQ(X)n, where X is any amino acid or amino acid derivative and wherein n is 0-30.
A method comprising the steps of:
- (a) admixing in an aqueous environment a guinea pig PAR-4 activating peptide (AP) with a cell or tissue;
- (b) incubating the PAR-4 activating peptide and the cell or tissue for a predetermined amount of time;
- and
- (c) measuring the interaction of the peptide with the cells.
A method for promoting human platelet aggregation comprising the step of combining an aggregating amount of an activating peptide with human platelets wherein the activating peptide comprises the sequence SFPGQ(X)n where x is any amino acid and n is a number between 0 and 30.
A method for determining whether a substance is capable of inhibiting guinea pig PAR-4 activity in a guinea pig comprising the steps of:
- giving a test substance and an activating pepetide to a guinea pig, wherein the activating peptide comprises the sequence SFPGQ(X)n where x is any amino acid and n is a number between 0 and 30;
- and
- measuring the ability of the test substance to inhibit PAR-4 activation in the guinea pig as compared to a control guinea pig that did not receive the test substance.
A method for determining whether a substance capable of interfering with the interaction between an activating peptide and a PAR-4 protein comprising the steps of:
- adding a test substance and an activating peptide to a cell membrane composition comprising a PAR-4 protein wherein the activating peptide comprises the sequence SFPGQ(X)n where x is any amino acid and n is a number between 0 and 30;
- measuring the amount of binding of the activating peptide to the cell membrane composition;
- and
- comparing the amount of binding of the activating peptide to the cell membrane composition with the amount of binding of the activating peptide in control cells receiving no test substance.
Dependencies are inferred from the claim text.