Biomaterial Database

Localization of factor IXa and factor VIIIa interactive sites. 1995

L M O'Brien, and L V Medved, and P J Fay

Department of Medicine, University of Rochester School of Medicine and Dentistry, New York 14642, USA.

The contribution of the catalytic and noncatalytic domains of factor IXa to the interaction with its cofactor, factor VIIIa, was evaluated. Two proteolytic fragments of factor IXa, lacking some or all of the serine protease domain, failed to mimic the ability of factor IXa to enhance the reconstitution of factor VIIIa from isolated A1/A3-C1-C2 dimer and A2 subunit. Both fragments, however, inhibited this factor IXa-dependent activity. Selective thermal denaturation of the factor IXa serine protease domain eliminated its effect on factor VIIIa reconstitution. Modification of factor IXa with dansyl-Glu-Gly-Arg chloromethyl ketone (DEGR-IXa) stabilized this domain, and heat-treated DEGR-IXa retained its ability to enhance factor VIIIa reconstitution. These results indicate the importance of the serine protease domain as well as structures residing in the factor IXa light chain (gamma-carboxyglutamic acid and/or epidermal growth factor domains) for cofactor stabilizing activity. In the presence of phospholipid, the A1/A3-C1-C2 dimer produced a saturable increase in the fluorescence anisotropy of fluorescein-Phe-Phe-Arg chloromethyl ketone-modified factor IXa (Fl-FFR-IXa). This effect was inhibited by a factor IXa fragment comprised of the gamma-carboxyglutamic acid and epidermal growth factor domains. The difference in Fl-FFR-IXa anisotropy in the presence of A1/A3-C1-C2 dimer (delta r = 0.043) compared with factor VIIIa (delta r = 0.069) represented the contribution of the A2 subunit, A peptide corresponding to factor VIII A2 domain residues 558-565 decreased the factor VIIIa dependent-anisotropy of Fl-FFR-IXa to a value similar to that observed with the A1/A3-C1-C2 dimer. These results support a model of multiple interactive sites in the association of the enzyme-cofactor complex and localize sites for the A1/A3-C1-C2 dimer and the A2 subunit to the factor IXa light chain and serine protease domain, respectively.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010446	Peptide Fragments	Partial proteins formed by partial hydrolysis of complete proteins or generated through PROTEIN ENGINEERING techniques.	Peptide Fragment,Fragment, Peptide,Fragments, Peptide
D011487	Protein Conformation	The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).	Conformation, Protein,Conformations, Protein,Protein Conformations
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003619	Dansyl Compounds	Compounds that contain a 1-dimethylaminonaphthalene-5-sulfonyl group.	Dimethylaminonaphthalenesulfonyl Compounds,Compounds, Dansyl,Compounds, Dimethylaminonaphthalenesulfonyl
D005454	Fluorescence Polarization	Measurement of the polarization of fluorescent light from solutions or microscopic specimens. It is used to provide information concerning molecular size, shape, and conformation, molecular anisotropy, electronic energy transfer, molecular interaction, including dye and coenzyme binding, and the antigen-antibody reaction.	Anisotropy, Fluorescence,Fluorescence Anisotropy,Polarization, Fluorescence,Anisotropies, Fluorescence,Fluorescence Anisotropies,Fluorescence Polarizations,Polarizations, Fluorescence
D005456	Fluorescent Dyes	Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags.	Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000590	Amino Acid Chloromethyl Ketones	Inhibitors of SERINE ENDOPEPTIDASES and sulfhydryl group-containing enzymes. They act as alkylating agents and are known to interfere in the translation process.	Peptide Chloromethyl Ketones,Chloromethyl Ketones, Peptide,Ketones, Peptide Chloromethyl
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein