Biomaterial Database

Isolation and characterization of proteolytic fragments of human factor VIIa which inhibit the tissue factor-enhanced amidolytic activity of factor VIIa. 1993

Y Kazama, and A Pastuszyn, and P Wildgoose, and T Hamamoto, and W Kisiel

Department of Pathology, University of New Mexico School of Medicine, Albuquerque 87131-5301.

The interaction of circulating factor VII/VIIa with tissue factor presented by cells in extravascular tissues represents the initial event in the extrinsic pathway of blood coagulation. To determine the tissue factor binding domains in human factor VIIa, we have subjected recombinant human factor VIIa to tryptic digestion and isolated two proteolytic fragments (molecular mass = 32 and 20 kDa) by a combination of immunoaffinity chromatography and reversed phase high performance liquid chromatography (HPLC) which strongly inhibits the tissue factor-enhanced amidolytic activity of factor VIIa and inhibits the activation of factor X by factor VIIa in the presence of tissue factor. The 32-kDa factor VIIa fragment consisted of residues 1-137/143 from the light chain of factor VIIa connected by a disulfide bond to residues 153-277 from the heavy chain. The 20-kDa factor VIIa fragment consisted of residues 1-137 of the light chain of factor VIIa in disulfide linkage with residues 248-266 of the heavy chain. The 32- and 20-kDa factor VIIa fragments inhibited the tissue factor apoprotein-enhanced factor VIIa amidolytic activity with Ki values of 35 and 65 nM, respectively. The Ki values for the inhibition of relipidated tissue factor apoprotein-enhanced factor VIIa amidolytic activity by the 32- and 20-kDa factor VIIa fragments were 70 and 610 nM, respectively. Factor X activation by factor VIIa-relipidated tissue factor was inhibited half-maximally by the 32- and 20-kDa factor VIIa fragments at 65 and 680 nM concentrations, respectively. Equilibrium binding studies indicated that the 32- and 20-kDa factor VIIa fragments interacted with cell surface tissue factor expressed on J82 cells in a specific and saturable manner with Kd values of 30 and 64 nM, respectively. In addition, a peptide consisting of residues 1-109 from the light chain of factor VIIa obtained by reduction and HPLC of the 20-kDa factor VIIa fragment retained inhibitory activity, but the selective removal of the gamma-carboxyglutamic acid domain from the 20-kDa factor VIIa fragment by cathepsin G cleavage resulted in the complete loss of inhibitory activity in this fragment. Our data strongly suggest that the epidermal growth factor-like domains covalently linked to the gamma-carboxyglutamic acid domain in factor VIIa constitute the high affinity tissue factor binding domain in this molecule.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
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
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
D002403	Cathepsins	A group of lysosomal proteinases or endopeptidases found in aqueous extracts of a variety of animal tissues. They function optimally within an acidic pH range. The cathepsins occur as a variety of enzyme subtypes including SERINE PROTEASES; ASPARTIC PROTEINASES; and CYSTEINE PROTEASES.	Cathepsin
D004591	Electrophoresis, Polyacrylamide Gel	Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.	Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
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
D000596	Amino Acids	Organic compounds that generally contain an amino (-NH2) and a carboxyl (-COOH) group. Twenty alpha-amino acids are the subunits which are polymerized to form proteins.	Amino Acid,Acid, Amino,Acids, Amino
D012697	Serine Endopeptidases	Any member of the group of ENDOPEPTIDASES containing at the active site a serine residue involved in catalysis.	Serine Endopeptidase,Endopeptidase, Serine,Endopeptidases, Serine