Structural requirements for the interaction between tissue factor and factor VII: characterization of chymotrypsin-derived tissue factor polypeptides.
Tissue Factor (TF) is the cellular receptor for coagulation Factor VII/VIIa (FVII/VIIa). TF binds to FVIIa and promotes the rapid activation of the zymogen substrates Factors IX and X (FIX and FX) to the respective serine proteinases. In order to probe structure-function relationships in TF, we have subjected the truncated membrane-bound variant, TF 1-243, to proteolytic digestion in SDS-containing gels. Three major polypeptide fragments were generated by proteolysis of TF 1-243 with chymotrypsin, producing cleavages C-terminal to residues 34, 76 and 103. All three polypeptides, TF 35-243, 77-243 and 104-243, bound biotinylated human FVII in a highly specific ligand blot assay. High-performance electrophoretic chromatography was used to isolated chymotrypsin-derived fragments of TF. These purified fragments bound FVII in ligand blots, and two of the three polypeptides exhibited much reduced, but significant, procoagulant activity in a chromogenic assay for the generation of Factor Xa in the presence of FVIIa and Ca2+. The smallest chymotrypsin-derived TF polypeptide, TF 104-243, showed reduced binding of FVII in ligand blot analyses, inhibited the activity of the full-length molecule, but had no procoagulant activity. These data suggest that a part of the binding site for FVII is contained within the TF sequence 104-243. The sequence TF 1-34 either contains a part of the FVII-binding domain or its removal leads to dysfunctional folding, disrupting binding sites elsewhere in the molecule.
Haemostasis Research Group, Clinical Research Centre, Harrow, Middx., U.K.
D P
DP
O'Brien
J S
JS
Anderson
D M
DM
Martin
P G
PG
Byfield
E G
EG
Tuddenham
D000595:Amino Acid Sequence; D001777:Blood Coagulation; D015153:Blotting, Western; D002853:Chromatography, Liquid / Q000379:methods; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D005167:Factor VII / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013925:Thromboplastin / Q000737:chemistry* / Q000378:metabolism
D010446:Peptide Fragments; D005167:Factor VII; D013925:Thromboplastin; D002918:Chymotrypsin