Characterization of functionally important regions of tissue factor by using monoclonal antibodies.
Tissue factor is a cellular membrane glycoprotein that acts as a cofactor for Factor VIIa-catalyzed activation of Factors X and IX in the extrinsic blood coagulation pathway. We prepared fourteen murine monoclonal antibodies to human tissue factor (designated MTF-1 to -14), and purified tissue factor from human placenta by immunoaffinity chromatography using the antibody-coupled Sepharose 4B. The antibodies were characterized for inhibitory activity against the activation of Factor X by a complex of Factor VIIa and purified placental tissue factor or a recombinant mutant tissue factor whose transmembrane and cytoplasmic domains were deleted. Four MTFs (MTF-6, -7, -8, and -14) markedly inhibited the cofactor activity of tissue factor by directly blocking the interaction with Factor VIIa. Three other MTFs (MTF-1, -4, and -9) moderately inhibited the cofactor activity of the mutant tissue factor, presumably by diminishing the interaction between the extracellular region of the tissue factor and phospholipids. Both groups of MTFs inhibited the cofactor activity of the mutant tissue factor in Factor X activation by Factor VIIa in the absence of phospholipids. These findings suggest that the former four MTFs recognize the binding site for Factor VIIa and the latter three MTFs recognize the site for Factor X in the extracellular region of tissue factor. These MTFs are useful to purify tissue factor by immunoaffinity chromatography and to study the structure-function relationship of tissue factor, and may be useful to establish an immunoassay of tissue factor in body fluids.
Ito
T
T
Department of Molecular Biology on Genetic Disease, Mie University School of Medicine.
Hiraiwa
J
J
Nishioka
J
J
Iino
M
M
Suzuki
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D001665:Binding Sites; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D015942:Factor VIIa / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D006801:Humans; D051379:Mice; D009154:Mutation; D010743:Phospholipids; D010920:Placenta / Q000737:chemistry; D013925:Thromboplastin / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology
D000911:Antibodies, Monoclonal; D010743:Phospholipids; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa