The tissue factor region that interacts with factor Xa in the activation of factor VII.
Tissue factor is the cell membrane-anchored cofactor for factor VIIa and triggers the coagulation reactions. The initial step is the conversion of factor VII to factor VIIa which, in vitro, is efficiently catalyzed by low concentrations of factor Xa. To identify the tissue factor region that interacts with the activator factor Xa during this process, we evaluated a panel of soluble tissue factor (1-219) mutants for their ability to support factor Xa-mediated activation of factor VII. The tissue factor residues identified as most important for this interaction (Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, and Tyr185) were identical to those found to be important for the interaction of substrate factor X with the tissue factor.factor VIIa complex. The residues form a continuous surface-exposed patch with an area of about 500 A(2), which appears to be located outside the tissue factor-factor VII contact zone. In agreement, the two monoclonal antibodies 5G6 and D3H44-F(ab')(2), whose epitopes overlap with this identified region, inhibited the rates of factor VII activation by 86% and 95%, respectively. These antibodies also strongly inhibited the conversion of (125)I-labeled factor VII when cell membrane-expressed, full-length tissue factor (1-263) was employed. Together the results suggest the usage of a common surface region of tissue factor in its dual role-as a cofactor for factor Xa-mediated factor VII activation and as a cofactor for factor VIIa-mediated factor X activation. The finding that factor Xa and factor X may engage in similar, if not identical, molecular interactions with tissue factor further indicates that factor Xa and factor X are similarly oriented toward their respective interaction partners in the ternary catalytic complexes.
Kirchhofer
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Department of Cardiovascular Research, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA. dak@gene.com
Eigenbrot
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Lipari
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Moran
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Peek
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Kelley
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D015055:1-Carboxyglutamic Acid / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D005167:Factor VII / Q000378:metabolism*; D015942:Factor VIIa / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012995:Solubility; D020349:Surface Plasmon Resonance; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism*
D004792:Enzyme Precursors; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D015055:1-Carboxyglutamic Acid; D005167:Factor VII; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa