Tissue factor pathway inhibitor: the carboxy-terminus is required for optimal inhibition of factor Xa.
Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that binds to and inactivates factor Xa directly, and in a factor Xa-dependent fashion inhibits the factor VIIa/tissue factor catalytic complex. TFPI is a slow, tight-binding, competitive, and reversible inhibitor of factor Xa, in which the formation of an initial encounter complex between TFPI and factor Xa is followed by slow isomerization to a final, tightened complex. Wild-type recombinant TFPI (rTFPI), expressed in mouse C127 cells, separates into two forms on heparin-agarose chromatography that elute at 0.3 mol/L and 0.6 mol/L NaCl. Western blot analysis shows that both forms contain the N-terminus of full-length TFPI, but only rTFPI(0.6) is recognized by an antibody directed against the C-terminus. rTFPI(0.3) and rTFPI(0.6) inhibit factor Xa with 1:1 stoichiometry and inhibit factor VIIa/tissue factor equally in an endpoint-type assay. However, rTFPI(0.6) is a more potent inhibitor than rTFPI(0.3) of coagulation in normal plasma induced by either factor Xa or tissue factor. The initial inhibition of factor Xa (less than 5 seconds) produced by rTFPI(0.6) is several-fold greater than that produced by rTFPI(0.3), presumably reflecting a lower Ki of the immediate encounter complex between factor Xa and TFPI. The differential effect of these forms of TFPI on tissue factor-induced coagulation in normal plasma appears to be directly related to their ability to inhibit factor Xa. To confirm the role of the C-terminal region of TFPI in optimal factor Xa inhibition, a carboxy-terminal mutant of rTFPI, which is truncated after leucine 252 and thus lacks the basic sequence K T K R K R K K Q R V K (residues 254-265), was expressed in C127 cells. This form of rTFPI elutes from heparin-agarose at 0.28 mol/L NaCl and inhibits factor Xa at a rate that is slower than rTFPI(0.3). The Ki(final)s for factor Xa inhibition by rTFPI(0.6), rTFPI(0.3), and rTFPI1-252 are 3.1 +/- 0.6, 19.6 +/- 0.8, and 19.6 +/- 3.0 pmol/L, respectively.
Wesselschmidt
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Division of Hematology/Oncology, Jewish Hospital, Washington University Medical Center, St Louis, MO 63110.
Likert
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Girard
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Wun
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Broze
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001777:Blood Coagulation / Q000187:drug effects; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005167:Factor VII / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D015951:Factor Xa / Q000378:metabolism; D065427:Factor Xa Inhibitors; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D013925:Thromboplastin / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection
D000906:Antibodies; D065427:Factor Xa Inhibitors; D008074:Lipoproteins; D010455:Peptides; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D005167:Factor VII; D013925:Thromboplastin; D015951:Factor Xa