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.

[1]  Burton E. Sobel,et al.  Prevention of Arterial Reocclusion After Thrombolysis With Recombinant Lipoprotein‐Associated Coagulation Inhibitor , 1991, Circulation.

[2]  J. Miletich,et al.  Purification and properties of heparin-releasable lipoprotein-associated coagulation inhibitor. , 1991, Blood.

[3]  P. Barr,et al.  Mammalian subtilisins: The long-sought dibasic processing endoproteases , 1991, Cell.

[4]  M. L. Larsen,et al.  Extrinsic pathway inhibitor (EPI) released to the blood by heparin is a more powerful coagulation inhibitor than is recombinant EPI. , 1991, Thrombosis research.

[5]  T. Wun,et al.  Recombinant lipoprotein-associated coagulation inhibitor inhibits tissue thromboplastin-induced intravascular coagulation in the rabbit. , 1990, Blood.

[6]  W. Novotny,et al.  Regulation of coagulation by a multivalent Kunitz-type inhibitor. , 1990, Biochemistry.

[7]  B. Bejcek,et al.  Identification of the 1.4 kb and 4.0 kb messages for the lipoprotein associated coagulation inhibitor and expression of the encoded protein. , 1989, Thrombosis research.

[8]  Susan G. Brown,et al.  Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor , 1989, Nature.

[9]  J. Miletich,et al.  Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains. , 1988, The Journal of biological chemistry.

[10]  J. Miletich,et al.  Isolation of the lipoprotein associated coagulation inhibitor produced by HepG2 (human hepatoma) cells using bovine factor Xa affinity chromatography. , 1987, Thrombosis research.

[11]  S. Rapaport,et al.  Studies of a mechanism inhibiting the initiation of the extrinsic pathway of coagulation. , 1987, Blood.

[12]  M. Bernatowicz,et al.  Preparation of peptide-protein immunogens using N-succinimidyl bromoacetate as a heterobifunctional crosslinking reagent. , 1986, Analytical biochemistry.

[13]  J. Bieth,et al.  In vivo significance of kinetic constants of protein proteinase inhibitors. , 1984, Biochemical medicine.

[14]  P. Majerus,et al.  Purification and properties of human coagulation factor VII. , 1980, The Journal of biological chemistry.

[15]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[16]  James,et al.  The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action. , 1988, Blood.

[17]  J. Vaitukaitis.  Production of antisera with small doses of immunogen: multiple intradermal injections. , 1981, Methods in enzymology.

[18]  E. Shaw,et al.  [2] Titration of trypsin, plasmin, and thrombin with p-nitrophenyl p′-guanidinobenzoate HCl☆ , 1970 .