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Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in vivo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in vivo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.

Tissue factor (TF) is a transmembrane protein that functions in the initiation of blood coagulation in vivo. At sites of vascular injury, TF serves as a cell-surface receptor for the serine protease factor VIIa (FVIIa), forming an enzyme--cofactor complex and enhancing the catalytic activity of FVIIa. Tissue factor, along with the receptors for alpha- and gamma-interferons, is a member of the class 2 cytokine receptor superfamily. Crystallographic analysis demonstrated that the extracellular domain of TF consists of two immunoglobulin-like domains joined by a linker region. Each domain is comprised of two antiparallel beta-sheets containing seven conserved beta-strands separated by more variable loop regions. Extensive mutagenesis has been performed in order to map the FVIIa binding site on TF. Results indicated that the discontinuous binding site for FVIIa lies at the domain--domain interface and includes residues from extended loops and beta-strands within both the N- and C-terminal domains. Our previous study provided evidence that three consecutive residues (D44, W45, K46) within the TF loop region between beta-strands C and C' of the N-terminal domain were important for interactions with FVIIa. We have presently extended our alanine-scanning mutagenesis to include the residues within the flanking beta-strands. Thirteen sTF mutants were screened for their ability to enhance FVIIa activity. Three residues within strand C (Y34, Q37, I38) and two residues within C' (K48, Y51) were shown to be important for TF cofactor function.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue factor (TF) is a membrane-bound glycoprotein that functions as a cofactor for coagulation factor VIIa (VIIa) and initiates blood coagulation at sites of vascular injury. On the basis of sequence alignments, TF was predicted to be a member of the cytokine receptor superfamily. Utilizing the structural information available for the cytokine receptor superfamily, we have used site-directed mutagenesis to identify the binding site on TF for VIIa. The predicted loop regions in TF were systematically replaced with the homologous loops from the gamma-interferon receptor (gamma-IFN-R), the protein most related to TF in the superfamily of cytokine receptors. Six discontinuous regions (residues 16-20, 40-46, 60-69, 101-111, 129-151, 193-207) were identified that are required for interaction with VIIa and enhancement of activity. Individual substitution of 68 residues within these loops with alanine revealed eight residues (K20, D44, W45, K46, Q110, R135, F140, V207) that are required for cofactor activity. These residues fall into two groups, those that are required only for interactions with VIIa (K46, Q110, R135, F140, V207) and those that are also required to induce the conformational change in VIIa required for enhanced activity (K20, D44, W45). The discontinuous regions of TF required for interactions with VIIa form a single binding surface for VIIa that is analogous to the interface defined by the crystal structure of the complex between growth hormone and its receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue factor (TF) is a 263 amino acid membrane-bound procoagulant protein that serves as a cofactor for the serine protease factor VII (fVII). Recombinant human TF (rTF) produced in both human kidney 293 cells and Escherichia coli has been immunoaffinity purified by using a TF-specific monoclonal antibody. Recombinant TF produced in 293 cells is glycosylated and migrates on reducing SDS-PAGE with an apparent molecular weight (Mr) of 45K. Some interchain disulfide-bonded rTF dimers are observed under nonreducing conditions. The E. coli produced rTF has a molecular weight of 33K and 35K, with the 33K band missing nine amino acids at the carboxy terminus. Although the E. coli produced rTF does not contain any carbohydrate, it is fully functional in both a chromogenic assay and a one-stage prothrombin time assay. A variant has been constructed wherein the cytoplasmic cysteine (residue 245) has been mutagenized to a serine residue. The amount of disulfide-linked aggregates is dramatically reduced following immunoaffinity purification of this four-cysteine variant (C2455), which is active in the chromogenic and prothrombin time assays.