Kunitz domain inhibitors of tissue factor-factor VIIa. I. Potent inhibitors selected from libraries by phage display

Potent active-site inhibitors of human tissue factor-Factor VIIa (TF.FVIIa) have been selected from Alzheimer's amyloid beta-protein precursor inhibitor (APPI) Kunitz domain libraries displayed on phage. Eight randomized positions on the extended primary binding loop (P5 through P4') and positions 34 and 39 were examined in three separate libraries. Libraries contained from 3.2 x 10(5) to 3.2 x 10(6) potential variants resulting from replacing up to 5 positions with all 20 amino acids. Following 4 rounds of selection against FVIIa associated with immobilized tissue factor (TF), 12 clones from each library were sequenced. Variants were purified by trypsin affinity chromatography and reverse-phase high performance liquid chromatography, and characterized for their ability to inhibit TF.FVIIa chromogenic activity. Measured apparent equilibrium dissociation constants (Ki*) ranged from about 10 to 500 nM. From sequence and activity data, an overall consensus sequence, TF7I-C, was constructed by site-directed mutagenesis. TF7I-C differed from APPI at 4 key residues, T11P, M17L, S19L, and G39Y, and inhibited TF.FVIIa with a Ki* = 1.9 +/- 0.4 nM, which represented an increase in binding affinity of more than 150-fold compared to APPI. At 40 microM, TF7I-C prolonged the clotting times 3.5-fold in a prothrombin time assay and > 10-fold at 7 microM in an activated partial thromboplastin time assay. Prolongation of the activated partial thromboplastin time correlates with potent inhibition of FXIa (Ki* = 0.8 nM) and plasma kallikrein (Ki* = 1.2 nM). TF7I-C also inhibited plasmin (Ki* = 40 nM) and FXa (Ki* = 55 nM), but not activated protein C, thrombin, or FXIIa (Ki* > 10 microM each).