Incorporation of an Active Site Inhibitor in Factor VIIa Alters the Affinity for Tissue Factor

Abstract

Recent studies showed that the administration of active site-inhibited factor VIIa blocked factor VIIa/tissue factor-induced fibrin and thrombus formation in ex vivo and in vivo model systems. These studies suggest that inactivated factor VIIa competes efficiently with plasma factor VII(a) for a limited number of tissue factor sites. In the present study, we compared the interactions of factor VIIa and active site-inhibited factor VIIa with tissue factor. Competition studies of factor VIIa and active site-inhibited factor VIIa in a factor X activation assay showed that the affinity of the latter for relipidated tissue factor was 5-fold higher than that of factor VIIa. Radioligand binding studies with a human bladder carcinoma cell line (J82) and surface plasmon resonance studies using soluble tissue factor demonstrated a faster association and a slower dissociation for the active siteinhibited factor VIIa. Studies of equilibrium binding to cell surface tissue factor showed that the affinity of active site-inhibited VIIa was 5-fold higher than that of factor VIIa to non-functional tissue factor sites, whereas both inactivated factor VIIa and factor VIIa bound to functional tissue factor sites with the same high affinity. Comparison of the CD spectra of factor VIIa and active site-inactivated factor VIIa revealed structural differences in the protease domain. The potential physiological implications of these findings are discussed.The in vivo initiation of the coagulation cascade is triggered by the binding of plasma factor VIIa (FVIIa) 1 to the cell surface receptor tissue factor (TF) (1). TF is normally expressed in adventitial cells and pericytes surrounding blood vessels but not in cells that come in contact with blood, such as monocytes and endothelial cells (2, 3). Tissue injury disrupting the endothelial cell barrier is normally required for FVIIa to come in contact with TF. However, under pathological conditions monocytes and endothelial cells could be perturbed to induce TF (4 -7).Factor VII circulates as a single chain zymogen, and the binding to TF markedly increases the susceptibility of factor VII to cleavage at Arg 152 resulting in formation of a two-chain serine protease, FVIIa. TF is a glycoprotein consisting of 263 amino acids with a 219-amino acid extracellular part. The extracellular part is structured in two fibronectin type III-like domains (8, 9). The crystal structure of D-Phe-L-Phe-L-Arg (FFR)-FVIIa-soluble TF (sTF) complex has recently been determined (10). In this complex, FVIIa has been shown to adopt an extended confirmation and wrap around TF with the Gla domain near the cell membrane and the catalytic domain distal to it.Administration of inactivated FVIIa was shown to reduce angiographic restenosis and decrease neointimal hyperplasia in a rabbit atherosclerotic injury model (11) and also to reduce thrombus formation at sites of vascular injury in a baboon femoral balloon artery angioplasty model (12). In a preliminary study, it was shown that infusion of a low concentration of inac...

Mutagenesis of the γ-Carboxyglutamic Acid Domain of Human Factor VII to Generate Maximum Enhancement of the Membrane Contact Site

“…The WT-FVIIai concentrations required to reach 50% inhibition (FVIIai⅐TF/FVIIa⅐TF ϭ 1.0) are reported in Table I. In agreement with the higher affinity of WT-FVIIai for TF (15,24,25), its concentration at 50% inhibition was lower than that of WT-FVIIa.…”

Factor VIIa-induced p44/42 Mitogen-activated Protein Kinase Activation Requires the Proteolytic Activity of Factor VIIa and Is Independent of the Tissue Factor Cytoplasmic Domain

“…FFR-FVIIa was prepared as described (21). Human thrombin, human FX and FXa were from Enzyme Research Laboratory Inc. (Lafayette, IN).…”

“…Measurements of Cell Surface FVIIa Binding Capacity and FVIIamediated FX Activation-Surface-exposed TF on transfected BHK cells was determined by FVIIa-mediated FXa generation and measurement of 125 I-FVIIa binding as described previously (21). Curve-fitting to a one-binding-site model using the Grafit (Erithacus Software Limited) program was applied for determination of the maximal number, B max , of 125 I-FVIIa binding sites.…”

Initial Inhibition of Tissue Factor Signalling Reduces Chronic Vascular Changes in Isogenic Rat Aortic Transplants

“…Recombinant FVIIai was prepared at Novo Nordisk, Gentofte, Denmark, as described previously (37). In brief, the active site of recombinant FVIIa was inactivated by incubation with the tripeptide D-Phe-L-Phe-L-Arg chloromethyl ketone which forms covalent bonds to the active sites His-193 and Ser-344, thereby blocking these.…”

“…This agent inhibits the formation of functional TF/ FVIIa complexes by competing with native plasma FVII(a) for binding sites on TF. The formed complexes are catalytically inactive, thereby preventing generation of FXa and thrombin with subsequent fibrin precipitation (37). A high dose of rFVIIai (1 mg/kg), which previously was shown to prevent local thrombosis and re-stenosis, was used (28,38).…”