Mutations of TFPI-binding exosites on factor VII cause bleeding phenotypes in factor VII deficiency

Tissue factor (TF) pathway inhibitor (TFPI) is a Kunitz-type anticoagulation protein that inhibits activated factor VII (FVIIa)/TF complex. Incidentally, many different F7 gene variants, including TFPI-binding exosite mutations, have been reported in patients with congenital FVII deficiency and clinical bleeding variabilities. Here, TFPI-binding exosites (R147 and K192) on FVII zymogen were selectively disrupted to understand their roles in the pathogenesis of bleeding phenotypes. Expression of recombinant FVII variants (R147A, K192A, and R147A/K192A) demonstrated markedly reduced secretion of FVII owing to intracellular retention in the endoplasmic reticulum, as demonstrated by upregulation of the unfolded protein response genes in all FVII variants. FVII variants showed a similar FVII activation pattern and FVIIa amidolytic activity than FVII wild-type (WT). In contrast to FVII activation, R147A and K192A showed a 90% reduction in FX activation relative to WT, whereas the R147A/K192A variant demonstrated a 99% decrease in FX activation. The clotting time was markedly prolonged with R147A and K192A than WT, and no FVII coagulant activity was detected in R147A/K192A. In addition, the thrombin generation assay revealed a significant prolongation of lag time in all FVII variants. Our study explains how mutations of TFPI-binding exosites of FVII can lead to bleeding phenotypes in individuals carrying these aberrancies.