Evaluation of Bleeding Phenotype of Inherited Factor VII Deficiency in Children With a Bleeding Assessment Tool and Global Assays.

INTRODUCTION: Inherited factor VII (FVII) deficiency is the most common of the rare bleeding disorders and shows a heterogenous distribution of bleeding phenotypes independent of factor activity level. The bleeding score (BS) evaluates the phenotype of patients with rare bleeding disorders. Thromboelastography (TEG) and thrombin generation assays (TGAs) are 2 methods to evaluate global hemostasis, and controversially both tests are useful for identifying different bleeding tendency phenotypes. The purpose of this study was to investigate the use of the BS and global assays (TEG and TGAs) to predict the bleeding phenotype of inherited FVII deficiency. MATERIALS AND METHODS: A total of 27 patients with FVII deficiency were evaluated with the BS and global hemostasis assays. RESULTS: The BS was compatible with disease severity according to the FVII activity level (P<0.05) but the BS and bleeding grade of patients did not show a statistically significant correlation with factor activity level (P>0.05). No significant correlation was observed between the factor activity level and any TEG parameter (P>0.05). The factor activity level was negatively correlated with the lag time of the TGA on the contrary positively correlated with the peak thrombin time of the TGA (P<0.05). CONCLUSIONS: The global assays do not successfully predict the bleeding phenotype. The BS is a more suitable tool than conventional and global assays for predicting the bleeding phenotype.

N-Glycan-calnexin interactions in human factor VII secretion and deficiency.

Factor VII (FVII) is a key serine protease in blood coagulation. N-glycosylation in FVII has been shown to be critical for protein secretion. To date, however, the underlying biochemical mechanism remains unclear. Recently, we found that N-glycans in the transmembrane serine protease corin are critical for calnexin-assisted protein folding and extracellular expression. In this study, we tested the hypothesis that N-glycans in the FVII protease domain mediate calnexin-assisted protein folding and that naturally occurring F7 mutations abolishing N-glycosylation impair FVII secretion. We expressed human FVII wild-type (WT) and mutant proteins lacking one or both N-glycosylation sites in HEK293 and HepG2 cells in the presence or absence of a glucosidase inhibitor. FVII expression, secretion and binding to endoplasmic reticulum chaperones were examined by immune staining, co-immunoprecipitation, Western blotting, and ELISA. We found that N-glycosylation at N360 in the protease domain, but not N183 in the pro-peptide domain, of human FVII is required for protein secretion. Elimination of N-glycosylation at N360 impaired calnexin-assisted FVII folding and secretion. Similar results were observed in WT FVII when N-glycan-calnexin interaction was blocked by glucosidase inhibition. Naturally occurring F7 mutations abolishing N-glycosylation at N360 reduced FVII secretion in HEK293 and HepG2 cells. These results indicate that N-glycans in the FVII protease domain mediate calnexin-assisted protein folding and subsequent extracellular expression. Naturally occurring F7 mutations abolishing N-glycosylation in FVII may impair this mechanism, thereby reducing FVII levels in patients.

An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes.

Tailored approaches to restore defective transcription responsible for severe diseases have been poorly explored. We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model. In this model, the deficiency is caused by the -94C > G or -61T > G mutation, which abrogate the binding of Sp1 or HNF-4 transcription factors. Reporter assays in hepatoma HepG2 cells naturally expressing FVII identified a single TALE-TF (TF4) that, by targeting the region between mutations, specifically trans-activated both the variant (>100-fold) and wild-type (20-40-fold) F7 promoters. Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene. The ectopic F7 expression in renal HEK293 cells was modestly affected by TF4 or by TALE-TF combinations. These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations.

Acquired factor VII deficiency associated with Wilms tumor.

We present the case of a 2-year-old female with Wilms tumor whose initial evaluation revealed a prolonged prothrombin time (PT) and normal activated partial thromboplastin time. Mixing studies demonstrated correction of the PT and the Factor VII activity was 17% in the absence of a Factor VII inhibitor. She underwent successful resection of the tumor with fresh frozen plasma support and no excessive bleeding. Post-operative testing demonstrated normal PT at 3 days and 1-month. Although acquired von Willebrand factor deficiency has a known association with Wilms tumor, paraneoplastic factor VII deficiency associated with Wilms tumor is previously unreported.