SummaryPolymorphic differences in the 5’ flanking region of the gene encoding procoagulant protein Factor VII (FVII) are associated with variations in FVII coagulant activity (FVII:C) and FVII antigen (FVII:Ag) levels. A decanucleotide insert polymorphism (CCTATATCCT) at 323 bp upstream of the start site of translation correlates with a decrease of approximately 20% FVII:C levels per allele containing this insert. However, linkage disequilibrium of the decanucleotide polymorphism with two single nucleotide polymorphisms (SNPs) at –122 and –401 have made it difficult to pinpoint the functional role, if any, of these genetic changes in lowering FVII levels. In vitro reporter gene studies in HepG2 cells analyzing the 8 possible combinations of polymorphic sites at –401, –323, and –122 reveal the necessity of the presence of the three concurrent polymorphic changes to maximally decrease promoter strength. In addition, these in vitro results are supported by in vivo studies in 89 individuals of African heritage, 34% of whom display a new haplotype that shows the polymorphic changes at -323 and -401 but lacks the change at -122.

Proteolytic cleavage of the peptide bond between Arg(152) and Ile(153) converts the procoagulant protein Factor VII (FVII) to an activated two-chain form (FVIIa). The formation of a salt bridge between Ile(153) and Asp(343) drives the conversion of FVIIa from being zymogen-like to the active form. In the present paper, we describe the novel FVII mutant V154G (Val(154)-->Gly mutation; residue 17 in the chymotrypsin numbering system), found in three FVII-deficient patients, which models a zymogen-like form of FVIIa. Recombinant V154G FVIIa, although normally cleaved, shows markedly reduced activity towards peptidyl substrate and undetectable activity towards macromolecular substrates. Susceptibility of Ile(153) to chemical modification, in either the presence or the absence of tissue factor (TF), suggests that the reduced V154G FVIIa activity is caused by impaired salt-bridge formation, thus resulting in a zymogen-like FVIIa form. The TF-mediated protection from chemical modification of V154A indicated that Gly(154) is responsible for this peculiar feature, and suggests that this region, proximal to the heavy chain N-terminus, is directly involved in the conversion of FVII into FVIIa. V154G FVII was exploited to study the FVII-TF interaction, together with three additional FVII variants that were expressed to serve as models for different FVII forms. The comparison of binding affinities of full-length TF after relipidation in L-alpha-phosphatidylcholine for the zymogen FVII (Arg(152)-->Gln, K (d)=1.04+/-0.27 nM), inactive FVIIa (Ser(344)-->Ala, K (d)=0.27+/-0.06 nM) and a zymogen-like FVIIa (V154G, K (d)=1.15+/-0.16 nM) supports the hypothesis that preferential binding of TF to active FVIIa is insufficient to drive the 10(5)-fold enhancement of FVIIa activity. In addition, the inability of V154G FVIIa to accommodate an inhibitor in the active site, indicating an improperly shaped specificity pocket, would explain the low activity of the zymogen-like form of FVIIa, which is predominant in the absence of TF.

Background: Symptomatology in congenital human FVII deficiency with FVII:C (Factor VII Coagulant Activity) levels < 1% ranges from asymptomatic to severe hemorrhagic problems. Additionally, FVII:C differs markedly depending upon the source of tissue factor (TF) in the thromboplastin utilized for measuring FVII:C. FVII deficiency reported in 1978 in patients from Padua, Italy was later shown to be associated with an arginine (R) to glutamine (Q) mutation at FVII amino acid 304. A study by Triplett et al. from 1985 reported FVII deficiency in 26 patients including 16 Caucasians, 1 Hispanic and 9 blacks. Of these patients, all 9 black patients were asymptomatic whereas the other 17 patients had a clinically relevant bleeding disorder. Since that time there have been no reports describing the genetic cause of FVII deficiency in asymptomatic African American individuals. Methods: Plasma was obtained from patient blood samples obtained in 3.2% sodium citrate. FVII:C levels were performed using one-stage clotting assays with either rabbit brain thromboplastin (Simplastin Excel, Biomerieux, Durham, NC) or lyophilized recombinant human TF (Innovin, Dade Behring, Inc., Newark, DE). Factor VII antigen levels (VII:Ag) were measured using an enzyme-linked immunoabsorbent assay (American Bioproducts Co, Parsippany, NJ). A normal pool was constructed by mixing equal volumes of plasma from greater than 20 healthy control subjects. Results Patient # Prothrombin Time in seconds FVII:C Simplastin Excel FVII:C Innovin FVII:Ag Genotype Phenotype 1-African American 15.6 16% 33% 61% Heterozygote R304Q Minor Nose Bleeds 2- African American Prolonged 26% 54% 67% Heterozygote R304Q Asymptomatic for bleeding 3- African American 14.5 21% 64% NA Heterozygote R304Q Asymptomatic for bleeding Conclusions: We show that FVII deficiency in three asymptomatic African American patients at our specialty laboratory was due to FVII-Padua (R304Q). An additional ten African American patients showed prolonged prothrombin times and FVII:C values consistent with the laboratory phenotype of FVII-Padua. This diagnosis prevented inappropriate blood transfusions prior to surgery in several of these patients.

A novel T to C point mutation at −60 in the gene for coagulant Factor VII results in life-long severe coagulant Factor VII deficiency in post-pubertal twin males. The clinical course of these patients provides an informative in vivo example of the regulation of expression of vitamin K-dependent clotting protein Factor VII. An analogous point mutation in the HNF4 binding site in the Factor IX gene results in the clinical phenotype Hemophilia B Leyden, a sex-linked antigen-negative Factor IX deficiency that resolves post-puberty. The affected Factor VII deficient patients have prolonged prothrombin times (46 and 52 secs), normal aPTTs and decreased FVII levels of FVII:Coagulant activity: < 1% and FVII:Antigen: < 3%. The −60 mutation, ACTTTG → ACTCTG occurs 9 base pairs before the start site of transcription and 59 bps before the before the start site of translation. The twins are compound heterozygotes and also possess a mutation in exon 8 at amino acid 348, a mutation that has previously been reported to cause FVII deficiency. Both affected individuals have recurrent target joint hemorrhage (shoulder, elbow, ankle) requiring replacement therapy 6–12 times/year. Results: Gel mobility shift assays using a radio-labeled probe spanning from −76 to −46 in the FVII promoter region demonstrate the loss of binding of transcription factor HNF-4. Transient transfection assays in HepG2 cells using 186 bps of the mutant and the wildtype promoters (−185 to +1) revealed a loss of expression with the mutant allele. Co-transfection with an HNF4 expression plasmid resulted in an increase in expression of the wildetype construct in HeLa cells, a non-hepatic cell line. However, co-transfection of the HNF4 expression plasmid failed to increase expression with the construct containing the mutant allele sequence. Conclusion: The lack of phenotypic change of the FVII:C in 19 yo twin boys provides dynamic support of the necessity of an overlapping androgen binding site in the homologous Factor IX gene as responsible for the phenotypic resolution of Factor IX deficiency (Hemophilia B Leyden) post-puberty. It is of interest that an increase in FVII:C did not occur with advancing age in FVII deficiency due to this HNF4 binding site mutation.