We investigated the role of thrombophilic mutations as possible modifiers of the clinical phenotype in severe factor VII (FVII) deficiency. Among 7 patients homozygous for a cross-reacting materialnegative (CRM ؊ ) FVII defect (9726؉5G>A, FVII Lazio), the only asymptomatic individual carried FV Leiden. Differential modulation of FVII levels by intragenic polymorphisms was excluded by a FVII to factor X (FX) gene haplotype analysis. The coagulation efficiency in the FV Leiden carrier and a noncarrier was evaluated by measuring FXa, FVa, and thrombin generation after extrinsic activation of plasma in the absence and presence of activated protein C (APC). In both patients coagulation factor activation was much slower and resulted in significantly lower amounts of FXa and thrombin than in a normal control. However, more FXa and thrombin were formed in the plasma of the patient carrying FV Leiden than in the noncarrier, especially in the presence of APC. These results were confirmed in FV-FVII doubly deficient plasma reconsti- IntroductionCoagulation factor VII (FVII) is a vitamin K-dependent glycoprotein that plays a key role in the initiation of coagulation. 1 Following vascular damage, membrane-bound tissue factor (TF) forms a Ca 2ϩ -dependent complex either with FVII (which is then converted to FVIIa by a single proteolytic cleavage) or directly with circulating FVIIa, present in blood at a very low concentration. TF-bound FVIIa activates factor X (FX) to FXa, which, together with its cofactor factor Va (FVa), converts prothrombin to thrombin. In addition, the TF/FVIIa complex can also activate factor IX to FIXa, which, after forming a complex with factor VIIIa (FVIIIa), contributes to FXa generation and thereby to thrombin formation via the intrinsic pathway. In plasma, the activity of the TF/FVIIa complex is down-regulated by the tissue factor pathway inhibitor (TFPI), which acts via the formation of a quaternary complex with FXa, FVIIa, and TF. 2 Thrombin generation is ultimately shut down by activated protein C (APC), which proteolytically inactivates FVa and FVIIIa, the essential cofactors of the prothrombin-and intrinsic FX-activating complexes. 3 Severe FVII deficiency 4,5 affects about 1 in 500 000 individuals in the general population and is inherited as an autosomal recessive trait with variable penetrance. Severely affected patients may develop life-threatening hemorrhages and require substitutive treatment with plasma concentrates or recombinant FVIIa. 6 Several intragenic mutations that impair gene expression or protein secretion (CRM Ϫ deficiency) or alter protein function (CRM ϩ deficiency) have been described (FVII Mutation Database, http://europium.csc.mrc.ac.uk). Moreover, a few intragenic polymorphisms 7-10 that modulate plasma levels of FVII 8,[10][11][12] are known.The FVII gene is located on chromosome 13 (13q34-qter), next to the FX gene, 13,14 and comprises 9 exons and 8 introns. 15 A polymorphic minisatellite, consisting of a variable number of 37-nucleotide tandem repeats, spans the 3Ј ...
Two cross-reacting material-positive (CRM ؉ ) factor VII (FVII) mutations, associated with similar reductions in coagulant activity (2.5%) but with mild to asymptomatic (Gly331Ser, c184 [in chymotrypsin numbering]) or severe (Gly283Ser, c140) hemorrhagic phenotypes, were investigated. The affected glycines belong to structurally conserved regions in the c184 through c193 and c140s activation domain loops, respectively. The natural mutants 331Ser-FVII and 283Ser-FVII were expressed, and in addition 331Ala-FVII and 283Ala-FVII were expressed because 3 functional serine-proteases bear alanine at these positions. The 331Ser-FVII, present in several asymptomatic subjects, showed detectable factor Xa generation activity in patient plasma (0.7% ؎ 0.2%) and in reconstituted system with the recombinant molecules (2.7% ؎ 1.1%). The reduced activity of recombinant 283Ala-FVII (7.2% ؎ 2.2%) indicates that the full function of FVII requires glycine at this position, and the undetectable activity of 283Ser-FVII suggests that the oxydrile group of Ser283 participates in causing severe CRM ؉ deficiency. Furthermore, in a plasma system with limiting thromboplastin concentration, 283Ser-FVII inhibited wild-type FVIIa activity in a dosedependent manner. (Blood. 2002;99: 1495-1497
Previous studies have established that factor VII gene (F7) polymorphisms (5′F7 and R353Q) contribute about one-third of factor VII (FVII) level variation in plasma. However, F7 genotyping in patients with cardiovascular disease has produced conflicting results. Population and expression studies were used to investigate the role of intron 7 (IVS7 ) polymorphisms, including repeat and sequence variations, in controlling activated FVII (FVIIa) and antigen (FVIIag) levels. Genotype–phenotype studies performed in 438 Italian subjects suggested a positive relation between the IVS7 repeat number and FVII levels. The lowest values were associated with theIVS7 + 7G allele. The screening of 52 patients with mild FVII deficiency showed an 8-fold increase in frequency (8%) of this allele, and among heterozygotes for identical mutations, lower FVII levels were observed in the IVS7 + 7G carriers. This frequent genetic component participates in the phenotypic heterogeneity of FVII deficiency. The evaluation of the individual contribution of polymorphisms was assisted by the expression of each IVS7variant, as a minigene, in eukaryotic cells. The novel quantitative analysis revealed that higher numbers of repeats were associated with higher mRNA expression levels and that the IVS7 + 7Gallele, previously defined as a functionally silent polymorphism, was responsible for the lowest relative mRNA expression. Taken together, these findings indicate that the IVS7 polymorphisms contribute to the plasmatic variance of FVII levels via differential efficiency of mRNA splicing. These studies provide further elements to understand the control of FVII levels, which could be of importance to ensure the hemostatic balance under pathologic conditions.
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