Sepideh Akhavan

Rare coagulation deficiencies

Summary.  Deficiencies of coagulation factors (other than factor VIII and factor IX) that cause a bleeding disorder are inherited as autosomal recessive traits and are generally rare, with prevalences in the general population varying between 1 : 500 000 and 1 : 2 000 000. In the last few years, the number of patients with recessively transmitted coagulation deficiencies has increased in European countries with a high rate of immigration of Islamic populations, because in these populations, consanguineous marriages are frequent. Owing to the relative rarity of these deficiencies, the type and severity of bleeding symptoms, the underlying molecular defects and the actual management of bleeding episodes are not as well established as for haemophilia A and B. This article reviews these disorders in terms of their clinical manifestations and characterization of the molecular defects involved. The general principles of management are also discussed.Keywords: afibrinogenaemia, autosomal recessive disorders, factor VIII, factor XI, factor XIII.

Interaction Between the G20210A Mutation of the Prothrombin Gene and Oral Contraceptive Use in Deep Vein Thrombosis

Single-point mutations in the gene coding for prothrombin (factor II:A20210) or factor V (factor V:A1691) are associated with an increased risk of venous thromboembolism. The use of oral contraceptives is also a strong and independent risk factor for the disease, and the interaction between factor V:A1691 and oral contraceptives greatly increases the risk. No information is available about the interaction between oral contraceptives and mutant prothrombin. We investigated 148 women with a first, objectively confirmed episode of deep vein thrombosis and 277 healthy women as controls. Fourteen patients (9.4%) were carriers of factor II:A20210, 24 (16.2%) of factor V:A1691, and 4 (2.7%) of both defects. Among controls, the prevalence was 2.5% for either factor II:A20210 or factor V:A1691, and there was no carrier of both the mutations. The relative risk of thrombosis was 6-fold for factor II:A20210 and 9-fold for factor V:A1691. The most prevalent circumstantial risk factor in patients and the only one observed in controls was oral contraceptive use, which per se conferred a 6-fold increased risk of thrombosis. The risk increased to 16.3 and 20.0 when women with factor II:A20210 or factor V:A1691 who used oral contraceptives were compared with noncarriers and nonusers. These figures indicate a multiplicative interaction between the genetic risk factors and oral contraceptives. No difference in the type of oral contraceptives was observed between patients and controls, those of third generation being the most frequently used (73% and 80%). We conclude that carriers of the prothrombin mutation who use oral contraceptives have a markedly increased risk of deep vein thrombosis, much higher than the risk conferred by either factor alone.

Gene mutations and three-dimensional structural analysis in 13 families with severe factor X deficiency

Summary.  Factor X (FX) deficiency is a rare autosomal recessive disorder. The phenotype and genotype of 15 Iranian patients with FX deficiency from 13 unrelated families with a high frequency of consanguinity were analysed. Five different assays identified four patients from three families with a discrepancy between low‐FX coagulant activity (FX:C) and higher‐FX antigen (FX:Ag) (a type II deficiency). The remaining 11 patients had parallel reductions of FX:C and FX:Ag (a type I deficiency). Nine different homozygous candidate mutations were identified, of which eight were novel. The four type II cases were associated with an Arg(−1)Thr missense mutation in the prepropeptide: Arg(−1) is highly conserved in all vitamin K‐dependent proteins. Four type I mutations (Gly78Asp, Cys81Tyr, Gly94Arg and Asp95Glu) were localized to the EGF‐1 and EGF‐2 domains, for which molecular views showed that the protein folding would be disrupted. The type I mutation Gly222Asp was localized in the catalytic domain of FX, and is sufficiently close to the Asp‐His‐Ser catalytic triad to disrupt its correct protein folding. The two type I splice site mutations were IVS1+3, A→T and IVS2–3, T→G. These novel homozygous FX mutations were consistent with their phenotypes and agree with experimental data from knockout mice, indicating that FX is an essential protein for survival.

The natural mutation by deletion of Lys9 in the thrombin A-chain affects the pKa value of catalytic residues, the overall enzyme's stability and conformational transitions linked to Na + binding

The catalytic competence of the natural thrombin mutant with deletion of the Lys9 residue in the A‐chain (ΔK9) was found to be severely impaired, most likely due to modification of the 60‐loop conformation and catalytic triad geometry, as supported by long molecular dynamics (MD) simulations in explicit water solvent. In this study, the pH dependence of the catalytic activity and binding of the low‐molecular mass inhibitor N‐α‐(2‐naphthylsulfonyl‐glycyl)‐4‐amidinophenylalanine‐piperidine (α‐NAPAP) to the wild‐type (WT) and ΔK9 thrombin forms were investigated, along with their overall structural stabilities and conformational properties. Two ionizable groups were found to similarly affect the activity of both thrombins. The pKa value of the first ionizable group, assigned to the catalytic His57 residue, was found to be 7.5 and 6.9 in ligand‐free ΔK9 and WT thrombin, respectively. Urea‐induced denaturation studies showed higher instability of the ΔK9 mutant compared with WT thrombin, and disulfide scrambling experiments proved weakening of the interchain interactions, causing faster release of the reduced A‐chain in the mutant enzyme. The sodium ion binding affinity was not significantly perturbed by Lys9 deletion, although the linked increase in intrinsic fluorescence was lower in the mutant. Essential dynamics (ED) analysis highlighted different conformational properties of the two thrombins in agreement with the experimental conformational stability data. Globally, these findings enhanced our understanding of the perturbations triggered by Lys9 deletion, which reduces the overall stability of the molecule, weakens the A–B interchain interactions, and allosterically perturbs the geometry and protonation state of catalytic residues of the enzyme.

A novel polymorphism in intron 1a of the human factor VII gene (G73A): study of a healthy Italian population and of 190 young survivors of myocardial infarction

We have identified a novel polymorphism located in intron 1a of the human factor VII gene, caused by the nucleotide change G to A at position + 73. In a population of 128 healthy individuals from northern Italy, the variant A73 allele had a frequency of 0.21, whereas the frequency of the previously reported 10 bp insertion allele located at −323 in the promoter region was 0.17 and that of the Q353 allele in the catalytic region of the factor VII gene was 0.20. In 75% of the healthy individuals, the A73 allele was present together with the 10 bp insertion and the Q353 alleles, indicating a strong linkage disequilibrium. The concomitant presence of A73 with both the 10 bp and the Q353 alleles was associated with the lowest factor VII levels, measured as coagulant activity, activated factor VII and factor VII antigen. The G73A polymorphism was also evaluated in 190 survivors of myocardial infarction who had experienced the event before the age of 45 years and in 179 individuals with a negative exercise test matched with patients for sex, age and geographical origin. Patients carrying the A73 allele associated with lower factor VII levels tended to have a lower risk of myocardial infarction (adjusted odds ratio 0.54; 95% confidence intervals 0.29–0.99). In conclusion, we found a novel variant allele in intron 1a of the human factor VII gene that is often associated in healthy individuals with the 10 bp and Q353 alleles in the promoter and catalytic region of the same gene. This intronic mutation, alone or in association with other factor VII gene polymorphisms, might confer protection against myocardial infarction in the young.

Phenotypic and genetic analysis of a compound heterozygote for dys- and hypoprothrombinaemia.

Summary. We studied a 2‐year‐old boy with a phenotype of combined hypo‐ and dysprothrombinaemia. Sequencing of polymerase‐chain‐reaction‐amplified genomic DNA revealed three different mutations in heterozygosity, a G to A transition at position 7312, resulting in the replacement of arginine 271 by histidine, an A to G transition at position 20058, resulting in the replacement of histidine 562 by arginine, and a 2‐bp deletion at 20062–20063, causing a frameshift and a premature stop codon in exon 14. The first two mutations are compatible with the dysprothrombinaemia phenotype, whereas the small deletion is thought to be the cause of hypoprothrombinaemia.

Gly319 Arg substitution in the dysfunctional prothrombin Segovia

The molecular defect of a congenitally dysfunctional form of prothrombin, prothrombin Segovia, was identified in a patient with a severe bleeding tendency, reduced prothrombin coagulant activity, and normal antigen level. Nucleotide sequencing of amplified DNA revealed a G → A change at nucleotide 7539 of exon 9 of the prothrombin gene. This resulted in the substitution of Gly319 by Arg. The proband was homozygous for this mutation, his father and brother were heterozygous. We surmised that the substitution, which occurs near the site of cleavage of prothrombin by factor Xa (Arg320–Ile321), altered the conformation of the protein making the cleavage site inaccessible.

A critical role for Gly25 in the B chain of human thrombin

Summary.  We have recently identified (Akhavan S et al., Thromb Haemost 2000; 84: 989–97) a patient with a mild bleeding diathesis associated to an homozygous mutation in the thrombin B chain (Gly25Ser, chymotrypsinogen numbering, i.e. position 330 in human prothrombin numbering). Transient transfection of wild‐type prothrombin (FII‐WT) and mutant prothrombin (designated FII‐G25(330)S) cDNA in COS‐7 cells showed a mild reduction (50%) in FII‐G25(330)S production. Recombinant proteins, stably expressed in Chinese hamster ovary cells, were isolated and activated by Taïpan snake or Echis carinatus venoms. We show that the G25(330)S mutation results in a decrease in the rate of prothrombin proteolytic activation. The mutation also significantly decreases (i) the catalytic activity of thrombin with a 9‐fold reduction in catalytic efficiency of the mutant toward S‐2238; (ii) the interaction with benzamidine; (iii) the rate of inhibition by TLCK and antithrombin; and (iv) the rate of hydrolysis of macromolecular substrates (fibrinogen, protein C). In contrast, exosite I does not appear to be affected by the molecular defect. These results, together with molecular modeling and dynamics, indicate that Gly25(330) is important for proper expression and probably proper folding of prothrombin, and also plays a critical role in both the alignment of the catalytic triad and the flexibility of one of the activation segments of prothrombin.

The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency.

We report the results of in vitro expression and biochemical characterization of the naturally occurring type II mutation Pro303Thr (P303T) in the factor VII (FVII) gene. Recombinant activated mutated FVII (FVIIa303T), compared with the activated wild‐type FVII (FVIIaWT), showed reduced amidase activity toward synthetic substrates, especially when the observed reduced binding affinity for human soluble tissue factor (TF) (Kd from 4·4 nmol/l for FVIIaWT to 17·3 nmol/l for FVIIa303T) was overcome by a fully saturating TF concentration. Likewise, factor X (FX) hydrolysis by FVIIa303T showed a reduced activity in the absence (and more severely in the presence) of TF (kcat/Km from 2·3 × 107/mol/l s for FVIIaWT to 8·7 × 105/mol/l s for FVIIa303T). These results showed that the mutant FVIIa is more shifted toward a zymogen‐like form compared to FVIIaWT, suggesting that P303 facilitates the conformational transitions that stabilize the active form of FVIIa. The alteration of these allosteric equilibria is especially evident in the presence of TF, which was unable to shift the equilibrium toward a fully active FVIIa form. Additional experiments showed that both TF‐catalysed FVII303T autoactivation and FVII303T activation by activated FX in the presence of TF were severely impaired, mainly because of an increase of the Km value. Altogether, these defects may explain the severe bleeding symptoms in a patient carrying the FVIIP303T mutation.