Martine Alhenc-Gelas

Factors V Leiden and II 20210A in patients with symptomatic pulmonary embolism and deep vein thrombosis

PURPOSEnFactor V Leiden and factor II 20210A are inherited disorders of the clotting system that occur frequently in patients with deep vein thrombosis. We conducted this study to determine whether these factors are also common in patients with pulmonary embolism.nnnSUBJECTS AND METHODSnWe determined the prevalence of factor V Leiden and factor II 20210A in 773 consecutive patients with objectively documented symptomatic deep vein thrombosis or symptomatic pulmonary embolism, or with a combination of these disorders.nnnRESULTSnIsolated symptomatic deep vein thrombosis occurred in 345 patients; isolated symptomatic pulmonary embolism occurred in 236; and both anomalies occurred in 192. Factor V Leiden was present in 21 (9%) of the patients with isolated symptomatic pulmonary embolism, in 30 (16%) with both manifestations, and in 63 (18%) with isolated symptomatic deep vein thrombosis (P = 0.007). Factor V Leiden was more common among patients with deep vein thrombosis (odds ratio [OR] = 2.1; 95% confidence interval [CI]: 1.2 to 3.7; P = 0.006) or both pulmonary embolism and deep vein thrombosis (OR = 1.8; 95% CI: 1.0 to 3.3; P = 0.07) than among patients with isolated pulmonary embolism. Factor V Leiden was less common in massive pulmonary embolism (5% [7 of 127]) than in submassive pulmonary embolism (13% [21 of 155], P = 0.03). We found no significant difference in the prevalence of factor II 20210A among the three groups.nnnCONCLUSIONnFactors V Leiden and II 20210A vary in prevalence among patients with pulmonary embolism and deep vein thrombosis, suggesting that the risk of pulmonary embolization may vary among patients who have different causes of venous thromboses.

Three novel mutations of antithrombin inducing high-molecular-mass compounds.

We have identified three novel mutations of the antithrombin (AT) gene in patients with thrombotic complications: a Cys 128 --> Tyr mutations, a G --> A mutation in the intervening sequence 4 (IVS4) 14 nucleotide 5 to exon 5, and a 9 bp deletion in the 3 end of exon 6 resulting in a short aberrant sequence after Arg 425. The latter mutation was associated with an Arg 47 --> His mutation in two compound heterozygous brothers. These three mutations led to the expression in the circulation of small amounts of inactive molecules with a high molecular mass in immunoblot analysis. In reducing conditions, these variant molecules had a normal molecular mass, which led us to postulate that these mutations prevent the formation of one intramolecular disulfide bond and allow the formation of intermolecular disulfide bonds. Plasma from a heterozygous patients bearing the Cys 128 --> Tyr mutation and from a compound heterozygote bearing the Arg 47 --> His mutation and the 9 bp deletion in exon 6 were passed through a heparin-sepharose column. In both cases a population of high-molecular-weight AT molecules with no binding affinity and no AT activity was separated from a population of normal molecules in the first patient, together with a population of molecules with a reduced binding affinity for heparin due to the substitution of Arg 47, in the compound heterozygote. The common feature of these three mutations is that they lead to partial misfolding and to the formation of intermolecular disulfide bonds with other plasma components, inducing the pleiotropic phenotypes observed.

Molecular bases of antithrombin deficiency in French families: identification of seven novel mutations in the antithrombin gene

We have investigated the molecular bases of familial antithrombin deficiency in eight French families. Eight mutations in the antithrombin coding exons were identified, seven of which were novel mutations. In all cases, individuals were heterozygous for the mutation. We found two small frameshift deletions in exon 3a, leading to type I deficiency. Five missense mutations in exons 3b or 5 also caused type I deficiency and their potential consequences on the antithrombin three‐dimensional structure were analysed. The last mutation in exon 4 was associated with a type II ‘reactive site’ deficiency: a dysfunctional antithrombin that is affected in its interaction with thrombin was present in circulation.

Molecular basis for hereditary antithrombin III quantitative deficiencies : a stop codon in exon IIIa and a frameshift in exon VI

Summary. Antithrombin III (AT III) is an inhibitor of serine protease (serpin) comprising 432 amino acids. Quantitative AT III deficiencies are associated with a high risk of thrombotic disease. Although this risk is smaller in patients with qualitative AT III deficiencies, the molecular defects characterizing the latter have been the subject of many studies. However, in quantitative AT III deficiencies, only three mutations have been described: Pro 407 to Leu and Ala 404 to Thr (both located in the C‐terminal part of the AT III molecule) and also a frameshift in exon IIIa.

A phenylalanine 402 to leucine mutation is responsible for a stable inactive conformation of antithrombin

In a South African family with antithrombin deficiency and unexplained thrombosis, genomic DNA analysis revealed a substitution of Phe 402 by Leu. This mutation involves an amino acid located in the carboxyterminal side of the antithrombin reactive loop and has already been observed in a French family (antithrombin Maisons-Laffitte). In both cases, the expression of the mutation is pleiotropic, i.e. results in a reduction in the circulating concentration of antithrombin and impairs both its anti-thrombin activity and its ability to bind heparin. The effect of a denaturing agent (sodium dodecyl sulfate) on the recognition of the plasma antithrombin by a polyclonal antibody was studied in an immuno-enzymatic assay. The Phe to Leu mutation decreased the sensitivity to denaturation, suggesting that the mutation increases the stability of the protein. Whether this stable conformation is due to a partial insertion of the amino-terminal side of the reactive loop, which would explain how both protease binding and heparin binding are affected, remains to be determined.