[Cerebral-vein thrombosis: retrospective study of twenty seven cases]. / Thrombophlébite cérébrale: étude rétrospective de vingt-sept cas.

PURPOSE: Among the locations of venous thrombosis, even if rare, cerebral-vein thrombosis is a severe event with a high mortality rate. No aetiology is found in 20 to 30% of the cases. In recent years, inherited coagulation disorders have been described, as risk factors for venous thrombosis. We report the results of a retrospective study of 27 patients who suffered cerebral-vein thrombosis, in which coagulation abnormalities have been searched for. METHOD: The patients were referred to the haemostasis laboratory of the Henri Mondor hospital between august 1982 and June 1988, after a cerebral-vein thrombosis. The predisposing factors, personal and family history of thromboembolism, clinical presentation, thrombosis location, evolution under treatment and long-term outcome, have been noted. Deficiencies in antithrombine, protein C, protein S, the Factor V Leiden and the G20210A prothrombin-gene mutation, the presence of lupus anticoagulant, of anticardiolipin antibodies as well as a hyperhomocysteinaemia have been searched, either at the initial presentation, or a posteriori. RESULTS: Fourty-one percent of patients had a coagulation abnormality. The prevalence of the different abnormalities was: inherited deficiency in AT 7.4%, in PC 8%, in PS 12.5%, factor V Leiden mutation 12%, G20210A prothrombin-gene mutation 12%. Two patients had combined defects: AT and PC deficiency in one, F V Leiden and F II G20210A mutations in one. e of the patient had lupus anticoagulant. Three patients had a significant rate of anticardiolipin antibodies. Five patients out of eight displayed a moderate hyperhomocysteinaemia. Nothing (past history, age, predisposing factors) distinguished those patients bearing a coagulation disorder from the others. The venous thromboembolic relapse rate of 15 % (4/27 patients). Three of them had an inherited thrombophilic abnormality. CONCLUSION: We recommend an investigation of the haemostasis after every cerebral venous thrombosis.

A direct, automated, immuno-turbidimetric assay of free protein S antigen in plasma.

A new, fully automated, one-step, immuno-turbidimetric assay of free protein S (fPS) in plasma (STA Liatest Free Protein S; Diagnostica Stago, Asnières, France) has been developed for STA analysers. This technique combines the advantages of a direct assay of fPS using two monoclonal antibodies, which specifically recognize fPS but not protein S (PS)-C4b-binding protein complexes, and the advantages of automation. The assay has good analytical performances, with intra- and inter-assay variation coefficients below 5% for normal values, and slightly higher for abnormal values. In a comparison study with a one-step enzyme-linked immunosorbent assay for fPS (Asserachrom Free Protein S; Diagnostica Stago), a correlation coefficient of 0.93 with a regression line close to 1 was found between the two techniques (n = 166 normal or PS-deficient plasma samples collected from healthy subjects and individuals with a personal or family history of thrombosis). This new technique is specific, reproducible, easy to perform, and provides a useful tool in the diagnosis of PS deficiency.

[Extracorporeal circulation with danaparoid sodium for valve replacement in thrombocytopenia induced by type II heparin]. / Circulation extracorporelle sous danaparoïde sodique pour remplacement valvulaire au cours d'une thrombopénie induite par l'héparine de type II.

A type II heparin-induced thrombocytopenia (HIT) was diagnosed in a 64-year-old woman at day 20 of intravenous unfractionated heparin (UFH) therapy, given after myocardial infarction treated by angioplasty and intracoronary stent. The infarction was complicated by a mitral insufficiency that led to a mitral valve replacement. Cardiopulmonary bypass was successfully performed with sodium danaparoid (Orgaran), as an alternative to UFH, without thrombotic or haemorrhagic complications and the follow-up was uneventful.

Inhibitor against coagulation factor V after liver transplantation.

A new case of anti-factor V inhibitor is described in a 46-year-old man, who received a liver transplantation for hepatocellular carcinoma, without exposure to bovine thrombin or fibrin glue during the operative course. The inhibition occurred on the 14th postoperative day, while the patient was being treated with oxacillin, azathioprine, and a new immunosuppressive drug, FK506. The inhibition was of short duration (3 days), and no bleeding complication occurred despite a very low plasmatic level of factor V activity and antigen (<5%). Plasma samples drawn after cessation of FK506 disclosed a dose-dependent inhibitory activity when alcoholic solutions of FK506 were exogeneously added; this suggests a possible role of the FK506 drug in the occurrence of this anti-factor V inhibitor.

Genetic modulation of plasma protein S levels by two frequent dimorphisms in the PROS1 gene.

We studied two polymorphisms located close to or within the 3'-untranslated (3'-UT) region of the PROS1 gene [an A to G transition at nt 2148 (Pro 626) and an A to C substitution at nt 2698] in 110 healthy volunteers. The allele frequency of the nt 2148 G variant was 35%, and that of the nt 2698 A variant was 27%. We found a relationship between the two dimorphisms (both separately and together) and the plasma total protein S antigen (tPS) level. The impact of the neutral Pro 626 dimorphism was more significant than that of nt 2698 C/A (p = 0.0003 and p = 0.013, respectively). The lowest tPS values were observed in subjects with the Pro 626;nt 2698 GG;CC genotype, and the highest values in those with the AA;AA genotype. Both polymorphisms acted independently of sex and age. The mechanisms by which the two polymorphisms regulate tPS synthesis were not revealed by the studies of platelet mRNA. This study provides the first evidence of a genetic modulation of tPS levels, which, in addition to age and sex, contributes to the wide normal range of tPS in plasma. Determination of these two polymorphisms could be a valuable additional tool for studying PS.

Chilblains and Raynaud phenomenon are usually not a sign of hereditary protein C and S deficiencies.

Hereditary protein C and S deficiencies are risk factors for thrombosis. They are associated with purpura fulminans and coumarin-induced skin necrosis. Recently, necrotic livedo of the extremities, severe chilblains and severe frostbite have been observed in protein C or S deficient patients. Our study was designed to evaluate the prevalence of cold-induced acral manifestations in patients with protein C or S deficiency. One-hundred-and-six patients with protein C or S deficiency and controls matched for sex and age were studied by questionnaire. Data included any history of acral manifestation possibly related to cold exposure, i.e. chilblains, Raynaud phenomenon, acrocyanosis and possible associated factors. Assessment of the diagnosis by a dermatologist was recorded. No difference was found in the prevalence of acral manifestations between patients and controls. This study suggests that protein C and S deficiencies are not risk factors for cold-induced acral manifestations.

A mutation of the active protein S gene leading to an EGF1-lacking protein in a family with qualitative (type II) deficiency.

The genomic analysis of a 70-year-old man with recurrent deep venous thrombosis having a protein S (PS)-deficient phenotype corresponding to both type III and type II evidenced two different mutations: a +5 g-->a mutation in the donor splice site of intron e (ivs e) and a ser 460 to Pro mutation. The propositus' son, who had a type II PS deficiency phenotype, only bore the ivs e +5 g-->a mutation. The study of platelet PS mRNA prepared from this subject showed that the ivs e, +5 g-->a mutation led to the generation of two abnormal transcripts, one lacking exon 5 and the other lacking exons 5 and 6. The presence of an additional PS band with a decreased molecular mass on immunoblots performed in reducing conditions suggested the presence of truncated PS lacking EGF1 (encoded by exon 5). Two monoclonal antibodies (MoAbs) were used to further characterize the nonfunctional plasma PS. Comparison of PS levels measured with each of these MoAbs and PS levels in conventional assays was consistent with the presence of an abnormal inactive protein in the plasma of both patients bearing the ivs e, +5 g-->a mutation, suggesting that variant PS lacking EGF1 is secreted but is devoid of activated protein C cofactor activity.

Evaluation of a new chronometric assay for factor V Leiden-dependent APC resistance.

Since the first description by Dahlbäck et al (1) of a hereditary defect in the plasma of three unrelated thrombophilic patients characterized by a poor anticoagulant response to activated protein C (APC), many reports have shown a high prevalence of the APC resistance in both the general and thrombophilic populations (2-6). In almost all cases APC resistance is due to a single point mutation in the factor V gene, (a G to A substitution at nucleotide position 1691), which predicts the synthesis of a factor V molecule (FVQ506 or FV Leiden) in which the Arg 506 in one of the APC cleavage sites is replaced by Gln (7), rendering factor Va resistant to inactivation by APC. The FVQ506 mutation is the most frequent hereditary risk factor for venous thrombosis (2, 5, 6, 8-10) making an assay for APC resistance with a high predictive value for the mutation highly desirable, especially when molecular diagnosis at the gene level is not possible. The aim of this study was to evaluate the performance of a new factor Xa-based assay for the FV Leiden-dependent APC resistance.

The 1691 G-->A mutation in the factor V gene: relationship to activated protein C (APC) resistance and thrombosis in 65 patients.

Four hundred fifty subjects were screened for the 1691 G-->A mutation in the factor V gene. Two hundred ninety-seven patients were referred to us for unexplained thrombosis, 133 were family members of these patients and 20 were normal subjects. We studied the relationships between the mutation, resistance to APC and thrombosis. Among the 450 subjects tested, 65 belonging to 42 families were found to have the 1691 G-->A mutation in one (n = 61) or both alleles (n = 4). The prevalence of the mutation in the thrombotic patients was 13%. Resistance to APC was tested for in 247 subjects not on anticoagulant treatment (4 homozygous and 44 heterozygous for the mutation, and 199 individuals without the mutation). Incomplete cosegregation of heterozygosity for the 1691 G-->A mutation with APC resistance (APC-SR < 2.4 or n-APC-SR < 0.75) was observed, showing that the functional assay alone is insufficient for a firm diagnosis. In patients carrying the mutation, elevated levels of prothrombin fragment 1 + 2 and D-dimers pointed to increased thrombin generation in vivo. Clinical manifestations in the heterozygous subjects were very similar to those reported in heterozygous PC or PS deficiencies, but the first thrombotic event occurred later than in PC- or PS-deficient patients. Homozygosity for the factor V gene mutation appears to be a far more benign thrombotic disorder than homozygous PC and PS deficiencies.

[Resistance to activated protein C disclosed by postphlebitic disease. Two cases]. / Résistance à la protéine activée révélée par une maladie post-phlébitique. Deux cas.

INTRODUCTION: Recently, resistance to activated protein C has been discovered. Resistance to activated protein C appears to be the main cause of familial thrombosis. CASE REPORT: Two consecutive patients have been studied. Both have a venous insufficiency associated with eczema in one patient and venous ulceration in the second patient. The two patients had a personal and familial history of venous thrombosis. In both patients, a resistance to activated protein C was found associated with a mutation in the factor V gene in residue 506. DISCUSSION: When a personal or familial history of the venous thrombosis is associated with symptoms of venous insufficiency, resistance to activated protein C must be added to the search for proteins C, S and anti-thrombin III deficiency.

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.

Inherited protein S deficiency: clinical manifestations and laboratory findings in 63 patients.

To further characterize inherited heterozygous protein S (PS) deficiencies, we studied 63 patients belonging to 33 families. Diagnosis of PS deficiency was based on protein S activity (PS Act) and/or free PS antigen (FPS Ag) levels below the lower limit of the normal range in patients not on oral anticoagulation. Depending on the level of total PS antigen (TPS Ag), two subpopulations could be distinguished: in the first one (25 patients belonging to 11 families) level of TPS Ag was reduced whereas in the second one (38 patients belonging to 22 families), TPS Ag was normal. In none of the families studied the two types of PS deficiency coexisted suggesting that they are different entities. In the 63 patients, thromboembolic events occurred in 57% of cases and were recurrent in 36.5% of patients. Age at the time of the first thrombosis ranged from 14 to 74 years, and was below 40 years in 69% of symptomatic cases. Thrombotic events were spontaneous in 64% of cases, and were associated with other risk factors in 36%. There was no apparent relationship between clinical status, symptomatic or asymptomatic, and the type or degree of the PS deficiency. Long-term anticoagulation prevented the recurrence of thrombosis in every case but one, and led to a decrease in circulating levels of C4b-binding protein suggesting the existence of a regulation between C4b-BP and PS concentrations. Together with previous reports, these findings underline the clinical and biological heterogeneity of inherited protein S deficiency.

First frameshift mutation in the active protein S gene associated with a quantitative hereditary deficiency.

The authors used a strategy combining the amplification-refractory mutations system (ARMS) and denaturing gradient gel electrophoresis (DGGE) to screen the active protein S (PS) gene in a family with PS deficiency, and found a frameshift mutation in exon V. The protein, if expressed, would have an aberrant amino acid sequence from positions 82 to 90 and a premature stop codon in position 91. The mutation co-segregated with the deficient phenotype and was not found in 120 normal chromosomes. It is proposed that the deletion of a T in the codon corresponding to Pro 82 described here is responsible for the deficient phenotype.

Influence of conditions of blood sampling on coagulation activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin complexes and D-dimers) measurements.

In this study, we evaluated the effects of anticoagulants used in blood sampling on the measurements of coagulation activation markers F1 + 2, TAT, D-Dimers by Elisa methods. The study was carried out on normal subjects and patients with inherited deficiency of coagulation inhibitors, antithrombin III (ATIII) protein C (PC) and protein S (PS). Three different anticoagulant solutions were compared: 1) ACD/EDTA/adenosine/heparin, 2) EDTA/aprotinin/a synthetic thrombin inhibitor and 3) sodium citrate. The results showed that sodium citrate, commonly used in coagulation laboratories, is a suitable anticoagulant for the study of coagulation activation markers. In addition, the type of tubes (plastic tubes vs glass Vacutainer R tubes) used for blood sampling as well as the order of sampling (early or late after the phlebotomy procedure) did not influence the results. We concluded that assays of coagulation activation markers F1 + 2 and D-Dimers can be performed in samples collected routinely by haemostasis laboratory staff using Vacutainer R tubes with sodium citrate. Further investigations are needed to understand why TAT measurements gave a pattern of results quite different from F1 + 2 or D-Di measurements.