Hydration status is critical for erythrocyte survival and is mainly determined by intracellular cation content. Active pumps, passive transporters, and ion channels are the key components of volume homeostasis, whereas water passively fits ionic movements. Whenever cation content increases, erythrocyte swells, whereas it shrinks when cation content decreases. Thus, inappropriate cation leak causes erythrocyte hydration disorders, hemolytic anemia, and characteristic red cell shape abnormalities named stomatocytosis. All types of stomatocytosis either overhydrated or dehydrated are linked to inherited or de novo mutations in genes encoding ion transporters or channels. Although intracellular ion content can be assessed by experimental methods, laboratory diagnosis is guided by a combination of red blood cell parameters and deformability measurement when possible, and confirmed by sequencing of the putative genes. A better knowledge of the mechanisms underlying erythrocyte hydration imbalance will further lead to therapeutic improvements.

Parmi les causes rares de surcharge martiale se trouvent certaines anémies hémolytiques constitutionnelles, dont le diagnostic précis peut rester longtemps incertain. Un homme né en 1961 consultait en médecine interne en 2007 (46 ans) pour asthénie, arthralgies et découverte d’une ferritinémie à 1600 μg/L. Il était connu comme porteur d’une grosse rate depuis son service militaire et faisait des poussées d’ictère, tout comme son père et sa tante. Une hémolyse constitutionnelle avait été affirmée en 1987 (26 ans) mais n’avait pu être typée précisément. Le patient avait refusé la splénectomie alors proposée. La fille du patient, née en 2004, présentait également une anémie hémolytique, dont la nature précise n’avait pas été déterminée, ayant nécessité à 3 reprises des transfusions depuis sa naissance. Chez le patient, dont l’hémoglobine était à 120 g/L, les réticulocytes à 200 G/L, et la saturation de la transferrine à 54 %, une recherche génétique d’hémochromatose liée à HFE s’était avérée négative, mais l’IRM hépatique objectivait une surcharge martiale majeure, estimée à plus de 360 μmol/g. Les hémochromatoses rares (acœruloplasminémie, mutations des gènes codant l’hémojuvéline, l’hepcidine, la transferrine, et la ferroportine) étaient éliminées après avis du centre de référence breton. La biopsie hépatique confirmait l’existence d’une hépatosidérose presque pure marquée, peu fibrosante (stade 4 de Searle-Schaeuer). Un mécanisme d’hyperabsorption intestinale de fer généré par la baisse de l’hepcidine induite par l’hyperhémolyse était retenu. Parallèlement, le diagnostic provisoirement retenu chez sa fille après une nouvelle ektacytométrie était celui de stomatocytose à cellules déshydratées (xérocytose). Des saignées étaient proposées au patient mais s’avéraient mal tolérées et étaient vite abandonnées. Le malade refusait un traitement chélateur du fer et était perdu de vue. Il était revu 3 ans plus tard (50 ans) à la suite de la découverte d’une ostéoporose après la survenue d’une fracture traumatique du fémur (T-score fémoral à −2,5, vertébral à −4). Il n’était pas retrouvé d’étiologie spécifique à cette ostéoporose, en dehors d’une discrète insuffisance androgénique. Une IRM cardiaque était réalisée, qui n’objectivait pas de surcharge martiale notable au niveau du myocarde. L’hémoglobine était à 125 g/L, la ferritinémie à 1700 μg/L, et la saturation de la transferrine à 90 %. Une nouvelle ektacytométrie ne permettait toujours pas d’élucider la nature de l’hémolyse constitutionnelle. Un traitement chélateur du fer était à nouveau refusé par le patient. C’est finalement la génétique moléculaire qui permettait, en 2017, d’identifier chez le propositus et sa fille une mutation dans l’exon 6 du gène KCNN4 (c.1055 G>A) à l’état hétérozygote codant le canal Gardos, associée à une forme récemment décrite de xérocytose. Les stomatocytoses sont de rares anémies hémolytiques congénitales de transmission autosomique dominante, qu’on sait désormais secondaires à des anomalies des canaux ioniques membranaires induisant une hyperhydratation ou une déshydratation des hématies, dont l’aspect évocateur mais inconstant sur le frottis est celui de lèvres (stomatocytes). Elles sont réputées cliniquement par l’importance de la surcharge martiale, y compris au niveau cardiaque, alors que l’anémie est rarement sévère, et par le risque thromboembolique majeur après splénectomie. Un traitement chélateur du fer est indispensable. Deux canaux ioniques ont été impliqués dans cette pathologie : Piezo1 (canal cationique non spécifique activé par des forces mécaniques) et Gardos (canal potassique dépendant du Calcium). Une molécule testée dans la drépanocytose serait susceptible de corriger la dysfonction des canaux ioniques de la xérocytose liée aux mutations gain de fonction des gènes codant pour ces canaux ioniques, le senicapoc. Certaines anémies hémolytiques constitutionnelles peuvent se révéler par une surcharge martiale sévère plutôt que par l’hémolyse elle-même. La xérocytose en est un exemple typique, dont les causes moléculaires ont été récemment identifiées, même si les mécanismes responsables de l’importance de la surcharge martiale restent incertains. Pour le clinicien, il est important de rappeler que la splénectomie est contre indiquée dans cette pathologie.

IntroductionHereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary stomatocytosis (HSt) are inherited red cell disorders caused by defects in various membrane proteins. The heterogeneous clinical presentation, biochemical and genetic abnormalities in HS and HE have been well documented. The need to raise the awareness of HSt, albeit its much lower prevalence than HS, is due to the undesirable outcome of splenectomy in these patients.MethodsThe scope of this guideline is to identify the characteristic clinical features, the red cell parameters (including red cell morphology) for these red cell disorders associated, respectively, with defective cytoskeleton (HS and HE) and abnormal cation permeability in the lipid bilayer (HSt) of the red cell. The current screening tests for HS are described, and their limitations are highlighted.ResultsAn appropriate diagnosis can often be made when the screening test result(s) is reviewed together with the patient's clinical/family history, blood count results, reticulocyte count, red cell morphology, and chemistry results. SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins, monovalent cation flux measurement, and molecular analysis of membrane protein genes are specialist tests for further investigation.ConclusionSpecialist tests provide additional evidence in supporting the diagnosis and that will facilitate the management of the patient. In the case of a patient's clinical phenotype being more severe than the affected members within the immediate family, molecular testing of all family members is useful for confirming the diagnosis and allows an insight into the molecular basis of the abnormality such as a recessive mode of inheritance or a de novo mutation.

Fig. 1. (a) Pedigrees of the two families with hereditary xerocytosis (HX). The pedigrees show cosegregation of the HX phenotype and heterozygous mutation in PIEZO1 . +/+, patients with wild-type alleles; +/−, patients with heterozygous mutation. Not available (NA) indicates that biochemical and genetic analyzes are not available. No hyperkalemia, no gallstones were diagnosed in any members of the two families. (b) Hematological analyzes of patients with HX. At left, erythrocytes morphology showing target and hyperchromic cells with rare stomatocytes. At right, eosin5-maleimide (EMA) staining in flow cytometry showing increased fixation of the stain (black peak). (c) Sequence chromatogram showing the mutation found in the two families. At the top, sequencing data from an affected individual (III-5 of family 1), representing the normal and the mutant PIEZO1 alleles. A G->A transition changes the codon for arginine (CGC) in position 2456 of the PIEZO1 protein to the codon for histidine (CAC).

SummaryVenous thromboembolism [TE] is a multifactorial disease and antithrombin deficiency [ATD] constitutes a major risk factor. In the present study the prevalence of ATD and the clinical presentation at TE onset in a cohort of paediatric index cases are reported. In 319 un - selected paediatric patients (0.1–18 years) from 313 families, recruited between July 1996 and December 2013, a comprehensive thrombophilia screening was performed along with recording of anamnestic data. 21 of 319 paediatric patients (6.6%), corresponding to 16 of 313 families (5.1%), were AT-deficient with confirmed underlying AT gene mutations. Mean age at first TE onset was 14 years (range 0.1 to 17). Thrombotic locations were renal veins (n=2), cerebral veins (n=5), deep veins (DVT) of the leg (n=9), DVT & pulmonary embolism (n=4) and pelvic veins (n=1). ATD co-occurred with the factor- V-Leiden mutation in one and the prothrombin G20210A mutation in two children. In 57.2% of patients a concomitant risk factor for TE was identified, whereas 42.8% of patients developed TE spontaneously. A second TE event within primarily healthy siblings occurred in three of 313 families and a third event among siblings was observed in one family. In an unselected cohort of paediatric patients with symptomatic TE, the prevalence of ATD adjusted for family status was 5.1%. Given its clinical implication for patients and family members, thrombophilia testing should be performed and the benefit of medical or educational interventions should be evaluated in this high risk population.

BACKGROUND:Heparin and its analogs, mediating their anticoagulant activity through antithrombin (AT) activation, remain largely used for the preventive and curative treatment of thrombosis. The major adverse reaction of these drugs is the bleeding risk associated with overdose. Unfractionnated heparin (UFH) can be efficiently and rapidly neutralized by protamine sulfate, but this reversal partially neutralizes low-molecular-weight heparin (LMWH) and is inefficient in reversing fondaparinux. To secure administration of AT-mediated anticoagulants and counteract bleeding disorders, we previously designed a recombinant inactive AT as an antidote to heparin derivatives.OBJECTIVES:To get around the limited production level of recombinant AT, we propose in this study an alternative strategy to produce a chemically modified inactive AT, exhibiting increased heparin affinity, as an antagonist of heparin analogs.METHODS:Plasma-derived AT was chemically modified with 2,3 butanedione, a diketone known to specifically react with the arginine side chain. The chemical reaction was conducted in the presence of heparin to preserve basic residues within the heparin binding site from modifications.RESULTS:AT treated by butanedione and selected for its high heparin affinity (AT-BD) was indeed modified on reactive Arg393 and thus exhibited decreased anticoagulant activity and increased heparin affinity. AT-BD was able to neutralize anticoagulant activity of heparin derivatives in vitro and in vivo and was devoid of intrinsic anticoagulant activity, as assessed by activated partial thromboplastin time assay.CONCLUSIONS:AT-BD appears to be as efficient as protamine to neutralize UFH in vivo but could be more largely used because it also reverses fondaparinux and LMWH.

La tumeur fibreuse solitaire est une tumeur peu fréquente, longtemps observée au niveau de la plèvre et maintenant décrite dans de nombreux tissus et organes. Nous rapportons un cas de tumeur localisée au rétropéritoine, révélée par des douleurs abdominales et une hypoglycémie, chez un homme de 34 ans. Cette pathologie inhabituelle est de diagnostic anatomopathologique et amène à discuter les autres tumeurs des tissus mous rencontrées dans cette localisation. La plupart des tumeurs fibreuses solitaires extrathoraciques sont bénignes, mais peuvent récidiver ou métastaser après exérèse complète, même en l'absence de signes histopathologiques de malignité. Une surveillance attentive au long cours est donc recommandée.Solitary fibrous tumour is unusual, arising most commonly in the pleura and can also occur in a large number of other sites. We report the case of a 34-year-old man with a retroperitoneal solitary-fibrous tumour, revealed by abdominal pain and hypoglycaemia. We describe the histopathological and immunohistochemical features. Solitary-fibrous tumour should be included in the differential diagnosis of spindle cell tumours in this location. Despite complete local excision, local recurrence and metastasis are seen. The behaviour of theses tumours is unpredictable and patients with solitary fibrous tumour require careful and long-term follow-up.

Inherited antithrombin (AT) deficiency is a major risk factor for venous thromboembolism (VTE). Type I AT deficiency is characterized by equally low functional and antigenic AT (about 50%) and type II deficiency by the synthesis of a variant protein with altered function, with type IIRS corresponding to reactive site defects, type IIHBS to heparin binding site defects and type IIPE (pleiotropic) caused by mutations initially clustered in the s1C-s4B region. AT Cambridge II (Ala416Ser) (mutation annotated using the HGVS numbering system) is a variant associated with borderline or mildly reduced activity, but normal immunologic level. Classification is of clinical importance because patients heterozygous for a HBS mutation are at reduced risk of VTE compared with type I, type IIRS or IIPE heterozygotes [1]. Moreover, whether the AT Cambridge II mutation increases the risk of VT remains doubtful. Initially, this mutation was described in asymptomatic subjects from the UK [2, 3]; in contrast, subsequent findings from a large Spanish case–control study [4] suggested that the mutation may be a VT risk factor and the most frequent cause of AT deficiency in Caucasian populations. However, results obtained in three independent large French cohorts [5, 6] are not in agreement with this hypothesis. Calibrated automated thrombinography (CAT) is an efficient method for studying in vitro thrombin generation (TG) [7]; TG when triggered by tissue factor appears to be sensitive to plasma AT level variation. Thus, Wilders et al., [8] using first generation TG tests (TGTs), observed increased endogenous thrombin potential (ETP) in eight patients with a thrombotic tendency as a result of AT-inherited deficiency. Recently, Dielis et al. [9] demonstrated a significant negative influence of AT on ETP and thrombin peak height (PH) in healthy subjects. A significant influence of AT was also observed in TGTs performed in plasmas mixtures with different AT concentrations [10]. In addition, available data strongly suggest that thrombinography is an interesting tool in evaluating the VTE risk, with several reports of positive associations between ETP and VT [11-13]. In the present study, we used thrombinography in order to gain insight into the respective influence of SERPINC1 mutations responsible for different AT phenotypes on the coagulability level and on the risk of thrombosis. We studied plasma from 43 patients whose DNA had been previously screened for SERPINC1 mutations in our laboratory, in the context of AT-inherited deficiency suspicion. Written informed consent had been obtained from every subject according to the declaration of Helsinki. Twenty-two patients presented with a type I deficiency or borderline AT level, 17 with a type IIHBS deficiency and four with a type IIRS deficiency. Of the 43 subjects, 37 were heterozygous for previously described detrimental mutations which fully explain AT plasma phenotype (Table 1). Interestingly, one of these patients with a type IIRS phenotype was heterozygous for Phe434Leu, an already reported type IIPE mutation. Novel mutations [−5C>T, c.1157T>C (Ile386Thr), c.791del leading to Met283Stop] were found in four patients. These mutations were probably the cause of type I AT deficiency accounting for their position, their putative detrimental consequences and results of the family studies. In one (type I) patient, no mutation could be identified despite extensive gene analysis of the coding regions and searching for large gene rearrangement using multiplex ligation-dependent probe amplification (MLPA). However, her sister also presented with a type I phenotype, suggesting a genetic defect. The thrombin generation test (TGT) was also performed on plasma from nine subjects with normal AT levels, no other biological risk factor for thrombosis, normal global clotting tests and no personal thrombosis antecedents, investigated in our laboratory within the same period. The main characteristics of the subjects are reported in Table 1. Age was similar in the five groups. As expected, AT cofactor activity was borderline in Cambridge II carriers and clearly low in carriers of other mutations. Six subjects were factor (F)V Leiden or F2 20210 G>A mutation carriers. In addition, we studied a patient, aged 20 years, with AT cofactor activity 30%, progressive activity 76%, antigen 65%, who was homozygous for a type IIHBS mutation (Leu131Phe), and suffered from a stroke during pregnancy, at age 17. No other biological VT risk factor was present in these 44 individuals. Plasma obtained from citrated blood after double centrifugation at 2500 ×g for 15 min at 15–22 °C was kept frozen at < −30 °C. Blood was sampled at a distance from VT in 16 of the 19 symptomatic subjects. In order to verify that storage had no detrimental effect on studied parameters, we checked each sample separately for FV activity or PT/APTT values and determined that these values were in the normal range. CAT was performed on the same samples, using reagents from Thrombinoscope (Maastricht, The Netherlands), according to the manufacturer’s instructions. Final concentrations of TF and phospholipids were 5 pmol L−1 and 4 umol L−1, respectively. The parameters derived from CAT are lag time (LT), ETP, PH and start-tail (ST), the time at which thrombin generation has come to an end. Between-run coefficients of variation, calculated using results obtained for a plasma pool tested in 13 runs, were < 10% for ETP, PH and ST, 15% for peak time (PT) and 30% for LT. Table 1 shows the influence of AT defects on CAT parameters. Carriers of type I or type IIRS/PE defects were combined in the analysis as no significant differences in descriptive characteristics or TG parameters were found among them. Means were adjusted for gender, age, personal history of VTE and for FII and fibrinogen which had a significant influence on PH and PT (r = 0.44, P = 0.005) or LT and PT (r = 0.52, P < 0.001, r = 0.36, P = 0.009), respectively. Overall comparison demonstrated significant differences for ETP (P < 0.001) and ST (P < 0.001), as a result of higher ETP and ST in the type I+IIRS/PE group, and no significant difference for LT, PH and PT. Results were not different after excluding the six FV Leiden or F2 20210G>A carriers, or the three patients studied at the time of VT. No significant differences between Cambridge II heterozygotes, type IIHBS heterozygotes or controls were observed. In the type IIHBS homozygote, ETP (3706 nm min) and PH (512 nm) were similar to those found in type I or IIRS/PE heterozygotes. Our findings are in accordance with previous observations of a significant influence of AT on ETP [8-10]. To the best of our knowledge, the influence of different types of AT deficiency on TG studied using CAT was previously investigated in a single study, which comprised four groups of patients (13 without AT defect, 13 with a type I, 6 with a type II plasma deficiency and 10 with the Cambridge II mutation) [6]. Classification was based mainly on plasma phenotype. No genotyping was performed except in the last group. In the present study, genotyping was performed in every subject providing for the first time information on the consequences of distinct natural SERPINC1 mutations responsible for either quantitative or qualitative AT deficiency. No increase in ETP was found in type IIHBS carriers, which is consistent with type IIHBS heterozygosity being associated with a low risk of VTE. Mean ETP was increased in patients heterozygous for type I or type IIRS/PE mutations, in agreement with these mutations being risk factors for thrombosis. Interestingly, the right part of the area under the thrombogram curve [PH × (ST−PT)/2] explained 80% of the difference in ETP between type I or IIRS/PE carriers and the other patients. Higher ETP was therefore mainly explained by slower neutralization of thrombin. Homozygous AT gene defects are extremely rare and have only been described in a few patients with type IIHBS mutations. All these patients presented with severe venous and/or arterial thromboembolism early in life (for review, see [14]). ETP and PH levels in the Leu131Phe homozygote studied here were in the expected range for a patient with a high risk of thrombosis. Sanchez et al. [6] found a significant association between Cambridge II mutation carrying and ETP or PH despite no influence on VT risk. However, this association tended to be milder than that observed in type I AT deficiency. In contrast, in the present study, we found no influence of Cambridge II mutation on the thrombogram, in accordance with the lack of clinical influence. The present findings underline the importance of phenotyping (using the usual means) inherited AT deficiency. Clinical management of the patients will indeed be different whether type I or RS mutations which significantly increase the coagulability level and the risk of VT, or mutations with milder effects such as AT Cambridge II or type IIHBS, are present. In our hands, thrombinography was helpful in studying the influence of AT defects on the clotting system, but at the individual level, it did not fully discriminate between subjects with thrombogenic defects and the others. ‘Normal’ ETP levels observed in eight out of the 18 heterozygotes for type I/IIRS/PE patients might be in part explained by age (mean 25 in these eight patients vs. 40 in the 10 others) or by borderline FII level (70%–75%) observed in four patients. Both parameters have indeed a positive influence on ETP [15]. These findings do not argue for the routine use of thrombinography in screening for AT thrombogenic mutations. We thank the following colleagues for giving us the opportunity to study their patients: M. Dreyfus, M. H. Horellou, I. Martin-Toutain, E. Mazoyer, A. Robert, (AP-HP Paris), C. Boinot (CHU Poitiers), C. Boiteux-Vergne (CHUBordeaux), D. Brunet (CHU Marseille), E. de Raucourt (Poissy), V. Girault (Lyon), T. Lecompte and J. Devisgnes (CHU Nancy), F. Lellouche (Quimper), C. Ternisien (CHU Nantes), N. Trillot (CHU Lille) and N. Ochat and I. Présot for excellent technical assistance. This study was in part supported by the DHOS (Direction de l’Hospitalisation et de l’Organisation des Soins) programme ‘Soutien financier en faveur des laboratoires pratiquant le diagnostic par génétique moléculaire des maladies rares’. The authors state that they have no conflict of interest.

Antithrombin Rouen VI (N187D) is a rare conformational thermolabile variant. The unique symptomatic carrier reported in the literature developed 3 thrombotic events during pregnancy, in each case in a context of pyrexial infection. In fresh plasma, antithrombin activity and antigen level were normal but in vitro experiments demonstrated the presence of a thermolabile variant, suggesting that fever could be a trigger for thrombosis in N187D carriers. The RouenVI variant was further found in two asymptomatic brothers. In these subjects, it was associated with normal antigen level but reduced activity. In order to better delineate the functional and clinical consequences of the N187 variants, we have studied a series of seven subjects from two distinct families heterozygous for the Rouen VI mutation. Antithrombin levels were normal or borderline in these patients. Thermostability of plasma antithrombin was normal. We have also studied six subjects heterozygous for a new mutation, 6462C>G,which results in an asparagine to lysine substitution at residue 187. In these patients, the N187K mutation is associated with a clear type II deficiency and decreased thermostability of the plasma protein has been demonstrated. That the N187D mutation has milder consequences on plasma antithrombin activity than the N187K mutation is in agreement with structural predictions. About 50% of the N187 carriers studied have suffered venous thrombotic events, strongly suggesting that both mutations are risk factors for thrombosis, but none occurred during pyrexial infections.

Dear Sir, Inherited antithrombin (AT) deficiency is a prominent risk factor for venous thrombosis, found in between 4 and 6% of young patients presenting with venous thrombosis [1Hirsh J. Piovella F. Pini M. Congenital antithrombin III deficiency. Incidence and clinical features.Am J Med. 1989; 87Abstract Full Text PDF PubMed Scopus (153) Google Scholar]. AT deficiency is classified as type I, quantitative, and type II, presence of a variant protein with altered function. According to which function of the protein is affected, three type II subtypes are defined: reactive site, heparin binding site (HBS) or pleiotropic variant [2Lane DA, Bayston T. Olds R.J. Fitches A.C. Cooper D.N. Millar D.S. Jochmans K. Perry D.J. Okajima K. Thein S.L. Emmerich J. Antithrombin mutation database: 2nd (1997) update. For the Plasma Coagulation Inhibitors Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis.Thromb Haemost. 1997; 77: 197-211Crossref PubMed Scopus (239) Google Scholar]. Classification of AT deficiency is clinically relevant because heterozygous individuals with type II HBS deficiency carry a lowered, if any, thrombotic risk than other inherited AT deficiencies. Eleven AT variants responsible for type II HBS deficiency have been reported and several are recurrent (Pro41Leu, Arg47Cys, Arg47His) [2Lane DA, Bayston T. Olds R.J. Fitches A.C. Cooper D.N. Millar D.S. Jochmans K. Perry D.J. Okajima K. Thein S.L. Emmerich J. Antithrombin mutation database: 2nd (1997) update. For the Plasma Coagulation Inhibitors Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis.Thromb Haemost. 1997; 77: 197-211Crossref PubMed Scopus (239) Google Scholar]. We describe here two new type II HBS mutations that indicate a role for residues Lys114 and Arg13 in heparin binding. Type II HBS AT deficiency was identified from decreased plasma heparin cofactor activity compared to normal AT antigen level and was further investigated by crossed immunoelectrophoresis in the presence of heparin in the first dimension and AT gene analysis (Table 1). The seven exons and intron–exon junctions of the gene were sequenced as previously described [3Picard V. Bura A. Emmerich J. Alhenc-Gelas M. Biron C. Houbouyan-Reveillard L.L. Molho P. Labatide-Alanore A. Sie P. Toulon P. Verdy E. Aiach M. Molecular bases of antithrombin deficiency in French families: identification of seven novel mutations in the antithrombin gene.Br J Haematol. 2000; 110: 731-4Crossref PubMed Scopus (17) Google Scholar]. Other genetic risk factors for venous thrombosis were investigated, including factor V Arg506 Gln mutation, prothrombin gene G20210A mutation, protein S and protein C deficiency.Table 1AT plasma levels and genetic thrombosis risk factors for three individuals with new type II HBS antithrombin deficiency. (NR: normal range)IndividualA1A2BAT plasma level Heparin cofactor activity (NR 80-120%)58%72%48% Progressive activity (NR 80-120%)75%94%100% Antigen level (NR 80-120%)83%115%100%AT gene MutationC2501TC2501TA5336G Amino acid changeR13WR13WK114EProtein C gene MutationC3390TC3390T Amino acid changeR116WR116WFII G20210AGGGGGA Open table in a new tab In family A, a type II HBS deficiency was identified in the proband and her sister (Table 1). It was confirmed by crossed immunoelectrophoresis, which showed two peaks: one major normal peak and a smaller one with slower migration in the presence of heparin. A C2501T substitution in exon 2 was identified, predicting a Arg13Trp substitution. The proband and her sister were heterozygous for the mutation. In addition, both sisters carried a heterozygous quantitative protein C deficiency (Table 1). Neither the propositus nor her sister, who are 26 and 22 years old, respectively, suffered from thrombosis, but their mother had a history of venous thrombosis. Subject B was a 30-year-old female heterozygous for the prothrombin gene G20210A mutation. Thrombophilic markers were investigated because of an intracranial hypertension episode occurring during her second pregnancy, although she never suffered from thrombosis. A type II HBS AT deficiency was identified (Table 1) and confirmed by crossed immunoelectrophoresis which showed a small normal peak and a major peak with low heparin affinity. AT gene analysis demonstrated a A5336G substitution in exon 3a, leading to a Lys114Glu substitution. Subject B was heterozygous for the mutation, no family member was available. Characterization of AT natural variants has contributed to defining the AT heparin binding site and a more accurate picture of this binding site is now emerging. Role of both Arg13 and Lys114 in heparin binding is supported by examination of the crystal structure of antithrombin complexed to a high-affinity pentasaccharide [4]. As predicted, the structure shows that basic AT residues interact with pentasaccharide sulfates and carboxylates. Interacting AT residues are located on helix A and helix D, but also on the AT N-terminal cleft (Arg13 and Lys11) and on a newly formed helix P (Lys114 and Glu113) [4]. Results obtained with recombinant AT variants also indicated a role for Arg13 and Lys114. Indeed, a recombinant Arg13Ala AT had a decreased affinity for matrix-bound heparin and characterization of several Lys114 recombinant ATs (Lys114 to Glu, Ala or Met) converged to demonstrate that Lys114 is the most important contributor to heparin binding [5Ersdal-Badju E. Lu A. Zuo Y. Picard V. Bock S.C. Identification of the antithrombin III heparin binding site.J Biol Chem. 1997; 272: 19393-400Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 6Kridel S.J. Knauer D.J. Lysine residue 114 in human antithrombin III is required for heparin pentasaccharide-mediated activation.J Biol Chem. 1997; 272: 7656-60Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 7Arocas V. Bock S.C. Raja S. Olson S.T. Bjork I. Lysine 114 of antithrombin is of crucial importance for the affinity and kinetics of heparin pentasaccharide binding.J Biol Chem. 2001; 276: 43809-17Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar]. In agreement with these data, our findings do support the role of Arg13 and Lys114 for heparin binding of plasma AT: both substitutions eliminate a positively charged residue, changed to a hydrophobic and a negatively charged residue respectively, and in both cases an impaired interaction with the negatively charged pentasaccharide can be predicted. In addition, substitution of the exposed Arg13 by a large hydrophobic residue might destabilize the AT N-terminal cleft. Heterozygous type II HBS AT deficiency is considered to be a weak thrombosis risk factor [2Lane DA, Bayston T. Olds R.J. Fitches A.C. Cooper D.N. Millar D.S. Jochmans K. Perry D.J. Okajima K. Thein S.L. Emmerich J. Antithrombin mutation database: 2nd (1997) update. For the Plasma Coagulation Inhibitors Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis.Thromb Haemost. 1997; 77: 197-211Crossref PubMed Scopus (239) Google Scholar]. In fact, all three asymptomatic individuals described here carry a heterozygous type II HBS deficiency plus an additional genetic thrombosis risk factor, a quantitative protein C deficiency in family A and a heterozygous prothrombin gene G20210A mutation in subject B. A more severe clinical course could have been expected, at least for subject B, as the substitution involves the most important AT residue contributing to heparin binding and suggests a strongly impaired AT–heparin interaction. After submission of the letter, a Lys114Glu AT variant with complete loss of heparin affinity has been described in another family (see Mushunje A, Zhou A, Huntington JA, Conard J, Carrell RW. Antithrombin 'DREUX' (Lys 114Glu): a variant with complete loss of heparin affinity. Thromb Haemost 2002; 88: 436–43).

Fatal cerebral hemorrhage involving the left thalamus in a neonate was attributed to deep cerebral vein thrombosis. Although antithrombin levels were at the lower end of the normal range, family and genetic studies showed constitutional type I antithrombin deficiency related to a novel missense mutation in the antithrombin gene. (J Pediatr 2001;139:741-3)

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.

The present study was designed to analyze the thrombomodulin proximal promoter region spanning nucleotides -293 to -12 to search for polymorphisms that could modify thrombomodulin gene expression in patients with venous thromboembolic disease. The study population comprised 205 patients and 394 healthy subjects of similar age and sex distribution. No polymorphisms and only 1 point mutation (G-33A) were found. The G-33A mutation was present at the heterozygous state in 2 patients and in 1 control. Being more frequent in the patients (0.97%) than in the controls (0.25%), the G-33A mutation might be a risk factor for venous thrombosis. To investigate the effect of this mutation on the thrombomodulin promoter activity, the proximal promoter region of the gene (bearing or not bearing the G-33A mutation) was inserted into a promotorless expression vector, upstream of the firefly luciferase gene, and transiently transfected into EA.hy926 endothelial cells. Under the conditions of the assay, the G-33A mutation mildly decreased the promoter activity. This study confirms that abnormalities of the thrombomodulin proximal promoter are not frequent in patients with venous thromboembolism.

Solving the structure of the stable complex between a serine protease inhibitor (serpin) and its target has been a long standing goal. We describe herein the characterization of a monoclonal antibody that selectively recognizes antithrombin in complex with either thrombin, factor Xa, or a synthetic peptide corresponding to residues P-14 to P-9 of the serpin's reactive center loop (RCL, ultimately cleaved between the P-1 and P'(1) residues), Accordingly, this antibody reacts with none of the monomeric conformers of antithrombin (native, latent, and RCL-cleaved) and does not recognize heparin-activated antithrombin or antithrombin bound to a non-catalytic mutant of thrombin (S195A, in which the serine of the charge stabilizing system has been swapped for alanine). The neoepitope encompasses the motif DAFHX, located in native antithrombin on strand 4 of beta-sheet A, which becomes strand 5 of beta-sheet A in the RCL-cleaved and latent conformers. The inferences on the structure of the antithrombin-protease stable complex are that either a major remodeling of antithrombin accompanies the final elaboration of the complex or that, within the complex, at the most residues P-14 to P-9 of the RCL are inserted into beta-sheet A. These conclusions limit drastically the possible locations of the defeated protease within the complex.

Oligonucleotide-directed mutagenesis techniques are extensively used for studying gene regulation and DNA and protein structure/function relationships. A number of PCR-based mutagenesis methods have been developed recently (, , , , , , , ). In general, these PCR-based approaches are simpler and faster than classical mutagenesis methods employing in vivo DNA synthesis, and several of them are now widely practiced.