Hypoglycosylation is a common finding in antithrombin deficiency in the absence of a SERPINC1 gene defect.

UNLABELLED: Essentials We investigated the molecular base of antithrombin deficiency in cases without SERPINC1 defects. 27% of cases presented hypoglycosylation, transient in 62% and not restricted to antithrombin. Variations in genes involved in N-glycosylation underline this phenotype. These results support a new form of thrombophilia. Click here to listen to Dr Huntington's perspective on thrombin inhibition by the serpins SUMMARY: Background Since the discovery of antithrombin deficiency, 50 years ago, few new thrombophilic defects have been identified, all with weaker risk of thrombosis than antithrombin deficiency. Objective To identify new thrombophilic mechanisms. Patients/methods We studied 30 patients with antithrombin deficiency but no defects in the gene encoding this key anticoagulant (SERPINC1). Results A high proportion of these patients (8/30: 27%) had increased hypoglycosylated forms of antithrombin. All N-glycoproteins tested in these patients (α1-antitrypsin, FXI and transferrin) had electrophoretic, HPLC and Q-TOF patterns indistinguishable from those of the congenital disorders of glycosylation (rare recessive multisystem disorders). However, all except one had no mental disability. Moreover, intermittent antithrombin deficiency and hypoglycosylation was recorded in five out of these eight patients, all associated with moderate alcohol intake. Genetic analysis, including whole exome sequencing, revealed mutations in different genes involved in the N-glycosylation pathway. Conclusions Our study provides substantial and novel mechanistic insights into two disease processes, with potential implications for diagnosis and clinical care. An aberrant N-glycosylation causing a recessive or transient antithrombin deficiency is a new form of thrombophilia. Our data suggest that congenital disorders of glycosylation are probably underestimated, especially in cases with thrombosis as the main or only clinical manifestation.

A new method to quantify ß-antithrombin glycoform in plasma reveals increased levels during the acute stroke event.

INTRODUCTION: ß-antithrombin, the minor antithrombin glycoform in plasma, is probably the major thrombin inhibitor in vivo because of its high heparin affinity. The levels and variability of this glycoform in general population and its relevance in thromboembolic diseases is unknown since there is no specific method to measure this glycoform in clinical samples. METHODS: Plasma and recombinant α- and ß-antithrombins were purified by heparin affinity chromatography. An anti-FXa chromogenic method in presence of pentassacharide was used with two NaCl concentrations (15mM and 1.1M). This method was applied to plasma samples from 97 healthy subjects and 117 consecutive patients with ischemic cerebrovascular disease during the acute event and one year later. SERPINC1 sequencing was done in cases with antithrombin deficiency. RESULTS: High salt concentrations specifically restricted the pentassacharide-induced activation of antithrombin to the ß glycoform. ß-antithrombin displayed a normal distribution in the general population (89.5%-103.5%), with no significant variations according to age or sex. In patients, whole antithrombin values remained within the normal range. Only five cases had antithrombin deficiency during the thrombotic event, one carrying the L99F mutation in SERPINC1. Interestingly, both ß-antithrombin and the ß/whole antithrombin ratio were significantly higher in patients during the acute event but normalized after one year. CONCLUSIONS: We have developed a rapid, simple, sensitive and specific method to quantify ß-antithrombin activity using 1µL of plasma. ß-antithrombin significantly increases in patients with ischemic cerebrovascular disease during the acute event, probably by its release from the vasculature.

An atypical IgM class platelet cold agglutinin induces GPVI-dependent aggregation of human platelets.

Platelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbß3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and αIIbß3-dependent decrease of platelet count in allogeneic donor citrated platelet-rich plasma (PRP), but not in PRP from Glanzmann's thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, (14)C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding.

Role of the C-sheet in the maturation of N-glycans on antithrombin: functional relevance of pleiotropic mutations.

BACKGROUND: The characterization of natural mutants identified in patients with antithrombin deficiency has helped to identify functional domains or regions of this key anticoagulant and the mechanisms involved in the deficiency, as well as to define the clinical prognosis. Recently, we described an abnormal glycosylation in a pleiotropic mutant (K241E) that explained the impaired heparin affinity and the mild risk of thrombosis in carriers. OBJECTIVES: To evaluate the effects of different natural pleiotropic mutations on the glycosylation of antithrombin and their functional effects. METHODS: Five pleiotropic mutations identified in patients with antithrombin deficiency and located at each one of the strands of the C-sheet were selected (K241E, M251I, M315K, F402L, and P429L). Recombinant mutants were generated and purified. Glycoform heterogeneity and conformational sensitivity were studied with electrophoresis, proteomic analysis, and glycomic analysis. Heparin affinity was evaluated from intrinsic fluorescence. Reactivity assays with factor Xa, thrombin and neutrophil elastase in the presence or absence of heparin were also performed. RESULTS AND CONCLUSIONS: Pleiotropic mutants, except for that with the M315K mutation, which affects a non-exposed residue, showed two glycoforms. Variant 1, with abnormal glycosylation, had reduced heparin affinity and severely affected reactivity with the target proteases. In contrast, variant 2, with similar electrophoretic mobility and heparin affinity to wild-type antithrombin, had impaired inhibitory activity that was partially compensated for by activation with heparin. Our results suggest the C-sheet of antithrombin as a new region that is relevant for proper maturation of the N-glycans. Therefore, pleiotropic mutations lead to glycosylation defects that are responsible for the reduced heparin affinity.

Type II antithrombin deficiency caused by a large in-frame insertion: structural, functional and pathological relevance.

BACKGROUND: The metastable native conformation of serpins is required for their protease inhibition mechanism, but also renders them vulnerable to missense mutations that promote protein misfolding with pathological consequences. OBJECTIVE: To characterize the first antithrombin deficiency caused by a large in-frame insertion. PATIENTS/METHODS: Functional, biochemical and molecular analysis of the proband and relatives was performed. Recombinant antithrombin was expressed in HEK-EBNA cells. Plasma and recombinant antithrombins were purified and sequenced by Edman degradation. The stability was evaluated by calorimetry. Reactive centre loop (RCL) exposure was determined by thrombin cleavage. Mutant antithrombin was crystallized as a dimer with latent plasma antithrombin. RESULTS: The patient, with a spontaneous pulmonary embolism, belongs to a family with significant thrombotic history. We identified a complex heterozygous in-frame insertion of 24 bp in SERPINC1, affecting strand 3 of ß-sheet A, a region highly conserved in serpins. Surprisingly, the insertion resulted in a type II antithrombin deficiency with heparin binding defect. The mutant antithrombin, with a molecular weight of 59 kDa, had a proteolytic cleavage at W49 but maintained the N-terminal disulphide bonds, and was conformationally sensitive. The variant was non-inhibitory. Analysis of the crystal structure of the hyperstable recombinant protein showed that the inserted sequence annealed into ß-sheet A as the fourth strand, and maintained a native RCL. CONCLUSIONS: This is the first case of a large in frame-insertion that allows correct folding, glycosylation, and secretion of a serpin, resulting in a conformationally sensitive non-inhibitory variant, which acquires a hyperstable conformation with a native RCL.

In silico discovery of a compound with nanomolar affinity to antithrombin causing partial activation and increased heparin affinity.

The medical and socioeconomic relevance of thromboembolic disorders promotes an ongoing effort to develop new anticoagulants. Heparin is widely used as activator of antithrombin but incurs side effects. We screened a large database in silico to find alternative molecules and predicted d-myo-inositol 3,4,5,6-tetrakisphosphate (TMI) to strongly interact with antithrombin. Isothermal titration calorimetry confirmed a TMI affinity of 45 nM, higher than the heparin affinity (273 nM). Functional studies, fluorescence analysis, and citrullination experiments revealed that TMI induced a partial activation of antithrombin that facilitated the interaction with heparin and low affinity heparins. TMI improved antithrombin inhibitory function of plasma from homozygous patients with antithrombin deficiency with a heparin binding defect and also in a model with endothelial cells. Our in silico screen identified a new, non-polysaccharide scaffold able to interact with the heparin binding domain of antithrombin. The functional consequences of this interaction were experimentally characterized and suggest potential anticoagulant therapeutic applications.

Identification of miRNAs as potential modulators of tissue factor expression in patients with systemic lupus erythematosus and antiphospholipid syndrome.

BACKGROUND: Tissue factor (TF) is the main initiator of the coagulation cascade and elements that may upregulate its expression might provoke thrombotic events. Systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are autoimmune diseases characterized by a high TF expression in monocytes. OBJECTIVES: To examine the role of microRNAs (miRNAs) in TF expression and to evaluate their levels in SLE and APS patients. METHODS: An in silico search was performed to find potential putative binding sites of miRNAs in TF mRNA. In vitro validation was performed transfecting cells expressing TF (THP-1 and MDA-MB-231) with oligonucleotide miRNA precursors and inhibitors. Additionally, reporter assays were performed to test for the binding of miR-20a to TF mRNA. Levels of miRNAs and TF were measured by quantitative (qRT-PCR) in patients with APS and SLE. RESULTS: Overexpression of miRNA precursors, but not inhibitors, of two of the members of cluster miR-17∼92, for example miR-19b and miR-20a, in cells expressing TF decreased TF mRNA, protein levels, and procoagulant activity between 30% and 60%. Reporter assays showed that miR-20a binds to TF mRNA. Finally, we measured levels of miR-19b and miR-20a in monocytes from patients with APS and SLE and observed significantly lower miRNAs levels in comparison with healthy subjects inversely correlated with the levels of TF. CONCLUSIONS: Down-regulation of miR-19b and miR-20a observed in patients with SLE and APS could contribute to increased TF expression and thus provoke the hypercoagulable state characteristic of these patients.

Rare homozygous status of P43 ß1-tubulin polymorphism causes alterations in platelet ultrastructure.

ß1-tubulin is the main constituent of the platelet marginal band and studies with deficient mice showed that it maintains discoid shape and it is required for normal platelet formation. TUBB1 Q43P polymorphism is associated with decreased ß1-tubulin expression, diminished platelet reactivity, and partial loss of discoid shape in heterozygous carriers. However, to date no studies have been carried out on homozygous PP individuals. Our study included 19 subjects genotyped for TUBB1 Q43P polymorphism (4 QQ, 4 QP, and 2 PP). The two PP individuals were recruited after genotyping of 2073 individuals. Biochemical, microscopy, and molecular studies were performed. Real-time PCR showed a ~40% decrease in TUBB1 mRNA in the two PP individuals compared to four QQ subjects. Western blot analysis confirmed this reduction. Electron microscopy revealed a majority of normal discoid platelets in PP individuals, although platelets with loose, re-orientated or invaginated protofilaments, and an over-developed open canalicular system were observed. Such abnormalities were not observed in QQ subjects. Morphometric analyses showed no differences between PP and QQ individuals. Immunofluorescence confirmed the presence of a normal marginal band in a majority of platelets from PP subjects. Interestingly, both PP subjects had a 40% lower platelet count than QP and QQ. TUBB1 Q43P polymorphism in homozygosity mildly affects platelet ultrastructure and our data further suggest that high levels of ß1-tubulin might not be critical to sustain platelet discoid shape.

Heparin affinity of factor VIIa: implications on the physiological inhibition by antithrombin and clearance of recombinant factor VIIa.

Factor VIIa (FVIIa), a trypsin-like serine protease, plays an essential role in haemostasis by initiating the coagulation in complex with its cofactor, tissue factor (TF). The TF pathway inhibitor is the main physiological inhibitor of FVIIa-TF complex, but FVIIa can also be inhibited by antithrombin, although little is known about this process. Functional analyses by second order kinetic determination and identification of FVIIa-antithrombin complex by electrophoresis, evaluating the effect of different cofactors: pentasaccharide, low molecular weight heparin (LMWH) and unfractionated heparin (UFH), confirmed that any activation of antithrombin significantly enhanced the inhibition of FVIIa. The analysis of the binding of FVIIa to heparin by surface plasmon resonance identified a high affinity interaction under physiologic conditions (K(D)=3.38 µM, with 0.15M of ionic strength) strongly dependent on Ca(2+) and ionic strength. This interaction was verified in cell models, indicating that FVIIa also binds to the surface of endothelial cells with similar requirements. Structural modeling suggests the presence of a potential exosite II in FVIIa. However, the binding of heparin did not display significant changes on both the intrinsic fluorescence and the associated functional consequences of FVIIa. These results indicate that FVIIa binds to exposed glycosaminglycans of the endothelium through an exosite II, structurally similar to that reported for thrombin and suggested for FIXa. This binding may favor its inhibition by antithrombin in the absence of TF, contributing to the physiological control of this protease. This process may also play an important role in the clearance of recombinant FVIIa administered to patients.

Effect of citrullination on the function and conformation of antithrombin.

Antithrombin is an anticoagulant serpin with conformational sensitivity. Mutations and environmental factors may induce its polymerization by a mechanism involving domain swapping, which still requires verification. We have evaluated the functional and conformational effects on antithrombin of citrullination, a post-translational modification catalyzed by peptidylarginine deiminase (PAD), which changes arginine to citrulline. Purified antithrombin (native and latent forms) and plasma were incubated with PAD in the presence and absence of heparin. Citrullines were identified by proteomic analysis. Anti-activated factor X activity determination, IEF, SDS/PAGE and native PAGE were performed. The cleavage pattern with the metalloprotease AspN was studied, and its target residues were identified by Edman sequencing. We confirmed that citrullination of antithrombin abolished its activity; this abolition of activity was accelerated by heparin, which facilitated the early citrullination of Arg393 (P1 residue). Proteomic analyses revealed nine additional citrullines that caused a significant decrease in its electrostatic potential (from 5.95 to 5.06). It was demonstrated that citrullination of antithrombin caused its polymerization. The observation that these polymers, like heat-generated polymers, are cleaved by AspN in helix I is compatible with their linkage by domain swapping from strand 5 to strand 4 of beta-sheet A.

Effects of acrolein, a natural occurring aldehyde, on the anticoagulant serpin antithrombin.

We studied the effect of acrolein, an alpha,beta-unsaturated aldehyde that causes adduct-modification of lysine, cysteine, and histidine residues, on antithrombin, a key anticoagulant serpin. Intrinsic fluorescence, functionality (anti-FXa and anti-IIa activity), heparin affinity and conformational features of plasma and purified antithrombin were evaluated. In vivo experiments were carried out in mice. Intrinsic fluorescence showed a two-step conformational change. Acrolein, even at low dose, impaired the anticoagulant function of purified antithrombin by affecting its heparin affinity. However, higher concentrations of acrolein and long incubations are required to cause mild functional effects on plasma antithrombin and mice.

Dexamethasone induces a heat-stress response that ameliorates the conformational consequences on antithrombin of L-asparaginase treatment.

BACKGROUND: L-asparaginase (L-ASP) treatment of patients with acute lymphoblastic leukemia causes a severe antithrombin deficiency by intracellular retention of this serpin within the endoplasmic reticulum (ER) of hepatic cells, and a subsequent risk of thrombosis. Interestingly, co-administration of dexamethasone with L-ASP seems to reduce the risk of thrombosis. OBJECTIVES: We have investigated the effect of two corticoids, dexamethasone and prednisone, on the conformational consequences of L-ASP treatment on antithrombin. PATIENTS/METHODS: Levels, activity, conformation and immunohistological features of antithrombin were studied in patients, cell and mice models. Because of the importance of the steroid receptor-heat stress response (HSR) axis, and the role of unfolded protein response (UPR) in conformational diseases, we also evaluated Hsp27, Hsp70, Hsp90, HSF-1 and ER chaperons (Grp78 and Grp94). RESULTS: In all models, L-ASP alone or in combination with prednisone caused the intracellular retention of antithrombin associated with a severe deficiency. In contrast, the combination of L-ASP with dexamethasone ameliorated both the deficiency and intracellular retention of the serpin, which is associated with increased expression of heat shock proteins and ER-chaperons. CONCLUSIONS: These results suggest a protective effect of dexamethasone on the conformational consequences of L-ASP on antithrombin as a result of exacerbated HSR and UPR that help to explain the reduced risk of thrombosis reported in patients that follow this scheme of treatment.

S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum: cloning, overexpression, purification, and biochemical characterization.

The S-adenosylhomocysteine hydrolase gene (sahase) was cloned from the Gram-positive soil bacterium Corynebacterium glutamicum (ATCC 13032) and sequenced. The sahase gene possesses an open reading frame, which consists of 1,434 nucleotides that encode 478 amino acids. The sahase gene from C. glutamicum was expressed in Escherichia coli Rosetta cells by inserting the 1,434-bp fragment downstream from the isopropyl-beta-D-thiogalactopyranoside-inducible promoter of the pET28a+ expression vector. The recombinant S-adenosylhomocysteine hydrolase from C. glutamicum (CgrSAHase) was purified efficiently by a two-step procedure, tangential ultrafiltration and affinity chromatography. The molecular weight of the CgrSAHase, estimated by gel filtration, was about 210 kDa, while sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded a relative molecular mass of 52 +/- 1 kDa. The Michaelis-Menten constants for the natural substrates of the enzyme, S-adenosylhomocysteine (SAH), adenosine, and homocysteine, were determined to be 12, 1.4, and 40 microM, respectively. The overexpression of CgrSAHase was achieved at high level (>40 mg protein/g wet cells). Because of its high capacity to synthesize SAH, this enzyme is of high biotechnological interest.

Cloning, overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Corynebacterium efficiens YS-314.

The gene encoding S-adenosylhomocysteine hydrolase activity (SAHase: EC 3.3.1.1) from Corynebacterium efficiens (YS-314) was cloned and expressed as a fusion protein in Escherichia coli Rosetta (DE3). The analyzed nucleotide sequence of the cloned gene proved to be identical to those reported on the NCBI database. The recombinant enzyme is a tetramer, showing a molecular weight of approximately 210 kDa, as estimated by gel filtration. The K(M) values of the enzyme for S-adenosylhomocysteine (SAH), adenosine (Ado), and homocysteine (Hcy), were determined to be 1.4, 10, and 45 microM. The overexpression of the recombinant enzyme produced a high level of protein (>40 mg of protein per gram of wet cells) and revealed certain thermostability when characterized at temperatures above 40 degrees C. It also showed a high capacity for the synthesis of SAH, thermal stability, and high kinetic similarity to human SAHase, indicating a high biotechnological and pharmacological potential.

A colorimetric assay for S-adenosylhomocysteine hydrolase.

A colorimetric method for S-adenosyl-L-homocysteine hydrolase (SAHase) which uses S-adenosyl-L-homocysteine (SAH) as substrate is described. This method involves the hydrolytic conversion of SAH into adenosine (ADO) and L-homocysteine (HCY). The formation of HCY is quantified using Ellman's reagent and spectrophotometrical measured at 412 nm. Under these assay conditions, the product was followed continuously in a facile and quantitative manner until substrate conversion was complete. This method is an easy, cheap and shorter alternative to more complex methods and it is applicable to routine clinical analysis and in the assay and development of new S-nucleosidylhomocysteines to be used as therapeutic compounds.

[The usual diet of a group of adolescents from Valencia]. / Dieta habitual de un grupo de adolescentes valencianos.

Adolescents are considered a high nutritional risk group because their nutritional needs are increased with respect to other age groups, and because this period of life coincides with changes in life style that affect, often negatively, their eating habits. Our overall goal is to study the usual eating pattern of Valencian adolescents together with their drink and tobacco consumption, but the first stage focuses on setting up, validating and correcting methods to be applied. The present study was therefore carried out in a sample composed of 64 adolescents, ranging from 16 to 20 years of age. A self administered survey developed in our Department was used to explore their food preferences, eating habits, smoking habits and alcohol and coffee intake. The following results were obtained: The number of daily meals was of 3.7 +/- 0.9. The 91% takes the breakfast daily (milk with cereals or sweet rolls) and the majority of the students eat a second mid-morning breakfast. Lunch is of the traditional type, consisting of two courses. The first is rice or pasta, followed by meat, fish or eggs usually accompanied by a side dish or salad. Fresh fruit is the dessert eaten, almost daily by the majority of the sample. The most usual drink is water. The 70% of the sample have one supplementary afternoon intake "merienda". All of the surveyed people like fruits, pasta and chicken meat. While, liver and legumes are disliked by the majority. The intakes of soft drinks, snacks, alcohol, coffee and tobacco are moderate, being, all these products mainly consumed during the weekend. The mean diet offers an excess of proteins and saturated fat, while complex carbohydrates and dietetic fiber are scarce. Nutritional intakes of iron, magnesium and zinc in girls, and magnesium, folates and vitamin A in boys are estimated insufficient to fulfil their needs.

[Weight and height of school children from the L'Horta region (Valencia)]. / Peso y talla de los escolares de las comarcas de L'Horta (Valencia).

The growth and development of Valencian children from 6 to 14 years of age was studied using the weight and height of a representative sample (n = 2011) of school children in L'Horta de Valencia area. They represent 62% and 32.5% of the school population of the Valencia province and of the Comunidad Valenciana, respectively. The sample was proportional to the whole population in the geographical area and to sex and age. The measured children came from 10 schools, some of which were public and some private, drawn at random. After obtaining the permission from the school administrative board the measurements were taken, always by the same trained anthropometrist and with the same instruments, which were calibrated daily. The application of the Lilliefors test showed that the distribution of weight and height according to age was normal in the majority of the groups of age. Therefore, the Student t-test was applied to compare girls and boys, and given the fact that the symmetry was not complete in all age ranges, the results are expressed as percentiles. From 6 to 14 years girls and boys in Valencia show a similar weight evolution. Only in children in the highest percentiles, can be observed sex-related differences from the age of 12 on. In general, up to 13-14 years of age boys were slightly taller than girls of the same age. From this age on boys follow continue growing while girls stop their longitudinal growth.