María Eugenia de la Morena-Barrio

Amelioration of the severity of heparin-binding antithrombin mutations by posttranslational mosaicism

The balance between actions of procoagulant and anticoagulant factors protects organisms from bleeding and thrombosis. Thus, antithrombin deficiency increases the risk of thrombosis, and complete quantitative deficiency results in intrauterine lethality. However, patients homozygous for L99F or R47C antithrombin mutations are viable. These mutations do not modify the folding or secretion of the protein, but abolish the glycosaminoglycan-induced activation of antithrombin by affecting the heparin-binding domain. We speculated that the natural β-glycoform of antithrombin might compensate for the effect of heparin-binding mutations. We purified α- and β-antithrombin glycoforms from plasma of 2 homozygous L99F patients. Heparin affinity chromatography and intrinsic fluorescence kinetic analyses demonstrated that the reduced heparin affinity of the α-L99F glycoform (K(D), 107.9 ± 3nM) was restored in the β-L99F glycoform (K(D), 53.9 ± 5nM) to values close to the activity of α-wild type (K(D), 43.9 ± 0.4nM). Accordingly, the β-L99F glycoform was fully activated by heparin. Similar results were observed for recombinant R47C and P41L, other heparin-binding antithrombin mutants. In conclusion, we identified a new type of mosaicism associated with mutations causing heparin-binding defects in antithrombin. The presence of a fully functional β-glycoform together with the activity retained by these variants helps to explain the viability of homozygous and the milder thrombotic risk of heterozygous patients with these specific antithrombin mutations.

GPI-anchor and GPI-anchored protein expression in PMM2-CDG patients

BackgroundMutations in PMM2 impair phosphomannomutase-2 activity and cause the most frequent congenital disorder of glycosylation, PMM2-CDG. Mannose-1-phosphate, that is deficient in this disorder, is also implicated in the biosynthesis of glycosylphosphatidyl inositol (GPI) anchors.ObjectiveTo evaluate whether GPI-anchor and GPI-anchored proteins are defective in PMM2-CDG patients.MethodsThe expression of GPI-anchor and seven GPI-anchored proteins was evaluated by flow cytometry in different cell types from twelve PMM2-CDG patients. Additionally, neutrophil CD16 and plasma hepatic proteins were studied by Western blot. Transferrin glycoforms were evaluated by HPLC.ResultsPatients and controls had similar surface expression of GPI-anchor and most GPI-anchored proteins. Nevertheless, patients displayed a significantly diminished binding of two anti-CD16 antibodies (3G8 and KD1) to neutrophils and also of anti-CD14 (61D3) to monocytes. Interestingly, CD16 immunostaining and asialotransferrin levels significantly correlated with patients’ age. Analysis by flow cytometry of CD14 with MΦP9, and CD16 expression in neutrophils by Western blot using H-80 ruled out deficiencies of these antigens.ConclusionsPMM2 mutations do not impair GPI-anchor or GPI-anchored protein expression. However, the glycosylation anomalies caused by PMM2 mutations might affect the immunoreactivity of monoclonal antibodies and lead to incorrect conclusions about the expression of different proteins, including GPI-anchored proteins. Neutrophils and monocytes are sensitive to PMM2 mutations, leading to abnormal glycosylation in immune receptors, which might potentially affect their affinity to their ligands, and contribute to infection. This study also confirms less severe hypoglycosylation defects in older PMM2-CDG patients.

The Infective Polymerization of Conformationally Unstable Antithrombin Mutants May Play a Role in the Clinical Severity of Antithrombin Deficiency

Mutations affecting mobile domains of antithrombin induce conformational instability resulting in protein polymerization that associates with a severe clinical phenotype, probably by an unknown gain of function. By homology with other conformational diseases, we speculated that these variants might infect wild-type (WT) monomers reducing the anticoagulant capacity. Infective polymerization of WT polymers and different P1 mutants (p.R425del, p.R425C and p.R425H) were evaluated by using native gels and radiolabeled WT monomers and functional assays. Human embryonic kidney cells expressing the Epstein-Barr nuclear antigen 1 (HEK-EBNA) cells expressing inducible (p.R425del) or two novel constitutive (p.F271S and p.M370T) conformational variants were used to evaluate intracellular and secreted antithrombin under mild stress (pH 6.5 and 39°C for 5 h). We demonstrated the conformational sensitivity of antithrombin London (p.R425del) to form polymers under mild heating. Under these conditions purified antithrombin London recruited WT monomers into growing polymers, reducing the anticoagulant activity. This process was also observed in the plasma of patients with p.R425del, p.R425C and p.R425H mutations. Under moderate stress, coexpression of WT and conformational variants in HEK-EBNA cells increased the intracellular retention of antithrombin and the formation of disulfide-linked polymers, which correlated with impaired secretion and reduction of anticoagulant activity in the medium. Therefore, mutations inducing conformational instability in antithrombin allow its polymerization with the subsequent loss of function, which under stress could sequestrate WT monomers, resulting in a new prothrombotic gain of function, particularly relevant for intracellular antithrombin. The in vitro results suggest a temporal and severe plasma antithrombin deficiency that may contribute to the development of the thrombotic event and to the clinical severity of these mutations.

Control of post-translational modifications in antithrombin during murine post-natal development by miR-200a

BackgroundDevelopmental haemostatic studies may help identifying new elements involved in the control of key haemostatic proteins like antithrombin, the most relevant endogenous anticoagulant.ResultsIn this study, we showed a significant reduction of sialic acid content in neonatal antithrombin compared with adult antithrombin in mice. mRNA levels of St3gal3 and St3gal4, two sialyltransferases potentially involved in antithrombin sialylation, were 85% lower in neonates in comparison with adults. In silico analysis of miRNAs overexpressed in neonates revealed that mir-200a might target these sialyltransferases. Moreover, in vitro studies in murine primary hepatocytes sustain this potential control.ConclusionsThese data suggest that in addition to the direct protein regulation, microRNAs may also modulate qualitative traits of selected proteins by an indirect control of post-translational processes.

Protective role of antithrombin in mouse models of liver injury

BACKGROUND & AIMS Coagulopathy caused by an imbalance of hemostatic factors is associated with the pathophysiology of liver disease. We have investigated the role of antithrombin (AT), a key anticoagulant serpin, in the onset of liver disease. METHODS Liver injury was induced by CCl(4) injection and bile duct ligation (BDL) in wild type (WT) and AT-deficient (AT(+/-)) mice. Twenty-four hours after CCl(4) treatment, aspartate-transaminase, alanine-transaminase, liver lesion size, leukocyte infiltration, and apoptosis were reduced in WT animals compared to AT(+/-) mice. RESULTS Administration of exogenous AT in AT(+/-) animals did not restore the values observed in WT mice, suggesting that intrahepatic AT might also offer protection against CCl(4). In the BDL model, increased liver injury was also evident in AT(+/-) compared to WT mice. An 85 kDa covalent complex involving AT was identified in immunoblottings of liver lysates from CCl(4)-treated animals. This complex was also present in anoikis hepatocytes and H(2)O(2)-treated HepG2 cells, suggesting a role for AT in apoptosis. Expression of recombinant WT-AT by HEK-EBNA cells increased cell survival while expression of AT mutants, ΔR393 and R47C, did not modify viability. Finally, plasma anti-FXa activity was attenuated by liver injury, with AT(+/-) animals showing a greater reduction than WT mice. CONCLUSIONS Our study reveals a protective role of AT against liver injury due to its recognized anticoagulant and anti-inflammatory action. AT may also act via a previously unrecognized antiapoptotic effect. The clinical implications of AT deficiency in patients with liver disease should be further addressed.

Identification of antithrombin-modulating genes. Role of LARGE, a gene encoding a bifunctional glycosyltransferase, in the secretion of proteins?

The haemostatic relevance of antithrombin together with the low genetic variability of SERPINC1, and the high heritability of plasma levels encourage the search for modulating genes. We used a hypothesis-free approach to identify these genes, evaluating associations between plasma antithrombin and 307,984 polymorphisms in the GAIT study (352 individuals from 21 Spanish families). Despite no SNP reaching the genome wide significance threshold, we verified milder positive associations in 307 blood donors from a different cohort. This validation study suggested LARGE, a gene encoding a protein with xylosyltransferase and glucuronyltransferase activities that forms heparin-like linear polysaccharides, as a potential modulator of antithrombin based on the significant association of one SNPs, rs762057, with anti-FXa activity, particularly after adjustment for age, sex and SERPINC1 rs2227589 genotype, all factors influencing antithrombin levels (p = 0.02). Additional results sustained this association. LARGE silencing inHepG2 and HEK-EBNA cells did not affect SERPINC1 mRNA levels but significantly reduced the secretion of antithrombin with moderate intracellular retention. Milder effects were observed on α1-antitrypsin, prothrombin and transferrin. Our study suggests LARGE as the first known modifier of plasma antithrombin, and proposes a new role for LARGE in modulating extracellular secretion of certain glycoproteins.

First case with antithrombin deficiency, mesenteric vein thrombosis and pregnancy: Multidisciplinary diagnosis and successful management

Mesenteric venous thrombosis (MVT) is a rare localization of thrombosis that is difficult to diagnose due to its non-specific clinical manifestations [1]. The main deleterious consequence of MVT is intestinal ischemia, which has a mortality of 20–50% [1]. Different prothrombotic states caused by heritable or acquired factors associated with MVT [1]. Antithrombin deficiency is the congenital risk factormost frequently associated with MVT [2]. However, as the levels of this key endogenous anticoagulant may be low in patients with acute thrombosis due to its fast consumption, deficiency of antithrombin in MVT may be difficult to be diagnosed by using functional methods. Accordingly, a correct identification of antithrombin deficiency in MVT should be performed by careful follow-up studies with serial antithrombin estimation, familial studies, or genetic tests. Pregnancy is an acquired hypercoagulable state that is worsened with underlying thrombophilia [3]. Thrombosis during pregnancy mainly locates at lower limbs, and only 12 cases with MVT have been described in pregnant women. We here present an exceptional case of a 7 week pregnant woman, with MVT and a new mutation in SERPINC1 responsible for a type I antithrombin deficiency.

Identification of Regulatory Mutations in SERPINC1 Affecting Vitamin D Response Elements Associated with Antithrombin Deficiency.

Antithrombin is a crucial anticoagulant serpin whose even moderate deficiency significantly increases the risk of thrombosis. Most cases with antithrombin deficiency carried genetic defects affecting exons or flanking regions of SERPINC1.We aimed to identify regulatory mutations inSERPINC1 through sequencing the promoter, intron 1 and 2 of this gene in 23 patients with antithrombin deficiency but without known genetic defects. Three cases with moderate antithrombin deficiency (63–78%) carried potential regulatory mutations. One located 200 bp before the initiation ATG and two in intron 1. These mutations disrupted two out of five potential vitamin D receptor elements (VDRE) identified in SERPINC1 with different software. One genetic defect, c.42-1060_-1057dupTTGA, was a new low prevalent polymorphism (MAF: 0.01) with functional consequences on plasma antithrombin levels. The relevance of the vitamin D pathway on the regulation of SERPINC1 was confirmed in a cell model. Incubation of HepG2 with paricalcitol, a vitamin D analog, increased dose-dependently the levels of SERPINC1transcripts and antithrombin released to the conditioned medium. This study shows further evidence of the transcriptional regulation of SERPINC1 by vitamin D and first describes the functional and pathological relevance of mutations affecting VDRE of this gene. Our study opens new perspectives in the search of new genetic defects involved in antithrombin deficiency and the risk of thrombosis as well as in the design of new antithrombotic treatments.

Natural Killer Cell Receptors and Cytotoxic Activity in Phosphomannomutase 2 Deficiency (PMM2-CDG)

Background PMM2-CDG is the most common N-glycosylation defect and shows an increased risk of recurrent and/or severe, sometimes fatal, infections in early life. We hypothesized that natural killer (NK) cells, as important mediators of the immune response against microbial pathogens and regulators of adaptive immunity, might be affected in this genetic disorder. Objective To evaluate possible defects on PMM2-CDG NK peripheral blood cell number, killing activity and expression of membrane receptors. Methods We studied fresh and activated NK cells from twelve PMM2-CDG cells. The number and expression of lymphoid surface receptors were studied by flow cytometry. The NK responsiveness (frequency of degranulated NK cells) and killing activity against K562 target cells was determined in the NK cytotoxicity assay. Results We found an increase of blood NK cells in three patients with a severe phenotype. Two of them, who had suffered from moderate/severe viral infections during their first year of life, also had reduced T lymphocyte numbers. Patient activated NK cells showed increased expression of CD54 adhesion molecule and NKG2D and NKp46 activating receptors. NKp46 and 2B4 expression was inversely correlated with the expression of NKG2D in activated PMM2-CDG cells. Maximal NK activity against K562 target cells was similar in control and PMM2-CDG cells. Interestingly, the NK cell responsiveness was higher in patient cells. NKG2D and specially CD54 increased surface expression significantly correlated with the increased NK cell cytolytic activity according to the modulation of the killer activity by expression of triggering receptors and adhesion molecules. Conclusions Our results indicate that hypoglycosylation in PMM2-CDG altered NK cell reactivity against target cells and the expression of CD54 and NKG2D, NKp46 and 2B4 activating receptors during NK cell activation. This suggests a defective control of NK cell killing activity and the overall anti-viral immune response in PMM2-CDG patients. The present work improves our understanding of the immunological functions in PMM2-CDG and possibly in other CDG-I types.

Assessment of two contact activation reagents for the diagnosis of congenital factor XI deficiency

INTRODUCTION Congenital FXI deficiency, a coagulopathy associated with low bleeding risk but thrombotic protection, is usually diagnosed by prolonged APTT and confirmed by coagulation assays. Recent evidences suggest that FXI deficiency might be underestimated. Sensitive and reliable methods to detect FXI deficiency are required. AIM To examine the sensitivity of two methods and two contact activators on FXI deficiency screening. METHODS 140 cases with FXI deficiency, 9 severe and 131 moderate, caused by 11different mutations were recruited. APTT and FXI:C were assessed in ACL-TOP 500coagulometer with silica-based (SynthASil) and ellagic acid-based (SynthAFax) reagents. F12 rs1801020 SNP was genotyped with Taqman probes. RESULTS Severe FXI deficiency significantly prolonged APTT with both reagents. However, a high proportion of moderate deficiencies would not be detected using APTT, with false negatives of 22% for SynthASil and 12% for SynthAFax. False negatives results mainly corresponded to cases with qualitative deficiency (CRM+: p.Pro538Leu), which also had higher FXI coagulant activity. Using SynthASil, the common F12 rs1801020 variant, associated to low FXII levels, significantly prolonged APTT in moderate FXI deficiency subjects. FXI:C values were significantly higher with SynthAFax than with SynthASil (47.7±12.7 vs. 40.4±14.9), so SynthAFax rendered higher rate of false negatives than SynthASil (7% vs.2%). CONCLUSIONS Moderate FXI deficiency, particularly CRM+, might be underestimated using current diagnostic methods. The activator, FXI and FXII levels may contribute to a higher rate of false negatives using APTT. Our results suggests that the best screening method for FXI deficiency is FXI:C using silica.