Nataliya Bohdan

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.

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.

Antithrombin Dublin (p.Val30Glu): a relatively common variant with moderate thrombosis risk of causing transient antithrombin deficiency

The key haemostatic role of antithrombin and the risk of thrombosis associated with its deficiency support that the low incidence of antithrombin deficiency among patients with thrombosis might be explained by underestimation of this disorder. It was our aim to identify mutations in SERPINC1 causing transient antithrombin deficiency. SERPINC1 was sequenced in 214 cases with a positive test for antithrombin deficiency, including 67 with no deficiency in the sample delivered to our laboratory. The p.Val30Glu mutation (Antithrombin Dublin) was identified in five out of these 67 cases, as well as in three out of 127 cases with other SERPINC1 mutations. Genotyping in 1593 patients with venous thrombosis and 2592 controls from two populations, revealed a low prevalent polymorphism (0.3 %) that moderately increased the risk of venous thrombosis (OR: 2.9; 95 % CI: 1.07-8.09; p= 0.03) and identified one homozygous patient with an early thrombotic event. Carriers had normal anti-FXa activity, and plasma antithrombin was not sensitive to heat stress or proteolytic cleavage. Analysis of one sample with transient deficit revealed a type I deficiency, without aberrant or increased latent forms. The recombinant variant, which lacked the two amino-terminal residues, had reduced secretion from HEK-EBNA cells, formed hyperstable disulphide-linked polymers, and had negligible activity. In conclusion, p.Val30Glu by affecting the cleavage of antithrombins signal peptide, results in a mature protein lacking the N-terminal dipeptide with no functional consequences in normal conditions, but that increases the sensitivity to be folded intracellularly into polymers, facilitating transient antithrombin deficiency and the subsequent risk of thrombosis.

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

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.

Antithrombin controls tumor migration, invasion and angiogenesis by inhibition of enteropeptidase.

Antithrombin is a key inhibitor of the coagulation cascade, but it may also function as an anti-inflammatory, anti-angiogenic, anti-viral and anti-apoptotic protein. Here, we report a novel function of antithrombin as a modulator of tumor cell migration and invasion. Antithrombin inhibited enteropeptidase on the membrane surface of HT-29, A549 and U-87 MG cells. The inhibitory process required the activation of antithrombin by heparin, and the reactive center loop and the heparin binding domain were essential. Surprisingly, antithrombin non-covalently inhibited enteropeptidase, revealing a novel mechanism of inhibition for this serpin. Moreover, as a consequence of this inhibition, antithrombin was cleaved, resulting in a molecule with anti-angiogenic properties that reduced vessel-like formation of endothelial cells. The addition of antithrombin and heparin to U-87 MG and A549 cells reduced motility in wound healing assays, inhibited the invasion in transwell assays and the degradation of a gelatin matrix mediated by invadopodia. These processes were controlled by enteropeptidase, as demonstrated by RNA interference experiments. Carcinoma cell xenografts in nude mice showed in vivo co-localization of enteropeptidase and antithrombin. Finally, treatment with heparin reduced experimental metastasis induced by HT29 cells in vivo. In conclusion, the inhibition of enteropeptidase by antithrombin may have a double anti-tumor effect through inhibiting a protease involved in metastasis and generating an anti-angiogenic molecule.

Identification of a new potential mechanism responsible of severe bleeding in myeloma: immunoglobulins bind the heparin binding domain of antithrombin activating this endogenous anticoagulant.

Bleeding is a relatively common complication in patients with multiple myeloma. Different factors have been involved, such as heparin-like anticoagulants, although the underlying mechanism remains obscure. The identification of a patient with a quiescent multiple myeloma IgG-γ that suffered a

Biochemical and cellular consequences of the antithrombin p.Met1? mutation identified in a severe thrombophilic family

Nature is always the best inspiration for basic research. A family with severe thrombosis and antithrombin deficiency, the strongest anticoagulant, carried a new mutation affecting the translation-start codon of SERPINC1, the gene encoding antithrombin. Expression of this variant in a eukaryotic cell system produced three different antithrombins. Two downstream methionines were used as alternative initiation codons, generating highly expressed small aglycosylated antithrombins with cytoplasmic localization. Wild-type antithrombin was generated by the use of the mutated AUU as initiation codon. Actually, any codon except for the three stop codons might be used to initiate translation in this strong Kozak context. We show unexpected consequences of natural mutations affecting translation-start codons. Downstream alternative initiation AUG codons may be used when the start codon is mutated, generating smaller molecules with potential different cell localization, biochemical features and unexplored consequences. Additionally, our data further support the use of other codons apart from AUG for initiation of translation in eukaryotes.

Heparanase Activates Antithrombin through the Binding to Its Heparin Binding Site.

Heparanase is an endoglycosidase that participates in morphogenesis, tissue repair, heparan sulphates turnover and immune response processes. It is over-expressed in tumor cells favoring the metastasis as it penetrates the endothelial layer that lines blood vessels and facilitates the metastasis by degradation of heparan sulphate proteoglycans of the extracellular matrix. Heparanase may also affect the hemostatic system in a non-enzymatic manner, up-regulating the expression of tissue factor, which is the initiator of blood coagulation, and dissociating tissue factor pathway inhibitor on the cell surface membrane of endothelial and tumor cells, thus resulting in a procoagulant state. Trying to check the effect of heparanase on heparin, a highly sulphated glycosaminoglycan, when it activates antithrombin, our results demonstrated that heparanase, but not proheparanase, interacted directly with antithrombin in a non-covalent manner. This interaction resulted in the activation of antithrombin, which is the most important endogenous anticoagulant. This activation mainly accelerated FXa inhibition, supporting an allosteric activation effect. Heparanase bound to the heparin binding site of antithrombin as the activation of Pro41Leu, Arg47Cys, Lys114Ala and Lys125Alaantithrombin mutants was impaired when it was compared to wild type antithrombin. Intrinsic fluorescence analysis showed that heparanase induced an activating conformational change in antithrombin similar to that induced by heparin and with a KD of 18.81 pM. In conclusion, under physiological pH and low levels of tissue factor, heparanase may exert a non-enzymatic function interacting and activating the inhibitory function of antithrombin.

Role on antithrombin deficiency and thrombotic risk of the beta glycoform: a post-translational mosaicism for heparin binding mutations

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.

Amelioration of the severity of heparin-binding antithrombin mutations by posttranslational mosaicism: a post-translational mosaicism for heparin binding mutations

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.