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Serine protease inhibitors (serpins) include thousands of structurally conserved proteins playing key roles in many organisms. Mutations affecting serpins may disturb their conformation, leading to inactive forms. Unfortunately, conformational consequences of serpin mutations are difficult to predict. In this study, we integrate experimental data of patients with mutations affecting one serpin with the predictions obtained by AlphaFold and molecular dynamics. Five SERPINC1 mutations causing antithrombin deficiency, the strongest congenital thrombophilia were selected from a cohort of 350 unrelated patients based on functional, biochemical, and crystallographic evidence supporting a folding defect. AlphaFold gave an accurate prediction for the wild-type structure. However, it also produced native structures for all variants, regardless of complexity or conformational consequences in vivo. Similarly, molecular dynamics of up to 1000 ns at temperatures causing conformational transitions did not show significant changes in the native structure of wild-type and variants. In conclusion, AlphaFold and molecular dynamics force predictions into the native conformation at conditions with experimental evidence supporting a conformational change to other structures. It is necessary to improve predictive strategies for serpins that consider the conformational sensitivity of these molecules., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Garrido-Rodríguez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The interaction between coagulation factors Xa and IXa and the activated state of their inhibitor, antithrombin (AT),have been investigated using X-ray diffraction studies. However, only mutagenesis data are available for non-activated AT. Our aim was to propose a model based on docking and advanced-sampling molecular dynamics simulations that can reveal the conformational behavior of the systems when AT is not binding a pentasaccharide. We built the initial structure for non-activated AT-FXa and AT-FIXa complexes using HADDOCK 2.4. The conformational behavior was studied using Gaussian accelerated molecular dynamics simulations. In addition to the docked complexes, two systems based on the X-ray structures were also simulated, with and without the ligand. The simulations revealed large variability in conformation for both factors. In the docking-based complex of AT-FIXa, conformations with stable Arg150-AT interactions can exist for longer time periods but the system also has a higher tendency for reaching states with very limited interaction with the "exosite" of AT. By comparing simulations with or without the pentasaccharide, we were able to gain insights into the effects of conformational activation on the Michaelis complexes. RMSF analysis and correlation calculations for the alpha-carbon atoms revealed important details of the allosteric mechanisms. Our simulations provide atomistic models for better understanding the conformational activation mechanism of AT against its target factors.

Glycosaminoglycans (GAGs) have high negative charge and are biologically and pharmaceutically important because their high charge promotes a strong interaction with many proteins. Due to the inherent heterogeneity of GAGs, multiple oligosaccharides, containing certain common domains, often can interact with clusters of basic amino acid residues on a target protein. The specificity of many GAG-protein interactions remains undiscovered since there is insufficient structural information on the interacting GAGs. Herein, we establish a cluster sequencing strategy to simultaneously deduce all major sequences of the affinity GAG oligosaccharides, leading to a definition of the consensus sequence they share that corresponds to the specific binding domain for the target protein. As a proof of concept, antithrombin III-binding oligosaccharides were examined, resulting in a heptasaccharide domain containing the well-established anticoagulant pentasaccharide sequence. Repeating this approach, a new pentasaccharide domain was discovered corresponding to the heparin motif responsible for binding interferon-γ (IFNγ). Our strategy is fundamentally important for the discovery of saccharide sequences needed in the development of novel GAG-based therapeutics.

Antithrombin (AT) is a serine protease inhibitor, its activity is highly accelerated by heparin. Mutations at the heparin-binding region lead to functional defect, type II heparin-binding site (IIHBS) AT deficiency. The aim of this study was to investigate and compare the molecular background of AT Budapest 3 (p.Leu131Phe, ATBp3), AT Basel (p.Pro73Leu), and AT Padua (p.Arg79His) mutations. Advanced in silico methods and heparin-binding studies of recombinant AT proteins using surface plasmon resonance method were used. Crossed immunoelectrophoresis and Differential Scanning Fluorimetry (NanoDSF) were performed in plasma samples. Heparin affinity of AT Padua was the lowest (KD = 1.08 × 10 -6 M) and had the most severe consequences affecting the allosteric pathways of activation, moreover significant destabilizing effects on AT were also observed. KD values for AT Basel, ATBp3 and wild-type AT were 7.64 × 10 -7 M, 2.15 × 10 -8 M and 6.4 × 10 -10 M, respectively. Heparin-binding of AT Basel was slower, however once the complex was formed the mutation had only minor effect on the secondary and tertiary structures. Allosteric activation of ATBp3 was altered, moreover decreased thermostability in ATBp3 homozygous plasma and increased fluctuations in multiple regions of ATBp3 were observed by in silico methods suggesting the presence of a quantitative component in the pathogenicity of this mutation due to molecular instability.

Heparan sulfate (HS) is a heterogeneous, extracellular glycan that interacts with proteins and other molecules affecting many biological processes. The specific binding motifs of HS interactions are of interest, but have not been extensively characterized. Glycan microarrays are valuable tools that can be used to probe the interactions between glycans and their ligands while relying on relatively small amounts of samples. Recently, chemoenzymatic synthesis of HS has been employed to produce specific HS structures that can otherwise be difficult to produce. In this study, a microarray of diverse chemoenzymatically synthesized HS structures was developed and HS interactions were characterized. Fluorescently labeled antithrombin III (AT) and fibroblast growth factor-2 (FGF2) were screened against 95 different HS structures under three different printing concentrations to confirm the utility of this microarray. Specific sulfation patterns were found to be important for binding to these proteins and results are consistent with previous specificity studies. Furthermore, the binding affinities (KD,surf) of AT and FGF2 to multiple HS structures were determined using a microarray technique and is consistent with previous reports. Lastly, the 95-compound HS microarray was used to determine the distinct binding profiles for interleukin 12 and platelet factor 4. This technique is ideal for rapid expansion and will be pivotal to the high-throughput characterization of biologically important structure/function relationships., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background:  Hereditary antithrombin deficiency is a rare autosomal-dominant disorder predisposing to recurrent venous thromboembolism (VTE). To date, only two founder mutations have been described., Objectives:  We investigated the antithrombin p.Thr147Ala variant, found in 12 patients of African origin. This variant is known as rs2227606 with minor allele frequency of 0.5% in Africans and absent in Europeans. A possible founder effect was investigated., Methods:  Phenotypical characterization was established through immunological and functional methods, both under basal and stress conditions. Recombinant antithrombin molecules were constructed by site-directed mutagenesis and expressed in HEK-293T cells. Secreted antithrombin was purified and functionally characterized. Structural modeling was performed to predict the impact of the mutation on protein structure. A novel nanopore sequencing approach was used for haplotype investigation., Results:  Ten patients experienced VTE, stroke, or obstetric complications. Antithrombin antigen levels and anti-IIa activity were normal or slightly reduced while anti-Xa activity was reduced with only one commercial assay. On crossed immunoelectrophoresis, an increase of antithrombin fractions with reduced heparin affinity was observed under high ionic strength conditions but not under physiological conditions. The recombinant p.Thr147Ala protein displayed a reduced anti-Xa activity. Structural modeling revealed that residue Thr147 forms three hydrogen bonds that are abolished when mutated to alanine. The investigated patients shared a common haplotype involving 13 SERPINC1 intragenic single nucleotide polymorphisms., Conclusion:  Antithrombin p.Thr147Ala, responsible for antithrombin type II heparin binding site deficiency, is the first founder mutation reported in people of African ancestry. This study further emphasizes the limitations of commercial methods to diagnose this specific subtype., Competing Interests: None declared., (Thieme. All rights reserved.)

Humans express seven heparan sulfate (HS) 3- O -sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3- O -sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3- O -sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS., Competing Interests: The authors declare no competing interest.

N-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its deficiency significantly increases thrombotic risk. In this study, we used the introduction of N-glycosylation sites as a tool to explore what effect this glycosylation has on the protein folding, secretion, and function of this key anticoagulant. To accomplish this task, we introduced an additional N-glycosylation sequence in each strand. Interestingly, all regions that likely fold rapidly or were surrounded by lysines were not glycosylated even though an N-glycosylation sequon was present. The new sequon in the strands of the A- and B-sheets reduced secretion, and the B-sheet was more sensitive to these changes. However, the mutations in the strands of the C-sheet allowed correct folding and secretion, which resulted in functional variants. Therefore, our study revealed crucial regions for antithrombin secretion and could potentially apply to all serpins. These results could also help us understand the functional effects of natural variants causing type-I deficiencies.

Background: Antithrombin (AT) is the primary physiological anticoagulant of normal hemostasis. Hereditary AT deficiency, an autosomal dominant thrombotic disease caused by mutations in the AT gene (SERPINC1), is associated with venous thromboembolism., Objective: We investigated the phenotypes, genotypes, and pathogenesis of hereditary AT deficiency in a 12-year-old boy (proband) who developed a pulmonary embolism and a subsequent deep vein thrombosis., Methods: The AT activity and AT antigen level of the proband and his family members were measured. Mutation sites in all seven exons of SERPINC1 were identified. Analysis of conserved regions around codon 462 of the SERPINC1 gene and functional predictions were performed using bioinformatics tools., Results: The proband, his father, and his paternal grandmother demonstrated reduced AT activity and antigen levels consistent with Type I AT deficiency. A novel heterozygous missense mutation, c.1385G>A (Cys462Tyr) was identified in all three symptomatic family members. This missense mutation causes disruption of the 279Cys-462Cys disulfide bond and leads to type Ⅰ hereditary AT deficiency., Conclusion: A SERPINC1 missense mutation (Cys462Tyr) causing damage to the 279Cys-462Cys disulfide bond of the AT protein appears to be the cause of Type I AT deficiency in this family. These findings indicate one pathological mechanism associated with hereditary AT deficiency., (Copyright © 2020. Published by Elsevier Inc.)

Acylated chitosan sulfate (ChS1), a sulfated polysaccharide with high anticoagulant activity, was chemically synthesized and structurally characterized using FT-IR analysis. The beneficial structural properties and high availability of the sulfate group in ChS1 led to greater anticoagulant activity through both the intrinsic and common pathways with antithrombin III (AT III)-mediated inhibition, particularly involving coagulation factors FXa and FIIa. The analysis of the binding affinities using surface plasma resonance found that the equilibrium dissociation constant (K D ) of ChS1 for FXa and FIIa in the presence of AT III was 67.4 nM and 112.6 nM, respectively, indicating the stronger interaction of the AT III/ChS1 complex with the ligands and the inhibition of activated FX and FII. The results of amidolytic assays further demonstrated the stronger inhibition of the proteolytic conversion of factor X by the intrinsic FXase complex and of FII by the prothrombinase complex. Molecular docking analysis further validated the protein-ligand interactions of ChS1 with AT III and their binding affinity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Heparins are the most pharmaceutically important polysaccharides. These heparin-based anticoagulant/antithrombotic agents include unfractionated heparins, low molecular weight heparins (LMWHs) and ultralow molecular weight heparins (ULMWHs). Heparins exhibit their pharmacological and biological activities through interaction with heparin-binding proteins. The prototypical heparin-binding protein is antithrombin III (AT), responsible for heparin's anticoagulant/antithrombotic activity. This study describes a filter-trapping method to isolate the chains in enoxaparin, a LMWH, which bind to AT. We demonstrate this method using the ULMWH, fondaparinux, which consists of a single well defined AT binding site. The interacting chains of enoxaparin are then characterized by activity assays, top-down liquid chromatography-mass spectrometry, and capillary zone electrophoresis mass spectrometry. This filter-trapping assay is an improvement over affinity chromatography for isolating heparin chains interacting with heparin binding proteins., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Antithrombin (AT) deficiency is a rare but strong risk factor for the thrombosis development. Mutations in gene encoding AT (SERPINC1) have provided a detailed understanding of AT deficiency and subsequent development of thrombotic complications. In the present study, we describe a case of thrombotic patient with reduced AT activity and normal AT antigen levels. AT deficiency in the patient was explained by the presence of heterozygous mutation g.13397A>G (Ala427Thr) in exon 6 of SERPINC1. Reduced APTT and TT with normal PT were observed. The mutation was found to be absent in healthy controls (n = 62). In vitro purification and characterization of variant AT showed significant decrease in fluorescence emission intensity, decreased bis-ANS fluorescence emission, changes in secondary structure and presence of polymerized AT in patient's plasma as assessed by fluorescence, circular dichroism and transmission electron microscopy respectively. Furthermore, molecular dynamics simulation studies showed altered conformation due to Ala427Thr substitution. Our study shows that genetic screening should be carried out in AT deficient patients in addition to the routinely used functional assays to understand the molecular basis of disease development.

The detection of proteases and their complexes with inhibitor proteins is of great importance for diagnosis and medical-treatment applications. In this study, we report a fingerprint-based sensor using an array of single-stranded DNAs (ssDNAs) labeled with environment-responsive 3'-carboxytetramethylrhodamine (TAMRA) for the identification of proteases. Four TAMRA-modified ssDNAs with different sequences solubilized in two different buffer solutions were incorporated in an array that was capable of generating fluorescent fingerprints unique to the proteases through diverse cross-reactive interactions, allowing the discrimination of (i) 8 proteases and (ii) 12 different mixtures of trypsin and its inhibitor protein (α 1 -antitrypsin) by multivariate analysis. Constructing an array with TAMRA-modified DNA aptamers that bind to different sites of human thrombin provides fluorescence fingerprints that reflect a reduction of the exposed surface area of thrombin upon complexation with antithrombin III, even in the presence of human serum. We finally demonstrate the potential of hybridization with complementary DNAs as an effective means to easily double the fingerprint information for proteases. Our approach based on the cross-reactive capability of ssDNAs enables high-throughput fingerprint-based sensing that can be flexibly designed and easily constructed, not only for the identification of a variety of proteins including proteases but also for the evaluation of their complexation ability.

Antithrombin (AT3) is one of the most important inhibitors of blood coagulation proteases that belong to the serpin family of protease inhibitors. In this study, a novel alternatively spliced isoform of AT3 was identified, both at transcript and protein level. This novel transcript contains an additional region in the continuation of exon 3b that was included in the transcript due to use of an alternate 5' splice site. The existence of the novel transcript was confirmed in human brain and liver through RT-PCR. An analysis of the complete transcript indicated that the native reactive centre loop (RCL) of AT3 is maintained; however the novel amino acid sequence projects out as an additional loop as evident from MD simulation studies. A unique amino acid sequence present in the novel isoform was used for the development of polyclonal antibody. The expression of novel isoform was confirmed in human brain and liver tissue using Western blot analysis. Interestingly an alignment of RCL like domain with other inhibitory serpins showed significant similarity with the neuroserpin RCL. To the best of our knowledge, this is the first evidence of alternatively spliced AT3 sequence containing an additional loop and could have physiological relevance., (Copyright © 2019 Elsevier Inc. All rights reserved.)

SERine Protease INhibitorS (Serpins) are a superfamily of proteins that are characterized by having a similar three-dimensional structure. The native conformation is not most thermodynamically stable, so polymerization is the main consequence when its stability is altered as a result of certain mutations. The polymerization of serpins has been a research topic for many years. Different mechanisms have been proposed and in the same way different compounds or strategies have been studied to prevent polymerization. A recent paper published in Bioscience Reports by Naseem et al. [ Biosci. Rep (2019) 5 , 39] studies the role of trehalose in the prevention of the polymerization of antithrombin, which belongs to the serpin superfamily. The main consequence of the antithrombin polymerization is the increased thrombotic risk, since antithrombin is the main inhibitor of the coagulation cascade. The authors demonstrate that trehalose is able to prevent the in vitro polymerization of antithrombin, under conditions in which it usually tends to polymerize, and demonstrate it by using different techniques. However, the binding site of trehalose in antithrombin should be defined by site-directed mutagenesis. On the other hand, it is not clear if all serpins polymerize in vivo through the same mechanism and it is also not clear if the same serpin can even polymerize through different mechanisms. Therefore, there are still doubts about the potential of trehalose or its derivatives to prevent in vivo antithrombin polymerization and, therefore, reduce thrombotic risk, as well as whether trehalose would be able to reduce polymerization in other serpins., (© 2019 The Author(s).)

Antithrombin is a key endogenous anticoagulant whose deficiency constitutes a strong risk factor for thrombosis. The study of antithrombin deficiency has generated excellent, and in some cases, surprising results that may be extrapolated to other thrombophilia and genetic disorders. Routine diagnosis of antithrombin deficiency is based on functional assays. Few specialized laboratories also perform genetic analysis, even though nowadays it is a simple, fast and cheap process that generates relevant information with clinical usefulness. Molecular analysis of SERPINC1, the gene encoding antithrombin, has been restricted so far to cases with confirmed or familial antithrombin deficiency. However, some pathogenic mutations are not detected by current functional methods and other gene defects may have functional consequences only observed under specific conditions. Thus, molecular analysis may be the best method to identify antithrombin deficiency. Up to 80% of patients with antithrombin deficiency have SERPINC1 gene defects, mostly (90% of the 315 gene defects described so far) point mutations or small deletions or insertions affecting the 7 exons or flanking regions. The description of new SERPINC1 gene defects may reveal new residues with functional or structural relevance and new mechanisms causing deficiency of this endogenous anticoagulant. Moreover, other genes and mechanisms may also be involved in antithrombin deficiency. Thus, disorders of N-glycosylation explain up to 5% of cases with antithrombin deficiency. However, there are still up to 10-15% of cases with antithrombin deficiency of unknown cause, whose study may reveal new genes and mechanisms involved in thrombosis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Essentials Heparin-binding site (HBS) variants of antithrombin (AT) are associated with thrombosis risk. HSB variants have, in general, normal progressive inhibitory activity but reduced heparin affinity. Thrombosis in HSB carriers has been primarily attributed to the loss of heparin cofactor activity. Results here demonstrate that HSB variants of AT also lack anti-inflammatory signaling functions., Summary: Background Several heparin-binding site (HBS) variants of antithrombin (AT) have been identified that predispose carriers to a higher incidence of thrombosis. Thrombosis in carriers of HBS variants has been primarily attributed to a loss in their heparin-dependent anticoagulant function. Objective The objective of this study was to determine whether HSB mutations affect the anti-inflammatory functions of variants. Methods Two HBS variants of AT (AT-I7N and AT-L99F), which are known to be associated with a higher incidence of thrombosis, were expressed in mammalian cells and purified to homogeneity. These variants were characterized by kinetic assays followed by analysis of their activities in established cellular and/or in vivo inflammatory models. The possible effects of mutations on AT structure were also evaluated by molecular modeling. Results The results indicated that, whereas progressive inhibitory activities of variants were minimally affected, their heparin affinity and inhibitory activity in the presence of heparin were markedly decreased. Unlike wild-type AT, neither AT variant was capable of inhibiting activation of nuclear factor-κB or downregulation of expression of cell adhesion molecules in response to lipopolysaccharide (LPS). Similarly, neither variant elicited barrier protective activity in response to LPS. Structural analysis suggested that the L99F substitution locally destabilizes AT structure. Conclusions It is concluded that the L99F mutation of AT is associated with destabilization of the serpin structure, and that the loss of anti-inflammatory signaling function of the HBS variants may also contribute to enhanced thrombosis in carriers of HBS mutations., (© 2017 International Society on Thrombosis and Haemostasis.)

Biochemical fluorogenic and chromogenic assays facilitate real-time study of enzyme function. Based on the principle of enzymatic inhibition, these kinetic assays can be adapted to measure the function of serpins. Compared to traditional, electrophoretic study of serpin inhibitory complex formation, kinetic assays allow for finer temporal resolution as well as more quantitative comparisons between different conditions. This chapter describes methodology for performing real-time, kinetic measurement of serpin inhibitory activity by fluorogenic substrate conversion assay. Specifically, the methods covered include measurement of alpha-1-antitrypsin inhibitory activity against trypsin and heparin-dependent anti-thrombin III inhibitory activity against thrombin. These methods are scalable to small-volume, high-density format and can be applied for high-throughput screening of serpin activity modulators.

The antithrombin III (ATIII)-binding site, which contains a special 3-O-sulfated, N-sulfated glucosamine residue with or without 6-O-sulfation, is mainly responsible for the anticoagulant activity of heparin. Undergoing the chemical depolymerization process, the preservation of the ATIII-binding site in low molecular weight heparins (LMWHs) are varied leading to the fluctuation of the anticoagulant activity. Herein we report a capillary electrophoresis (CE) method in combination with heparinase digestion and affinity chromatography for the measurement of molar percentage of ATIII-binding site of LMWHs. After exhaustively digesting LMWHs with the mixture of heparinase I, II and III, almost all the resulting oligosaccharide building blocks, including the three 3-O-sulfated tetrasaccharides derived from the ATIII-binding site, were resolved by CE separation. The peak area of each building block permits quantification of the molar percentage of the ATIII-binding site. The peaks corresponding to the 3-O-sulfated tetrasaccharides were assigned based on the linear relationship between the electrophoretic mobilities of the oligosaccharides and their charge to mass ratios. The peak assignment was further confirmed by analysis of the high ATIII affinity fractions, which contains much high 3-O-sulfated tetrasaccharides. With the method, the molar percentage of the ATIII-binding site of enoxaparin from different batches and different manufactures were measured and compared. It was demonstrated that the CE method provides more precise data for assessing the anti-FXa activity than that of the biochemical assay method., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Fondaparinux sodium is a synthetic pentasaccharide representing the high affinity antithrombin III binding site in heparin. It is the active pharmaceutical ingredient of the anticoagulant drug Arixtra ® . The single crystal X-ray structure of Fondaparinux sodium is reported, unequivocally confirming both structure and absolute configuration. The iduronic acid adopts a somewhat distorted chair conformation. Due to the presence of many sulfur atoms in the highly sulfated pentasaccharide, anomalous dispersion could be applied to determine the absolute configuration. A comparison with the conformation of Fondaparinux in solution, as well as complexed with proteins is presented. The content of the solution reference standard was determined by quantitative NMR using an internal standard both in 1999 and in 2016. A comparison of the results allows the conclusion that this method shows remarkable precision over time, instrumentation and analysts., Competing Interests: B.Ü., W.d.W., M.L., A.L. and E.K. hold positions in Aspen which commercially manufactures Fondaparinux.

Antithrombin is an anticoagulant serpin that efficiently inhibits multiple procoagulant proteases. The cost for the structural flexibility required for this function is the vulnerability to mutations that impact its folding pathway. Most conformational mutations identified in serpins cause polymerisation. Only three mutations in SERPINC1 affecting two residues have been found to favour transformation to the latent conformation of antithrombin, another hyperstable non-anticoagulant form with strong antiangiogenic activity that constitutes 3 % of plasma antithrombin in healthy subjects. The analysis of latent antithrombin in 141 unrelated patients with antithrombin deficiency carrying 89 different SERPINC1 mutations identified four cases with higher levels than that of controls: p.Pro439Thr, p.Pro461Ser, p.Met283Val, and p.His401Tyr, the last also with circulating polymers. Heating of plasma at 42ºC exacerbated the transformation to the latent conformation in p.Pro439Thr and p.Pro461Ser. The conformational effect of p.Met283Val, the mutation associated with the highest levels of latent antithrombin detected in four members of a family, was verified in a recombinant model. Antithrombin deficiency in these cases should be classified as pleiotropic based on the impaired reactivity and low heparin affinity of the variant. Despite high levels of latent antithrombin (up to 80 µg/ml in p.Met283Val carriers), no vascular defects were described in carriers of these mutations. In conclusion, our study identifies new residues involved in the structural stability of antithrombin (and potentially of all serpins). High levels of endogenous latent antithrombin seem to play a minor antiangiogenic effect. Finally, pleiotropic deficiencies may be caused by mutations inducing transformation to the latent conformation.

Heparin is a polysaccharide that is widely used as an anticoagulant drug. The mechanism for heparin's anticoagulant activity is primarily through its interaction with a serine protease inhibitor, antithrombin III (AT), that enhances its ability to inactivate blood coagulation serine proteases, including thrombin (factor IIa) and factor Xa. The AT-binding site in the heparin is one of the most well-studied carbohydrate-protein binding sites and its structure is the basis for the synthesis of the heparin pentasaccharide drug, fondaparinux. Despite our understanding of the structural requirements for the heparin pentasaccharide AT-binding site, there is a lack of data on the natural variability of these binding sites in heparins extracted from animal tissues. The present work provides a detailed study on the structural variants of the tetrasaccharide fragments of this binding site afforded following treatment of a heparin with heparin lyase II. The 5 most commonly observed tetrasaccharide fragments of the AT-binding site are fully characterized, and a method for their quantification in heparin and low-molecular-weight heparin products is described., (Copyright © 2017 American Pharmacists Association®. All rights reserved.)

Essentials Glucocorticoids are associated with an increased risk of thrombosis. Healthy volunteers received dexamethasone or placebo in an endotoxin lung instillation model. Dexamethasone suppressed thrombin generation in bronchoalveolar lavage. Glucocorticoids inhibit endotoxin induced pulmonary coagulopathy., Summary: Background Activation of local and systemic coagulation is a common finding in patients with pneumonia. There is evidence that glucocorticoids have procoagulant activity in the circulation, particularly in the context of inflammation. The effects of glucocorticoids on local pulmonary coagulation have not yet been investigated. Objective To use a human model of lung inflammation based on the local instillation of endotoxin in order to investigate whether glucocorticoids alter pulmonary coagulation. Methods Twenty-four healthy volunteers were randomized to receive either dexamethasone or placebo in a double-blind trial. Endotoxin was instilled via bronchoscope into right or left lung segments, followed by saline into the contralateral site. Six hours later, a bilateral bronchoalveolar lavage (BAL) was performed and coagulation parameters were measured. Results Endotoxin induced activation of coagulation in the bronchoalveolar compartment: the level of prothrombin fragment 1 + 2 (F 1 + 2 ) was increased three-fold (248 pmol L -1 , 95% confidence interval [CI] 43-454 versus 743 pmol L -1 , 95% CI 437-1050) and the level of thrombin-antithrombin complex (TATc) was increased by ~ 50% (31 μg L -1 , 95% CI 18-45 versus 49 μg L -1 , 95% CI 36-61) as compared with saline-challenged segments. Dexamethasone reduced F 1 + 2 (284 pmol L -1 , 95% CI 34-534) and TATc (9 μg L -1 , 95% CI 0.7-17) levels almost to those measured in BAL fluid from the saline-instilled segments in the placebo group. Dexamethasone even profoundly reduced F 1 + 2 levels (80%) in saline-instilled lung segments (50 pmol L -1 , 95% CI 12-87). In contrast, dexamethasone had no effect on systemic F 1 + 2 levels. Conclusions Dexamethasone inhibits endotoxin-induced coagulopathy in lungs. This trial is the first to provide insights into the effects of glucocorticoids on pulmonary coagulation in response to endotoxin., (© 2016 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Antithrombin (AT) deficiency is an autosomal dominant disorder, and identification of mutation AT variants would improve our understanding of the anticoagulant function of this serine protease inhibitor (SERPIN) and the molecular pathways underlying this disorder. In the present study, we performed whole-exome sequencing of a Chinese family with deep vein thrombosis, and identified a new small deletion that eliminates four amino acids (INEL) from exon 4 of SERPINC1 gene. This causes type I AT deficiency by enhancing the intracellular retention of this protein. AT retention leads to endoplasmic reticulum (ER) stress, which further inhibits AT release. In addition, ER stress activates ER-associated degradation, which promotes AT degradation. Suppression of ER stress enhanced the secretion of AT, while inhibition of ER-associated degradation suppressed AT release. Thus, our study identified a new mutation (INEL deletion) causing type I AT deficiency, and uncovered a novel mechanism for AT retention through enhanced ER stress, which may provide an innovative approach for treating AT deficiency.

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., (© 2016 International Society on Thrombosis and Haemostasis.)

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 antithrombin's 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.

Although proteins generally fold to their thermodynamically most stable state, some metastable proteins populate higher free energy states. Conformational changes from metastable higher free energy states to lower free energy states with greater stability can then generate the work required to perform physiologically important functions. However, how metastable proteins fold to these higher free energy states in the cell and avoid more stable but inactive conformations is poorly understood. The serpin family of metastable protease inhibitors uses large conformational changes that are downhill in free energy to inhibit target proteases by pulling apart the protease active site. The serpin antithrombin III (ATIII) targets thrombin and other proteases involved in blood coagulation, and ATIII misfolding can thus lead to thrombosis and other diseases. ATIII has three disulfide bonds, two near the N terminus and one near the C terminus. Our studies of ATIII in-cell folding reveal a surprising, biased order of disulfide bond formation, with early formation of the C-terminal disulfide, before formation of the N-terminal disulfides, critical for folding to the active, metastable state. Early folding of the predominantly β-sheet ATIII domain in this two-domain protein constrains the reactive center loop (RCL), which contains the protease-binding site, ensuring that the RCL remains accessible. N-linked glycans and carbohydrate-binding molecular chaperones contribute to the efficient folding and secretion of functional ATIII. The inability of a number of disease-associated ATIII variants to navigate the folding reaction helps to explain their disease phenotypes.

Heparin and related members of the glycosaminoglycan (GAG) family are highly polyanionic linear saccharides that play important roles in a variety of physiological processes ranging from blood coagulation to embryo- and oncogenesis, tissue regeneration, and immune response regulation. These diverse functions are executed via a variety of mechanisms, including protein sequestration, activation, and facilitation of their interactions with cell-surface receptors, but deciphering the specific molecular mechanisms is frequently impossible due to the extremely high degree of GAG heterogeneity. As a result, the vast majority of studies of heparin (or related GAGs) interactions with its client proteins use synthetically produced heparin mimetics with defined structure or short heparin fragments. In this work we use native electrospray ionization mass spectrometry (ESI MS) in combination with limited charge reduction in the gas phase to obtain meaningful information on noncovalent complexes formed by intact unfractionated heparin and antithrombin-III, interaction which is central to preventing blood clotting. Complexes of different stoichiometries are observed ranging from 1:1 to 1:3 (heparin/protein ratio). In addition to binding stoichiometry, the measurements allow the range of heparin chain lengths to be obtained for each complex and the contribution of each complex to the total ionic signal to be calculated. Incorporation of ion mobility measurements in the experimental workflow allows the total analysis time to be shortened very significantly and the charge state assignment for the charge-reduced species to be verified. The possibility to study interactions of intact unfractionated heparin with a client protein carried out directly by native ESI MS without the need to use relatively homogeneous surrogates demonstrated in this work opens up a host of new exciting opportunities and goes a long way toward ameliorating the persistent but outdated view of the intractability of such interactions.

Unlabelled: ESSENTIALS: Antithrombin III (AT)β binds heparin with higher affinity than ATα. A conformation-specific antibody against ATβ, TPP2009, was made to investigate ATβ in hemostasis. TPP2009 bound specifically to heparin-ATβ and greatly reduced the anticoagulant effect of AT. This antibody was effective in elucidating the importance of ATβ in hemostasis., Background: Antithrombin III (AT)β is an isoform of AT that lacks the post-translational carbohydrate modification at Asn135. This isoform binds heparin with greater affinity than ATα, and has been shown to target antithrombotic function to the extracellular vascular endothelial injury site., Objectives: To characterize a conformation-specific antibody against ATβ and begin to investigate the role of ATβ in maintaining hemostasis., Methods: Surface plasmon resonance (SPR), antigen binding and functional assays were conducted to characterize the mode of action of antibodies generated against heparin-bound ATβ (ATβ*H) by the use of phage display., Results: SPR and binding studies showed that one of the antibodies, TPP2009, bound specifically to ATβ*H and glycosaminoglycan-associated ATβ on endothelial cells. In diluted prothrombin and activated factor X (FXa)-induced clotting assays, TPP2009 dose-dependently reduced the anticoagulant effect of heparin in non-hemophilic and FVIII-deficient human plasma, with half-maximal effective concentrations (EC50 ) of 10.5 nm and 4.7 nm, respectively. In AT-deficient human plasma, TPP2009 dose-dependently inhibited the effects of exogenously added ATβ and heparin. In purified systems with ATβ and pentasaccharide, TPP2009 restored > 91% of FXa activity. TPP2009 dose-dependently reversed the effects of heparin in rabbit (EC50 , 25.7 nm) and cynomolgus monkey (EC50 , 21.5 nm) plasma, but not in mouse plasma. TPP2009 was also effective in partially restoring FXa activity in rabbit and cynomolgus monkey plasma treated with FVIII function-neutralizing antibodies., Conclusions: TPP2009 specifically targets a unique conformational epitope on ATβ*H and blocks ATβ-mediated anticoagulation. It effectively promotes coagulation in plasma, indicating the importance of ATβ in hemostasis., (© 2015 International Society on Thrombosis and Haemostasis.)

Venoms of invertebrates contain an enormous diversity of proteins, peptides, and other classes of substances. Insect venoms are characterized by a large interspecific variation resulting in extended lists of venom compounds. The venom composition of several hymenopterans also shows different intraspecific variation. For instance, venom from different honeybee castes, more specifically queens and workers, shows quantitative and qualitative variation, while the environment, like seasonal changes, also proves to be an important factor. The present study aimed at an in-depth analysis of the intraspecific variation in the honeybee venom proteome. In summer workers, the recent list of venom proteins resulted from merging combinatorial peptide ligand library sample pretreatment and targeted tandem mass spectrometry realized with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS/MS). Now, the same technique was used to determine the venom proteome of queens and winter bees, enabling us to compare it with that of summer bees. In total, 34 putative venom toxins were found, of which two were never described in honeybee venoms before. Venom from winter workers did not contain toxins that were not present in queens or summer workers, while winter worker venom lacked the allergen Api m 12, also known as vitellogenin. Venom from queen bees, on the other hand, was lacking six of the 34 venom toxins compared to worker bees, while it contained two new venom toxins, in particularly serine proteinase stubble and antithrombin-III. Although people are hardly stung by honeybees during winter or by queen bees, these newly identified toxins should be taken into account in the characterization of a putative allergic response against Apis mellifera stings.

We validate the utility of ion mobility to measure protein conformational changes induced by the binding of glycosaminoglycan ligands, using the well characterized system of Antithrombin III (ATIII) and Arixtra, a pharmaceutical agent with heparin (Hp) activity. Heparin has been used as a therapeutic anticoagulant drug for several decades through its interaction with ATIII, a serine protease inhibitor that plays a central role in the blood coagulation cascade. This interaction induces conformational changes within ATIII that dramatically enhance the ATIII-mediated inhibition rate. Arixtra is the smallest synthetic Hp containing the specific pentasaccharide sequence required to bind with ATIII. Here we report the first travelling wave ion mobility mass spectrometry (TWIMS) investigation of the conformational changes in ATIII induced by its interaction with Arixtra. Native electrospray ionization mass spectrometry allowed the gentle transfer of the native topology of ATIII and ATIII-Arixtra complex. IM measurements of ATIII and ATIII-Arixtra complex showed a single structure, with well-defined collisional cross section (CCS) values. An average 3.6% increase in CCS of ATIII occurred as a result of its interaction with Arixtra, which agrees closely with the theoretical estimation of the change in CCS based on protein crystal structures. A comparison of the binding behavior of ATIII under both denaturing and non-denaturing conditions confirmed the significance of a folded tertiary structure of ATIII for its biological activity. A Hp oligosaccharide whose structure is similar to Arixtra but missing the 3-O sulfo group on the central glucosamine residue showed a dramatic decrease in binding affinity towards ATIII, but no change in the mobility behavior of the complex, consistent with prior studies that suggested that 3-O sulfation affects the equilibrium constant for binding to ATIII, but not the mode of interaction. In contrast, nonspecific binding by a Hp tetrasaccharide showed more complex mobility behavior, suggesting more promiscuous interactions with ATIII. The effect of collisional activation of ATIII and ATIII-Arixtra complex were also assessed, revealing that the binding of Arixtra provided ATIII with additional stability against unfolding. Overall, our results validate the capability of TWIMS to retain the significant features of the solution structure of a protein-carbohydrate complex so that it can be used to study protein conformational changes induced by the binding of glycosaminoglycan ligands.

The present study details the development of a family of novel D-Ala(8) glucagon-like peptide-1 (GLP-1) peptide conjugates by site specific conjugation to an antithrombin III (ATIII) binding carrier pentasaccharide through tetraethylene glycol linkers. All conjugates were found to possess potent insulin-releasing activity. Peptides with short linkers (<25 atoms) conjugated at Lys(34) and Lys(37) displayed strong GLP-1 receptor (GLP-1-R) binding affinity. All D-Ala(8) GLP-1 conjugates exhibited prominent glucose-lowering action. Biological activity of the Lys(37) short-linker peptide was evident up to 72 h post-injection. In agreement, the pharmacokinetic profile of this conjugate (t1/2 , 11 h) was superior to that of the GLP-1-R agonist, exenatide. Once-daily injection of the Lys(37) short-linker peptide in ob/ob mice for 21 days significantly decreased food intake and improved HbA1c and glucose tolerance. Islet size was decreased, with no discernible change in islet number. The beneficial effects of the Lys(37) short-linker peptide were similar to or better than either exenatide or liraglutide, another GLP-1-R agonist. In conclusion, GLP-1 peptides conjugated to an ATIII binding carrier pentasaccharide have a substantially prolonged bioactive profile compatible for possible once-weekly treatment of type 2 diabetes in humans., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Centrifugation is an essential step for plasma preparation to remove residual elements in plasma, especially platelets and platelet-derived microparticles (PMPs). Our working hypothesis was that centrifugation as a preanalytical step may influence some coagulation parameters., Materials and Methods: Healthy young men were recruited (N=17). For centrifugation, two protocols were applied: (A) the first centrifugation at 2500xg for 15 min and (B) at 2500xg for 20 min at room temperature with a light brake. In protocol (A), the second centrifugation was carried out at 2500xg for 15 min, whereas in protocol (B), the second centrifugation involved a 10 min spin at 13,000 x g. Thrombin-antithrombin (TAT) and plasmin-antiplasmin (PAP) complexes concentrations were determined by enzyme-linked immunosorbent assays. PMPs were stained with CD41 antibody and annexin V, and analyzed by flow cytometry method. Procoagulant activity was assayed by the Calibrated Automated Thrombogram method as a slope of thrombin formation (CAT velocity)., Results: Median TAT and PAP concentrations did not differ between the centrifugation protocols. The high speed centrifugation reduced the median (IQR) PMP count in plasma from 1291 (841-1975) to 573 (391-1010) PMP/µL (P=0.001), and CAT velocity from 2.01 (1.31-2.88) to 0.97 (0.82-1.73) nM/min (P=0.049). Spearman's rank correlation analysis showed correlation between TAT and PMPs in the protocol A plasma which was (rho=0.52, P<0.050) and between PMPs and CAT for protocol A (rho=0.74, P<0.050) and protocol B (rho=0.78, P<0.050)., Conclusion: Centrifugation protocols do not influence the markers of plasminogen (PAP) and thrombin (TAT) generation but they do affect the PMP count and procoagulant activity.

Herein, the synthesis and characterization of heparin-based nanocapsules (NCs) as potential drug delivery systems is described. For the synthesis of the heparin-based NCs, the versatile method of miniemulsion polymerization at the droplets interface was achieved resulting in narrowly distributed NCs with 180 nm in diameter. Scanning and transmission electron microscopy images showed clearly NC morphology. A highly negative charge density for the heparin-based NCs was determined by measuring the electro-kinetic potential. Measuring the activated clotting time demonstrated the biological intactness of the polymeric shell. The ability of heparin-based NCs to bind to antithrombin (AT III) was investigated using isothermal titration calorimetry and dynamic light scattering experiments. The chemical stability of the NCs was studied in physiological protein-containing solutions and also in medically interesting fluids such as sodium chloride 0.9%, Ringer's solution, and phosphate buffer saline using dynamic light scattering and measuring the fluorescence intensity. The impressive uptake of NCs in different cells was confirmed by fluorescence-activated cell sorting, confocal laser scanning microscopy, and transmission electron microscopy. The low toxicity of all types of NCs was demonstrated., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Interaction with antithrombin and heparin regulates distribution, activity, and clearance of factor IXa (FIXa). Hemophilia B prophylaxis targets plasma FIX levels > 1% but neglects extravascular FIX, which colocalizes with antithrombin-heparan sulfate., Objective: Combined mutagenesis of FIX was undertaken to selectively disrupt heparin- and antithrombin-mediated regulation of the protease., Methods: Human FIX alanine substitutions in the heparin (K126A and K132A) and antithrombin (R150A) exosites were characterized with regard to coagulant activity, plasma thrombin generation, antithrombin inhibition, and plasma half-life., Results: Single or combined (K126A/R150A or K132A/R150A) exosite mutations variably reduced coagulant activity relative to wild-type (WT) for FIX (27-91%) and FIXa (25-91%). Double mutation in the heparin exosite (K126A/K132A and K126A/K132A/R150A) markedly reduced coagulant activity (7-21%) and plasma TG. In contrast to coagulant activity, FIX K126A (1.8-fold), R150 (1.6-fold), and K132A/R150A (1.3-fold) supported increased tissue factor-initiated plasma TG, while FIX K132A and K126A/R150A were similar to WT. FIXa K126A/R150A and K132A/R150A (1.5-fold) demonstrated significantly increased FIXa-initiated TG, while FIXa K132A, R150A, and K126A (0.8-0.9-fold) were similar to WT. Dual mutations in the heparin exosite or combined mutations in both exosites synergistically reduced the inhibition rate for antithrombin-heparin. The half-life of FIXa WT in FIX-deficient plasma was remarkably lengthy (40.9 ±1.4 min) and further prolonged for FIXa R150A, K126A/R150A, and K132A/R150A (> 2 h)., Conclusion: Selective disruption of exosite-mediated regulation by heparin and antithrombin can be achieved with preserved or enhanced thrombin generation capacity. These proteins should demonstrate enhanced therapeutic efficacy for hemophilia B., (© 2015 International Society on Thrombosis and Haemostasis.)

Antithrombin III (AT) is the main inhibitor of blood coagulation proteases like thrombin and factor Xa. In this study we report the identification and characterization of several variants of AT for the first time in Indian population. We screened 1950 deep vein thrombosis (DVT) patients for AT activity and antigen levels. DNA sequencing was further carried out in patients with low AT activity and/or antigen levels to identify variations in the AT gene. Two families, one with type I and the other with type II AT deficiency were identified. Three members of family I showed an increase in the coagulation rates and recurrent thrombosis in this family was solely attributed to the rs2227589 polymorphism. Four members of family II spanning two generations had normal antigen levels and decreased AT activity. A novel single nucleotide insertion, g.13362&#95;13363insA in this family in addition to g.2603T>C (p.R47C) mutation were identified. AT purified from patient's plasma on hi-trap heparin column showed a marked decrease in heparin affinity and thrombin inhibition rates. Western blot analysis showed the presence of aggregated AT. We also report a novel point mutation at position g.7549 A>G (p.T280A), that is highly conserved in serpin family. Variant protein isolated from patient plasma indicated loss of regulatory function due to in-vivo polymerization. In conclusion this is the first report of AT mutations in SERPINC1 gene in Indo-Aryan population where a novel point mutation p.T280A and a novel single nucleotide insertion g.13362&#95;13363insA are reported in addition to known variants like p.R47C, p.C4-X and polymorphisms of rs2227598, PstI and DdeI.

Aims: Interaction between blood and bio-surfaces is important in many medical fields. With the aim of studying blood-mediated reactions to cellular transplants, we developed a whole-blood model for incubation of small volumes for up to 48 h., Methods: Heparinized polyvinyl chloride tubing was cut in suitable lengths and sealed to create small bags. Multiple bags, with fresh venous blood, were incubated attached to a rotating wheel at 37°C. Physiological variables in blood were monitored: glucose, blood gases, mono- and divalent cations and chloride ions, osmolality, coagulation (platelet consumption, thrombin-antithrombin complexes (TAT)), and complement activation (C3a and SC5b-9), haemolysis, and leukocyte viability., Results: Basic glucose consumption was high. Glucose depletion resulted in successive elevation of extracellular potassium, while sodium and calcium ions decreased due to inhibition of energy-requiring ion pumps. Addition of glucose improved ion balance but led to metabolic acidosis. To maintain a balanced physiological environment beyond 6 h, glucose and sodium hydrogen carbonate were added regularly based on analyses of glucose, pH, ions, and osmotic pressure. With these additives haemolysis was prevented for up to 72 h and leukocyte viability better preserved. Despite using non-heparinized blood, coagulation and complement activation were lower during long-term incubations compared with addition of thromboplastin and collagen., Conclusion: A novel whole-blood model for studies of blood-mediated responses to a cellular transplant is presented allowing extended observations for up to 48 h and highlights the importance of stringent evaluations and adjustment of physiological conditions.

The inefficient glycosylation of consensus sequence on N135 in antithrombin explains the two glycoforms of this key anticoagulant serpin found in plasma: α and β, with four and three N-glycans, respectively. The lack of this N-glycan increases the heparin affinity of the β-glycoform. Recent studies have demonstrated that an aromatic sequon (Phe-Y-Asn-X-Thr) in reverse β-turns enhances N-glycosylation efficiency and stability of different proteins. We evaluated the effect of the aromatic sequon in this defective glycosylation site of antithrombin, despite of being located in a loop between the helix D and the strand 2A. We analyzed the biochemical and functional features of variants generated in a recombinant cell system (HEK-EBNA). Cells transfected with wild-type plasmid (K133-Y-N135-X-S137) generated 50% of α and β-antithrombin. The S137T, as previously reported, K133F, and the double mutant (K133F/S137T) had improved glycosylation efficiency, leading to the secretion of α-antithrombin, as shown by electrophoretic and mass analysis. The presence of the aromatic sequon did not significantly affect the stability of this conformationally sensitive serpin, as revealed by thermal denaturation assay. Moreover, the aromatic sequon hindered the activation induced by heparin, in which is involved the helix D. Accordingly, K133F and particularly K133F/S137T mutants had a reduced anticoagulant activity. Our data support that aromatic sequons in a different structural context from reverse turns might also improve the efficiency of N-glycosylation.

Antithrombin III (ATIII) is a key antiproteinase involved in blood coagulation. Previous investigations have shown that ATIII is degraded by Staphylococcus aureus V8 protease, leading to release of heparin binding fragments derived from its D helix. As heparin binding and antimicrobial activity of peptides frequently overlap, we here set out to explore possible antibacterial effects of intact and degraded ATIII. In contrast to intact ATIII, the results showed that extensive degradation of the molecule yielded fragments with antimicrobial activity. Correspondingly, the heparin-binding, helix D-derived, peptide FFFAKLNCRLYRKANKSSKLV (FFF21) of human ATIII, was found to be antimicrobial against particularly the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Fluorescence microscopy and electron microscopy studies demonstrated that FFF21 binds to and permeabilizes bacterial membranes. Analogously, FFF21 was found to induce membrane leakage of model anionic liposomes. In vivo, FFF21 significantly reduced P. aeruginosa infection in mice. Additionally, FFF21 displayed anti-endotoxic effects in vitro. Taken together, our results suggest novel roles for ATIII-derived peptide fragments in host defense., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

A novel dual nanoparticle amplification approach is introduced for the enhanced surface plasmon resonance (SPR) detection of a target protein at subattomolar concentrations. Thrombin was used as a model target protein as part of a sandwich assay involving an antithrombin (anti-Th) modified SPR chip surface and a thrombin specific DNA aptamer (Th-aptamer) whose sequence also includes a polyadenine (A30) tail. Dual nanoparticle (NP) enhancement was achieved with the controlled hybridization adsorption of first polythymine-NP conjugates (T20-NPs) followed by polyadenine-NPs (A30-NPs). Two different nanoparticle shapes (nanorod and quasi-spherical) were explored resulting in four different NP pair combinations being directly compared. It was found that both the order and NP shape were important in optimizing the assay performance. The use of real-time SPR measurements to detect target concentrations as low as 0.1 aM is a 10-fold improvement compared to single NP-enhanced SPR detection methods.

Allosteric conformational changes in antithrombin induced by binding a specific heparin pentasaccharide result in very large increases in the rates of inhibition of factors IXa and Xa but not of thrombin. These are accompanied by CD, fluorescence, and NMR spectroscopic changes. X-ray structures show that heparin binding results in extension of helix D in the region 131-136 with coincident, and possibly coupled, expulsion of the hinge of the reactive center loop. To examine the importance of helix D extension, we have introduced strong helix-promoting mutations in the 131-136 region of antithrombin (YRKAQK to LEEAAE). The resulting variant has endogenous fluorescence indistinguishable from WT antithrombin yet, in the absence of heparin, shows massive enhancements in rates of inhibition of factors IXa and Xa (114- and 110-fold, respectively), but not of thrombin, together with changes in near- and far-UV CD and (1)H NMR spectra. Heparin binding gives only ∼3-4-fold further rate enhancement but increases tryptophan fluorescence by ∼23% without major additional CD or NMR changes. Variants with subsets of these mutations show intermediate activation in the absence of heparin, again with basal fluorescence similar to WT and large increases upon heparin binding. These findings suggest that in WT antithrombin there are two major complementary sources of conformational activation of antithrombin, probably involving altered contacts of side chains of Tyr-131 and Ala-134 with core hydrophobic residues, whereas the reactive center loop hinge expulsion plays only a minor additional role.

In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

Antithrombin III (ATIII) performs a critical anticoagulant function by precluding the activation of blood clotting proteinases, aided by its two cofactors, heparin and heparan sulfate. Though several studies have been carried out on physiological, biochemical and structural perspectives on ATIII, so far there are limited studies on the molecular evolution of ATIII. Herein, we carried out molecular phylogenetic analyses of ATIII genes, combining gene structures, synteny and sequence-structural features for ATIII spanning 50 vertebrate genomes. ATIII is maintained over 450 MY on same genomic loci in vertebrates with few changes in ray-finned fishes and lost one intron 262c in tetrapods and coelacanth. In ray-finned fishes, ATIII gene has additional intron at the position 262c, which shared by group V1 members, corroborating that it is lost in other vertebrates and also in lobed-finned fish coelacanth (Latimeria chalumnae). We found that heparin binding basic residues, hD helix, three pairs of Cys-Cys salt bridges, N-glycosylation sites, serpin motifs and inhibitory reactive center loop (RCL) of ATIII protein are highly conserved. Using 1092 human genomes available from 1000G project, we also compiled 1997 ATIII variants, which reveals 76.2% single nucleotide polymorphisms (SNPs), 11.8% deletions and 8.1% insertions as three major classes of gene variations. These understandings may have medical importance as well., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Introduction: Antithrombin (AT) deficiency is associated with an increasing risk of thrombosis., Materials and Methods: 15 unrelated patients with AT deficiency defined by thrombophilic assays were recruited and detailed clinical information about patients, focusing on the personal and family history of thromboembolism (TE), were recorded. Mutation analysis was performed by direct sequencing of an AT gene (SERPINC1) in the patients and their family members., Results: A total of 15 heterozygous causative mutations, each being identified in one family, were identified. Five mutations (33.3%) were reported here for the first time, including three null mutations (Ser36X, Lys70X and Try307X) and two missense mutations (Phe123Cys and Leu340Phe) probably impairing the structural integrity and stability of protein based on the AT structural analysis. Of the 15 patients, 33.3% (5/15) had additional risk factors and only one patient presented with additional genetic alteration causing an early onset of thrombosis. Fourteen patients (93.9%) suffered from multisite recurrent thrombotic episodes after a first episode of thrombosis. 93.3% of the patients experienced deep vein thrombosis (DVT) and 40.0% presented with mesenteric venous thrombosis (MVT). In addition, both venous and arterial thrombosis was present in two unrelated patients. 51.0% subjects with AT deficiency in the 15 unrelated pedigrees experienced TE events., Conclusions: Prophylactic anticoagulation may be suggested in AT-deficient patients to avoid the recurrent and multisite thrombosis. The association of primary MVT and AT deficiency is highlighted., (© 2013.)

Heparan sulfate (HS) represents a large class of linear polysaccharides that are required for the function of all mammalian physiological systems. HS is characterized by a repeating disaccharide backbone that is subject to a wide range of modifications, making this class of macromolecules arguably the most information dense in all of biology. The majority of HS functions are associated with the ability to bind and regulate a wide range of proteins. Indeed, recent years have seen an explosion in the discovery of new activities for HS where it is now recognized that this class of glycans functions as co-receptors for growth factors and cytokines, modulates cellular uptake of lipoproteins, regulates protease activity, is critical to amyloid plaque formation, is used by opportunistic pathogens to enter cells, and may even participate in epigenetic regulation. This review will discuss the current state of understanding regarding the specificity of HS-protein binding and will describe the concept that protein binding to HS depends on the overall organization of domains within HS rather than fine structure.

Furin, PACE4, PC5/6 and PC7 are members of the subtilisin-like proprotein convertase (SPC) family. Although these enzymes are known to play critical roles in various physiological and pathological events including cell differentiation, tumor growth, virus replication and the activation of bacterial toxins, their distinct functions are yet to be fully delineated. α1-PDX is an engineered α1-antitrypsin variant carrying the RXXR consensus motif for furin within its reactive site loop. However, α1-PDX inhibits other SPCs in addition to furin. In this work, we prepared various rat α1-antitrypsin variants containing Arg at the P1 site within the reactive site loop, and examined their respective selectivity. The novel α1-antitrypsin variant AVNR (AVPM(352)/AVNR) was identified as a highly selective inhibitor of furin. This variant formed a sodium dodecyl sulfate- and heat-stable furin/α1-antitrypsin complex and inhibited furin activity ex vivo and in vitro. Other SPC members including PACE4, PC5/6 and PC7 were not inhibited by the AVNR variant. Furin-mediated maturation of bone morphogenetic protein-4 was completely inhibited by ectopic expression of the AVNR variant. The AVNR variant should prove to be a useful inhibitor in identifying the specific role of furin.