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PubMed2005

QCM-FIA with PGMA coating for dynamic interaction study of heparin and antithrombin III.

In this work, we describe a method of constructing a film of linear poly(glycidyl methacrylate) (PGMA) polymer onto the surface of quartz crystal microbalance (QCM) electrode as a coating material that allows easy coupling of heparin molecules onto the electrode and facilitates the determination of the interaction between heparin and antithrombin III (AT III). The PGMA film was characterized with atomic force microscopy (AFM) and infra-red spectroscopy. The coupling of heparin was accomplished in one step solution reaction. A home-made quartz crystal microbalance-flow injection analysis (QCM-FIA) system with data analysis software developed in our laboratory was used to determine the interaction. The interactions between immobilized heparin and AT III were studied with various concentrations under various conditions. The obtained constants are kass=(1.49+/-0.12)x10(3)mol-1ls-1, kdiss=(3.94+/-0.63)x10(-2)s-1, KA=(3.82+/-0.33)x10(4)mol-1l.

Adsorption↗
PubMed2005

A novel application of multi-wavelength TIRF spectroscopy for real time monitoring of antithrombin interactions with immobilized heparin.

Real time interactions of antithrombin (AT) with Corline Heparin Surfaces (CHS) with one and two layers of heparin conjugate have been examined using a multi-wavelength TIRF spectroscopy technique with continuous flow. Fluorescently labeled AT, adsorbed from citrated human blood plasma, showed significantly higher signals on CHS compared to the cationic surface used to attach the heparin conjugate. The AT binding to CHS was very stable, also after exposure to soluble heparin at a concentration of 1.5 IU/mL. Only a few percent of the bound AT were displaced from the surfaces by AT present in plasma after long-term exposure to plasma. In contrast, larger amounts of the freshly added AT had adsorbed to the surfaces, especially to the surface with two layers of heparin conjugate, indicating the presence of unsaturated AT binding sites. The amount of AT bound to the different surfaces was quantified after elution using an enzyme immunoassay (EIA). Characteristic emission spectra of proteins and fluorophores of labeled proteins, obtained at the surfaces after a long-term exposure to plasma, confirmed their presence at the surfaces. The multi-wavelength TIRF technique proved to be a useful tool when combined with other techniques to study the time course of interactions of fluorescently labeled proteins with biomaterials, even in a complex environment such as plasma.

Antithrombins↗
PubMed2006

Factor V Leiden mutation, deficiency of antithrombin III and elevation of factor VIII in a child with ischemic stroke: a case report.

A 4-year-old boy was admitted with left hemiplegia. Thrombophilia marker examination resulted with factor V Leiden mutation heterozygosity, a deficiency of antithrombin III and a high level of factor VIII. Cranial computed tomography scan revealed an ischemic infarct in the region of right anterior cerebral artery. In the literature, combinations of multiple thrombophilia risk factors that trigger cerebral ischemic stroke in children have been emphasized. To our knowledge, this is the first child with these combinations of thrombophilia risk factors and ischemic stroke to be reported in the literature.

Antithrombin III Deficiency↗
PubMed2005

Constitutional trisomy 8 mosaicism with myelodysplastic syndrome complicated by intestinal Behcet disease and antithrombin III deficiency.

Trisomy 8 is the most common acquired chromosomal abnormality associated with myeloid malignancy. As a constitutional trisomy 8 mosaicism (T8M), it exhibits an extremely variable phenotype. In addition, Behcet disease (BD) has been reported as an unusual complication of myelodysplastic syndrome (MDS). To our knowledge, 12 case reports of various hematologic malignancies in patients with T8M and 18 case reports of MDS with acquired trisomy 8 complicated by BD have been published to date. We report a case of constitutional T8M with MDS complicated by intestinal BD and antithrombin III deficiency.

Antithrombin III Deficiency↗
PubMed2006

Thrombin inhibition by antithrombin in the presence of oversulfated dermatan sulfates.

DSS1 and DSS2 are two oversulfated dermatan sulfate derivatives with sulfur contents of 7.8% and 11.5%, respectively. DSS1 and DSS2 both enhanced the rate at which antithrombin (AT) inactivates thrombin according to a concentration dependent manner. The analysis of the experimental data, using our previously described kinetic model [Biomaterials1997, 18, 203] (i) suggested that both DSS1 and DSS2 catalyzed the thrombin-AT reaction according to a mechanism in which the oversulfated derivative quickly formed with AT a complex, which was more reactive towards thrombin than the free inhibitor and (ii) allowed us to determine the dissociation constants of the polysaccharide-inhibitor complexes, which were (1.15 +/- 0.74) x 10(-7) and (7.17 +/- 0.65) x 10(-9) M, and the catalyzed reaction rate constants, which were (2.29 +/- 0.15) x 10(8) and (8.71 +/- 0.08) x 10(8) M(-1) min(-1), for DSS1 and DSS2, respectively. These data suggested that the oversulfation confers an affinity for AT to dermatan sulfate and that the higher the sulfur content the higher the affinity for AT. They also suggested that the reactivities of the polysaccharide-AT complexes formed towards the protease increased with the sulfur content.

Antithrombins↗
PubMed2005

On the variation of glycosylation in human plasma derived antithrombin.

The paper presents data on the primary structure of the glycan variants present in human antithrombin (AT) isoforms obtained from a plasma pool. The analysis is conducted on the level of glycopeptides gained by tryptic digestion. The glycopeptides were pre-separated by lectin-affinity chromatography and analyzed by means of electrospray ionization-tandem mass spectrometry involving collision-induced dissociation. Variations of the canonical biantennary complex-type structure were present with relative abundances of about 1-5% and most of them were found site-specifically. Core fucosylation was observed at one single glycopeptide only (peptide containing N155), triantennary glycan structures with two glycopeptides (containing N155 and N135). Deficiency of one terminal sialic acid was observed as not site-specific. Fucosylation was not yet reported to be present in human AT from plasma, opposite to recombinant human AT from baby hamster kidney cells, which was reported as fully core fucosylated. In total, the variability in the carbohydrate structure of plasma derived AT appears as being quite limited. This might be of significance in the context of the reported correlation between glycosylation and physiological activity.

Antithrombins↗
PubMed2006

Determination of the binding parameters for antithrombin-heparin fragment systems by affinity and frontal analysis continuous capillary electrophoresis.

The suitability of affinity capillary electrophoresis (ACE) and frontal analysis continuous capillary electrophoresis (FACCE) for binding constant determination was investigated for complexes between heparin fragments and antithrombin III, one of the main target proteins in the coagulation cascade. In a 100 mM ionic strength phosphate buffer (pH 7.4), ACE was suitable to determine weak to medium interactions developed by short oligomeric heparin fragments, but it failed for decasaccharide, which presents a more complex irreversible interaction. However FACCE allowed evaluating the binding constant for these longer oligomeric fragments. Both experimental approaches were complementary for a wide variety of heparinic fragments.

Antithrombins↗
PubMed2006

Allosteric activation of antithrombin is independent of charge neutralization or reversal in the heparin binding site.

We investigate the hypothesis that heparin activates antithrombin (AT) by relieving electrostatic strain within helix D. Mutation of residues K125 and R129 to either Ala or Glu abrogated heparin binding, but did not activate AT towards inhibition of factors IXa or Xa. However, substitution of residues C-terminal to helix D (R132 and K133) to Ala had minimal effect on heparin affinity but resulted in appreciable activation. We conclude that charge neutralization or reversal in the heparin binding site does not drive the activating conformational change of AT, and that the role of helix D elongation is to stabilize the activated state.

Allosteric Regulation↗
PubMed2005

A novel matrix for high performance affinity chromatography and its application in the purification of antithrombin III.

Viscose fiber, a regenerated cellulose, was evaluated for using as a novel matrix for high performance affinity chromatography. With a one-step activation with epichlorohydrin, heparin can be readily covalently attached to the matrix. This heparin-viscose fiber material was used for purifying antithrombin III (AT III) from human plasma. The purity of the AT III from this one-step purification is 93% as measured by SDS-PAGE and the protein recovery yield is about 90%. This column is highly specific as described by the dissociation constant of the complex of immobilized heparin and AT III, which was 2.83 x 10(-5)mol/L. And more important, this viscose fiber material demonstrated its excellent mechanical property that allows the flow rate to reach up to 900 cm/h or more.

Antithrombin III↗
PubMed2005

Insights into the induced fit mechanism in antithrombin-heparin interaction using molecular dynamics simulations.

Heparin was isolated in the beginning of the 20th century and until today remains as one of the most important drugs able to interfere with the haemostatic process. Due to the side effects produced by heparin therapy, new promising drugs have been developed, as the synthetic pentasaccharide (synthetically derived from the sequence GlcN-GlcA-GlcN-IdoA-GlcN). The anticoagulant activity of this compound is based on potentiation of antithrombin (AT) inhibitory activity upon serine proteinases of clotting cascade, a mechanism based on the conformational modification of AT. In this context, we present here a molecular dynamics (MD) study of the interaction between the synthetic pentasaccharide and AT. The obtained data correctly predicted an induced fit mechanism in AT-pentasaccharide interaction, showing a solvent-exposed P1 residue instead of a hided conformation. Also, the specific contribution of important amino acid residues to the overall process was also characterized, both in (2)S(0) and (1)C(4) conformations of IdoA residue, suggesting that there is no conformational requirement to the interaction of this residue with AT. Altogether, the results show that MD simulations could be used to characterize and quantify the interaction of synthetic compounds with AT, predicting its specific capacity to induce conformational changes in AT structure. Thus, MD simulations of heparin (and heparin-derived)-AT interactions are proposed here as a powerful tool to assist and support drug design of new antithrombotic agents.

Allosteric Regulation↗
PubMed2005

Mechanism of antithrombotic effect of heparin and antithrombin in balloon-injured arteries.

The mechanism of the antithrombotic effects of heparin and the synthetic antithrombin agent argatroban was evaluated in a dog model. Thrombus formation following balloon injury was evaluated by angioscopy in the right iliac arteries of 20 dogs to serve as a control. After the evaluation of the growth of thrombus on the control side, heparin (200 U/kg) or argatroban (0.2 mg/kg) was infused intravenously, and the distal site of the contralateral left iliac artery was injured in the same manner. At 30 to 120 min before the final examination, the proximal site of the left iliac artery also was injured. After antithrombotic drug infusion, the percent angioscopic stenosis at the distal site was much lower (P<0.0001) than that of the control site (mean stenosis index: 0.67 in heparin vs. 3.8 in control, and 0.25 in argatroban vs. 4.3 in control); however, thrombus formation was observed at the proximal site. With local delivery of a low dose of either antithrombotic drug (n=10), an antithrombotic effect was maintained until 4 h after the infusion. A very weak fluorescence of FITC-heparin was detected at the injured artery 2 h after infusion. In a shunt experiment involving 5 dogs, carotid arteries were injured and incubated in oxygenated Krebs--Henseleit solution before auto grafting into the femoral artery. At 2 h after the grafting, no thrombus was formed in the grafted vessels incubated for 4 h, but was formed in those incubated for 10 min. These results indicate that the relatively long antithrombotic effect of these drugs may be due to a local drug effect at the injured artery, as well as the recovery of the anti-thrombogenicity of the injured artery.

Angiography↗
PubMed2006

[Homozygous antithrombin type HBS deficiency; a family study].

Congenital antithrombin (AT) deficiency is the most thrombotic genetic abnormality of haemostasis. Total quantitative deficits are lethal as early as life intra-uterine. Only homozygous mutations concerning the heparin-binding site are compatible with life. We report here the case of an 18 years old patient with recurrent deep venous thrombosis of the inferior members. Haemostasis exploration shows a decreased AT activity (11%) in the presence of heparin while AT progressive activity and AT antigen are normal. Two other homozygous sisters are identified in this family study. Molecular study of AT gene show Arg47-Cys substitution, already reported in the literature with patients of different geographic origins. Treatment of patients with homozygous AT type HBS deficiency is similar that for patients with heterozygous AT deficiency; a continuous prophylactic anticoagulant treatment is always necessary and AT concentrates infusions are required in all situations needing curative heparin treatment.

Adolescent↗
PubMed2005

Purification and characterization of recombinant human antithrombin containing the prelatent form in Chinese hamster ovary cells.

Antithrombin (AT) is a serine proteinase inhibitor and a major regulator of the blood coagulation cascade. AT in human plasma has two isoforms, a predominant alpha-isoform and a minor beta-isoform; the latter lacks N-glycosylation at Asn 135 and has a higher heparin affinity. From the difference in its folding states, the AT molecule can be separated into three forms: a native form, a denatured and inactive form known as the latent form, and a partially denatured form called the prelatent form. In this study, we purified and characterized recombinant human AT (rAT) containing the prelatent form produced by Chinese hamster ovary (CHO) cells. When rAT was purified at physiological pH, its specific activity was lower than that of plasma-derived human AT (pAT). The latent and prelatent forms were detected in rAT by using hydrophobic interaction chromatography analysis. However, when rAT was purified at alkaline pH, the prelatent form was reversibly folded to the native form and the inhibitory activity of rAT increased to a value similar to that of pAT. Highly purified rAT was analyzed and compared with pAT by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, amino acid composition, N-terminal sequence, monosaccharide composition, peptide mapping, and heparin-binding affinity. From these analyses, rAT was found to be structurally identical to pAT, except for carbohydrate side-chains. rAT in CHO cells had a high beta-isoform content and it caused a higher heparin affinity than by pAT and also pH-dependent reversible inhibitory activity.

Animals↗
PubMed2004

Heparin cofactor II is more sensitive than antithrombin to secretory impairment arising from mutations introduced into its carboxy-terminal region.

INTRODUCTION: Antithrombin (AT) and heparin cofactor II (HCII) are plasma glycoproteins and serpins that inhibit thrombin. We showed [Blood 86 (1995) 3461] that recombinant rabbit AT containing the Utah mutation of AT, P407L, was inefficiently secreted by transfected primate and rodent cultured cells. In the current study, the effects of P407L and related substitutions in human AT and human HCII were investigated. MATERIALS AND METHODS: Cultured cells were transfected transiently (COS-1) or permanently (CV-1) with AT and HCII expression vectors encoding the wild type or mutant serpins. The amount of protein secreted was determined immunologically, while RNA levels were assessed by reverse-transcription-PCR (RT-PCR). The kinetics of secretion were investigated by pulse-chase experiments, supplemented by endoglycosidase H or lactacystin treatment. RESULTS AND CONCLUSIONS: The F450L, P455L, P477L, P477*, and T446* (*=stop codon) mutations reduced HCII secretion 6.6- to 24-fold, while the F402L, A404T, and P407L mutations reduced AT secretion in COS-1 cells 1.7- to 5.2-fold. Homologous mutants HCII (P455L) and AT (P407L) were transcribed at similar levels in COS-1 cells, but were secreted less rapidly and less efficiently than their wild-type counterparts. HCII (P455L) exhibited intracellular proteasomal degradation in permanently transfected CV-1 cells, while AT (P407L) secretion was unaffected in this milieu. HCII secretion is thus more sensitive than that of AT to C-terminal mutations, as shown in two primate cell lines, likely reflecting a greater tendency to misfold during synthesis. We speculate that this difference may arise due to an interstrand s1C/s4B loop that is shorter in HCII than in AT.

Amino Acid Substitution↗
PubMed2005

Antithrombin replacement in neonates: is there any indication?

INTRODUCTION: Activation of the coagulation system and severe acquired antithrombin (AT) deficiency are common and prognostically important findings in sick and preterm neonates. It has been hypothesised that treatment of the acquired AT deficiency with AT concentrate may improve the outcome of conditions such as the neonatal respiratory distress syndrome (RDS), intracranial hemorrhage (ICH) and sepsis. MATERIALS AND METHODS: We performed a systematic review of randomised controlled trials (RCTs) of AT replacement therapy in newborn infants. RESULTS: Two full-length trial reports were found. Both were placebo-controlled. The first RCT examined the effects of AT therapy in 122 preterm infants with RDS. Administration of AT prolonged rather than shortened the duration of mechanical ventilation and oxygen therapy. The second RCT determined whether AT replacement decreased the incidence of ICH in 60 preterm infants who were born before 30 weeks of gestation. No beneficial effect on ICH was found. CONCLUSIONS: Preterm infants with RDS do not benefit from therapy with AT concentrate and may be harmed. There is also little evidence that the administration of AT reduces the risk of ICH. The role of AT replacement during neonatal sepsis remains uncertain.

Antithrombin III↗
PubMed2005

A collaborative study to establish a Korea national biological standard for antithrombin concentrate.

BACKGROUND AND OBJECTIVES: Six laboratories consisting of three manufacturers and three national control laboratories participated in a collaborative study to evaluate the suitability of a candidate material to serve as the first Korean National Standard for Antithrombin (AT) concentrate. MATERIALS AND METHODS: The potency of this candidate preparation was determined using the heparin cofactor chromogenic method. The method is described in the Minimum Requirements for Biological Products in Korea and in the European Pharmacopoeia. The candidate was calibrated against the second International Standard for AT concentrate, coded as 96/520. RESULTS: The participants contributed data from a total of 90 independent assays and the results were accepted as statistically valid when the outcome of the analysis exhibited linear dose-response relationships and intersected at a common point at zero dose in the slope-ratio model. The combined potency estimates were obtained by taking the geometric means of results from all assays at each laboratory, and overall potency estimates were calculated as unweighted geometric means of results from all laboratories. The results were expressed in the form of histograms with 95% confidence intervals. CONCLUSIONS: According to the results of the collaborative study, the candidate preparation showed excellent intra- and inter-laboratory correlations and is judged to be suitable to serve as the Korean National Standard for AT concentrate with the following potency: 51.9 IU/vial (95% confidence intervals=48.24 approximately 55.98 IU/vial).

Antithrombins↗
PubMed2006

Heparin chain-length dependence of factor Xa inhibition by antithrombin in plasma.

Heparin anticoagulants function by enhancing the inhibition of coagulation proteases by the serpin antithrombin (AT). A direct evaluation of the specific anti-factor Xa (fXa) activity of therapeutic heparins in the physiologically relevant plasma-based clotting assays has not been feasible since thrombin, the final protease of the cascade, is the primary target for inhibition by AT in the presence of heparin. To circumvent this problem, we developed an assay in which the native AT in plasma was replaced with an AT mutant which exhibits identical affinity for heparin and near normal reactivity for fXa, but does not react with thrombin and other coagulation proteases in either the absence or presence of heparin. This assay was used to distinguish the anti-fXa activity of different molecular weight heparins from their anti-thrombin activity in clotting assays which were initiated by the triggers of either the extrinsic or intrinsic coagulation pathway. The results suggest that the acceleration of fXa inhibition by AT exhibits a marked heparin chain-length dependence, with fondaparinux (a pentasaccharide) having the lowest and unfractionated heparin having the highest effect. Interestingly, comparative studies revealed that the fondaparinux-catalyzed acceleration of thrombin inhibition by AT also contributes to the prolongation of the clotting time, possibly suggesting that the anticoagulant function of the therapeutic pentasaccharide is mediated though the inhibition of both fXa and thrombin.

Anticoagulants↗
PubMed1990

Intravenous nitroglycerin-induced heparin resistance: a qualitative antithrombin III abnormality.

An ability of intravenous nitroglycerin to interfere with the anticoagulant properties of intravenous heparin would have profound clinical implications. To investigation nitroglycerin-heparin interactions, the following pilot study was performed. Patients (N = 18) admitted to the coronary care unit with a diagnosis of either acute myocardial infarction or unstable angina were divided into four treatment groups: (1) intravenous nitroglycerin and intravenous heparin; (2) intravenous nitroglycerin alone; (3) intravenous heparin alone; or (4) neither intravenous nitroglycerin nor intravenous heparin. Serial determinations of activated partial thromboplastin time (APTT), serum heparin concentration, antithrombin III (ATIII) antigen (ATA), and ATIII activity (ATC) were obtained over a 72-hour period. Overall, patients receiving intravenous nitroglycerin did not differ significantly from other patients in APTT, heparin dose, heparin concentration, ATA, ATC, or ATA/ATC ratio (ATR). However, patients receiving intravenous nitroglycerin at a rate exceeding 350 micrograms per minute had a lower APTT (p less than 0.05), lower ATC (p = 0.02), higher ATR (p = 0.004), and a larger heparin dose requirement than patients receiving lower infusion rates. ATR correlated directly (r = 0.91; p less than 0.05) and ATC inversely (r = -0.78; p less than 0.05) with the intravenous nitroglycerin dose. Serum heparin concentration did not correlate with the intravenous nitroglycerin dose. Intravenous nitroglycerin-induced heparin resistance occurs at a critical nitroglycerin dose. A nitroglycerin-induced qualitative ATIII abnormality may be the underlying mechanism.

Adult↗