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At least 145 records · Page 8Linked to original sources

PubMed1998

Antithrombin III in patients with severe sepsis. A randomized, placebo-controlled, double-blind multicenter trial plus a meta-analysis on all randomized, placebo-controlled, double-blind trials with antithrombin III in severe sepsis.

OBJECTIVES: To evaluate the safety and potential efficacy of antithrombin III (AT III) in reducing mortality in patients with severe sepsis. DESIGN: Prospective, randomized, placebo-controlled, double-blind, phase II, multicenter, multinational clinical trial. SETTING: Seven academic medical center intensive care units (ICU) in Belgium, Denmark, the Netherlands, Norway and Sweden. PATIENTS: 42 patients with severe sepsis who received standard supportive care and antimicrobial therapy, in addition to the administration of AT III or placebo. INTERVENTIONS: Patients received either an intravenous loading dose of 3000 IU AT III followed by a maintenance dose of 1500 IU every 12 h for 5 days or equivalent amounts of placebo. MEASUREMENTS AND RESULTS: All patients were evaluated for safety and for 30-day all-cause mortality. CONCLUSIONS: The administration of AT III was safe and well-tolerated. It was followed by a 39 % reduction in 30-day all-cause mortality (NS). The reduction in mortality was accompanied by a considerably shorter stay in the ICU. Patients treated with AT III exhibited a better performance in overall severity of illness and organ failure scores (Acute Physiology and Chronic Health Evaluation II, multiple organ failure, organ system failure), which was noticeable soon after initiation of treatment. Patients treated with AT III demonstrated a better resolution of pre-existing organ failures and a lower incidence of new organ failures during the observation period. A meta-analysis comprising this and two other double-blind, placebo-controlled trials with AT III with a total of 122 patients suffering from severe sepsis confirms the positive trend. The results of the meta-analysis demonstrate a 22.9 % reduction in 30-day all-cause mortality in patients treated with AT III. Although still too small to be confirmative, the meta-analysis clearly points to the fact that a sufficiently powered phase III trial is warranted to prove whether AT III has a beneficial role in the treatment of severe sepsis.

APACHE↗

PubMed1976

Hereditary antithrombin III deficiency. Effect of antithrombin III deficiency on platelet function.

Antithrombin III (AT III) is the main physiologic inhibitor of thrombin, and activated factors X and IX as well. Normal levels of AT III appear to be necessary to maintain blood fluidity and to prevent thrombosis. Four families with AT III deficiency and recurrent venous thromboembolism have been reported on. We present an additional family with AT III deficiency and a high incidence of thromboembolism. AT III levels were determined by both a functional and an immunologic assay. Results of platelet function tests, not previously reported in persons with AT III deficiency, were found to be normal. Following gel filtration, the platelets were very sensitive to thrombin. Thrombin-induced platelet aggregation appears to be dependent on a balance between the amount of thrombin and AT III present.

Adolescent↗

PubMed1992

Enzyme-linked immunosorbent assay for proteolytically inactivated antithrombin-III: use of sodium dodecyl sulfate to eliminate signal due to intact antithrombin-III.

Antithrombin III (AT-III) is a serine protease inhibitor (serpin) that can be catalytically inactivated by human neutrophil elastase (HNE) without inhibiting HNE activity. As with catalytic inactivation of most serpins, the cleaved form of the inhibitor is difficult to measure in the presence of active inhibitor. One major difference between the cleaved and intact forms of AT-III is that the cleaved form adopts a more stable conformation. Using sodium dodecyl sulfate (SDS), we were able to devise an enzyme-linked immunosorbent assay (ELISA) capable of detecting cleaved AT-III in the presence of intact AT-III. It seems likely that the SDS alters the intact AT-III so that it is not detected in the ELISA. As little as 5 micrograms/ml HNE-cleaved AT-III could be detected when spiked into human plasma; HNE-cleaved AT-III spiked into human plasma at different levels was recovered as expected. Thrombin-cleaved AT-III was also detected using this ELISA. The generation of cleaved AT-III in human plasma by HNE in the presence of heparin could be monitored as well. The cleaved AT-III ELISA is a novel, yet simple way to measure proteolytically inactivated AT-III in the presence of intact AT-III and should be useful for studying the role of proteolytic inactivation of serpins such as AT-III in vivo.

Antithrombin III↗

PubMed1992

Antithrombin Budapest 3. An antithrombin variant with reduced heparin affinity resulting from the substitution L99F.

The molecular basis and functional properties of a variant antithrombin (AT) protein. AT Budapest 3, were studied. A single base substitution was identified in codon 99, CTC----TTC, altering the normal leucine to phenylalanine. The proband presented with a history of venous thrombotic disease and was found to be homozygous for the mutation. The variant protein demonstrated reduced heparin affinity and reduced antiproteinase activity in the presence of either unfractionated heparin or the AT-binding heparin pentasaccharide, when compared to normal AT. A small change in the isoelectric point was also identified. The substituted amino acid residue of AT Budapest 3 is located near to the proposed AT heparin binding site, and it is suggested that reduced heparin affinity of the variant protein may result from substitution-induced distortion of positive charge geometry in the binding site and/or changes in its position relative to the rest of the inhibitor molecule.

Amino Acid Sequence↗

PubMed1983

Further studies of the turnover of dog antithrombin III. Study of 131I-labelled antithrombin protease complexes.

Fresh plasma containing 131I-antithrombin III (*I-AT) was coagulated and incubated at 37 degrees C for 2 hr. A "complex peak," separated on heparin-agarose contained AT and *I-AT antigen but no heparin cofactor activity. Crossed immunoelectrophoresis showed only AT complexes. SDS PAGE showed 80% of the *I-AT in a major band (approximately 80,000 daltons), 15% in a minor band (approximately 100,000 daltons) and the rest in trace bands (approximately 60,000 and/or 115,000 daltons). Ammonia treatment of the complex peak released alpha-thrombin. After i.v. injection 80% of the complexed *I-AT, chiefly as the major band, left the plasma with t 1/2 approximately 15 min and was almost immediately catabolized to low molecular weight breakdown products. A major catabolic site was the liver. A simple kinetic model describes the findings approximately.

Animals↗

PubMed1990

The effect of sulfation on the anticoagulant and antithrombin III-binding properties of a heparin fraction with low affinity for antithrombin III.

Heparin with low affinity for antithrombin III (ATIII) and devoid of anticoagulant activity was chemically oversulfated and fractionated by affinity for ATIII. The oversulfated material showed ATIII binding properties, as monitored by intrinsic fluorescence enhancement of ATIII. The fluorescence increase was comparable to that of the AT III high affinity fraction of native heparin. The estimated dissociation constants however, showed a 10-fold weaker binding of the oversulfated material to ATIII, Kd = 6.4 x 10(-8) M, as compared to native heparin, Kd = 0.63 x 10(-8) M. Concomitant with the binding-induced allosteric change in ATIII, the oversulfated material stimulated the ATIII-thrombin and ATIII-factor Xa reactions. The high affinity fractions of native heparin and the sulfated material were almost equally effective in enhancing the rate of thrombin neutralization by ATIII. However, a 3-fold faster rate of factor Xa inactivation was found with the native high affinity material.

Antithrombin III↗

PubMed1992

The pregnant antithrombin III deficient patient: management without antithrombin III concentrate.

Pregnant patients with antithrombin III (AT III) deficiency have an unacceptably high risk of venous thromboembolism (VTE). Antithrombotic therapy is therefore recommended. The reported clinical experience of such prophylaxis is limited. Some authors have recommended the use of AT III concentrate in addition to heparin in the management of these patients. We report successful management with heparin alone during pregnancy and the postpartum period in two patients with AT III deficiency. Both patients had experienced VTE during a prior pregnancy; one also experienced VTE during the reported pregnancy. Both patients were therefore at particularly high risk of further VTE. Based on the good results in these two patients, and a review of previously reported cases, we propose that heparin alone, in a dose to maintain the APTT in a therapeutic range, provides adequate prophylaxis and treatment for VTE during pregnancy and delivery in many AT III deficient subjects.

Adult↗

PubMed2003

Isoform composition of antithrombin in a covalent antithrombin-heparin complex.

Antithrombin (AT) circulates in two isoforms, alpha- (90-95%) and beta-AT (5-10%). AT inhibits clotting factors such as thrombin and factor Xa, a reaction catalyzed by heparin. Heparin has been used in many clinical situations but suffers from limitations such as a short intravenous half-life, bleeding risk, and the inability to inhibit thrombin bound to fibrin clots. In order to overcome some of heparin's limitations, we prepared a covalent AT-heparin complex (ATH) that has increased intravenous half-life, reduced bleeding risk, and can directly inhibit clot-bound thrombin. However, structural analysis is required to further develop this promising antithrombotic agent. It was found that the proportion of isoforms in ATH (55% alpha-AT, and 45% beta-AT) was significantly different than that in the commercial AT starting material (80% alpha-AT and 20% beta-AT). Further analysis of the rate of heparin-catalyzed inhibition of thrombin by AT isoforms prepared from ATH revealed that the beta-variant reacted approximately 2-fold faster.

Antithrombins↗

PubMed2004

Pasteurization of antithrombin without generation of the prelatent form of antithrombin.

Human antithrombin (AT) is the major inhibitor of blood coagulation and has also been shown to exert anti-inflammatory and anti-angiogenic effects. Pasteurization of pharmaceutical AT products is usually performed at 60 degrees C for 10h in the presence of sodium citrate as stabilizer, sometimes in combination with sucrose. These stabilizers significantly decrease the aggregation and denaturation of AT, but during the pasteurization, a small amount of latent AT (LAT), a partially denatured form, is usually generated, as is an equal amount of another latent form of AT, the so-called prelatent AT (PLAT). The LAT formed during pasteurization has a rather low affinity to heparin and is easily removed by using a second heparin affinity chromatography step in the production process. This is in contrast to the PLAT, which has a slightly lower affinity to heparin than does native AT, which makes it hard to remove. Hence, four commercial products of pasteurized AT were previously shown to contain about 4% of PLAT. In the present work, an alternative pasteurization method is presented, where 2M ammonium sulfate and 50% sucrose are used as stabilizers. During this pasteurization, no, or trace amounts ( < 0.5%), of PLAT may be generated with no formation of aggregates. Moreover, the pasteurized AT has the same specific thrombin-inhibiting activity when compared to incubation in the presence of citrate and sucrose. Heparin affinity high-performance liquid chromatography was used for the determination of PLAT, LAT, and AT.

Antithrombins↗

PubMed1995

Partial glycosylation of antithrombin III asparagine-135 is caused by the serine in the third position of its N-glycosylation consensus sequence and is responsible for production of the beta-antithrombin III isoform with enhanced heparin affinity.

Two antithrombin III (ATIII) isoforms occur naturally in human plasma. The alpha-ATIII isoform has four N-linked oligosaccharides attached to asparagines 96, 135, 155, and 192. The beta-ATIII isoform lacks carbohydrate on asparagine-135 (N135), which is near the heparin binding site, and binds heparin with higher affinity than does alpha-ATIII. Two isoforms are also produced when the normal human ATIII cDNA sequence is expressed in baculovirus-infected insect cells, and the recombinant beta' isoform similarly binds heparin with higher affinity than the recombinant alpha' isoform. Consensus sequences (CSs) of the ATIII N-glycosylation sites are N-X-S for 135 and N-X-T for 96, 155, and 192. On the basis of database and in vitro glycosylation studies suggesting that N-X-S CSs are utilized less efficiently than N-X-T CSs, we hypothesized that the beta-ATIII isoform might result from inefficient core glycosylation of the N135 N-X-S CS due to the presence of a serine, rather than a threonine, in the third position. ATIIIs with N-X-S, N-X-T, and N-X-A consensus sequences were expressed in baculovirus-infected insect cells. In contrast to the N-X-S sequence, which expressed a mixture of alpha' and beta' molecules, the N-X-T variant produced alpha' exclusively, while the N-X-A variant produced beta' exclusively. Thus, serine in the third position of the N135 CS is responsible for its "partial" glycosylation and leads to production of beta-ATIII.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2006

Antithrombin-S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation.

Regulation of blood coagulation is critical for maintaining blood flow, while preventing excessive bleeding or thrombosis. One of the principal regulatory mechanisms involves heparin activation of the serpin antithrombin (AT). Inhibition of several coagulation proteases is accelerated by up to 10,000-fold by heparin, either through bridging AT and the protease or by inducing allosteric changes in the properties of AT. The anticoagulant effect of short heparin chains, including the minimal AT-specific pentasaccharide, is mediated exclusively through the allosteric activation of AT towards efficient inhibition of coagulation factors (f) IXa and Xa. Here we present the crystallographic structure of the recognition (Michaelis) complex between heparin-activated AT and S195A fXa, revealing the extensive exosite contacts that confer specificity. The heparin-induced conformational change in AT is required to allow simultaneous contacts within the active site and two distinct exosites of fXa (36-loop and the autolysis loop). This structure explains the molecular basis of protease recognition by AT, and the mechanism of action of the important therapeutic low-molecular-weight heparins.

Allosteric Regulation↗

PubMed2003

Use of recombinant human antithrombin in patients with congenital antithrombin deficiency undergoing surgical procedures.

BACKGROUND: Hereditary antithrombin (AT) deficiency is associated with a significant risk of venous thromboembolism. Patients with this disorder frequently require long-term anticoagulation. Discontinuation of anticoagulation for childbirth or surgery may carry a substantial thrombotic risk. For this reason, replacement with AT concentrate has been used when anticoagulation is interrupted. A new recombinant human AT concentrate, produced using transgenic technology, has recently been developed. STUDY DESIGN AND METHODS: Human recombinant AT (rhAT) was provided by GTC Biotherapeutics, Inc. on a compassionate-use basis for five patients with hereditary AT deficiency who underwent six surgical procedures. Patients were treated perioperatively. Dosing was determined individually by the investigators with a goal of maintaining an AT activity of 80 to 150 percent. RESULTS: There was no clinical evidence of thrombosis or bleeding. Four of the five patients had postoperative duplex ultrasound studies of the lower extremities, which showed no evidence of acute thrombosis. Four patients were tested for antirecombinant rhAT antibodies postoperatively and were negative. CONCLUSION: These case reports indicate that rhAT can provide effective support for AT-deficient patients who undergo surgery. Further study of this product is needed to define optimal dosing and further assess clinical response.

Adult↗

PubMed2000

Stimulation of pulmonary big endothelin-1 and endothelin-1 by antithrombin III: a rationale for combined application of antithrombin III and endothelin antagonists in sepsis-related acute respiratory distress syndrome?

OBJECTIVES: Antithrombin (AT) III reduces lung damage in animal models of septic acute respiratory distress syndrome (ARDS), which is generally attributed to stimulation of endothelial prostacyclin synthesis. However, clinical studies have failed so far to demonstrate mortality reduction by application of AT III. We investigated whether AT III stimulates pulmonary prostacyclin release. In addition, we hypothesized that it may promote pulmonary endothelins, thereby mitigating its own protective effect in the course of ARDS. DESIGN: Controlled experiment using isolated organs. SETTING: Experimental laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Isolated lungs were perfused over 120 mins in recirculatory mode in the presence of 50 microg/mL endotoxin (n = 11), 2U/mL AT III (n = 10), 5 U/mL AT III (n = 13), endotoxin plus 2 U/mL AT III (n = 5), or vehicle alone (controls, n = 13), respectively. MEASUREMENTS AND MAIN RESULTS: We determined the effects of AT III on vascular release of thromboxane B2, 6-keto-prostaglandin-F1alpha, big endothelin-1, and endothelin-1. Control lungs released 59+/-23 pg/mL thromboxane B2, 1,480+/-364 pg/mL 6-keto-prostaglandin-F1alpha, 15.2+/-4.5 pg/mL big endothelin-1, and 0.46+/-0.13 pg/mL endothelin-1. Exposure to endotoxin increased thromboxane B2 release 2.9-fold, 6-keto-prostaglandin-F1alpha release 1.6-fold, and endothelin-1 1.6-fold (p < .05 each); levels of big endothelin-1 were unchanged. AT III at 2 U/mL elevated production of big endothelin-1 (1.7-fold) and endothelin-1 (1.2-fold) (p < .05 for both). AT III at 5 U/mL enhanced levels of big endothelin-1 (1.6-fold) and endothelin-1 (1.3-fold) (p < .05 for both). Neither dose of AT III affected thromboxane B2 or 6-keto-prostaglandin-F1alpha concentrations. Application of 2 U/mL AT III plus endotoxin stimulated big endothelin-1 production (2.6-fold) compared with endotoxin or AT III alone (p < .05 for both), but did not further elevate endothelin-1 release. CONCLUSIONS: AT III does not stimulate pulmonary prostacyclin, but promotes pulmonary release of big endothelin-1 and endothelin-1 under basal and, particularly, under septic conditions, which may blunt the AT III-induced lung protection during ARDS. Therefore, we suggest combined application of AT III and endothelin antagonists in animal models of septic ARDS.

Analysis of Variance↗

PubMed1996

Antithrombin III prevents renal dysfunction and hypertension induced by enhanced intravascular coagulation in pregnant rats: pharmacological confirmation of the benefits of treatment with antithrombin III in preeclampsia.

We tested the hypothesis that enhanced intravascular coagulation in pregnancy could produce clinical symptoms similar to those of preeclampsia, such as hypertension, proteinuria, and edema. Having confirmed this, we then examined whether the pathological changes caused by intravascular coagulation could be suppressed by administration of antithrombin III (AT III), an endogenous inhibitor active to thrombin and factor X a. Intravascular coagulation was induced in Wistar rats on day 16-20 of pregnancy by 1-h arterial infusion of tissue thromboplastin (TP) through the left ventricle of the heart. One hour after the end of the infusion period, organ blood flows were measured by the radioactive ((57)Co-labeled) microsphere method, and fibrin deposition in organs was measured by radiolabeling with [(125)I] fibrinogen injected before TP infusion. Infusion of TP produced fibrin deposition in the kidney, lung, and liver, but not in the myometrium and placenta, as well as an 80% decrease in renal blood flow (RBF), with oliguria and proteinuria. TP also caused an increase in blood pressure (BP) accompanied by an increase in plasma renin activity (PRA), both of which were suppressed by bilateral nephrectomy before TP infusion. The prophylactic administration of AT III concentrates (60 or 300 U/kg intravenously (i.v.), followed by infusion of 30 or 150 U/kg/2 h, respectively) prevented all pathological changes in a dose-dependent manner. AT III increased placental blood flow regardless of the state of coagulation. These findings suggest that intravascular coagulation plays a significant part in the pathophysiology of preeclampsia and that AT III concentrates may have therapeutic potential in the treatment of this condition.

Animals↗

PubMed2006

The cleaved and latent forms of antithrombin are normal constituents of blood plasma: a quantitative method to measure cleaved antithrombin.

Antithrombin (AT), a member of the serine protease inhibitor family, is the key regulator of thrombin activity in vivo. Thrombin inhibition is accomplished by the formation of covalent thrombin-AT (TAT) complex. The rate of inhibition is accelerated by heparin, which also leads to the formation of a substantial amount of cleaved AT. We produced a murine monoclonal antibody (mAb) (M9) that is specific for the two forms of AT, in which the reactive center loop is inserted into beta-sheet A, i.e. cleaved and latent AT. The antibody has no measurable affinity for native AT. Using M9 as a catcher antibody in conjunction with a mAb (M27) that does not bind latent AT, we developed a sandwich assay that measures cleaved AT without interference from latent and native AT. The concentration in healthy subjects was determined to be 1.3 mg L(-1) (range: 1.0-1.9), which was about 100-fold lower than the plasma concentration of native AT and 1000-fold higher than the concentration of the TAT complex. The cleaved AT concentration is higher than what would be expected from the rate of formation of cleaved AT in vitro in conjunction with TAT complex formation in the presence of heparin. The concentration of cleaved AT did not correlate with the TAT concentration in plasma from patients with venous thrombosis.

Animals↗

PubMed2006

An immunochemical method for quantitative determination of latent antithrombin, the reactive center loop-inserted uncleaved form of antithrombin.

Antithrombin (AT) is a serine protease inhibitor that has thrombin, factors IXa and Xa as target proteases. In addition to active native AT, two other forms have been identified in plasma: the reactive center loop inserted cleaved and latent, uncleaved forms. Both have been shown to be present in normal human blood. Latent AT forms a dimer with native AT in vitro, thus inactivating the native form. Here we describe a mouse monoclonal antibody, 8C8, that is specific for latent AT. The affinity of 8C8 was found to be 500-fold higher for latent than for native AT and 5000-fold higher for latent than for cleaved AT. A sandwich assay was developed to measure the concentration of latent AT in plasma, which was found to be approximately 4.8 mg L(-1) in healthy individuals. The K(D) of the interaction between native and latent AT was found to be 51 mum, i.e. far above the plasma concentration of both native and latent AT, indicating a negligible complex formation in blood.

Adult↗

PubMedDate not supplied

Quantification of antithrombin isoform proportions in plasma samples of healthy subjects, sepsis patients, and in antithrombin concentrates.

Antithrombin (AT) circulates in plasma in two isoforms, AT-alpha (90-95%) and AT-beta (5-10%). AT isoform proportions were measured in plasma samples of 17 healthy subjects and 26 posttraumatic or postoperative septic patients, as well as in 4 commercially available AT concentrates. Total AT was immune-purified from plasma and concentrates. Micellar electrokinetic chromatography was used to analytically separate and quantify the isoforms. Compared with plasma samples of healthy donors, septic plasmas revealed significantly reduced AT activity (p < 0.001) and beta-isoform content (p < 0.05). AT-beta correlated inversely with urea and creatinine serum concentrations (p < 0.01), indicating a relationship between better renal function and higher beta-isoform content. beta-Isoform neither correlated with age, gender, and 28-day mortality, nor with plasma concentrations of various inflammatory and organ function parameters. The commercial AT concentrate, which is equivalent to the current WHO standard, had an AT-beta content close to that found in plasma of healthy subjects. The availability of this novel quantitative AT isoform assay allows, for the first time, a closer look at the role of AT isoforms in hemostasis and sepsis pathophysiology.

Adult↗

PubMed2005

Molecular mechanisms of antithrombin deficiency in two Chinese families. One novel and one recurrent point mutation in the antithrombin gene causing venous thrombosis.

We investigated the molecular mechanisms responsible for type I congenital antithrombin (AT) deficiency in two unrelated Chinese pedigrees manifesting multiple site venous thrombosis. Phenotype analysis showed both probands had almost 50% of normal AT levels. Direct sequencing of amplified DNA revealed 2757C > T in proband 1 and 13328G > A in proband 2, predicting a heterozygous Thr98Ile (T981) and Ala404Thr (A404T), respectively. No proband had 20210A allele or factor V Leiden mutation. Transient expression of complementary DNA coding for the mutations in COS-7 cells showed impaired secretion of the mutant molecules. Real-time quantitative PCR indicated that the mutant AT mRNA was transcribed at a similar or even higher level as that of wild-type (wt). Pulse-chase labeling studies suggested both AT variants did not accumulate, but degraded intracellularly. Immunohistochemical staining of the transfected cells revealed that CHO cells expressing the AT-198 mutant were stained diffusely without perinuclear enhancement and cells expressing AT-T404 mutant mainly in the whole cytoplasm with weaker perinuclear enhancement. We conclude that the impaired secretion of the mutant AT molecules, due to intracellular degradation, is the molecular pathogenesis of AT deficiency caused by T981 and A404T mutation for the two families, respectively.

Animals↗