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PubMed2002

Antithrombin and near-fatal exertional heat stroke.

Heat waves result in excess deaths, excess emergency department visits, and intensive care unit admissions for heat stroke. We describe the clinical features and 3-month outcome of a patient with near-fatal heat stroke, admitted to our intensive care unit in July, 2001. After heavily working for hours at a construction site during a heat wave, the 28-year-old male presented with 41.4 degrees C body temperature and multiorgan failure, consisting of neurological impairment, rhabdomyolysis, acute renal failure, disseminated intravascular coagulation, and acute respiratory distress syndrome (ARDS). In the first week there was no evidence of infection. Treatment included cooling, aggressive volume resuscitation, administration of antithrombin-III concentrates and steroids. The patient survived and recovered normal neurological, renal, respiratory and haematological function, and no disability persisted. This case illustrates survival and complete recovery after multiorgan failure in heat stroke with vigorous intensive care. Treatment with antithrombin and steroids and may well have contributed to the favourable outcome. Correction of reduced antithrombin III levels to supranormal by therapeutic administration of antithrombin III concentrate in disseminated intravascular coagulation of heat stroke was not associated with any bleeding complications.

Adult↗

PubMed1990

Occult antithrombin III deficiency: a potentially lethal complication of the postphlebitic limb.

Chronic venous insufficiency is a frequent sequel to lower extremity venous thrombosis. A relatively uncommon, but potentially lethal, cause of the thrombosis is congenital antithrombin III deficiency. Recognition and treatment of this occult deficiency is critical. The following report describes a family treated by the authors for this problem. In one generation of nine siblings, three males had documentation of the disease with functional antithrombin III levels in the range of 50% to 60%. Before evaluation for the deficiency one female sibling died at the age of 20 years as a consequence of a proven pulmonary embolus. Antithrombin III levels in another female sibling, who was free of symptoms, were normal (80% to 120%). Four other siblings who were free of symptoms (one female, three males) refused evaluation. All three men with the deficiency had severe, chronic, bilateral, lower extremity, venous insufficiency manifested by pain, varicosities, edema, pigmentation, and ulceration. Despite chronic warfarin therapy, one experienced recurrent pulmonary embolization with eventual loss of perfusion of the entire right lung. Ascending venography in the symptomatic males with the deficiency revealed evidence of recurrent and diffuse venous thrombosis with partial recanalization. Recurrent lower extremity venous thrombosis consequent to antithrombin III deficiency causes a particularly fulminant postphlebitic syndrome with characteristic venographic findings. Although potentially lethal if unrecognized and treated simply as venous insufficiency, chronic therapy with warfarin offers palliation and prolongs life.

Adult↗

PubMed1983

Isolation and sequence characterization of a cDNA clone of human antithrombin III.

A human liver cDNA library was constructed by using poly(A)-containing RNA isolated from a human liver biopsy specimen. This library is comprised of 40,000 independent transformants with an average inserted DNA length of 1,200 base pairs. By using the previously cloned baboon antithrombin III cDNA as a specific hybridization probe, greater than 30 human antithrombin III cDNA clones were identified from this library. The clone with the longest DNA insert was selected for sequence analysis. This antithrombin III cDNA clone contains 1,479 base pairs of inserted human DNA and was designated phATIII 113. It contains DNA sequences that code for a signal peptide and the entire mature antithrombin III protein which is comprised of 432 amino acid residues.

Amino Acid Sequence↗

PubMed1987

Expression of functionally active human antithrombin III.

Human antithrombin III cDNA was cloned into an expression vector suitable for transient expression in COS cells. Upon transfection COS cells secreted a single immunoreactive 58-kDa protein. Quantitation of secretion levels by ELISA indicated that at 44 hr posttransfection cells were secreting 48 +/- 5 ng of antithrombin III per 10(6) cells per 24 hr. Heparin-agarose chromatography resulted in the elution of the COS-derived protein as a broad band between 0.3 and 1.0 M NaCl. 35S-labeled medium from transfected cells reacted with human thrombin (1.5 ng/ml) in the absence of heparin. In 40 min, greater than 80% of the immunoreactive material was found as a higher molecular weight species, consistent with stoichiometric covalent complex formation. In a two-stage chromogenic thrombin inactivation assay, under pseudo-first-order conditions, at 16 nM antithrombin III the t1/2 was 74 min and 50 min for plasma and COS cell-derived antithrombin III, respectively, in the absence of heparin. In the presence of 17.4 nM high-affinity heparin, the t1/2 was 5.2 min and 2.2 min, respectively.

Animals↗

PubMed1996

Role of arginine 132 and lysine 133 in heparin binding to and activation of antithrombin.

The binding of heparin to antithrombin greatly accelerates the rate of inhibition of the target proteinases thrombin and factor Xa. Acceleration of the rate of inhibition of factor Xa involves a conformational change in antithrombin that is translated from the heparin binding site to the reactive center loop. A mechanism has been proposed for generation and propagation of the conformational change in which the binding of the negatively charged heparin reduces ionic repulsions between positively charged residues on and adjacent to the D-helix in the heparin binding site of antithrombin (van Boeckel, C. A. A., Grootenhuis, P. D. J., and Visser, A. (1994) Nature Struct. Biol. 1, 423-425). This charge neutralization is proposed to elongate the D-helix and initiate the conformational change which is then translated to the reactive center loop. Several basic residues, including arginine 132 and lysine 133, were predicted to be important both in heparin binding and in this mechanism of heparin activation. To test both the helix extension mechanism and the role of these two residues in heparin binding and factor Xa inhibition, we individually changed arginine 132 and lysine 133 to uncharged methionine by site-directed mutagenesis. The Kd values for binding of R132M and K133M variants to the high affinity pentasaccharide were weakened only 2.3- and 4.5-fold respectively, suggesting a location for R132 and K133 peripheral to the main pentasaccharide binding site. However, the Kd values for long chain high affinity heparin were weakened at least 17-fold for both R132M and K133M, indicating involvement of each residue in binding extended chain heparin species. These reductions in affinity were ionic strength-dependent. The rates of inhibition of factor Xa and thrombin by each variant, however, were indistinguishable from those of control antithrombin, and the accelerations of the rate of inhibition produced by heparin were normal. We conclude that neither arginine 132 nor lysine 133 plays an important role in the binding of heparin pentasaccharide or in the mechanism of heparin activation, suggesting that D-helix extension through charge neutralization is not the mechanism for transmission of conformational change from the heparin binding site to the reactive center region. Arginine 132 and lysine 133 do, however, play a role in tight binding of longer chain heparin species through ionic interactions.

Antithrombin III↗

PubMed2000

The effect of a reducing-end extension on pentasaccharide binding by antithrombin.

Antithrombin requires heparin for efficient inhibition of the final two proteinases of the blood coagulation cascade, factor Xa and thrombin. Antithrombin binds heparin via a specific pentasaccharide domain in a two-step mechanism whereby initial weak binding is followed by a conformational change and subsequent tight binding. The goal of this study is to investigate the role of a reducing-end extension in the binding of the longer oligosaccharides that contain the cognate pentasaccharide sequence. We determined the antithrombin binding properties of a synthetic heptasaccharide containing the natural pentasaccharide sequence (DEFGH) and an additional reducing-end disaccharide (DEFGHG'H'). Binding at low ionic strength is unaffected by the disaccharide addition, but at ionic strengths >/=0.2 the mode of heptasaccharide binding changes resulting in a 2-fold increase in affinity due to a decrease in the off-rate caused by a greater nonionic contribution to binding. Molecular modeling of possible binding modes for the heptasaccharide at high ionic strength indicates a possible shift in position of the pentasaccharide domain to occupy the extended heparin-binding site. This conclusion supports the likely presence of a range of sequences that can bind to and activate antithrombin in the natural heparan sulfates that line the vascular endothelium.

Antithrombins↗

PubMed2000

The conformational activation of antithrombin. A 2.85-A structure of a fluorescein derivative reveals an electrostatic link between the hinge and heparin binding regions.

Antithrombin is unique among the serpins in that it circulates in a native conformation that is kinetically inactive toward its target proteinase, factor Xa. Activation occurs upon binding of a specific pentasaccharide sequence found in heparin that results in a rearrangement of the reactive center loop removing constraints on the active center P1 residue. We determined the crystal structure of an activated antithrombin variant, N135Q S380C-fluorescein (P14-fluorescein), in order to see how full activation is achieved in the absence of heparin and how the structural effects of the substitution in the hinge region are translated to the heparin binding region. The crystal structure resembles native antithrombin except in the hinge and heparin binding regions. The absence of global conformational change allows for identification of specific interactions, centered on Glu(381) (P13), that are responsible for maintenance of the solution equilibrium between the native and activated forms and establishes the existence of an electrostatic link between the hinge region and the heparin binding region. A revised model for the mechanism of the allosteric activation of antithrombin is proposed.

Amino Acid Sequence↗

PubMed2000

Critical role of the linker region between helix D and strand 2A in heparin activation of antithrombin.

The binding of pentasaccharide heparin to antithrombin induces a conformational change that is transmitted to the reactive center loop and increases the rate of inhibition of factor Xa by approximately 300-fold. The mechanism of such transmission is not known. To test the role of residues 134-137, which link helix D to beta-sheet A, in this signal transduction, we created variant antithrombins in which we removed amino acids 134-137 stepwise and cumulatively. Although the deletions did not compromise the fundamental ability of antithrombin to bind to heparin or to inhibit target proteinases thrombin and factor Xa, they did largely decouple conformational changes in the heparin-binding site from conformational activation of the reactive center loop. Because the variant with only Ala(134) removed was as compromised as variants with larger deletions, yet the variant with Ser(137) removed was normal, we concluded that the length of the linker is less important than the precise interrelationship between residues in this region and other residues involved in conformational activation of antithrombin.

Amino Acid Sequence↗

PubMed2000

Hypersulfated low molecular weight heparin with reduced affinity for antithrombin acts as an anticoagulant by inhibiting intrinsic tenase and prothrombinase.

In buffer systems, heparin and low molecular weight heparin (LMWH) directly inhibit the intrinsic factor X-activating complex (intrinsic tenase) but have no effect on the prothrombin-activating complex (prothrombinase). Although chemical modification of LMWH, to lower its affinity for antithrombin (LA-LMWH) has no effect on its ability to inhibit intrinsic tenase, N-desulfation of LMWH reduces its activity 12-fold. To further explore the role of sulfation, hypersulfated LA-LMWH was synthesized (sLA-LMWH). sLA-LMWH is not only a 32-fold more potent inhibitor of intrinsic tenase than LA-LMWH; it also acquires prothrombinase inhibitory activity. A direct correlation between the extent of sulfation of LA-LMWH and its inhibitory activity against intrinsic tenase and prothrombinase is observed. In plasma-based assays of tenase and prothrombinase, sLA-LMWH produces similar prolongation of clotting times in plasma depleted of antithrombin and/or heparin cofactor II as it does in control plasma. In contrast, heparin has no effect in antithrombin-depleted plasma. When the effect of sLA-LMWH on various components of tenase and prothrombinase was examined, its inhibitory activity was found to be cofactor-dependent (factors Va and VIIIa) and phospholipid-independent. These studies reveal that sLA-LMWH acts as a potent antithrombin-independent inhibitor of coagulation by attenuating intrinsic tenase and prothrombinase.

Anticoagulants↗

PubMed2001

Helix D elongation and allosteric activation of antithrombin.

Antithrombin requires allosteric activation by heparin for efficient inhibition of its target protease, factor Xa. A pentasaccharide sequence found in heparin activates antithrombin by inducing conformational changes that affect the reactive center of the inhibitor resulting in optimal recognition by factor Xa. The mechanism of transmission of the activating conformational change from the heparin-binding region to the reactive center loop remains unresolved. To investigate the role of helix D elongation in the allosteric activation of antithrombin, we substituted a proline residue for Lys(133). Heparin binding affinity was reduced by 25-fold for the proline variant compared with the control, and a significant decrease in the associated intrinsic fluorescence enhancement was also observed. Rapid kinetic studies revealed that the main reason for the reduced affinity for heparin was an increase in the rate of the reverse conformational change step. The pentasaccharide-accelerated rate of factor Xa inhibition for the proline variant was 10-fold lower than control, demonstrating that the proline variant cannot be fully activated toward factor Xa. We conclude that helix D elongation is critical for the full conversion of antithrombin to its high affinity, activated state, and we propose a mechanism to explain how helix D elongation is coupled to allosteric activation.

Allosteric Site↗

PubMed2003

The influence of hinge region residue Glu-381 on antithrombin allostery and metastability.

Antithrombin becomes an efficient inhibitor of factor Xa and thrombin by binding a specific pentasaccharide sequence found on a small fraction of the heparan sulfate proteoglycans lining the microvaculature. In the structure of native antithrombin, the reactive center loop is restrained due to the insertion of its hinge region into the main beta-sheet A, whereas in the heparin-activated state the reactive center loop is freed from beta-sheet A. In both structures, hinge region residue Glu-381 makes several stabilizing contacts. To determine the role of these contacts in the allosteric mechanism of antithrombin activation, we replaced Glu-381 with an alanine. This variant is less active toward its target proteases than control antithrombin, due to a perturbation of the equilibrium between the two forms, and to an increase in stoichiometry of inhibition. Pentasaccharide binding affinity is reduced 4-fold due to an increase in the off-rate. These data suggest that the main role of Glu-381 is to stabilize the activated conformation. Stability studies also showed that the E381A variant is resistant to continued insertion of its reactive center loop upon incubation at 50 degrees C, suggesting new stabilizing interactions in the native structure. To test this hypothesis, and to aid in the interpretation of the kinetic data we solved to 2.6 A the structure of the variant. We conclude that wild-type Glu-381 interactions stabilize the activated state and decreases the energy barrier to full loop insertion.

Allosteric Regulation↗

PubMed1979

Purification and properties of guinea pig antithrombin III.

Guinea pig antithrombin III has been purified from plasma by sequential heparin-Sepharose affinity chromatography, DE-52 cellulose chromatography, isoelectric focussing, and Sephadex G-100 gel filtration chromatography. The final product was homogeneous as judged by sodium dodecyl sulfate disc gel electrophoresis. Purification was 202-fold with a yield of 41%. Antiproteinase activity of antithrombin III was determined by progressive inactivation of thrombin coagulant and amidolytic activity. Heparin cofactor activity was demonstrated by immediate inactivation of thrombin by antithrombin III in the presence of minute quantities of heparin. It also could be demonstrated that thrombin inactivation by antithrombin III occurs by formation of a bimolecular complex whose rate of formation is markedly enhanced by minute quantities of heparin.

Animals↗

PubMed2004

Reference intervals for plasma levels of fibronectin, von Willebrand factor, free protein S and antithrombin during third-trimester pregnancy.

During pregnancy, significant changes occur in the hemostatic system and in the plasma levels for several plasma proteins, especially towards term. In this study changes occurring during normal pregnancy and immediately postpartum were investigated to establish adequate reference intervals for important hemostatic parameters. Blood samples were collected during pregnancy weeks 33, 36, 39 and 1-3 h after delivery from 153 healthy pregnant women with at least one previous normal pregnancy. The plasma samples were analyzed for antithrombin, von Willebrand factor (vWf), free protein S and fibronectin. Fibronectin and vWf are contact-promoting proteins responsible for adhesion and aggregation during primary hemostasis, but are also released from thrombocytes during activation of the coagulation process. Antithrombin is the most important primary physiological inhibitor of activated serine proteases related to the coagulation cascade. Protein S is a co-factor to protein C and in cooperation is also an important inhibitor of the coagulation cascade. During third-trimester pregnancy, vWf was higher than in non-pregnant women, and continued to increase postpartum. The fibronectin plasma level was mostly unchanged in comparison with non-pregnant values. Within this reference interval it gradually increased during the third trimester, but fell slightly postpartum. Antithrombin decreased slightly during the third trimester and even further, postpartum. Free protein S decreased markedly but to a stable level from week 33 to 39, decreasing even more postpartum. The present results are concordant with clinical knowledge of increased risk of thrombosis during pregnancy and early puerperium, with increased levels of vWf and fibronectin and decreased levels of antithrombin and free protein S. Clearly, current reference values based on healthy non-pregnant subjects are not usable during late pregnancy and immediately postpartum.

Adult↗

PubMed1984

The role of laser nephelometer in the study of abnormal clotting factors: characterization of two abnormal antithrombins (AT III Padua and AT III Padua2).

The immunologic concentration of two abnormal antithrombins III (AT III), namely antithrombin III Padua (AT III Padua) and antithrombin III Padua2 (AT III Padua2) and the kinetics of the reaction of these two ATs III with an anti AT III antiserum was investigated by means of a laser nephelometer. The immunologic concentration of these two AT III both in presence (0.2 IU/mL) or absence of heparin was normal. On the contrary, the analysis of kinetics behavior demonstrated that AT III Padua is radically different from pooled normal plasma both in presence or in absence of heparin. This was not the case for AT III Padua2, which showed no difference from pooled normal plasma regardless of the presence or absence of heparin. Both abnormal antithrombins III reached the plateau of the reaction at about the corresponding value of pooled normal plasma, indicating a normal antigen level. These experimental data were analyzed by means of a computer (Apple II 48 RAM) programmed to solve a four unknowns equation (Rodbard's equation). This analysis showed that the time needed to reach half of the maximum voltage, i.e., the parameter C, which is expressed in minutes, is clearly longer in the case of AT III Padua samples (heparinized or not) as compared with pooled normal plasma. Moreover, the time at which the maximum rate was reached was also longer. On the contrary, in the case of AT III Padua2 there is no difference from pooled normal plasma. These data confirm the view that a different kind of defect is present in these two AT III abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen-Antibody Reactions↗

PubMed1990

Contribution of chemical 6-O-sulfation of the aminodeoxyhexose residues in whale heparin with high affinity for antithrombin III to its anticoagulant properties.

The tributylammonium salt of whale (Balaenoptera borealis L.) intestinal heparin with high affinity for antithrombin III, whose degrees of sulfate-substitution in D-glucosamine and L-iduronic acid residues are GlcNS 0.738, GlcN6S 0.384, and IdoA2S 0.510 mol, was reacted with 2.5, 5.0, or 10.0 mol of pyridine-sulfur trioxide/mol of available hydroxyl groups in N,N-dimethylformamide at -10 degrees C for 1 h. Both chemical and NMR spectroscopic analyses revealed that an exclusive 6-O-sulfation of the D-glucosamine residues proceeded, according to the amount of the sulfating reagent used (GlcN6S: 0.476, 0.585, and 0.641 mol, respectively), the degree of sulfation at other natural substitution positions in the polysaccharide being unchanged, without any detectable unnatural sulfate-substitution. Biological examination of these products indicated that the 6-O-sulfation in the original whale heparin resulted in significant increases in blood clotting and anti-Factor IIa activities (maximal 43 and 82% increases, respectively), and in a moderate increase in the ability to bind antithrombin III, that is, in anti-Factor Xa activity and in intrinsic fluorescence enhancement of the protein (maximal 28 and 30% increases, respectively), together with a maximal 10% increase in the proportion of heparin species with higher affinity for antithrombin III, released with 1.0-3.0 M NaCl from antithrombin III-Sepharose.

Animals↗

PubMed1975

Inherited antithrombin-III deficiency causing mesenteric venous infarction: a new clinical entity.

Primary superior mesenteric venous thrombosis is sometimes preceded by peripheral thrombophlebitis. Inherited antithrombin-III deficiency is a recently recognized autosomal dominant trait, which is characterized by thrombophlebitis and pulmonary embolism. This case report illustrates many features of both entities and strongly suggest a causal relationship. While long-term therapy has yet to be established, prophylactic therapy is recommended when asymptomatic individuals with known antithrombin-III deficiency are at increased risk of thrombosis. The efficacy of heparin alone has been unreliable, whereas Coumadin has been encouraging. Antithrombin-III concentrates are being developed and theoretically should be helpful. Patients with thrombophlebitis or pulmonary embolism should be suspected of having antithrombin-III deficiency. Such individuals also represent one mechanism to explain "primary" mesenteric venous thrombosis.

Adult↗

PubMed1995

Activation of antithrombin III isoforms by heparan sulphate glycosaminoglycans and other sulphated polysaccharides.

Antithrombin III occurs naturally as two functionally distinct molecular species that differ in glycosylation at Asn135. Whereas the predominant, glycosylated isoform has high affinity for heparin, a quantitatively minor isoform lacking glycosylation at that site displays relatively higher affinity for both heparins and heparinoids. We characterized the ability of various sulphated polysaccharides to potentiate the rates of thrombin inhibition by the isoforms. High-molecular-weight dextran sulphate was the most effective of those studied, increasing thrombin inhibition by the higher-affinity antithrombin III isoform up to five-fold more efficiently than did heparin fractions with low-affinity for antithrombin III. In addition, dextran sulphate activated the higher-affinity isoform as much as twelve times more effectively than it did the lower-affinity isoform. Pentosan polysulphate was up to three-fold, and some heparan sulphate fractions up to two-fold, more effective with the higher, compared with the lower affinity, isoform. Heparan sulphate preparations less effectively increased the rate of thrombin inhibition than did the other low-affinity polysaccharides. Structure-function studies indicated positive correlations between activity and both polymer length and anionic group density of low-affinity sulphated polysaccharides. The observed effects of the heparan sulphates on this anticoagulant pathway, although of low potency, are consistent with the hypotheses that these substances naturally regulate blood homeostasis in vascular tissues and that much of this function may be mediated by the higher-affinity antithrombin III isoform.

Antithrombin III↗

PubMed1995

Prothrombin fragment 1 + 2 and thrombin-antithrombin III complex as markers of activation of blood coagulation in inflammatory bowel diseases.

OBJECTIVES AND METHODS: The aims of the present work were to assess the presence of thrombin generation in Crohn's disease and in ulcerative colitis by using the prothrombin fragment 1 + 2 and the thrombin-antithrombin III complex assays and to study the possible relationships between these markers and disease activity. RESULTS: Prothrombin fragment 1 + 2 and thrombin-antithrombin III complex were significantly raised in patients with Crohn's disease (n = 69) and with ulcerative colitis (n = 25) as compared with healthy controls (n = 50). In Crohn's disease these two markers of thrombin generation were correlated with the Van Hees index (P < 0.05 and P < 0.001, respectively); values were significantly different from controls even in the patient group displaying the lowest disease activity (P < 0.001). No correlation was found with tumour necrosis factor alpha and C-reactive protein; nevertheless patients with C-reactive protein less than or equal to 10 mg/l had significant lower values of prothrombin fragment 1 + 2 (P < 0.03). In ulcerative colitis prothrombin fragment 1 + 2 and thrombin-antithrombin III complex were significantly increased by comparison with controls, were higher in patients with pancolitis and correlated with C-reactive protein (P < 0.002 and P < 0.009, respectively). CONCLUSION: These data show that prothrombin fragment 1 + 2 and thrombin-antithrombin III complex are increased in inflammatory bowel diseases and suggest that thrombin generation might be an early event in their pathogenesis.

Adolescent↗