Biological implications of a 3 A structure of dimeric antithrombin

BACKGROUND: Antithrombin, a member of the serpin family of inhibitors, controls coagulation in human plasma by forming complexes with thrombin and other coagulation proteases in a process greatly accelerated by heparin. The structures of several serpins have been determined but not in their active conformations. We have determined the structure of intact antithrombin in order to study its mechanism of activation, particularly with respect to heparin, and the dysfunctions of this mechanism that predispose individuals to thrombotic disease. RESULTS: The crystal structure of a dimer of one active and one inactive molecule of antithrombin has been determined at 3 A. The first molecule has its reactive-centre loop in a predicted active conformation compatible with initial entry of two residues into the main beta-sheet of the molecule. The inactive molecule has a totally incorporated loop as in latent plasminogen activator inhibitor-1. The two molecules are linked by the reactive loop of the active molecule which has replaced a strand from another beta-sheet in the latent molecule. CONCLUSION: The structure, together with identified mutations affecting its heparin affinity, allows the placement of the heparin-binding site on the molecule. The conformation of the two forms of antithrombin demonstrates the extraordinary mobility of the reactive loop in the serpins and provides insights into the folding of the loop required for inhibitory activity together with the potential modification of this by heparin. The mechanism of dimerization is relevant to the polymerization that is observed in diseases associated with variant serpins.     

Thermal unfolding of three acclimation temperature-associated isoforms of carp light meromyosin expressed by recombinant DNAs

Differential scanning calorimetry (DSC) was performed to investigate thermodynamic properties of three carp fast skeletal light meromyosin (LMM) isoforms expressed in Escherichia coli by recombinant DNAs. Three isoforms were the 10 degreesC-, intermediate-, and 30 degreesC-type LMM predominantly expressed in carp acclimated to 10, 20, and 30 degreesC. The isoforms expressed in E. coli by recombinant DNAs exhibited a typical pattern of alpha-helix in CD spectroscopy with two minima at 222 and 208 nm. Moreover, the three isoforms formed paracrystals typical of LMM, suggesting that expressed proteins retained intact structural properties. When the LMM isoforms were subjected to DSC analysis, the 10 degreesC and 30 degreesC types showed endotherms having transition temperatures (Tm) at 35.1 and 39.5 degreesC, respectively, which are responsible for thermal unfolding of alpha-helix. The intermediate type exhibited two comparable endotherms with Tm values at 34.9 and 40.6 degreesC, implying that it has intermediate thermodynamic properties between those of 10 degreesC and 30 degreesC types. However, a chimeric LMM having the 10 degreesC and 30 degreesC type as N- and C-terminal halves, respectively, showed the DSC pattern typical of the whole 30 degreesC-type molecule. On the other hand, another chimeric LMM composed of the N-terminal 30 degreesC type and C-terminal 10 degreesC type gave the pattern of the full 10 degreesC type. These results suggest that thermodynamic properties of the C-terminal half largely account for thermal unfolding of the whole molecule.     

The N-terminal segment of antithrombin acts as a steric gate for the binding of heparin

The binding of heparin causes a conformational change in antithrombin to give an increased heparin binding affinity and activate the inhibition of thrombin and factor Xa. The areas of antithrombin involved in binding heparin and stabilizing the interaction in the high-affinity form have been partially resolved through the study of both recombinant and natural variants. The role of a section of the N-terminal segment of antithrombin, residues 22-46 (segment 22-46), in heparin binding was investigated using rapid kinetic analysis of the protein cleaved at residues 29-30 by limited proteolysis with thermolysin. The cleaved antithrombin had 5.5-fold lowered affinity for heparin pentasaccharide and 1.8-fold for full-length, high-affinity heparin. It was shown that, although the initial binding of heparin is slightly enhanced by the cleavage, it dissociates much faster from the cleaved form, giving rise to the overall decrease in heparin affinity. This implies that the segment constituting residues 22-46 in the N terminus of antithrombin hinders access to the binding site for heparin, hence the increased initial binding for the cleaved form, whereas, when heparin is bound, segment 22-46 is involved in the stabilization of the binding interaction, as indicated by the increased dissociation constant. When the heparin pentasaccharide is bound to antithrombin prior to incubation with thermolysin, it protects the N-terminal cleavage site, implying that segment 22-46 moves to interact with heparin in the conformational change and thus stabilizes the complex.     

Estrogen-containing oral contraceptive agents. A basis for their thrombogenicity

The mechanism whereby estrogen-containing contraceptives facilitate thrombosis is obscure, and published data concerning their effect on antithrombin III are conflicting. Plasma samples were examined for the quantity of antithrombin III and activated factor X (Xa) inhibitory activity among 57 women receiving oral contraceptives and 48 controls. The quantity of antithrombin III in both groups was normal. In contrast, Xa inhibitory activity was significantly reduced (P less than .001) among patients taking oral contraceptives, compared to controls. Heparin sodium added to plasma from patients taking contraceptives raised Xa inhibitory activity toward or above normal without changing the quantity of antithrombin III. The effect of estrogen is not to decrease the quantity of plasma antithrombin III, but rather to diminish plasma Xa inhibitory activity, an effect that can be abolished by heparin.     

The binding of 1-anilino-8-naphthalenesulfonate, heparin, salicylate and caprylate by human antithrombin III

The binding of 1-anilino-8-naphthalenesulfonate to human antithrombin III was studied by fluorescence enhancement of the fluorophor and fluorescence quenching of the protein emission. Two molecules of 1-anilino-8-naphthalenesulfonate were found to bind per antithrombin molecule with an average dissociation constant of 4.4-10(-5) M. The binding of heparin to antithrombin was studied by ultraviolet difference spectroscopy. The stoichiometry of the heparin binding indicated 1.8 binding sites with an average dissociation constant of 4.3 - 10(-6) M. Further the fluorometric competition experiments with 1-anilino-8-naphthalenesulfonate, heparin, salicylate and caprylate indicated two different classes of anion binding sites on the antithrombin molecule.     

Heparin-induced thrombopenia: rapid regression of thrombopenia and thrombosis after plasma exchange. Case report

INTRODUCTION: Heparin-induced thrombocytopenia with thrombosis is a rare but severe adverse reaction to heparin therapy, whose management is difficult. After heparin withdrawal, the initiation of an alternative anticoagulant therapy, such as recombinant hirudin or danaparoid, is strongly recommended before vitamin K antagonists are effective. Several reports of the efficacity of plasma exchanges in patients with life-threatening thrombosis have been made. EXEGESIS: We report on a patient with severe aortic thrombosis related to heparin therapy in whom a unique plasma exchange resulted in both dramatic improvement in the platelet count and marked reduction of the thrombosis. CONCLUSION: This case provides further evidence that plasma exchanges can be useful in the management of heparin-induced thrombocytopenia with thrombosis. They are rapidly efficient and can be used before heparin alternative treatment is effective.     

Structure, thermostability, and conformational flexibility of hen egg-white lysozyme dissolved in glycerol

Hen egg-white lysozyme dissolved in glycerol containing 1% water was studied by using CD and amide proton exchange monitored by two-dimensional 1H NMR. The far- and near-UV CD spectra of the protein showed that the secondary and tertiary structures of lysozyme in glycerol were similar to those in water. Thermal melting of lysozyme in glycerol followed by CD spectral changes indicated unfolding of the tertiary structure with a Tm of 76.0 +/- 0.2 degreesC and no appreciable loss of the secondary structure up to 85 degreesC. This is in contrast to the coincident denaturation of both tertiary and secondary structures with Tm values of 74.8 +/- 0.4 degreesC and 74.3 +/- 0.7 degreesC, respectively, under analogous conditions in water. Quenched amide proton exchange experiments revealed a greater structural protection of amide protons in glycerol than in water for a majority of the slowly exchanging protons. The results point to a highly ordered, native-like structure of lysozyme in glycerol, with the stability exceeding that in water.     

Low back pain among different groups of subjects exposed to hand-arm transmitted vibration

Two major glycoforms of recombinant antithrombin which differ 10-fold in their affinity for the effector glycosaminoglycan, heparin, were previously shown to be expressed in BHK or CHO mammalian cell lines (I. Bj?rk, et al., 1992, Biochem. J. 286, 793-800; B. Fan et al., 1993, J. Biol. Chem. 268, 17588-17596). To determine the source of the glycosylation heterogeneity responsible for these different heparin-affinity forms, each of the four Asn residue sites of glycosylation, residues 96, 135, 155, and 192, was mutated to Gln to block glycosylation at these sites. Heparin-agarose chromatography of the four antithrombin variants revealed that Gln 96, Gln 135, and Gln 192 variants still displayed the two functional heparin-affinity forms previously observed with the wild-type inhibitor, whereas the Gln 155 variant showed only a single functional high heparin affinity form. These results demonstrate that heterogeneous glycosylation of Asn 155 of recombinant antithrombin is responsible for generating the low heparin affinity glycoform. Analysis of heparin binding to the higher heparin affinity forms of the four variants showed that all exhibited increased heparin affinities of two- to sevenfold compared to wild-type higher heparin affinity form or to plasma antithrombin, with the Gln 135 variant showing the largest effect on this affinity. The extent of heparin-affinity enhancement was correlated with the distance of the mutated glycosylation site to the putative heparin-binding site in the X-ray structure of antithrombin. All variants displayed normal kinetics of thrombin inhibition in the absence and presence of saturating heparin, indicating that the carbohydrate chains solely affected heparin binding and not heparin-activation or proteinase-binding functions. These results indicate that all carbohydrate chains of recombinant antithrombin adversely affect heparin-binding affinity to an extent that correlates with their relative proximity to the putative heparin-binding site in antithrombin.     

Cellular and functional characterization of three recombinant antithrombin mutants that caused pleiotropic effect-type deficiency

Inherited antithrombin deficiency is associated with a predisposition for familial venous thromboembolic disease. Pleiotropic effect-type mutants of antithrombin that have an amino acid replacement in a distal hinge region including strands 1C, 4B, and 5B of the polypeptide chain are known to exhibit impaired interactions with both thrombin and heparin, coupled with a secretion defect. To examine the mechanism of pleiotropic effect-type antithrombin deficiency, we expressed three mutants, Oslo (Ala404-->Thr), Kyoto (Arg406-->Met), and Utah (Pro407-->Leu), in baby hamster kidney (BHK) cells, and compared their secretion rates, affinities for heparin and abilities to form thrombin-antithrombin (TAT) complexes with those of wild-type (Wt) antithrombin. Pulse-chase experiments showed that the Oslo- and Kyoto-mutants were secreted at rates similar to Wt antithrombin. In contrast, the Utah-mutant underwent partial intracellular degradation. The intracellular degradation of the Utah-mutant was not inhibited by lysosomotropic inhibitors, but by proteasome inhibitors such as carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (LLL) and lactacystin, indicating that a part of the Utah-mutant was degraded by proteasome through quality control in the endoplasmic reticulum (ER). Crossed immunoelectrophoresis in the presence of heparin showed that only the Oslo-mutant lacks heparin-binding ability. Incubation with thrombin showed that the Kyoto- and Utah-mutants, but not the Oslo-mutant, formed a weak but detectable TAT complex. Furthermore, heparin enhanced the TAT complex formation by the Kyoto- and Utah-mutants, suggesting heparin cofactor activities of these mutants. These results show that each of the Oslo-, Kyoto-, and Utah-mutants exhibits different properties as to secretion, intracellular degradation and functional activity, although they are grouped as pleiotropic effect-type mutants.     

Investigation of the structural basis for thermostability of DNA-binding protein HU from Bacillus stearothermophilus

Site-directed mutagenesis was used to identify amino acid residues essential for the thermostability of the DNA-binding protein HU from the thermophile Bacillus stearothermophilus (BstHU). Two mutants, BstHU-A27S and BstHU-V42I, in which Ala27 and Val42 in BstHU were replaced by the corresponding amino acids Ser27 and Ile42, respectively, in the homologue from a mesophile B. subtilis (BsuHU), were less stable than the wild-type BstHU (63.9 degreesC), showing Tm values of 58.4 degreesC and 60.1 degreesC, respectively, as estimated by circular dichroism (CD) analysis at pH 7.0. The denaturation of two mutants was further characterized using differential scanning calorimetry; the Tm values obtained by calorimetric analysis were in good agreement with those estimated by CD analysis. The results suggest that Ala27 and Val42 are partly responsible for enhancing the thermostability of BstHU. When considered together with previous results, it is revealed that Gly15, Ala27, Glu34, Lys38, and Val42 are essential for the thermostability of thermophilic protein BstHU. Moreover, five thermostabilizing mutations were simultaneously introduced into BsuHU, which resulted in a quintuple mutant with a Tm value of 71.3 degreesC, which is higher than that of BstHU, and also resulted in insusceptibility to proteinase digestion.     

Covalent antithrombin-heparin complexes with high anticoagulant activity. Intravenous, subcutaneous, and intratracheal administration

Although heparin has been used clinically for prophylaxis and treatment of thrombosis, it has suffered from problems such as short duration within compartments in vivo that require long term anticoagulation. A covalent antithrombin-heparin complex has been produced with high anticoagulant activity and a long half-life relative to heparin. The product had high anti-factor Xa and antithrombin activities compared with noncovalent mixtures of antithrombin and heparin (861 and 753 units/mg versus 209 and 198 units/mg, respectively). Reaction with thrombin was rapid with bimolecular and second order rate constants of 1.3 x 10(9) M-1 s-1 and 3.1 x 10(9) M-1 s-1, respectively. The intravenous half-life of the complex in rabbits was 2.6 h as compared with 0.32 h for similar loads of heparin. Subcutaneous injection of antithrombin-heparin resulted in plasma levels (peaking at 24-30 h) that were still detectable 96 h post-injection. Given the increased lifetime in these vascular and intravascular spaces, use of the covalent complex in the lung was investigated. Activity of antithrombin-heparin instilled into rabbit lungs remained for 48 h with no detection of any complex systemically. Thus, this highly active agent has features required for pulmonary sequestration as a possible treatment for thrombotic diseases such as respiratory distress syndrome.     

Mechanism of heparin activation of antithrombin: evidence for an induced-fit model of allosteric activation involving two interaction subsites

The anticoagulant activation of the serpin antithrombin by heparin pentasaccharide DEFGH was previously shown to involve trisaccharide DEF first binding and inducing activation of the serpin, followed by disaccharide GH binding and stabilizing the activated state [Petitou et al. (1997) Glycobiology 7, 323-327; Desai et al. (1998) J. Biol. Chem. 273, 7478-7487]. In the present study, the role of conformational changes and charged residues of the GH disaccharide in the allosteric activation mechanism was investigated with variant pentasaccharides modified in the GH disaccharide. Perturbation of the conformational equilibrium of iduronate residue G through replacement of the nonessential 3-OH of this residue with -H resulted in parallel decreases in the fraction of residue G in the skew boat conformer (from 64 to 24%) and in the association constant for pentasaccharide binding to antithrombin [(2.6 +/- 0.3)-fold], consistent with selective binding of the skew boat conformer to the serpin. Introduction of an additional sulfate group to the 3-OH of residue H flanking a putative charge cluster in the GH disaccharide greatly enhanced the affinity for the serpin by approximately 35-fold with only a small increase in the fraction of residue G in the skew boat conformation (from 64 to 85%). The salt dependence of binding, together with a recent X-ray structure of the antithrombin-pentasaccharide complex, suggested that the majority of the enhanced affinity of the latter pentasaccharide was due to direct electrostatic and hydrogen-bonding interactions of the H residue 3-O-sulfate with antithrombin. All variant pentasaccharides produced a normal enhancement of antithrombin fluoresence and normal acceleration of factor Xa inhibition by the serpin at saturating levels, indicating that conformational activation of antithrombin was not affected by the pentasaccharide modifications. Rapid kinetic studies were consistent with the altered affinities of the variant pentasaccharides resulting mostly from perturbed interactions of the reducing-end GH disaccharide with the activated antithrombin conformation and minimally to an altered binding of the nonreducing-end DEF trisaccharide to the native serpin conformation. Together, these results support a model in which the conformational flexibility of the G residue facilitates conversion to the skew boat conformer and thereby allows charged groups of the GH disaccharide to bind and stabilize the activated antithrombin conformation that is induced by the DEF trisaccharide.     

Laboratory diagnosis of immune inner ear disease

Inherited antithrombin deficiency is associated with an increased risk of thrombosis, primarily venous rather than arterial. Most affected individuals have inherited only a single copy of an abnormal antithrombin (AT) gene. Homozygously affected individuals, although rare, have a severe thrombotic history of early onset and often affecting the arteries. We report two new cases of type II HBS (heparin binding site) deficiency in which the propositi are homozygous for the previously reported mutation 99 Leu to Phe, and who have a severe thrombotic history. These cases are considered alongside existing homozygote and compound heterozygote cases.     

Natural anticoagulants, aging, and thromboembolism

The normal aging process alters blood coagulation system in humans; this may be of great concern in the view of the known association of vascular disease with advancing age. The plasma concentration of several coagulation factors, namely fibrinogen, factor VII, factor VIII, factor IX, high molecular-weight kininogen, and prekallikrein, increase in healthy humans, paralleling the physiological aging process. Plasma parameters of clotting activation in vivo, such as prothrombin fragment 1 + 2, fibrinopeptide A, thrombin-antithrombin III complex, and D-dimer, are positively correlated with age. Nevertheless, among centenarians, biochemical signs of marked hypercoagulability are associated with a healthy state. Natural anticoagulants, including antithrombin III, heparin cofactor II, protein C, protein S, and tissue factor pathway inhibitor, can modulate the reactions of blood coagulation system. The occurrence of menopause is accompanied by a significant increase in antithrombin III plasma level; the mean antithrombin III levels in older women exceed levels in male contemporaries. In healthy elderly subjects heparin cofactor II plasma concentrations are lower than in young subjects, independently of gender. Protein C levels raise with age in both sexes, as well as free protein S levels. In women, statistically significant increases in the plasma concentration of the tissue factor pathway inhibitor have been observed, whereas no significant age-related change has been found in men. The fact that many subjects with congenital defects of natural anticoagulants do not undergo thromboembolic events in young age suggests that in healthy individuals a raise in natural anticoagulants can balance the age-related increase of procoagulant factors.     

Control of contact activation on end-point immobilized heparin: the role of antithrombin and the specific antithrombin-binding sequence

The uptake and activation of FXII from blood plasma was studied in small-diameter polyethylene tubing, surface-modified by end-point immobilization of heparin. Two preparations of heparin were used to modify the contact-activating properties of the plastic tubing: unfractionated, functionally active heparin and low-affinity heparin, lacking the specific antithrombin-binding sequence and virtually devoid of anticoagulant activity. The uptakes of FXII on the two heparin surfaces were similar. No activated FXII could be demonstrated on the unfractionated heparin surface, whereas on the low-affinity heparin surface nearly all FXII underwent spontaneous activation. The suppression of FXII activation on the unfractionated heparin surface was investigated by using plasma depleted of antithrombin, complement C1 esterase inhibitor, or both. The removal of antithrombin resulted in extensive activation of FXII, whereas the depletion of C1 esterase inhibitor had only a minor effect. Experiments with recalcified plasma showed rapid clot formation during exposure to the low-affinity heparin surface. After depletion of antithrombin, but not complement C1 esterase inhibitor, the recalcified plasma clotted in contact with the unfractionated heparin surface as well. We conclude that antithrombin and the antithrombin-binding sequence in the surface-immobilized heparin are essential for the prevention of surface activation of FXII and triggering of the intrinsic coagulation system.     

Deconvolution of the fluorescence emission spectrum of human antithrombin and identification of the tryptophan residues that are responsive to heparin binding

Heparin causes an allosterically transmitted conformational change in the reactive center loop of antithrombin and a 40% enhancement of tryptophan fluorescence. We have expressed four human antithrombins containing single Trp --> Phe mutations and determined that the fluorescence of antithrombin is a linear combination of the four tryptophans. The contributions to the spectrum of native antithrombin at 340 nm were 8% for Trp-49, 10% for Trp-189, 19% for Trp-225, and 63% for Trp-307. Trp-225 and Trp-307 accounted for the majority of the heparin-induced fluorescence enhancement, contributing 37 and 36%, respectively. Trp-49 and Trp-225 underwent spectral shifts of 15 nm to blue and 5 nm to red, respectively, in the antithrombin-heparin complex. The blue shift for Trp-49 is consistent with partial burial by contact with heparin, whereas the red shift for Trp-225 and large enhancement probably result from increased solvent access upon heparin-induced displacement of the contact residue Ser-380. The enhancement for Trp-307 may result from the heparin-induced movement of helix H seen in the crystal structure. The time-resolved fluorescence properties of individual tryptophans of wild-type antithrombin were also determined using the four variants and showed that Trp-225 and Trp-307 experienced the largest change in lifetime upon heparin binding, providing support for the steady-state fluorescence deconvolution.     

Morphological changes in paraboloid glycogen of the accessory cone of the chick retina by exposure to light (author''s transl)

We studied five patients in whom severe thrombocytopenia developed during intermittent intravenous heparin treatment of arterial and venous thrombosis. Platelet aggregation was demonstrated when heparin (0.5 U per milliliter) was incubated with the patients' citrated platelet-rich plasma or with normal platelet-rich plasma in the presence of the patients' serum. Antiplatelet antibody was not detected in the patient globulin fractions prepared from serum collected within one week after heparin withdrawal by use of the platelet factor 3 availability technic. When the studies were repeated with modifications to detect heparin-dependent antiplatelet antibodies, positive results were obtained in four of five patients. The data suggest that a casual relation, mediated by an immune mechanism, existed between heparin therapy and thrombocytopenia, and that this syndrome may occur more often than has previously.     

Purpura fulminans in the newborn. Report of two cases successfully treated with heparin and antithrombin III

Purpura fulminans is a rare form of disseminated intravascular coagulation characterized by rapidly progressive purpuric lesions, hypotension and, in some cases, fever. In neonates, purpura fulminans usually develops following deficiency of anticoagulant protein C or S, although acquired forms have been described. The management of disseminated intravascular coagulation is still controversial, with some studies finding a positive effect of anticoagulants and others showing no effect or even a detrimental one. Therefore, at present, management is limited to the treatment of underlying disease and replacement of clotting factors. Personal experience is reported on the efficacy of heparin in combination with antithrombin III in the treatment of purpura fulminans in two preterm neonates who did not have inherited deficiency of protein C or S and developed the disease possibly following prolonged labor (36 hours) in the first case, and maternal neoplasia, in the second. Both neonates presented with widespread cyanotic areas rapidly evolving in purpuric lesions and hemorrhagic bullae. Laboratory tests (prolonged prothrombin and partial thromboplastin time, fibrinogen and antithrombin III concentrations below normal ranges, d-dimer highly positive) were consistent with disseminated intravascular coagulation. In both cases anticoagulant treatment with heparin (50 UI/kg in bolus followed by 15 UI/kg/h) and antithrombin III was associated with resolution of disseminated intravascular coagulation and prompt amelioration of the purpuric lesions, without apparent side effects.     

Cerebral thrombophlebitis, sural phlebitis, pulmonary embolisms and protein S deficiencies

The authors report about a patient with thrombosis of the transverse and sigmoid sinus who also suffered from multiple pulmonary embolism and deep leg vein thrombosis. The etiologic factor was a deficiency of the free (unbounded) and total protein S. The cerebral sinus thrombosis was diagnosed by MRI and angio-MRI. The first 4 weeks the patient was treated with heparin and later with phen-procoumon. The plasma protein S serves as a cofactor for protein C and plays an important role in the anticoagulation. Deficiencies of these proteins are either hereditary with an autosomal dominant trait or acquired in patients with severe hepatic diseases and coagulation disorders.