Mechanisms of the development of acquired antithrombin III deficiency in the experimental nephrotic syndromes

In the experiments on white rats was studied the role of excessive thrombinogenesis in the development of acquired antithrombin III deficiency in the experimental nephrotic syndrome. It was determined that excess thrombin generation induced the marked acceleration of 125I-antithrombin III clearance from blood stream in consequence formation of thrombin antithrombin III complexes with the following limited proteolysis of the inhibitor by enzyme. These results give evidence that apart from proteinuria the excess thrombin generation accompanied by nephrotic syndrome play a part in the development of acquired deficiency of antithrombin III in this experimental pathology.     

The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III.

The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III has been studied. Fragment 2 was found to slow the rate of inhibition of thrombin by antithrombin III about 3-fold. The effect of prothrombin fragment 2 on antithrombin III inhibition was examined by comparing its action in the presence of either thrombin or meizothrombin (des fragment 1). The second order rate constants for antithrombin III inhibition of thrombin with saturating fragment 2 and antithrombin III inhibition of meizothrombin (des fragment 1) were the same. Prothrombin fragment 2 had no effect on either antithrombin III inhibition of meizothrombin (des fragment 1) or Factor Xa. The effect of the fragment on the reaction mechanism of thrombin inhibition was evaluated to see if the fragment altered binding of antithrombin III to thrombin or inhibited the formation of the covalent complex. The fragment was found to have no inhibitory effect on the rate of covalent complex formation, indicating that the protective effect of the fragment is by inhibiting binding of antithrombin III to thrombin. These data suggest that prothrombin fragment 2 may be an important factor in controlling the localization of clot formation by regulating the interaction between thrombin and antithrombin III.     

Antiangiogenic antithrombin blocks the heparan sulfate-dependent binding of proangiogenic growth factors to their endothelial cell receptors: evidence for differential binding of antiangiogenic and anticoagulant forms of antithrombin to proangiogenic heparan sulfate domains

The anticoagulant serpin antithrombin acquires a potent antiangiogenic activity upon undergoing conformational alterations to cleaved or latent forms. Here we show that antithrombin antiangiogenic activity is mediated at least in part through the ability of the conformationally altered serpin to block the proangiogenic growth factors fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) from forming signaling competent ternary complexes with their protein receptors and heparan sulfate co-receptors on endothelial cells. Cleaved and latent but not native forms of antithrombin blocked the formation of FGF-2-FGF receptor-1 ectodomain-heparin ternary complexes, and the dimerization of these complexes in solution and similarly inhibited the formation of FGF-2-heparin binary complexes and their dimerization. Only antiangiogenic forms of antithrombin likewise inhibited (125)I-FGF-2 binding to its low affinity heparan sulfate co-receptor and blocked FGF receptor-1 autophosphorylation and p42/44 MAP kinase phosphorylation in cultured human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with heparinase III to specifically eliminate the FGF-2 heparan sulfate co-receptor suppressed the ability of antiangiogenic antithrombin to inhibit growth factor-stimulated proliferation. Antiangiogenic antithrombin inhibited full-length VEGF(165) stimulation of HUVEC proliferation but did not affect the stimulation of cells by the heparin-binding domain-deleted VEGF(121). Taken together, these results demonstrate that antiangiogenic forms of antithrombin block the proangiogenic effects of FGF-2 and VEGF on endothelial cells by competing with the growth factors for binding the heparan sulfate co-receptor, which mediates growth factor-receptor interactions. Moreover, the inability of native antithrombin to bind this co-receptor implies that native and conformationally altered forms of antithrombin differentially bind proangiogenic heparan sulfate domains.     

Antithrombin III activity in long-developing hypercoagulation in animals

Old rats aged 12-18 months and rats kept on an atherogenic diet for 3.5 months demonstrate high blood antithrombin III content at the initial period of the development of anticoagulation function suppression and of hypercoagulation. During long-developing hypercoagulation, the high content of antithrombin III might be regarded as compensatory reaction interfering with formation in the blood of thrombin microamounts. With hypercoagulation becoming more pronounced and with a further increase of blood thrombin concentration the content of antithrombin III progressively descends, which is accompanied by steady development of anticoagulation function suppression.     

Replacement therapy of acquired antithrombin III deficiency in experimental nephrotic syndrome

The influence of antithrombin III on hemostasis and renal function was studied in experiments on rats with nephrotic syndrome. The development of nephrotic syndrome was accompanied by the activation of blood coagulation and appearance of acquired antithrombin III deficiency due to its loss with the urine. The replacement therapy by bovine antithrombin III at a dose of 25 U/kg a day for 10 days decreased the signs of excessive thrombinogenesis in experimental animals and increased the amount of thrombin-antithrombin III complexes in the blood flow. The activation of coagulation in rats with nephrotic syndrome predominantly induced the disturbances of the excretory renal function which could be efficiently corrected by antithrombin III.     

A monoclonal antibody that triggers deacylation of an intermediate thrombin-antithrombin III complex

Upon incubation of antithrombin III with thrombin in the presence of a monoclonal antibody recognizing an epitope exposed on the heavy chain part of thrombin-cleaved two-chain antithrombin III, antithrombin III was preferentially cleaved by the enzyme as a substrate, rather than covalently complexed with the enzyme to form an equimolar, stable acyl complex. Once the stable acyl complex was formed between the enzyme and antithrombin III, however, no further liberation of two-chain antithrombin III was observed. Kinetic studies showed that heparin does not affect this reaction, although generation of thrombin-cleaved two-chain antithrombin III is apparently accelerated in accordance with the rate constant for heparin-enhanced thrombin-antithrombin III complex formation. Here we propose the term "switching antibody" for an antibody that triggers deacylation of an intermediate enzyme-inhibitor complex by switching the enzyme-inhibitor reaction from the major pathway of stable acyl complex formation to an alternative pathway of cleavage of the inhibitor as a substrate.     

Molecular weight analysis of antithrombin III-heparin and antithrombin III-thrombin-heparin complexes

The molecular interactions between components of the heparin-catalyzed antithrombin III/thrombin reaction were investigated by light scattering. When heparin was added to antithrombin III, the molecular weight increased to a maximum and then decreased to that of a 1:1 (antithrombin III X heparin) complex. The initial molecular weights at low heparin to antithrombin III ratios were consistent with the formation of a 2:1 (antithrombin III X heparin) complex in which only one antithrombin III molecule had undergone the conformational change measured by protein fluorescence enhancement. The peak molecular weight never reached that of a complete 2:1 complex. This behavior was observed for bovine and human antithrombin III in the presence of both unfractionated heparin and high molecular weight-high affinity heparin. Pentosane polysulfate also caused some multiple associations. Bovine antithrombin III and thrombin formed a 1:1 complex that underwent further aggregation within minutes, while the human proteins did not aggregate on this time scale after forming the 1:1 complex. In the presence of stoichiometric amounts of heparin, the bovine proteins formed an initial complex of Mr = 230,000 (corresponding to a dimer of heparin-antithrombin III-thrombin) which underwent further aggregation. The human proteins, however, formed a 1:1 (antithrombin III X thrombin) initial complex in the presence of heparin, followed by aggregation. These interactions of thrombin and antithrombin with heparin suggest complex interactions that could relate to heparin function.     

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.     

A novel anti-angiogenic form of antithrombin with retained proteinase binding ability and heparin affinity

Latent antithrombin, an inactive antithrombin form with low heparin affinity, has previously been shown to efficiently inhibit angiogenesis and tumor growth. We now show that heat treatment similar to that used for preparation of latent antithrombin also transforms antithrombin to another form, which we denote prelatent, with potent anti-angiogenic and anti-tumor activity but with retained proteinase- and heparin-binding properties. The ability of prelatent antithrombin to inhibit angiogenesis is presumably due to a limited conformational change, which may partially resemble that in latent antithrombin. Such a change is evidenced by a different cleavage pattern of prelatent than of native antithrombin by nontarget proteinases. Prelatent antithrombin exerts its anti-angiogenic effect by a similar mechanism as latent antithrombin, i.e. by inhibiting focal adhesion formation and focal adhesion kinase activity, thereby leading to decreased proliferation of endothelial cells. The proteinase inhibitory fractions in commercial antithrombin preparations, which have been heat treated during production, also have anti-angiogenic activity, comparable with that of the prelatent antithrombin form.     

Anticoagulant activities of heparin oligosaccharides and their neutralization by platelet factor 4.

Oligosaccharides of well-defined molecular size were prepared from heparin by nitrous acid depolymerization, affinity chromatography on immobilized antithrombin III (see footnote on Nomenclature) and gel chromatography on Sephadex G-50. High affinity (for antithrombin III) octa-, deca-, dodeca-, tetradeca-, hexadeca- and octadeca-saccharides were prepared, as well as oligosaccharides of larger size than octadecasaccharide. The inhibition of Factor Xa by antithrombin III was greatly accelerated by all of these oligosaccharides, the specific anti-Factor Xa activity being invariably greater than 1300 units/mumol. The anti-Factor Xa activity of the decasaccharide was not significantly decreased in the presence of platelet factor 4, even at high platelet factor 4/oligosaccharide ratios. Measurable but incomplete neutralization of the anti-Factor Xa activities of the tetradeca- and hexadeca-saccharides was observed, and complete neutralization of octadeca- and larger oligo-saccharides was achieved with excess platelet factor 4. The octa-, deca-, dodeca-, tetradeca- and hexadeca-saccharides had negligible effect on the inhibition of thrombin by antithrombin III, whereas specific anti-thrombin activity was expressed by the octadeca-saccharide and by the larger oligosaccharides. An octadecasaccharide is therefore the smallest heparin fragment (prepared by nitrous acid depolymerization) that can accelerate thrombin inhibition by antithrombin III. The anti-thrombin activities of the octadecasaccharide and larger oligosaccharides were more readily neutralized by platelet factor 4 than were their anti-Factor Xa activities. These findings are compatible with two alternative mechanisms for the action of platelet factor 4, both involving the binding of the protein molecule adjacent to the antithrombin III-binding site. Such binding results in either steric interference with the formation of antithrombin III-proteinase complexes or in displacement of the antithrombin III molecule from the heparin chain.     

Thrombophilia in blood-donation for plasma- and cytapheresis caused by antithrombin III depletion

Induced by the case of a blood-donor with more than 100 donations-mostly apheresis-who suffered acute thromboembolism caused by antithrombin III depletion, nearly 60 apheresis-donors were examined before, during and after aphereses, as well manual fourfold as computerized (CS-3000 TRAVENOL) cytaphereses with special methods of coagulation, mainly antithrombin III levels and fibrin-monomeres. The findings were described and compared, multifold in manual techniques happened breaks caused by occluded tubules were accompanied with decrease of antithrombin III and positive FM-tests. It was discussed, if apheresis-techniques for themselves (extracorporal multifold manipulation in manual techniques and contact with artificial surfaces in both kinds of techniques) can be the reason for a hypercoagulation. It seems to be important, how is the situation of apheresis-donor before starting, therefore we give the recommendation, to pay more attention in examination this kind of donors, additional with testing antithrombin III levels and tests for fibrin-monomers.     

Disorders of thrombin interaction with the blood vessel wall and its inactivation by antithrombin III in experimental nephrotic syndrome

In the experiments on white rats was conducted a comparative study of 125I-alpha-thrombin clearance and its inactivation by antithrombin III in animals of the control group and rats with the experimental nephrotic syndrome (Heymann nephritis). It was determined that alterations of thrombin binding to the vascular wall in the nephrotic syndrome induced the prolongation of the labelled enzyme half-life in the blood stream. The formation of 125I-alpha-thrombin complexes with antithrombin III was delayed in the nephrotic syndrome, that suggests the violation of mechanisms of thrombin inactivation by antithrombin III. The distortions of endothelium-mediated thrombin elimination and inactivation in the nephrotic syndrome resulted in the enzyme interaction with fibrinogen, which threatened organism by thrombosis.     

Inhibition of bovine factor IXa and factor Xabeta by antithrombin III.

Factor IXa and factor Xabeta are serine proteases which participate in the middle phase of blood coagulation. These two enzymes are inhibited by antithrombin III by the formation of an enzyme-inhibitor complex containing 1 mol of enzyme and 1 mol of antithrombin III. The complex was readily demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and loss of coagulant or esterase activity at increasing concentrations of inhibitor. The inactivation of factor IXa by antithrombin III was relatively slow, but the reaction was greatly accelerated by the addition of heparin.     

The role of the COOH-terminal region of antithrombin III. Evidence that the COOH-terminal region of the inhibitor enhances the reactivity of thrombin and factor Xa with the inhibitor.

To elucidate the role of the COOH-terminal region of antithrombin III, we studied the effects of synthetic peptides corresponding to its sequence on the amidolytic and proteolytic activities of thrombin and Factor Xa in the presence or absence of the inhibitor, antithrombin III. The peptides ANRPFLVFI and IIFMGRVANP corresponding to residues Ala404 to Ile412 and Ile420 to Pro429, respectively, blocked the inhibition by antithrombin III. The effect of IIFMGRVANP was reduced in the presence of heparin. Both peptides at a concentration of 1 mM blocked complex formation between antithrombin III and thrombin or Factor Xa. The two peptides, particularly IIFMGRVANP, directly enhanced the amidolytic activity of thrombin and Factor Xa on the synthetic substrate Boc-Ala-Gly-Arg-MCA (where Boc is t-butoxycarbonyl and MCA is 4-methylcoumarin), which corresponds to residues P3-P1 of the reactive site of antithrombin III, and also on other substrates due to increased Vmax. IIFMGRVANP also shortened the thrombin-induced fibrinogen clotting time, whereas ANRPFLVFI inhibited the thrombin-catalyzed activation of protein C both in the presence and absence of thrombomodulin. The direct effect of ANRPFLVFI and IIFMGRVANP on thrombin was confirmed by enhancement of the incorporation of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide into thrombin. These findings suggest that the COOH-terminal region of antithrombin III interacts with thrombin and Factor Xa to increase the reactivity of the enzyme, which may enhance acyl-bond formation between the inhibitor and the enzyme.     

A method to determine the affinity of heparin to thrombin and antithrombin III on equilibrium gel permeation chromatography

Equilibrium gel permeation chromatography was employed to determine the ability of heparin to form complexes with thrombin and antithrombin III. In the eluate from a Sephacryl S-200 column, heparin caused a peak and then a trough in the fluorescence of 48 nM antithrombin III or 63 nM thrombin. The peak-heights with known amounts of heparin were used for standard curves to determine the extent of complex formation by test heparin preparations. Only heparin species with high-affinity for antithrombin III specifically formed a complex with antithrombin III under the conditions used. The ability to form a complex of heparin preparations with different anticoagulant activities for thrombin and antithrombin III could be determined satisfactorily. The heparin species with different affinities for antithrombin III did not coincide those with different affinities for thrombin. Of 4 preparations with one low-affinity and three high-affinity subfractions of heparin for antithrombin III, the species with the lowest affinity for antithrombin III had the highest affinity for thrombin. All of these observations showed that the method could be used to determine the ability to form a complex of test heparin preparations.     

Antithrombin III regulates complement activity in vitro

Heparin, a polyion, exerts its main activity to inhibit coagulation through a serine protease inhibitor, antithrombin III. Previous studies have clearly shown that heparin in the absence of antithrombin III also has the capacity to regulate C activity. The present studies examined the ability of purified human antithrombin III to regulate classical and alternative pathways of C, alone and in the presence of heparin. Antithrombin III alone inhibited generation of both pathways in a dose-related manner; antithrombin III at 8 micrograms/10(7) cellular intermediates inhibited generation of the classical and alternative pathway convertases by 60 and 42%, respectively. Antithrombin III and heparin augmented each other's capacity to inhibit generation of both convertases in a dose-related manner. Antithrombin III did not appear to inhibit on the basis of charge because it is only slightly anionic (isoelectric pH value, 5.0); instead, antithrombin III may have acted as a serine protease inhibitor of the proteolytic enzymes of the C cascades. Antithrombin III acted only to inhibit formation of the alternative pathway convertase but had no activity on terminal lysis by this pathway; similarly, antithrombin III inhibited preformed EAC1,4b,2a,3b but had no activity on classical pathway cellular intermediates containing additional components. Finally, antithrombin III inhibited consumption of factor B hemolytic activity in a reaction mixture that also contained factor D and C3b, suggesting that factor D activity was also inhibited. These studies demonstrate the capacity of antithrombin III to regulate C and suggest that, in concert with heparin, antithrombin III may play an important role in the regulation of C in vivo.     

Thrombin generation and inactivation in the presence of antithrombin III and heparin

We have determined the rate constants of inactivation of factor Xa and thrombin by antithrombin III/heparin during the process of prothrombin activation. The second-order rate constant of inhibition of factor Xa alone by antithrombin III as determined by using the synthetic peptide substrate S-2337 was found to be 1.1 X 10(6) M-1 min-1. Factor Xa in prothrombin activation mixtures that contained prothrombin, and either saturating amounts of factor Va or phospholipid (20 mol % dioleoylphosphatidylserine/80 mol % dioleoylphosphatidylcholine, 10 microM), was inhibited by antithrombin III with a second-order rate constant that was essentially the same: 1.2 X 10(6) M-1 min-1. When both factor Va and phospholipid were present during prothrombin activation, factor Xa inhibition by antithrombin III was reduced about 10-fold, with a second-order rate constant of 1.3 X 10(5) M-1 min-1. Factor Xa in the prothrombin activation mixture that contained both factor Va and phospholipid was even more protected from inhibition by the antithrombin III-heparin complex. The first-order rate constants of these reactions at 200 nM antithrombin III and normalized to heparin at 1 microgram/mL were 0.33 and 9.5 min-1 in the presence and absence of factor Va and phospholipid, respectively. When the prothrombin concentration was varied widely around the Km for prothrombin, this had no effect on the first-order rate constants of inhibition. It is our conclusion that factor Xa when acting in prothrombinase on prothrombin is profoundly protected from inhibition by antithrombin III in the absence as well as in the presence of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Latent and polymeric antithrombin: clearance and potential thrombotic risk

Antithrombin, the most potent anticoagulant in vivo, displays a significant conformational flexibility. The native five-stranded anticoagulant form transforms under different conditions or mutations to inactive six-stranded conformations: latent or polymer. However, the function, potential deleterious effects, and clearance of these forms are not completely known. The dimerization of latent antithrombin with a native molecule has been suggested to have thrombotic potential. We have assessed the potential thrombogenicity of high amounts of latent and polymeric antithrombin by experiments performed in mice and human plasma. Moreover, we have analyzed the clearance of (125)I-labeled native, latent, polymer, and thrombin-complexed antithrombins in rat, as well as the clearance of latent antithrombin from plasma of patients treated with commercial concentrates. Our results show that high plasma levels of latent or polymeric antithrombin do not interfere with the anticoagulant function of native antithrombin. Moreover, we confirm that all monomeric forms of antithrombin have similar turnover. Finally, we show that polymers have the longest half-life of all conformers, being in circulation for prolonged periods of time. In conclusion, our data support that latent and polymeric antithrombin would not likely have a thrombotic effect, thus dispelling doubts about the potential harmful effect of latent antithrombin present in commercial concentrates for therapeutic use. Moreover, the suggested antiangiogenic role of latent antithrombin, together with its stability in plasma and its negligible thrombogenicity raises the possibility of its use as a new antiangiogenic drug.     

Monoclonal antibodies against antithrombin III. Identification of their epitopes and effects on antithrombin III activities

Four monoclonal antibodies with distinct epitopes were prepared against antithrombin III. None of them is directed against the heparin-binding region nor the active site, yet two mAb namely A36 and B108, interfere with antithrombin III inhibition of thrombin. The epitope of monoclonal antibody A36 is located within amino acid residues 1-393, at a site different from the active site since it recognizes antithrombin III and antithrombin-III-thrombin complexes with the same affinity. A36 partially prevents the intrinsic antithrombin III activity and has no effect on the heparin-enhanced antithrombin III activity when added to the antithrombin-III--heparin complex. If A36 is first reacted with antithrombin III and then heparin is added to the reaction mixture, A36 fixes the conformation of antithrombin III so that heparin binds to antithrombin III, but is not able to induce the conformational change in the antithrombin III molecule required for the enhanced activity. The epitope for monoclonal antibody B108 is located within residues 282-393, close to the active site. It does not recognize antithrombin-III-thrombin complexes by solid-phase radioimmunoassay. Its binding to antithrombin III induces a conformational change that enhances antithrombin III activity in a manner that resembles the heparin effect, but its effect is additive to the heparin effect, since when it was added to a reaction mixture which contained a saturating amount of heparin, inhibition of thrombin was enhanced. The epitope for monoclonal antibody A5 is located within residues 1-393, and its recognition of antithrombin III or antithrombin-III-thrombin is strongly dependent on the integrity of the disulfide bonds. A5 has no effect on antithrombin III activities. The epitope for monoclonal antibody A10 is well defined within a narrow range of 55 amino acid residues, 339-393, on the antithrombin III molecule, close to the active site, yet it has no effect on antithrombin III inhibitory activity. These monoclonal antibodies may be developed for various diagnostic or clinical purposes and offer a powerful tool for studying the conformational changes and structure/activity relationships in the antithrombin III molecule.     

EPR spectroscopy of soybean lipoxygenase-1. Determination of the zero-field splitting constants of high-spin Fe(III) signals from temperature and microwave frequency dependence

Antithrombin III-heparin cofactor has been isolated from normal rat plasma, purified to homogeneity on acrylamide gel electrophoresis and used to prepare a monospecific antiserum in rabbits. Measurements of rat antithrombin III were made by a single radial immunodiffusion assay. Net synthesis of antithrombin III was investigated during 12- or 24-h perfusions of the isolated rat liver. In perfusions performed under basal conditions cumulative synthesis of antithrombin-III was observed to occur at a rate sufficient to replace the total circulating plasma antithrombin III in about 6 h. In perfusions performed under full supplementation conditions which greatly enhanced synthesis of fibrinogen and alpha-2 (acute-phase) globulin (known acute-phase reactant proteins) net synthesis of antithrombin III was not significantly greater than that observed in control perfusions. Although these prolonged perfusion studies conclusively demonstrate net synthesis of antithrombin III by the isolated rat liver, they afford no evidence that this protein is an acute-phase reactant.