A new familial variant of antithrombin III: 'Antithrombin III Paris'

S ummary . A 59-year-old woman presented a recurrent history of thromboembolism. A qualitative defect of antithrombin III (AT III) was suggested by the discrepancy between a normal amount of AT III antigen and a decreased heparin cofactor activity. Six members of the same family showed a similar defect although clinically asymptomatic. The qualitative abnormality of AT III was confirmed by two-dimensional immunoelectrophoresis. In the absence of heparin, a single peak was obtained with both control and patients' plasmas. In the presence of heparin, two peaks of AT III were observed in the patients' plasmas: the mobility of one peak was similar to that of the control, whereas the other showed a decreased mobility, suggesting a lack of binding to heparin. The two populations of AT III were separated by affinity chromatography on heparin-agarose. 50% of the patients' AT III bound to the agarose beads. The remainder, recovered in the supernatant, migrated in two-dimensional immunoelectrophoresis as a single peak with the same mobility in the presence or absence of heparin, and was devoid of heparin cofactor activity. This familial AT III variant characterized by a reduced affinity for heparin is tentatively named 'Antithrombin III Paris'.     

Heparin binds to human monocytes and modulates their procoagulant activities and secretory phenotypes. Effects of histidine-rich glycoprotein

The binding of heparin to human monocytes and the monocytoid cell line U937 was characterized. Heparin binding was rapid, specific, saturable, and reversible. There was a single class of heparin binding sites, with an apparent dissociation constant of 0.19 mumol/L and 1.9 x 10(6) sites per cell. The binding was not dependent on the anticoagulant property of heparin. Analysis of surface-iodinated cell lysates by heparin affinity chromatography revealed a major 120 Kd cell surface heparin-binding protein. Histidine-rich glycoprotein, a potent heparin antagonist found in human plasma and platelets, decreased the affinity of heparin for cell binding. Cell surface bound heparin was functionally active and markedly accelerated the inactivation of thrombin by antithrombin III. Heparin induced the release of two monocyte secretory proteins of 160 and 17 Kd. Our study supports the thesis that heparin and related glycosaminoglycans interact with monocytes and macrophages, as well as endothelial cells and smooth muscle cells, and play an important and complex role in blood vessel wall biology.     

Investigations on Antithrombin III in Normal Plasma and Serum

Studies on antithrombin III (AT-III) were made by a modification of the two dimensional crossed immunoelectrophoresis technique and gel filtration. Mixing various quantities of heparin with agarose in the first phase of electrophoresis, AT-III from normal human plasma and serum revealed a heterogeneity which depended on the heparin concentration in the agarose gel. At heparin concentrations higher than 16 u/ml, AT-III displayed three components with different electrophoretic mobilities. The component with the highest mobility (designated immunoantithrombin III1 : IAT-III1) dominated in plasma. In normal serum, however, the quantity of this component was decreased and the two other peaks with a slower electrophoretic mobility (IAT-III2 and IAT-III3) became more evident. Normal human plasma and serum were filtered on Sephadex G-200 and the AT-III concentration measured in the fractions by rocket immunoelectrophoresis. The peaks of AT-111 were found in the same fractions for both plasma and serum and were coincident with the albumin peak of the plasma proteins. However, in the case of serum the AT-III concentration decreased less sharply in those fractions with higher molecular weight than in the corresponding plasma fractions. Analysis of these fractions by crossed immunoelectrophoresis revealed that the two components with slower electrophoretic mobility (IAT-III2 and IAT-III3) had higher molecular size than IAT-III1, that the concentration of IAT-III2 and IAT-III3 was significantly higher in serum, and that the high molecular weight components in plasma and serum were qualitatively identical. It is concluded that high molecular weight complexes between AT-III and activated coagulation factors may be present in normally circulating blood and that their detection and possibly quantitation can be achieved using the heparin/agarose crossed immunoelectrophoresis system.     

Specific binding between human neutrophils and heparin

Heparin binding on polymorphonuclear leucocytes (PMNL) was characterized. Heparin binding was specific, rapid, saturable and reversible. One single class of heparin binding sites was found with a dissociation constant of 1.22 mumol/l and 7.7 x 10(6) sites per PMNL. The binding was independent of the anticoagulant activity of heparin. Heparin affinity chromatography on radio-iodinated cell lysates followed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate revealed a 130 kD heparin binding protein. Heparin binding was inhibited by disodium salt of ethylenediamine tetraacetic acid. Cell surface bound heparin was functionally inactive and did not affect the inactivation of thrombin by antithrombin III. Our study demonstrates that heparin interacts with PMNL by a cell-surface binding protein. These instructions could be consistent with the modifications of some PMNL functional properties in the presence of heparin.     

Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.     

Apheresis of low density lipoproteins using a heparin-based sorbent with low antithrombin III binding capacity

A sorbent based on heparin fraction with low affinity for antithrombin III is proposed for low density lipoproteins apheresis in hypercholesterolemia. Heparin was fractionated on antithrombin III-Sepharose; fractions with high and low affinity for antithrombin III were immobilized on CNBr-activated Sepharose 4B. Both sorbents appeared to have an LDL-binding capacity essentially similar to that of the sorbent based on unfractionated heparin. However only plasmasorption on a low affinity heparin-containing sorbent did not reduce plasma antithrombin III. Hence the use of this sorbent may be advantageous over the currently applied sorbents with unfractionated heparin in the treatment of familial hypercholesterolemia.     

Antithrombin III kumamoto II; A single mutation at Arg393-his increased the affinity of antithrombin III for heparin

Abnormal antithrombin III (AT III) was found in a 30-year-old woman who suffered from recurrent thrombosis during pregnancy and the postpartum period. Among her family members, only her father had recurrent episodes of deep vein thrombosis of the lower extremities, from his youth. The antithrombin and antifactor Xa heparin cofactor activities of the proposita's plasma were 61% and 42% of normal, respectively. The progressive antithrombin and antifactor Xa activities were also decreased to 55% and 58% of normal, respectively. The immunoreactive level of AT III was within the normal range (23.1 mg/dl). Analysis of the proposita's plasma by crossed immunoelectrophoresis in the presence or absence of heparin and by affinity chromatography on heparin-Sepharose revealed that the proposita's AT III had apparently normal affinity for heparin. Nucleotide sequencing of 7 exons of the proposita's AT III gene amplified by polymerase chain reaction (PCR) disclosed that the second base of codon 393 comprised both G and A, indicating Arg393-His conversion. The base sequences of exons 1,2,3a, 3b, 4, and 5 were normal, excluding any other mutation. These findings indicated that the proposita's AT III was a variant of AT III at the thrombin binding site and that the proposita was a heterozygote for the abnormality. Heparin affinity of purified abnormal AT III from the proposita's plasma was demonstrated to be increased upon affinity chromatography using heparin-Sepharose, suggesting that the mutation (Arg393-His) per se could possibly increase the affinity of antithrombin III for heparin. For this variant AT III (Arg393-His), the name AT III Kumamoto II is proposed. ©1995 Wiley-Liss, Inc.     

Antithrombin III (AT III) Padua2: A “New” congenital abnormality with defective heparin co-factor activities but no thrombotic disease

Summary A “new” antithrombin III (AT III) abnormality is described in five members of the same family. None of the affected members showed thrombotic manifestations and no consanguinity was present in the family. The main laboratory features were: normal routine clotting tests, slightly decreased AT III activities in all assays carried out in the presence of heparin. In the absence of heparin, antithrombin III activities were instead within normal limitis. Progressive AT III activity and AT III antigen were also normal. Crossed immunoelectrophoresis in the absence of heparin showed a normal pattern both in plasma and serum. In the presence of heparin, the propositi's plasma showed a major, less anodal, abnormal peak and a smaller normal peak. Three peaks were present in the propositi's serum as compared with the two normal ones. This AT III abnormality is different from AT III Padua previosly described by us and we propose the toponym of Antithrombin Padua-₂ to define this condition. Supported by grants from the M.P.I. (grant 1592–1981), Rome and from the Veneto Regional Government, Venice     

The effect of molecular weight on heparin binding to platelets

Low molecular weight heparin is reported to be less reactive with platelets than larger heparins. We probed the molecular basis for this pattern of reactivity by characterizing the saturable platelet binding of [3H]heparin in plasma using heparins of different molecular weights (Mr approximately 3000, approximately 5000, approximately 10,000, approximately 15,000). Binding affinity increased with increasing molecular weight, as expressed by decreasing apparent dissociation constants (Kdapp approximately 1.3 microM for Mr approximately 3000, to Kdapp approximately 0.31 microM for Mr approximately 15,000). After adjusting for the effect of antithrombin III in the plasma, true dissociation constants (Kd) could be calculated and these showed the same trend with molecular weight (Kd approximately 1.1 microM for Mr approximately 3000 to Kd approximately 0.096 microM for Mr approximately 15,000). Platelet binding capacity for the different heparin fractions also increased with molecular weight, although this correlation appeared to lessen with the largest species. Heparin antithrombin III affinity was shown not to affect heparin binding to platelets. We propose a model in which heparin binding to platelets is mediated by charge interaction. Larger molecules with more charge bind with greater affinity and to sites with a broader range of electronegativity than do smaller, less     

Heparin cofactor activities in a family with hereditary antithrombin III deficiency: evidence for a second heparin cofactor in human plasma

Plasma levels of antithrombin-heparin cofactor, determined by heparin- dependent antithrombin assay, and antithrombin III antigen were measured in 22 members of a large kindred predisposed to venous thrombosis. While 11 members had reduced plasma levels of both antithrombin-heparin cofactor and antithrombin III antigen, the levels of antithrombin-heparin cofactor were always greater than the levels of antithrombin III antigen: 66% (+/- 7%) and 49% (+/- 5%) of normal plasma, respectively. Pooled normal plasma and plasma from one of the affected family members (60% antithrombin-heparin cofactor and 47% antithrombin III antigen) were fractionated by heparin-agarose affinity chromatography. Antithrombin-heparin cofactor, which eluted from heparin-agarose with buffer containing 0.4 M NaCl and did not cross- react with antibody specific for antithrombin III and did not inhibit factor Xa at an appreciable rate in the presence of heparin, was designated heparin cofactor A. Antithrombin-heparin cofactor, which eluted from heparin-agarose with buffer containing 2.0 M NaCl, was functionally and antigenically identified as antithrombin III. The concentrations of heparin cofactor A in normal and patient plasma were similar (4.5 x 10(-7) M), while the concentration of antithrombin III in patient plasma (8.0 x 10(-7) M) was only 50% of normal (1.6 x 10(-6) M). The functional properties of both heparin cofactor A and antithrombin III obtained from patient plasma were normal. From the results of the present study it would appear that the antithrombin- heparin cofactor concentrating measured in patient plasma reflects the combined concentrations of heparin cofactor A and antithrombin III. Since heparin cofactor A does not cross-react with antibody to antithrombin III, the concentration of antithrombin III antigen in patient plasma is thus lower than the concentration of antithrombin- heparin cofactor.     

Adverse effect of heparin in thrombin-antithrombin III interaction.

Thrombin, while reacting in the presence of hepatin, impairs the inhibitory capacity of antithrombin III so that subsequent inhibition of thrombin or factor Xa is decreased or abolished. This adverse effect of hepatin has been observed directly with at least 1.5 Iowa units of thrombin per each unit of purified human antithrombin III participating in the reaction. The inhibitory capacity was then totally destroyed and some residual thrombin remained in the active form. With a lower enzyme/inhibitor ratio inactivation of thrombin in the presence of hepatin was fast and complete, however, a significant decrease of inhibitory capacity below that found in reaction without heparin, has been established by measuring the residual antithrombin III activity. In defibrinated human plasma at least 2 units of thrombin per each antithrombin III unit were required to demonstrate directly the adverse effect of heparin but a fast depletion of inhibitory capacity has been also observed after repeated additions of small thrombin portions into plasma heparinized in vitro or in vivo. Portions of enzyme initially added disappeared with great velocity; subsequent portions, however, accumulated building up a high thrombin level not seen in the absence of heparin. The accumulation of residual enzyme was more extensive in plasma containing about 1 heparin unit per ml than anticoagulant at lower concentrations and was particularly noticeable in antithrombin III deficient plasma. These results may have some bearings on the approach to heparin therapy in the event when thrombin continuously generates or when a marked deficiency of antithrombin III exists.     

Automated antithrombin III assay with a centrifugal analyser

The factor Xa inactivating function of antithrombin III is measured automatically by an amidolytic method, adapted to a centrifugal analyser. Plasma is diluted in buffer with heparin. In stage I, diluted plasma is incubated with excess factor Xa. Heparin accelerates the saturation of antithrombin with factor Xa. In stage II, remaining factor Xa is determined with the chromogenic substrate Bz-Ile-Glu-Gly-Arg-pNA. The precision of the present assay compares favourably with that of the clotting assays and immunoassay. There is a close correlation (r = 0.82) between the results obtained with this assay and the immunoassay of antithrombin III.     

High dose of intravenous antithrombin III without heparin in the treatment of disseminated intravascular coagulation and organ failure in four children

In several animal experiments, high doses of antithrombin III concentrates have shown beneficial effects on mortality and reversal of coagulation abnormalities which had resulted from disseminated intravascular coagulation. Other experiments have suggested that antithrombin III infusion without heparin is effective in the treatment of organ failure. We clinically treated children suffering disseminated intravascular coagulation only with antithrombin concentrate. Four patients suffering disseminated intravascular coagulation with organ failure were selected. We started antithrombin III concentrate infusion as soon as the diagnosis was established. The dosage of antithrombin III was 120–250 units/kg/day for 2 or 3 days. Heparin was not used. All 4 patients recovered completely and quickly without any complications within 14 days. We suggest that the high-dose antithrombin III infusion without heparin is an effective and safe therapy for disseminated intravascular coagulation with organ failure. © 1996 Wiley-Liss, Inc.     

Respective role of antithrombin III and heparin cofactor II in the in vitro anticoagulant effect of heparin and of various sulphated polysaccharides

The in vitro anticoagulant effects of standard heparin (SH) and of seven other sulphated polysaccharides (SPS) were investigated by measuring activated partial thromboplastin time (APTT) prolongation of normal plasma and of plasmas selectively depleted of antithrombin III (AT III), of heparin cofactor II (HC II) and of both heparin cofactors. This allowed the determination of the relative contribution of each of the two heparin cofactors to the SPS anticoagulant effect. The SPS varied in their relative activities as catalysts of thrombin inhibition by purified AT III or HC II. The anticoagulant activities of heparin and dermatan sulphate were primarily attributable to their ability to enhance thrombin inhibition by AT III and HC II respectively. Heparin had an additional minor anticoagulant activity which was independent of both AT III and HC II. Pentosan polysulphate, high molecular weight dextran sulphate, heparin with low affinity for AT III and a sulphated heparin derivative had weaker anticoagulant activities in normal plasma than standard heparin. The anticoagulant activities of these last four SPS in plasma depleted of both AT III and HC II were similar to their respective activities in normal plasma. This suggests that these SPS act by directly preventing thrombin generation rather than by enhancing thrombin inhibition.     

On the interaction of rabbit antithrombin III with the luminal surface of the normal and deendothelialized rabbit thoracic aorta in vitro

Pure rabbit antithrombin III was isotope labeled (with 125I or 3H) by two different methods; neither procedure caused a loss of antithrombin activity although both methods affected the affinity of the protein for Sepharose-heparin. From segments from freshly excised rabbit aorta, the uptake of isotope-labeled antithrombin III by the endothelium was rapid and saturable, although relatively small compared to the uptake of thrombin; binding of 3H-antithrombin III to the endothelium resembled that of 125I-antithrombin III. Transendothelial passage of antithrombin III into the subendothelial layers (intima-media) was slow and progressive. Endothelium binding was not affected by pretreating the vessel with either heparin, thrombin, or glycosaminoglycan-specific enzymes. Endothelium-bound antithrombin III was not selectively displaced by either heparin or thrombin. In contrast, endothelium-bound thrombin was rapidly dislodged by antithrombin III as a thrombin- antithrombin III complex. The surface of the deendothelialized aorta (ie, subjected to a balloon catheter) bound antithrombin III avidly. Pretreatment of the deendothelialized vessel with glycosaminoglycan- specific enzymes, particularly heparitinase, decreased intima-media binding by up to 80%. 125I-antithrombin III, when bound to the deendothelialized vessel surface, was actively displaced by either heparin, thrombin, or by unlabeled antithrombin III. The relatively poor binding of antithrombin III compared with that of thrombin by the endothelium in vitro supports an earlier proposal (Lollar P, Owen WG: J Clin Invest 66:1222-1230, 1980) that thrombin bound to high-affinity sites, possibly pericellular proteoglycan, of the endothelium is inactivated by plasma antithrombin III in vivo. Such a situation probably holds for large arteries at least.     

Familial Thrombosis Due to Antithrombin III Deficiency

A large kindred from eastern Kentucky, withextensive history of recurrent venous thrombosis and pulmonary embolism, was studied.Low antithrombin III titers, ranging from 26%to 49% of normal values, were found in plasmaof nine members in three consecutive generations; another five members, four of whomwere not available for study, are suspected ofhaving the biochemical defect. There was agood correlation between clinical symptomsand antithrombin III deficiency, although threeof the younger members with the defect stillremained free of thrombosis. In serum of theaffected subjects antithrombin III was almostcompletely utilized, which indicates that stoichiometric binding to coagulation enzymes dominates under biological conditions. Antithrombinand antifactor Xa activities residing in themacroglobulin region of plasma and serum remained unchanged. The responsiveness to heparin in vitro and in vivo confirmed the evidencethat antithrombin III is the sole blood component through which heparin exerts its anticoagulant effect. In five affected memberstherapy with oral anticoagulants increased verysignificantly the level of antithrombin III inplasma and contributed to a remarkable increase of residual antithrombin III in serum.This objective improvement after warfarin therapy may create significant difficulties in thelaboratory diagnosis of antithrombin III deficiency.

Submitted on May 7, 1973 Revised on July 5, 1973 Accepted on July 16, 1973     

Two Progressive Inhibitors of Factor Xa in Human Blood

The limited ability of human blood to inactivate human factor Xa was found to be markedly increased in human plasma and serum intensively absorbed by aluminium hydroxide. Using gel-filtration analysis on Sephadex G-200 it was demonstrated that the activity brought about by adsorption resides in the macro-globulin fraction. This fraction, as obtained from nonabsorbed plasma or serum, has low antifactor-Xa activity; it represents a progressive inhibitor of this enzyme, additional to antithrombin III. The increase of inhibitory activity due to adsorption is probably based on removal of a substance associated with macroglobulin in vivo , and on uncovering new binding sites for factor Xa. Whereas antithrombin III and the antifactor Xa of lower molecular size are probably represented by a single protease inhibitor which may be potentiated by heparin, the macroglobular anti-factor Xa is not closely related to antithrombin activity and is unaffected by heparin.     

An abnormal antithrombin III (AT III) with low heparin affinity: AT III Clichy

We have identified an inherited qualitative deficiency of antithrombin III (AT III) in a family with apparently no increased incidence of venous thrombosis. Plasma antithrombin and anti-Xa activities were normal, but the interaction with heparin, heparan sulphate and low molecular weight heparin was uniformly decreased. An immunoblotting technique performed in plasma showed normal complex formation with thrombin. By using heparin-Sepharose affinity chromatography and crossed immunoelectrophoresis, the variant could be separated: at least two fractions of low affinity AT III were obtained. A minor one had no antiprotease activity; the other one was further purified to homogeneity and found to have normal specific activity in absence of heparin and a 50% decreased activity in presence of heparin. We propose to call this new variant AT III Clichy.     

Heparin adheres to the damaged arterial wall and inhibits its thrombogenicity.

Heparin binds to thrombogenic extracellular matrices as well as to smooth muscle cells of the vascular wall in vitro. The inhibitory effects of heparin on thrombogenicity of the damaged arterial wall were examined in vivo using small mesenteric arteries of rats and a video recording system attached to a microscope. To induce thrombosis, we damaged the vessel wall over a short segment by compression and exposed the media to the blood stream. A platelet-rich thrombus enlarged gradually at the damaged site, occluded the vascular lumen for a short period, and then flowed away. Compression damage induced such thrombus formation several times. Heparin (500 units/ml) was given in three different ways: intravenous and intra-arterial administration (both 300 units/kg) and intraluminal application under stopped-flow conditions (less than 0.01 ml) for 1-2 minutes with subsequent draining out. Intravenous heparin significantly decreased both the total duration and the number of thrombotic occlusions, whereas intra-arterial heparin abolished thrombotic occlusion. Both routes of heparin administration similarly prolonged the blood coagulation time. Intraluminal application of heparin significantly inhibited subsequent thrombus formation after restoring the flow without changes in the blood coagulation time. After an intra-arterial administration or intraluminal application of fluorescein isothiocyanate-bound heparin, strong fluorescence was observed only at the damaged vascular segment. A heparin fraction with low affinity to antithrombin III or chondroitin sulfate A did not inhibit thrombosis. To clarify anticoagulant activity of vascular wall-bound heparin, damaged carotid arterial segments of rats were incubated (inside out) in platelet-poor plasma with thrombin, and fibrin clot formation around the segments with or without heparin binding was measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of Elimination of Antithrombin III Concentrate in Heparinized Patients

The rate of elimination of injected antithrombin III (AT III) concentrate was investigated by measuring plasma AT III levels in two control subjects and four patients treated with continuous intravenous infusion of heparin. No significant differences were noted in the disappearance rate of injected AT III between controls and patients receiving heparin for several days. Their half-time of AT III elimination was 8·4-13·6 h. The patient in whom AT III concentrate was injected at the start of heparin therapy showed a very rapid decrease of AT III activity with a three-fold increase in the elimination rate constant. Inactive, modified AT III protein present in purified AT III concentrate was found in the circulation of all injected patients. No evidence was obtained that this altered AT III was eliminated more rapidly than the unaltered AT III or that it was subjected to accelerated clearance during heparin therapy.