Antithrombin concentrate

Plasma-derived antithrombin concentrates have been used medically since the 1980s for the treatment of hereditary and acquired antithrombin deficiency. Hereditary (genetic) deficiency is characterized by the presence of little/no native antithrombin activity in plasma and results in an increased risk of inappropriate blood clot/emboli formation. Acquired antithrombin deficiency can be induced by drugs (e.g. heparin and oestrogens), liver disease (decreased antithrombin... 

Conrad J, Brosstad F Larsen ML, Samama M, Abildgaard U. Molar antithrombin concentration in normal human plasma, Haemostasis 1983 13 363-368. 

Trials of antithrombin concentrate in the treatment of DlC from various causes show some beneficial effect on improving DlC score, decreasing duration of DlC, or improving end-organ function. " A meta-analysis of a number of well-designed studies demonstrated a trend toward decreased mortahty (from 56% to 44%). In addition to variable efficacy, antithrombin is an expensive product with only intermittent availability. Therefore restricting its use to patients at high risk for morbidity and mortahty should be considered. 

Affinity chromatography is used in the preparation of more highly purified Factor IX concentrates (53—55) as well as in the preparation of products such as antithrombin III [9000-94-6] (56,57). Heparin [9005-49-6], a sulfated polysaccharide (58), is the ligand used most commonly in these appHcations because it possesses specific binding sites for a number of plasma proteins (59,60). 

Figure 5. Fluorescence anisotropy of F-D labelled heparin-antithrombin interaction. F-D-heparin (0.02 fluoresceins per uronic acid) at 0.1 mg/ml was incubated with different concentrations of antithrombin (open circles) or bovine serum albumin (solid diamonds) in 20 mM sodium phosphate buffer, pH 7.4.

Antithrombin, already mentioned in the context of heparin, is the most abundantly occurring natural inhibitor of coagulation. It is a single-chain 432 amino acid glycoprotein displaying four oligosaccharide side chains and an approximate molecular mass of 58 kDa. It is present in plasma at concentrations of 150 pig ml 1 and is a potent inhibitor of thrombin (factor Ha), as well as of factors IXa and Xa. It inhibits thrombin by binding directly to it in a 1 1 stoichiometric complex. 

Hypercoagulable states include malignancy activated protein C resistance deficiency of protein C, protein S, or antithrombin factor VIII or XI excess antiphospholipid antibodies and other situations. Estrogens and selective estrogen receptor modulators have been linked to venous thrombosis, perhaps due in part to increased serum clotting factor concentrations. Although a thrombus can form in any part of the venous circulation, the majority of thrombi begin in the lower extremities. Once formed, a venous... 

Antithrombin and activated protein C concentrates are available for the appropriate indications that include thrombosis in the setting of antithrombin deficiency and sepsis respectively. 

The formation of aptamer-substrate complexes was also followed by the use of redox-active intercalators73 (Fig. 12.18d). A nucleic acid hairpin structure that contained in its single-stranded loop the antithrombin base sequence was assembled on a Au electrode, and methylene blue was intercalated as a redox label in the double-stranded stem of the hairpin structure. The hairpin was, then, opened in the presence of thrombin, by generating the respective G-quadruplex-thrombin complex, and as a result, the redox label was removed from the nucleic structure, showing a decrease in the voltammetric response with the increase in the concentration of thrombin. This method enabled the analysis of thrombin with a detection limit that corresponded to... 

Another method for the analysis of aptamer-protein complexes involved the use of a positively charged ferrocene-tethered polythiophene, (19), as redox label reporting unit (Fig. 12.19). The antithrombin aptamer was immobilized on an electrode surface, and the electrostatic binding of the redox polymer (19) to the aptamer monolayer resulted in a supramolecular complex that revealed electrical contact between the polymer and the electrode.74 The formation of the aptamer-thrombin complex removed the polymer from the surface and blocked the electrical contact between the polymer label and the electrode. As a result, higher concentrations of thrombin increased the surface coverage of the aptamer-thrombin complex on the electrode, and this decreased the amperometric responses of the sensing device. 

Subsequent investigations on the structure of heparin concentrated on the isolation and structural determination of larger oligosaccharides, in order to determine the structural elements involved in anti-blood-clotting activity associated with the binding to antithrombin. 

In 50 postpartum women who took chlorotrianisene for lactation suppression in a double-blind, randomized, placebo-controlled study, antithrombin III concentrations were significantly lower on the third day postpartum compared with placebo (2). 

The risk of thromboembolic complications when diethylstilbestrol is used in treating prostatic cancer is well documented, but there has been some doubt as to the mechanisms involved. Oral diethylstilbestrol diphosphate 300 mg/day has been compared with LR-RH agonist therapy or no treatment in 35 patients with prostatic cancer (5). Diethylstilbestrol reduced the concentrations of protein S to below the lower limit of normal in 24 of the 35 cases. There was also some reduction in antithrombin III concentrations. These results were consistently confirmed in a follow-up group of eight further patients who took diethylstilbestrol. Since these very low concentrations of protein S are virtually the same as those found in congenital deficiency, it seems likely that this plays a role in the development of cardiovascular complications during diethylstilbestrol treatment. 

Despite the variations that are found, the overall conclusion is that oral contraceptives cause an increase in coagulation factors I (fibrinogen), II, VII, IX, X, and XII, and a reduction in antithrombin III concentrations, which would be expected to predispose to venous thromboembolism, especially if not counterbalanced by an increase either in fibrinolytic activity or of other inhibitory proteins of the coagulation, such as protein C (70). 

The effect of continuously administered low-dose 17-beta-estradiol (E2) + norethisterone acetate (NETA) on coagulation and fibrinolytic factors has been studied in 120 menopausal women, using two dosage variations (1 mg of E2 with 0.25 mg or 0.5 mg of NETA) compared with placebo over a year (53). In either dose, the combination significantly lowered plasma concentrations of factor VII, fibrinogen, antithrombin, and plasminogen activator inhibitor-1 (PAI-1) compared with placebo. These changes appear favorable, since they may lead to increased fibrinolytic activity and could reduce the risk of coronary heart disease. However, antithrombin activity was also reduced, which may increase the risk of venous thromboembolism. 

Cushman M, Psaty BM, Meilahn EN, Dobs AS, Kuller LH. Post-menopausal hormone therapy and concentrations of protein C and antithrombin in elderly women. Br J Haematol 2001 114(l) 162-8. 

Another label-free optical detection method—FTIR-ATR—has been applied for detection of thrombin by means of DNA aptamers [73], The antithrombin DNA aptamer previously developed by Tasset et al. [17] was immobilized covalently onto Si surface using UV irradiation method. As a quantitative measure, the area of N-H and CH2 bands was used. This method allowed to detect thrombin with a sensitivity around 10 nmol/L. The specificity of binding of protein to aptamer was also investigated using DNA with no binding site for thrombin. It has been noted that for effective binding study by FTIR-ATR method, the concentration of protein should be kept lower than 100 nmol/L. 

Further, it will be assumed that E is proportional to the concentration of the heparin antithrombin III cofactor... 

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