In vitro Characterization of Antithrombin III Concentrates – A Single-Blind Study

Abstract

Twenty-three lots of five antithrombin III (AT III) concentrates from four manufacturers were analyzed in a single-blind study. All the preparations had been virus-inactivated by pasteurization, and one concentrate had also been treated with solvent/detergent (S/D). AT III activities were determined using two thrombin-based and one factor Xa-based chromo-genic substrate assays. AT III antigen was measured by kinetic nephelometry. All AT III assays were tested against the first international reference preparation coded 72/1. In addition, AT III was characterized by crossed immunoelectrophoresis in the presence of heparin and by gel filtration. The following were quantified: heparin cofactor II activity and antigen content, heparin activity, thrombin-AT III complexes, AT III-protease complexes, total protein, albumin, immunoglobulins, glucose and pH. The AT III concentrates differed markedly in terms of their purity and potency. The specific activities of AT III and the ratios of AT III activity to antigen content ranged from 3.4 to 6.9 and from 0.63 to 0.84, respectively. The highest values were found in five lots of the concentrate that had been treated by both pasteurization and S/D. This preparation was the only one that was virtually free of denaturated AT III, as judged by crossed immunoelectrophoresis. Marked batch-to-batch variation in AT III potencies was found in two out of the five preparations analyzed. In two out of five lots from one manufacturer, the measured potencies were more than 10% lower than the declared potencies. None of the analyzed preparations contained heparin cofactor II, and heparin activities were very low in all lots.

Quantification of Antithrombin Isoform Proportions in Plasma Samples of Healthy Subjects, Sepsis Patients, and in Antithrombin Concentrates

“…Regarding clinically important differences between the available AT concentrates, only few clinical studies showing considerable differences in the in vivo AT recovery during supplementation have been published so far [30]. However, purity, which is a result from different production procedures, also differs considerably between different AT concentrates [31,32]. According to our study, significant variations in the ß-isoform contents of different concentrates are obvious.…”

When antithrombin substitution strikes back

“…Increased temperature/fever, freeze drying, ultrafiltration, and citrate speed up transformation. [9][10][11][12] In the process of this conformational change, NAT turns into pre-LAT which still interacts with heparin and serine proteinases and also has high anti-angiogenic properties (Figure 1). 6 Subsequently the hyper-stable LAT molecule is formed when the reactive loop of NAT is irreversibly folded into the protein and the interaction with heparin and serine proteases is lost.…”

“…Nearly all antithrombin products available for clinical use are produced from fractioned human plasma. In this process, heat treatment at 60°C during up to 10 hours to neutralize viruses, 9,10 the presence of citrate, 10 and freeze drying is part, 12 all which contribute to increased transformation of NAT to LAT. These plasma-based products contain up to 40% LAT.…”