A Single‐Step Method for the Isolation of Antithrombin III^1,2

Abstract

A single-step method is described for the isolation of a highly purified antithrombin III (AT III) concentrate at a recovery of over 30% using affinity chromatography on heparin-Sepharose (HS). The polyethylene glycol precipitation step frequently employed in the preparation of AT III concentrates for clinical use has been eliminated and purification is accomplished entirely by optimizing the salt concentration in the HS washing buffer to enhance the desorption of impurities prior to elution of AT III. Pasteurization of the AT III concentrate in the presence of 0.5 M sodium citrate to minimize the risk of hepatitis decreases the recovery by about 20% and induces changes in the patterns obtained by polyacrylamide gel electrophoresis and by crossed immunoelectrophoresis in heparinized agarose gel.

A Highly Purified Antithrombin III Concentrate Prepared From Human Plasma Fraction IV-1 by Affinity Chromatography

“…This was removed by the second He parin Hyper D column. A similar result was observed by Wickerhauser and Williams [10], Known reasons for lowaffinity interaction of AT-III to heparin are: (a) cleavage of AT-III at the active site [24]; (b) folding into putative latent states by heat or chaotropes [22][23][24]; (c) aggregation fol lowing dénaturation [25]; (d) modification of the N-asparaginyl oligosaccharides from bianterinary to tetra-antennary structures in in vitro culture [26], and (e) point mutations of critical amino acids in the heparin binding region [1,27]. The latter two possibilities are not relevant to this situation.…”

A heparin binding synthetic peptide from human HIP / RPL29 fails to specifically differentiate between anticoagulantly active and inactive species of heparin

“…Antithrombin III was purified from human plasma as described in Wickerhauser and Williams [20]. The purified ATIII was then linked to CNBr activated Sepahrose in the presence of excess N-acetylated Hp as in Höök et al ., [2].…”

A Pasteurized Antithrombin III Concentrate for Clinical Use

“…Judging by the recovery of AT III activity and its appearance on two-dimensional immunoelectrophoresis, 0.35 M citrate added directly to the HS eluate (approximately 1.2 M NaCl) preserves AT III during pas teurization and gives results comparable with 0.5-1.0 M citrate in 0.15 M saline phos phate [18]. Since viruses, as well as AT III, may be protected by high concentrations of citrate and sodium chloride, it seems pru dent to use these in the lowest possible con centration.…”

“…Since viruses, as well as AT III, may be protected by high concentrations of citrate and sodium chloride, it seems pru dent to use these in the lowest possible con centration. Only trials in chimpanzees or patients will prove whether this strategy is as effective in inactivating hepatitis viruses as is that used by Tabor et al [19], Wickerhauser and Williams [18] found that precipitation with 20% PEG removed all detectable HBsAg from the HS eluate and included this step as a precaution in addition to pasteuri zation. Since precipitation with PEG was not used in our method, there is an addi tional element of uncertainty about com plete inactivation of viruses and it cannot yet be claimed that this concentrate will not transmit hepatitis.…”