Summary
The preparation of antithrombin concentrate for clinical use requires a viral inactivation
step. In most commercial preparations this is achieved by heat pasteurisation. This
process would be expected to alter the conformation of antithrombin from the active
native species to an inactive latent (L-form) state (1, 2). To determine if this occurs
during commercial preparation and to identify the proportion of the product in the
inactive state, we examined the various antithrombin conformations within a therapeutic
concentrate. The antithrombin concentrate was separated into five fractions by heparin-Sepharose
chromatography. The fraction with the highest heparin affinity retained full activity,
whereas the four fractions with reduced heparin affinity (~40% of the total antithrombin)
had lost their inhibitory function. These inactive antithrombins were intact, monomeric,
thermostable and resistant to unfolding in 8 M urea. Moreover, the protein patterns
on isoelectric focusing and non-denaturing-PAGE showed that there were at least two
different L-forms with isoelectric points separate from the native active species.
Our findings demonstrate that approximately 40% of the antithrombin preparation examined
exists as inactive l-forms. The clinical significance of administering this altered
material is uncertain.
