Purification and further characterization of antithrombin III Milano: Lack of reactivity with thrombin

M. Wolf, C. Boyer-Neumann, D. Meyer, A. Tripodi, P. M. Mannucci, M. J. Larrieu

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Abstract

The functional abnormality of Antithrombin III 'Milano', a previously described variant with monomeric and dimeric forms of abnormal AT III, has been further characterized. Affinity chromatography on heparin-Sepharose led to the separation and purification of two distinct fractions: fraction I is identical to normal AT III; fraction II (abnormal AT III) reproduced the abnormalities of the AT III 'Milano', i.e. lack of thrombin inhibition, increased mobility by two-dimensional immunoelectrophoresis in the absence of heparin and migration as two bands with molecular weights of 60 K and 120 K by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The interaction of both fractions with purified α-thrombin was studied by the formation of complexes as well as by affinity chromatography on thrombin-Sepharose. No thrombin-AT III complexes could be demonstrated with either the monomeric or dimeric forms of purified variant AT III at both concentrations of thrombin used. Similarly, no binding to thrombin-Sepharose was observed, thus indicating that the molecular defect of AT III Milano is related to its absence of reactivity with thrombin.

Original languageEnglish
Pages (from-to)888-892
Number of pages5
JournalThrombosis and Haemostasis
Volume58
Issue number3
Publication statusPublished - 1987

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ASJC Scopus subject areas

  • Hematology

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