Summary
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 tractions: fraction I is identical to normal AT III; fraction II (abnormal
AT III) reproduces the abnormalities of the AT III “Milano”, i.e. lack of thrombin
inhibition, incieased mobility by two-dimensional immunoclectrophoresis in the absence
of heparin and migration as two bands with molecular weights of 60 K and 120 K by
sodium dodecyl sulfate polyaeiylamide gel electrophoresis (SDS-PAGE). The interaction
of both fiactions with purified u-thrombin was studied by the formation of complexes
as well as by affinity chromatography on thrombin Seplmiose. 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.
Keywords
Antithrombin III - Variant - Thrombin binding site
