Purification and further characterization of antithrombin III Milano: lack of reactivity with thrombin.
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) reproduces 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 alpha-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.
Wolf
M
M
INSERM U. 143, Hpital Bicêtre, Le Kremlin-Bicêtre, France.
Boyer-Neumann
C
C
Meyer
D
D
Tripodi
A
A
Mannucci
P M
PM
Larrieu
M J
MJ
D000990:Antithrombin III / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D014644:Genetic Variation; D006579:Heterozygote; D006801:Humans; D013917:Thrombin / Q000037:antagonists & inhibitors*
C054797:antithrombin III Milano; D000990:Antithrombin III; D013917:Thrombin