Related Experiment Videos
Purification and further characterization of antithrombin III Milano: lack of reactivity with thrombin
M Wolf1, C Boyer-Neumann, D Meyer
1INSERM U. 143, Hpital Bicêtre, Le Kremlin-Bicêtre, France.
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) 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.
Insights
Antithrombin III "Milano" is a variant protein that cannot inhibit thrombin. Further studies confirm this defect is due to an inability to react with thrombin.
Area of Science:
- Biochemistry
- Hematology
Background:
- Antithrombin III (AT III) is a critical protein in regulating blood coagulation.
- The AT III "Milano" variant exhibits abnormal monomeric and dimeric forms.
- Previous characterization indicated functional abnormalities in this variant.
Purpose of the Study:
- To further characterize the functional abnormality of the Antithrombin III "Milano" variant.
- To investigate the interaction between the AT III "Milano" variant and alpha-thrombin.
Main Methods:
- Affinity chromatography using heparin-Sepharose to separate AT III fractions.
- Two-dimensional immunoelectrophoresis to assess mobility.
- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine molecular weight.
- Studying complex formation and affinity chromatography on thrombin-Sepharose to analyze thrombin interaction.
Main Results:
- Two distinct fractions of AT III were purified: normal AT III (fraction I) and abnormal AT III "Milano" (fraction II).
- Fraction II showed lack of thrombin inhibition, altered mobility, and molecular weights of 60 K and 120 K on SDS-PAGE.
- No thrombin-AT III complexes were formed with the variant AT III, and no binding to thrombin-Sepharose was observed.
Conclusions:
- The molecular defect in AT III "Milano" is its absence of reactivity with thrombin.
- This lack of reactivity explains the observed functional abnormalities, including impaired thrombin inhibition.