Related Experiment Videos
Sequence variation in heparin octasaccharides with high affinity for antithrombin III.
Biochemistry
|November 20, 1984
Summary
Two heparin octasaccharide fragments were isolated and characterized for their binding affinity to antithrombin (AT). Both fragments bind AT, influencing its conformation and enhancing factor Xa inhibition, crucial for understanding heparin
Area of Science:
- Biochemistry
- Glycobiology
- Pharmacology
Background:
- Heparin is a complex polysaccharide with anticoagulant properties mediated by its interaction with antithrombin (AT).
- Understanding the specific structural features of heparin responsible for AT binding is crucial for developing targeted anticoagulant therapies.
Purpose of the Study:
- To isolate and characterize heparin-derived octasaccharide fragments with high affinity for human antithrombin.
- To elucidate the structural basis of heparin-antithrombin interaction and its functional consequences on protease inhibition.
Main Methods:
- Nitrous acid cleavage of heparin to generate octasaccharide fragments.
- High-affinity binding assays to determine dissociation constants with antithrombin.
- Enzymatic degradation studies using Flavobacterium heparinase and platelet heparitinase.
- Stopped-flow fluorometry to monitor antithrombin conformational changes and factor Xa inhibition rates.
Main Results:
- Two major octasaccharide fragments, S and R, were isolated, exhibiting high affinity for antithrombin (Kd values in the range of 10^-8 to 10^-7 M).
- Octasaccharide S was sensitive to heparinase degradation, while Octasaccharide R was resistant.
- Structural variations, including N-sulfated glucosamine and iduronic acid substitutions, had minimal impact on AT binding.
- Both octasaccharides induced a 40% enhancement in antithrombin's intrinsic fluorescence and facilitated rapid inhibition of human factor Xa (5 x 10^5 M^-1 s^-1).
Conclusions:
- Heparin octasaccharides with high affinity for antithrombin can be generated through specific structural arrangements.
- The binding of these octasaccharides induces conformational changes in antithrombin, leading to enhanced anticoagulant activity.
- These findings provide insights into the structure-activity relationships of heparin and its interaction with antithrombin, relevant for anticoagulant drug development.