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Published on: June 29, 2015
[The functional study of antithrombin L99 mutation]
Tingting Yu1, Jing Dai1, Qiulan Ding1
1Clinical Laboratory Department, Ruijin Hospital of Shanghai Jiaotong University School of Medicine, Shanghai 200025, China.
Insights
The L99 mutation in antithrombin (AT) impairs its binding to heparin, thrombin, and FXa, leading to reduced AT activity and inherited AT deficiency. This study elucidates the molecular basis of this condition.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Context:
- Inherited antithrombin (AT) deficiency is a genetic disorder predisposing individuals to thrombosis.
- The L99 mutation is implicated in AT deficiency, but its precise molecular mechanisms require elucidation.
Purpose:
- To investigate the molecular mechanisms underlying inherited antithrombin (AT) deficiency caused by the AT L99 mutation.
- To assess the impact of specific L99 mutations (L99V, L99A, L99I, L99S) on AT's binding affinities and activity.
Summary:
- Wild-type (WT) and mutant AT proteins (L99V, L99A, L99I, L99S) were expressed and purified.
- Binding assays revealed significantly reduced affinity of mutant AT for heparin, thrombin (FIIa), and coagulation factor Xa (FXa).
- Chromogenic assays demonstrated a marked decrease in AT activity (AT:A) for most L99 mutants.
Impact:
- The L99 mutation disrupts critical interactions of AT with heparin and its targets, leading to impaired anticoagulant function.
- Understanding these molecular defects provides insights into the pathogenesis of inherited AT deficiency.
- This research contributes to the molecular understanding of thrombophilia and may inform future therapeutic strategies.
Objective:
To study the molecular mechanisms of inherited antithrombin (AT) deficiency caused by AT L99 mutation.
Methods:
Wild type (WT), L99V, L99A, L99I and L99S AT were purified from drosophila expression system. The binding capacity of AT and the low molecular weight heparin sodium was analyzed by the heparin binding assay. Surface plasmon resonance (SPR) was used to detect the binding ability of AT to thrombin (FIIa) or AT to coagulation factor Xa (FXa). The activity of AT(AT∶A)was detected by chromogenic assay.
Results:
The purified WT and mutant AT were at the same size. No additional band was observed by coomassie blue staining and western blot assay. Compared to the WT AT, the binding abilities of the low molecular weight heparin sodium to the AT L99V, L99A, L99I and L99S were (44.8±3.6)%, (118.9±14.0)%, (15.2±8.8)%, and(23.0±8.2)%, respectively. The binding abilities of FIIa to AT L99V, L99A, L99I and L99S were 13%, 57%, 3%, and 29%, while the binding of FXa to AT L99V, L99A, L99I and L99S were 7%, 51%, 1%, and 25%. The AT∶A of WT, L99V, L99A, L99I and L99S AT were 146.5%, 21.4%, 120.9%, 10.8%, and 39.0%, respectively.
Conclusion:
The binding abilities of AT to heparin, FIIa and FXa were damaged by the L99 mutation, which resulted in decreased AT∶A and inherited AT deficiency.
