Abnormal antithrombin III with defective serine protease binding (antithrombin III "Denver")

Area of Science:

• Biochemistry
• Hematology
• Genetics

Background:

• Hereditary antithrombin III (AT-III) deficiency is a known risk factor for venous thromboembolism.
• Previous studies have identified various AT-III mutations affecting its anticoagulant function.
• Understanding the molecular basis of AT-III dysfunction is crucial for diagnosing and managing thrombotic disorders.

Purpose of the Study:

• To characterize a novel hereditary antithrombin III (AT-III) deficiency in a family with normal AT-III antigen levels but reduced anticoagulant activity.
• To investigate the functional properties of the identified AT-III variant, termed AT-III "Denver".

Main Methods:

• Plasma assays including heparin cofactor activity, progressive antithrombin activity, and anti-Xa activity.
• Crossed immunoelectrophoresis (CIE) with and without heparin.
• Heparin-sepharose affinity chromatography for AT-III purification.
• SDS-polyacrylamide gel electrophoresis (PAGE) for thrombin-antithrombin (TAT) complex formation analysis.
• Turnover studies using autologous 131I-AT-III.

Main Results:

• Patients presented with normal AT-III antigen levels but significantly reduced heparin cofactor and antithrombin activities.
• Purified AT-III
• Denver
• showed normal recovery and elution via heparin-sepharose chromatography but exhibited only 50% of normal thrombin neutralization and anti-Xa activity.
• SDS-PAGE revealed that AT-III
• Denver
• formed TAT complexes with only half the efficiency of normal AT-III.
• Purified AT-III
• Denver
• demonstrated normal CIE and SDS-PAGE migration but failed to bind thrombin or Xa.
• Turnover studies suggested a slightly increased plasma clearance rate for AT-III
• Denver
• .

Conclusions:

• The identified hereditary AT-III deficiency, AT-III
• Denver
• , is characterized by a molecular defect leading to impaired binding and inhibition of thrombin and factor Xa.
• Despite normal heparin interaction, this variant AT-III molecule exhibits reduced anticoagulant efficacy.
• Heparin-sepharose affinity chromatography can be utilized to isolate this non-reactive AT-III variant, aiding in its characterization.