Related Experiment Videos
Subunit structure of thrombin-activated porcine factor VIII
1Department of Medicine, University of Vermont, Burlington 05405.
Biochemistry
|January 24, 1989
Summary
This study shows that activated Factor VIII (fVIII) forms a stable heterotrimer of three fragments. This finding clarifies the structure of active fVIII, crucial for understanding blood coagulation.
Area of Science:
- Biochemistry
- Hematology
- Protein Chemistry
Background:
- Factor VIII (fVIII) is a key protein in the blood coagulation cascade.
- Thrombin cleavage of fVIII generates active fragments, but their association is not fully understood.
Purpose of the Study:
- To characterize the molecular assembly of thrombin-activated porcine Factor VIII.
- To determine the quaternary structure of the active fVIII complex.
Main Methods:
- Proteolytic cleavage of fVIII by thrombin.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
- Cation-exchange chromatography (Mono S).
- Analytical ultracentrifugation (equilibrium and velocity sedimentation).
Main Results:
- Activated fVIII yielded three distinct fragments: fVIIIA1, fVIIIA2, and fVIIIA3-C1-C2.
- These fragments co-eluted as a single, stable peak with high coagulant activity.
- Analytical ultracentrifugation confirmed the formation of a heterotrimer (fVIIIA1/A2/A3-C1-C2) with an apparent molecular weight consistent with the sum of its subunits.
Conclusions:
- The major species of activated porcine Factor VIII is a stable heterotrimer.
- This structural characterization provides insights into the functional conformation of active Factor VIII.