Heterogeneity in human prothrombin: analysis of cause

Area of Science:

• Biochemistry
• Proteomics
• Human Physiology

Background:

• Human prothrombin exists in multiple forms in plasma.
• Understanding prothrombin heterogeneity is crucial for coagulation studies.

Purpose of the Study:

• To isolate and characterize distinct human prothrombin fractions.
• To elucidate the molecular basis of prothrombin charge heterogeneity.

Main Methods:

• Heparin-agarose chromatography for prothrombin isolation from single-donor plasma.
• Sodium dodecyl sulfate gel electrophoresis and nondenaturing electrophoresis for analyzing protein forms.
• Isoelectric focusing in urea to determine isoelectric points.
• Amino acid analysis and quantification of neutral sugar and sialic acid residues.

Main Results:

• Two prothrombin fractions, H-II1 and H-II2, were isolated in a 4:1 ratio.
• Fractions exhibited identical migration in SDS-PAGE but distinct mobilities under nondenaturing conditions.
• H-II1 showed two components (H-II1a, H-II1b) with different isoelectric points (pI).
• Prothrombin fractions shared identical amino-terminal residues and gamma-carboxyglutamic acid content but differed in neutral sugar and sialic acid content.
• Neuraminidase treatment eliminated electrophoretic heterogeneity, confirming sialic acid's role.

Conclusions:

• Human prothrombin exhibits charge heterogeneity due to variations in sialic acid residues.
• Sialic acid content directly influences the electrophoretic properties of prothrombin variants.
• These findings clarify the molecular basis of prothrombin heterogeneity in normal human plasma.