Purification of human brain tissue factor.

Area of Science:

• Biochemistry
• Hematology

Background:

• Tissue factor (factor III) is a critical lipoprotein cofactor.
• It significantly enhances the catalytic activity of coagulation factor VIIa on factors IX and X.

Purpose of the Study:

• To purify human tissue factor apoprotein from brain tissue.
• To characterize its properties and determine optimal relipidation conditions for restoring clotting activity.

Main Methods:

• Purification involved acetone delipidation, Triton X-100 extraction, and affinity chromatography using factor VII-agarose.
• Characterization included SDS-PAGE for molecular weight determination and amino acid sequencing.
• Relipidation studies assessed clotting activity at varying phospholipid/apoprotein ratios.

Main Results:

• Human tissue factor apoprotein was purified 53,000-fold to homogeneity.
• The purified apoprotein has an apparent molecular weight of 44,000 Da and a determined NH2-terminal amino acid sequence.
• Optimal relipidation, achieved at a phospholipid/apoprotein ratio > 600 (w/w), resulted in a 5000-fold enhancement of clotting activity.

Conclusions:

• The study successfully purified and characterized human tissue factor apoprotein.
• Restoring the lipid environment is essential for the cofactor activity of tissue factor in blood coagulation.