Purification and Characterization of Human Coagulation Factor

We have purified human coagulation Factor V 6,000fold to homogeneity from citrated plasma using polyethylene glycol 6000 precipitation, adsorption of Factor V to barium citrate, DEAE-Sepharose chromatography, and gel filtration on Ultrogel AcA 34 (yield 21%). Human Factor V is a single polypeptide chain before and after disulfide bond reduction with an apparent M, = 335,000 as determined by electrophoresis on 5% acrylamide sodium dodecyl sulfate gels. Human Factor V is a glycoprotein containing 13% by weight carbohydrate and there is a high content of sialic acid (86 residues/mol) compared to the other sugars. When human Factor V is treated with thrombin, coagulation activity increases 25to 30-fold to a specific activity of 1.7 to 2.0 units/pg. Thrombin activation is accompanied by the cleavage of three bonds in the Factor V molecule. We have detected activation intermediates with apparent M, = 295,000 and 248,000 and final products with apparent M, = 150,000,121,000, and a doublet at 95,000-91,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final products of thrombin activation of human Factor V and bovine Factor V are similar, yet the intermediates observed are different. This suggests that cleavages are made at similar locations in bovine and human Factor V, but that they occur in a different sequence. When human Factor V is treated with the Factor V activator from Russell’s viper venom, it is split into two components with apparent M, = 303,000 and 95,00001,000 and is fully activated. The increase in coagulation activity observed upon treatment of human Factor V with thrombin or the Factor V activator from Russell’s viper venom seems to correlate with the generation of the doublet M, = 95,000-91,000 component.