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Human plasma FVIIa (pFVIIa) and human recombinant FVIIa (rFVIIa) were both purified by immune adsorption chromatography using a calcium dependent monoclonal antibody. The FVII obtained is highly purified and contains only trace contaminants as revealed by SDS-PAGE and reverse phase HPLC chromatography. FVII was fully activated during the purification procedure. A FVIIa activity assay has been developed in microplates using human FX as a substrate and methoxycarbonyl-D-cyclohexal-alanyl-glycyl-arginine-pNA as a chromogenic substrate for the FXa generated. The assay was linear at FVIIa concentrations between 0.5 and 10 nM. The concentration of the chromogenic substrate was 0.5 mM. A pH optimum at about 8 was found. An apparent Km=0.2 μM for FX was found for both pFVIIa and rFVIIa.The results suggest that the kinetics of human FX activation by pFVIIa and rFVIIa are identical. The FVIIa activity was found to be calcium dependent with maximal activity at about 0.25 mM, while the activities at 1 and 2 mM were 20% and 3%, respectively. When rabbit brain extract is used, the well-known dramatic enhancement effect of thromboplastin could be demonstrated with both FVII preparations. Also this reaction is calcium-dependent; however, the profile of the curve is distinctly different. Poly-D-lysine (MW 160,000) was found to enhance the FVIIa activity in a concentration dependent manner. Maximum stimulation (fivefold) was obtained at a concentration of about 10 mg/l.

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