Purification and characterization of natural and recombinant human plasminogen activator inhibitor‐1 (PAI‐1)

Open Access
Publisher Website
Google Scholar
Abstract
Human plasminogen activator inhibitor‐1 (PAI‐1) was purified from the conditioned medium of endotoxin‐stimulated umbilical vein endothelial cell cultures by combinations of zinc‐chelate‐Sepharose chromatography, gel filtration on Sephacryl S‐300 and immunoadsorption on an insolubilized murine monoclonal antibody (MA‐7D4). The final product was obtained with a recovery of approximately 20% from conditioned medium containing about 3 μ;g/ml PAI‐1. The yield of PAI‐1 was 15 – 100 μ;g/umbilical cord, depending on the culture and harvest conditions. SDS gel electrophoresis revealed a main band with Mr= 46000 both under reducing and non‐reducing conditions. On gel filtration on Sephacryl S‐300, however, the material was separated in two fractions, one eluting at the void volume, which contains active PAI‐1, and one with Mr= 46000 containing inactive material that could be reactivated with 12 M urea. SDS gel electrophoresis of the isolated high‐Mr fraction revealed several bands including a main 46000‐Mr component, which reacted with anti‐(PAI‐1) antibodies on immunoblotting and neutralized tissue‐type plasminogen activator (t‐PA). The active high‐Mr fraction and the reactivated low‐Mr fraction of PAI‐1 inhibited t‐PA very rapidly with an apparent second‐order rate constant of (1.5–4) × 107 M−1 S−1. The cDNA of endothelial cell PAI‐1 was cloned and expressed in Chinese hamster ovary cells. The translation product, purified from conditioned medium of transfected cells, also revealed a high‐Mr and a low‐Mr fraction on gel filtration, which were indistinguishable from the natural proteins by physicochemical, immunochemical and functional analysis. On reduced SDS gel electrophoresis, the high‐Mr fraction was separated into the Mr‐46000 low‐Mr PAI‐1 and two other components with Mr 65000 and one barely entering the gel. When reactivated low‐Mr PAI‐1 was added to plasma, PAI activity and PAI‐1 antigen eluted with an apparent Mr 300000 on gel filtration, indicating that active PAI‐1 complexes with one or more binding proteins in plasma.