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

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 micrograms/ml PAI-1. The yield of PAI-1 was 15-100 micrograms/umbilical cord, depending on the culture and harvest conditions. SDS gel electrophoresis revealed a main band with Mr = 46,000 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 = 46,000 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 46,000-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) x 10(7) 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-46,000 low-Mr PAI-1 and two other components with Mr 65,000 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 greater than or equal to 300,000 on gel filtration, indicating that active PAI-1 complexes with one or more binding proteins in plasma.