Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. Identification as a multimeric form of S protein (vitronectin).

Abstract

A binding protein for plasminogen activator inhibitor 1 (PAI-1-BP) was isolated from human plasma by a four-step procedure. 1) The 7 S globulin fraction of plasma was isolated by gel filtration on Sephacryl S-300. 2) Human endothelial cell-type plasminogen activator inhibitor (PAI-1), pretreated with 12 M urea, was added to this fraction (22 micrograms of PAI-1/ml of plasma), and a PAI-1 antigen peak with apparent mass 450 kDa (representing 65% of PAI-1 antigen and 85% of PAI activity) was isolated by gel filtration of this mixture. 3) The PAI-1.PAI-1-BP complex was further purified by immunoadsorption on an immobilized murine monoclonal antibody directed against PAI-1 (MA-7D4) and by elution with 4 M KSCN. 4) The complex was then dissociated by addition of excess human tissue-type plasminogen activator (t-PA), and t-PA and PAI-1 antigen (t-PA.PAI-1 complexes and free t-PA and PAI-1) were removed by immunoadsorption on monoclonal antibodies directed against t-PA (MA-62E8) and against PAI-1 (MA-7D4 and MA-12A4). Sodium dodecyl sulfate-gel electrophoresis of the purified material under nonreducing conditions revealed two bands with apparent mass approximately equal to 150 kDa and two bands with mass 74 and 68 kDa. Reduced sodium dodecyl sulfate-gel electrophoresis displayed two main bands with apparent masses of 73 and 64 kDa. The PAI-1-BP reacts with urea-treated, but not with inactive PAI-1. t-PA dissociates the complex between PAI-1 and PAI-1-BP. PAI-1 in complex with PAI-1-BP is 2-3-fold more stable at 37 degrees C than purified PAI-1, suggesting that PAI-1-BP may stabilize PAI-1 in blood. The concentration of PAI-1-BP in plasma determined by titration with PAI-1 is approximately 130 mg/liter. The isolated PAI-1-BP was shown to be identical to S protein (vitronectin) both by cross-reactivity with monospecific rabbit antisera and by NH2-terminal amino acid sequence analysis. The gel filtration behavior, mobility on sodium dodecyl sulfate-gel electrophoresis, and concentration in plasma suggest that PAI-1-BP is a multimer (presumably a dimer) of S protein accounting for approximately 35% of the S protein in plasma.