Expression, purification, and characterization of recombinant rat plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1) is a physiologically important regulatory protein of the fibrinolytic system. To study in vivo its influence on a variety of biological events (thrombosis, atherosclerosis, tumour progression) a number of different experimental models in various animals, including rats, have been established. A major drawback of these models is the lack of the purified endogenous proteins and/or specific reagents for their detection in each particular species. In this study we describe the expression, in Escherichia coli (E. coli) cells, the purification, and the characterization of rat PAI-1. As expected, sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed a protein with an apparent molecular mass of similar to 45 kDa. Recombinant rat PAI-I had a specific inhibitory activity of 434000+/-74000 U/mg (mean+/-SD, n=11) towards human tissue-type plasminogen activator (t-PA), corresponding to 58+/-10% of the theoretical maximum value. Evaluation of the reaction products formed upon interaction between recombinant rat PAI-1 and its target proteinases t-PA or urokinase-type plasminogen activator revealed the presence of large amounts of covalent complex, small amounts of cleaved PAI-1 and residual latent PAI-1, Active recombinant rat PAI-1 converted spontaneously to the latent form with a half-life of 2+/-0.2 h (n=6). The second-order rate constant of inhibition of human t-PA by recombinant rat PAI-1 was two-fold lower compared to that by recombinant human PAI-1 (0.6+/-0.02x10(7) M-1 S-1 VS 1.4+/-0.14x10(7) M-1 S-1 respectively, P<0.0001). Comparative gel filtration experiments of recombinant rat PAI-1 either in the presence or in the absence of rat plasma demonstrated the formation of a high molecular weight form (M-r similar to 400 kDa) of active PAI-1, indicative for its association with vitronectin. The current data demonstrate the biochemical equivalence between rat PAI-1 and human PAI-1. The availability of purified recombinant rat PAI-1 will allow the development of research tools required to evaluate the in vivo role of PAI-1 in various rat models.