EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT RAT PLASMINOGEN-ACTIVATOR INHIBITOR-1

Plasminogen activator inhibitor-1 (PAI-1) is a physiologically importa nt regulatory protein of the fibrinolytic system. To study in vivo its influence on a variety of biological events (thrombosis, atherosclero sis, tumour progression) a number of different experimental models in various animals, including rats, have been established. A major drawba ck 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 e xpected, sodium dodecyl sulphate polyacrylamide gel electrophoresis re vealed 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 act ivator (t-PA), corresponding to 58+/-10% of the theoretical maximum va lue. Evaluation of the reaction products formed upon interaction betwe en recombinant rat PAI-1 and its target proteinases t-PA or urokinase- type plasminogen activator revealed the presence of large amounts of c ovalent complex, small amounts of cleaved PAI-1 and residual latent PA I-1, Active recombinant rat PAI-1 converted spontaneously to the laten t form with a half-life of 2+/-0.2 h (n=6). The second-order rate cons tant 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). Comparativ e gel filtration experiments of recombinant rat PAI-1 either in the pr esence 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 demo nstrate the biochemical equivalence between rat PAI-1 and human PAI-1. The availability of purified recombinant rat PAI-1 will allow the dev elopment of research tools required to evaluate the in vivo role of PA I-1 in various rat models.