EXPRESSION AND CHARACTERIZATION OF RECOMBINANT PORCINE PLASMINOGEN-ACTIVATOR INHIBITOR-1

Porcine models are, among other animal models, very suitable for in vi vo investigations in the vascular field especially with respect to the possible relationship between atherosclerosis and thrombosis. In orde r to use this model to define the in vivo role of PAI-1, the character ization of porcine PAI-1 and its availability for the generation of im munological tools are a prerequisite. Porcine plasminogen activator in hibitor-1 (poPAI-1) cDNA was isolated from a cDNA library prepared fro m cultured porcine aortic cells and characterized in comparison with P AI-I cDNA's from other species including human, bovine, rabbit, rat an d murine. Subsequently the DNA sequence coding the mature protein was cloned into an appropriate vector for expression in Escherichia coli a nd recombinant porcine PAI-1 was purified and characterized. On SDS-PA GE the apparent molecular weight was estimated to be 45 kDa, identical to the molecular weight of human PAI-1. The purified recombinant porc ine PAI-1 (rpoPAI-1) had a specific activity of 508,800 +/- 800 U/mg ( mean +/- SD, n = 3) towards human tissue-type plasminogen activator (h t-PA) and a functional half-life in vitro of 2.1 +/- 0.8 h (n = 3). In cubation with a two fold molar excess of ht-PA (n = 3) or human urokin ase-type plasminogen activator (hu-PA, n = 2) followed by analysis by SDS-PAGE revealed reaction products corresponding to active (71 +/- 7% resp. 96 +/- 3.6%), latent (12 +/- 0.4% resp. 2.6 +/- 2.4%) and subst rate (16.6 +/- 6.8% resp. 1.5 +/- 1,3) forms. Inactivated samples of p orcine PAI-1 could be reactivated with guanidinium chloride up to 52% of its original specific activity towards t-PA and u-PA. The second or der rate constant of inhibition of ht-PA was 1.64 +/- 0.37 10(7) M(-1) s(-1) (n = 9). In gel filtration rpoPAI-1 in buffer eluted at a volum e corresponding to 24 kDa, whereas in the presence of porcine plasma, the molecular form containing PAI-1 activity eluted at a volume corres ponding to 330 kDa, presumably as a consequence of binding of active P AI-1 to vitronectin. Taken together, these data demonstrate that no ob vious functional differences exist between human and porcine PAI-1.