Avian urokinase-type plasminogen activator (u-PA) lacks the putative binding site for plasminogen activator inhibitor (PAI) and is resistant to inhibition by human PAI-1 and PAI-2

Abstract

Summary In mammalian cultures, the activation and catalytic activity of both urokinase-type plasminogen activator (u-PA) and tissue-type PA (t-PA) is regulated in part by naturally occurring plasminogen activator inhibitors PAI-1 and PAI-2, members of the serpin family of serine protease inhibitors. Interaction of human PAIs with human u-PA (h-u-PA) and t-PA appears to be mediated by a putative PAI binding sequence in the enzymes. Comparison of the amino acid sequences of human, murine, porcine, bovine, simian and avian u-PA revealed specific sequences in the mammalian enzymes that are homologous to the recently discovered PAI-binding site in human u-PA and t-PA which is characterized by conserved basic amino acids. However, no homology was seen in the same region in chicken u-PA (c-u-PA), which is devoid of the consensus arginine and/or lysine residues. The chicken enzyme is a close structural and catalytic homolog of mammalian u-PA. The lack of the putative PAI binding sequence would predict that c-u-PA cannot interact with and be inhibited by human PAIs. To test this hypothesis, the effects of human PAI-1 and PAI-2 on avian u-PA were examined, using purified enzyme and inhibitors. Under conditions where h-u-PA activity is inhibited by human recombinant PAI-1 (rPAI-1), c-u-PA retains 95% activity. c-u-PA was also resistant to inhibition by human PAI-2. High molecular weight, SDS-stable complexes do not form readily when c-u-PA is incubated with PAI-1 or PAI-2, while human as well as murine u-PA formed complexes with both PAIs under the identical conditions. The c-u-PA molecule, thus, is a naturally occurring u-PA variant which lacks a PAI-binding region. The resistance of this enzyme to inhibition by human PAI-1 and PAI-2 supports the functional relevance of this sequence in mammalian u-PA and t-PA.