PROTECTIVE EFFECT OF VITRONECTIN ON OXIDANT-INDUCED INACTIVATION OF RECOMBINANT PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 (RPAI-1) - STRUCTURAL AND FUNCTIONAL-ANALYSIS

This study was undertaken to further characterize the nature of the pr otective effect of vitronectin (VN) on the inhibition of an active for m of recombinant plasminogen activator inhibitor type 1 (rPAI-1) by va rious oxidants. The activity of rPAI-1, as determined by its ability t o neutralize the enzymatic activity of tissue plasminogen activator (t -PA) in a chromogenic assay, was inhibited by chloramine-T (CT) in con centration-dependent fashion with an IC50 of 30 uM. This inhibition wa s antagonized by pretreatment of rPAI-1 with VN (1 to 30 ug/ml) a prot ective effect that was dependent on the concentration of both the oxid ant and VN. Interestingly, no other antioxidants including a-tocophero l, ascorbate, butylated hydroxytoluene, or amino acids such as L-argin ine or cysteine were able to prevent the inhibition of rPAI-1. To exam ine the structural basis for the VN effect on rPAI-1, fluorescense spe ctroscopic studies were performed to determine the effect of CT on the tryptophan emission intensity (TEI) of rPAI-1. Treatment of rPAI-1 wi th CT resulted in significant quenching of the TEI of rPAI-1, which is correlated with the loss of activity of rPAI-1 in neutralizing t-PA. VN suppressed the quenching effect of CT on the TEI of rPAI-1, indicat ing that VN may exert its protective effect by maintaining rPAI-1 in a conformation in which the tryptophans are protected. These results su ggest a specific mechanism for the protection of rPAI-1 by VN.