Neutralization by plasminogen activator inhibitor-1 of mutants of tissue plasminogen activator.

Abstract

Deletion mutants of human tissue plasminogen activator (tPA) were expressed in Chinese hamster ovary cells. These cells had been transfected with genes that encoded tPA but included restriction sites that allowed the deletion of DNA encoding specific structural domains of the tPA molecule's heavy chain. Purified, two-chain mutant tPAs, or analogues of tPA, lacking one or several structural domains, along with Bowes melanoma tPA were studied in order to determine their susceptibility to inhibition by plasminogen activator inhibitor-1 (PAI-1). The full-length analogue of tPA, designated by its domains FGK1K2P, as well as analogues GK1K2P, FK2P, and FGK1P were treated with various amounts of PAI-1. When the amounts of added tPA and analogues were standardized so that each generated the same absorbance in a chromogenic assay containing S-2251, plasminogen, and fibrinogen fragments, there was a significant difference in the way in which the analogues were titrated by the inhibitor. The melanoma tPA and FGK1K2P were the most susceptible, FK2P slightly less sensitive, and GK1K2P and FGK1P the least sensitive to inhibition. In contrast, when the amounts of enzyme used were standardized on the basis of absorbance generated in a direct assay employing the chromogenic substrate S-2288 and then titrated with PAI-1, these differences in susceptibility to inhibition were not observed. Based on these data, the differential susceptibility to inhibition observed in the plasminogen-dependent assay was attributed to the extent to which the activity of a given analogue is enhanced by fibrinogen fragments, and thus reflected the different amounts of enzymes added in order to standardize the assay on the basis of absorbance.(ABSTRACT TRUNCATED AT 250 WORDS)