Functional analysis of the human tissue-type plasminogen activator protein: the light chain

A pBR322::Rous sarcoma virus(RSV)-based shuttle vector was used to insert fused genes, composed of the amino-terminal portion of the bacterial chloramphenicol-acetyltransferase gene (cat) and the entire coding region for the C-terminally derived light (L) chain of human tissue-type plasminogen activator (t-PA) cDNA. Cotransfection of rat 3Y1 cells with pRSVneo DNA and pRSVcat/t-PA DNA yielded stably integrated G418-resistant transfectants which contain unrearranged copies of pRSVcat/t-PA DNA. These transfectants synthesize cat/t-PA L-chain mRNA, apparently correctly initiated and terminated. With the help of an enzyme-linked immunosorbent assay (ELISA), it is demonstrated that these cells produce human t-PA antigen. Furthermore, pRSVcat/t-PA L-chain cDNA-containing rat 3Y1 cells synthesize a plasminogen-dependent amidolytic activity which is suppressed by specific anti-human t-PA antibodies. This activity cannot be stimulated by fibrin, a property displayed by native t-PA. It is concluded that the t-PA L-chain cDNA contains the complete genetic information for the plasminogen activator activity.