P.A. Andreasen

Plasminogen activator inhibitor type‐1 : reactive center and amino‐terminal heterogeneity determined by protein and cDNA sequencing

Both the urokinase‐type and tissue‐type plasminogen activator can convert their 5̃4 kDa type‐1 inhibitor (PAI‐1) to an inactive form with a lower apparent molecular mass. We have determined the amino‐terminal amino acid sequences of human native and converted PAI‐1, and isolated PAI‐1 cDNA and determined the nucleotide sequence in regions corresponding to the amino‐terminus and the cleavage site. The data show that the conversion of the inhibitor consists of cleavage of an Arg‐Met bond 33 residues from the carboxy‐terminus, thus localizing the reactive center of the inhibitor to that position. In addition, a heterogeneity was found at the amino‐terminus, with a Ser‐Ala‐Val‐His‐His form and a two‐residue shorter form (Val‐His‐His‐) occurring in approximately equal quantities.

Plasminogen activator inhibitor-type 1 in Lewis lung carcinoma.

SummaryPlasminogen activator inhibitor-type 1 (PAI-1) was identified in extracts of Lewis lung carcinoma, and its immunohistochemical localization was studied together with that of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators. All primary tumors (n=11) contained heterogeneously distributed immunoreactivity against each of the three components. Most often, areas that contained u-PA immunoreactivity also contained PAI-1 immunoreactivity. However, several areas showed a strong u-PA immunoreactivity, but no or low PAI-1 immunoreactivity. The latter staining pattern was only found in periferal areas, and usually in areas with histological signs of tissue destruction. Lung metastases always contained u-PA immunoreactivity, while PAI-1 immunoreactivity was found in most, but not all, metastases. t-PA immunoreactivity was found in a few scattered tumor cells, in primary carcinomas as well as metastases. Controls that included absorption with highly purified antigen preparations and immunoblotting, indicated that all the immunoreactivity represented genuine PAI-1, u-PA and t-PA, respectively. The results are consistent with an assumption that the plasminogen activation system, and particularly u-PA and PAI-1, plays a role in regulation of breakdown of extracellular matrix proteins during invasive growth in this carcinoma.

Plasminogen activator inhibitor type 1 biosynthesis and mRNA level are increased by dexamethasone in human fibrosarcoma cells.

Dexamethasone increases type 1 plasminogen activator inhibitor (PAI-1) activity released from the human fibrosarcoma cell line HT-1080. We demonstrated that dexamethasone caused about 10-fold increases in the intracellular and extracellular levels of PAI-1 protein, as measured by an enzyme-linked immunosorbent assay, in the rate of PAI-1 biosynthesis, and in the PAI-1 mRNA level. The effects on PAI-1 biosynthesis and mRNA level were detectable within 4 h and were maximal 16 to 24 h after the addition of dexamethasone. Cycloheximide did not inhibit the dexamethasone-induced increases in the capacity of the cells to synthesize PAI-1 and in the PAI-1 mRNA level.

Plasminogen activators catalyse conversion of inhibitor from fibrosarcoma cells to an inactive form with a lower apparent molecular mass

Purified 54 kDa plasminogen activator inhibitor from human fibrosarcoma cells was converted to an inactive form with slightly higher electrophoretic mobility by incubation with catalytic amounts of urokinase‐type or tissue‐type plasminogen activator. Serine proteinase inhibitors and a monoclonal antibody against urokinase‐type plasminogen activator inhibited the conversion, indicating that it was caused by plasminogen activator‐catalyzed proteolysis. These findings represent the first demonstration of a well‐defined protein apart from plasminogen, constituting a substrate for plasminogen activators.

General detection of proteins after electroblotting by trinitrobenzene sulphonic acid derivatization and immunochemical staining with a monoclonal antibody against the trinitrophenyl group

A sensitive method for the general detection of proteins electroblotted onto nitrocellulose sheets after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. The proteins on the blots were reacted with 2,4,6-trinitrobenzene sulphonic acid. The resulting trinitrophenyl groups on the proteins were rendered visible by immunochemical staining with a monoclonal anti-trinitrophenyl antibody, and a peroxidase-conjugated second antibody. Using various proteins, the method was compared to the amidoblack method for staining of protein blots. The method was 10-100-fold more sensitive than the amidoblack method. Amounts as low as 1 ng of human serum albumin could be detected.