Free tPA Research Articles

A Monoclonal Antibody that Does Not Recognize Tissue-Type Plasminogen Activator Bound to Its Naturally Occurring Inhibitor

Hybridoma clones specific for tissue-type plasminogen activator (tPA) were screened in solid-phase radioimmunoassays for their reactivity toward free tPA and tPA complexed to the endothelial cell-derived, beta-migrating plasminogen activator inhibitor (beta-PAI). Two monoclonal antibodies (MABs) were identified with quite distinct properties. The first, MAB LI72D1, bound to free tPA but did not recognize tPA complexed to the beta-PAI, whereas the second, MAB HI72C1, bound both to free tPA and to tPA in complex with beta-PAI. These properties were maintained when the MABs were immobilized to plastic microtiter wells, thus permitting the development of immunoradiometric assays (IRMAs) to quantitate free tPA and total tPA antigen in various samples. The IRMAs were employed to analyze the tPA in media conditioned by several human cell types. The results indicate that in some cases, tPA may be present entirely as a free and active enzyme, while in others it apparently exists entirely in complex with beta-PAI. Interestingly, some cells appear to contain both forms of tPA.

Turnover of tissue plasminogen activator in normal and hepatectomized rabbits

The distribution, and clearance of the tissue plasminogen activator (tPA) was studied in rabbits, rats and mice. Following an intravenous injection of I-labelled tPA a large portion of the radioactive dose was rapidly accumulated in the liver. After one hour the radioactivity in the liver was less than 5 per cent of the injected dose. The highest activity was then found in the intestine, stomach and in the blood. Gel filtration of plasma taken one hour after the injection revealed that the major part (60%) of the radioactivity was present as low molecular- weight metabolites or free iodine The remaining activity was present as high molecular weight inhibitor complexes (26%) or as free tPA (15%). In order to study in vivo reactions between tPA and plasma inhibitors without hepatic interference, plasma turnover was also studied in hepatectomized rabbits. High amounts of radioactivity remained in the plasma after one hour. Gel filtration of this plasma revealed that 54 per cent of the radioactivity was bound to inhibitors, 34 per cent circulated as free tPA while a minor portion (12%) was found as free iodine or metabolites. The half-life of fibrinolytically active tPA in hepatectomized rabbits was 40 minutes compared to 2 minutes in intact rabbits. The increase in the half-life of tPA in hepatectomized rabbits also resulted in an improved thrombolytic effect after treatment with 0.5 mg tPA.