Jeff Robinson

Redesigning t-PA for improved thrombolytic therapy.

Attempts are being made to redesign the structure of tissue-type plasminogen activator (t-PA) in order to increase its plasma half-life, increase its fibrin affinity or decrease its rate of interaction with plasma inhibitors. The principal strategies employed so far have been to construct hybrid enzymes, to mutate the polypeptide sequence of t-PA or to add extra fibrin-binding elements. It has been relatively easy to alter the half-life of t-PA but more difficult to do this with retention of the full specific activity of the molecule; the most promising molecules will have to be evaluated in the clinic before we know whether the redesign of t-PA has been truly successful.

The importance of genome analysis to the drug discovery process

The sequencing of partial human expressed genes and of microbial genomes is leading to the discovery of many new targets for drug discovery. As more targets become known, drugs can be designed that have greater selectivity than was possible using traditional pharmacological methods. The promise of the new approach is that drugs discovered in this way will be highly efficacious and will have a reduced tendency for unwanted side-effects.

Identification of the calcium-inducible plasminogen activators secreted by a human diploid fibroblast cell line (MRC-5)

Human embryonic lung fibroblast (MRC5) cells secreted small amounts of plasminogen activators into normal, serum-free harvest medium. Stimulation with calcium led to markedly enhanced levels of activator. The major species of plasminogen activator in the harvest medium of the stimulated cultures resembled u-PA when analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by fibrin zymography. This activator was partially-purified using controlled-pore glass and Blue Sepharose CL6B. Characterisation by fibrin zymography in the presence of specific antibody, by 3H-DFP labelling and by fibrin-binding ability indicated that the activator was indistinguishable from high molecular weight u-PA. The possible physiological role of the production of relatively large amounts of u-PA by a lung cell capable of producing both u-PA and t-PA is discussed.

Induction of plasminogen activator in a human diploid fibroblast cell line (MRC-5) by conditions which cause induction of interferon: The role of calcium ions

The human diploid fibroblast cell line, MRC-5, derived from embryo lung tissue produced only small quantities of plasminogen activator (PA) when harvested using a standard nutrient medium (Eagles Minimal Essential Medium, MEM). Use of a schedule designed to induce high concentrations of fibroblast interferon in these cells also resulted in production of considerably enhanced levels of PA. The kinetics of PA production differed from those of interferon production; specifically, PA was produced for at least 6 days following induction despite the toxicity of the inducers whereas interferon synthesis continued for only 1 day. Further investigation of the induction conditions for PA revealed that double-stranded RNA which was absolutely required for interferon production was not required for induction of PA. Indeed, the stimulus for enhancement of PA production appeared to be solely an elevated concentration of calcium ions in the extracellular medium. The possible physiological relevance of this induction of PA by elevated concentrations of calcium ions is discussed.