Robinson J H, Browne M J, Carey J E, Chamberlain P D, Chapman C G, Cronk D W, Dodd I, Entwisle C, Esmail A F, Kalindjian S B
Department of Biotechnology, SmithKline Beecham Pharmaceuticals, Surrey, England, UK.
Circulation. 1992 Aug;86(2):548-52. doi: 10.1161/01.cir.86.2.548.
Early intervention with thrombolytic agents has been shown unequivocally to reduce mortality after acute myocardial infarction. Presently used agents have disadvantages such as short half-life, immunogenicity, hypotension, and bleeding complications. Therefore, there is a need to develop improved thrombolytic drugs with novel mechanisms of action leading to improved properties.
Hybrid plasminogen/tissue-type plasminogen activator (t-PA) complementary DNA was constructed and expressed in Chinese hamster ovary cells. The chimeric protein, comprising the fibrin-binding domains of plasminogen covalently linked to the catalytic domain of t-PA, was purified and evaluated in vitro and in vivo. The hybrid was inhibited rapidly in human and animal plasmas. The mediator of this rapid inhibition was shown to be alpha 2-antiplasmin. The active center of the hybrid could be protected by reversible active center acylation with a novel inverse acylating agent, 4'-amidinophenyl-4-chloroanthranilic acid (AP-CLAN). An acylated (CLAN-) hybrid was cleared from the bloodstream of guinea pigs at 0.35 +/- 0.02 ml/min.kg-1 compared with a clearance rate of 36 +/- 4 ml/min.kg-1 for t-PA. The CLAN-plasminogen/t-PA hybrid was evaluated in a quantitative, "humanized" guinea pig pulmonary embolism model and shown to be approximately threefold more potent when given by bolus than an infusion of t-PA. Furthermore, the acylated hybrid was more fibrin selective than t-PA as determined by the relation between clot lysis and fibrinogen degradation.
An acylated, recombinant plasminogen/t-PA hybrid has sufficiently slow clearance to be administered by bolus and is more potent and fibrin selective than t-PA in vivo.
