PRODUCTION OF A RECOMBINANT ANTITHROMBOTIC AND FIBRINOLYTIC PROTEIN, PLATSAK, IN ESCHERICHIA-COLI

The three main components involved in thrombosis and haemostasis are t hrombin, platelets, and plasmin. Almost all inhibitors of thrombosis a re focused either on the inhibition of thrombin or on the inhibition o f platelets. We designed a construct using the fibrinolytic activity o f staphylokinase, fused via a cleavable linker to an antithrombotic pe ptide of 29 amino acids. The peptide was designed to include three inh ibitory regions: (1) the Arg-Gly-Asp (RGD) amino acid sequence to prev ent fibrinogen binding to platelets; (2) a part of fibrinopeptide A, a n inhibitor of thrombin; and (3) the tail of hirudin, a potent direct antithrombin. The amino acid sequence of the 29 amino acid peptide was reverse translated, and the gene was chemically synthesised and clone d into an expression vector as a 3' fusion to the staphylokinase gene. Gene expression was induced in E. coli Top 10 cells and the fusion pr otein, designated PLATSAK, was purified using metal affinity chromatog raphy. The purified fusion protein significantly lengthened the activa ted partial thromboplastin time and thrombin time and inhibited the am idolytic activity of thrombin. The fibrinolytic activity was almost eq ual to that of recombinant staphylokinase as measured with a thrombela stograph. Platelet aggregation was not markedly inhibited by PLATSAK, probably due to the unfavourable three dimensional structure, with the Arg-Gly-Asp sequence buried inside. Our results confirm that it is fe asible to design and produce a hybrid multifunctional protein that tar gets various components of the haemostatic process. (C) 1998 Elsevier Science Ltd.