Isolation, Identification and Pharmacokinetic Properties of Human Tissue-Type Plasminogen Activator Species: Possible Localisation of a Clearance Recognition Site

Abstract

Purified preparations of recombinant tissue-type plasminogen activator (t-PA) from the recombinant Bowes melanoma cell line TRBM6 were shown to contain multiple species of plasminogen activator. Using a combination of chromatography on Sephadex G25, Sephadex G75 and Heparin Sepharose CL6B we have isolated two fibrinolytically active species, which, under non-reduced SDS PAGE, have apparent Mr = 38,000 and 56,000. Double immunodiffusion studies indicated that both species were closely related to both the t-PA B chain and t-PA itself. N-terminal sequencing identified the Mr = 38,000 species as ala160- t-PA (essentially delta FGKI t-PA) and the Mr = 56,000 species as ser1-tyr2-gln3-glyx-cys51 t-PA (delta F t-PA), the latter probably produced by alternative splicing of the t-PA gene. The pharmacokinetic properties of N,N dimethyl-4-aminobenzoyl (DAB) derivatives of these activators and native t-PA were determined in the guinea pig. Whereas DAB----delta F t-PA showed a similar, rapid plasma disappearance profile to that of DAB----t-PA, DAB----delta FGKI t-PA was cleared significantly slower. These results suggest that a rapid clearance recognition site resides on either the growth factor or kringle 1, or both, domains of t-PA.