Alternative Lys277 cleavage modulates tissue plasminogen activator processing and fibrinolysis

Background Tissue plasminogen activator (tPA) is secreted as a single-chain enzyme and converted to its canonical two-chain form by cleavage at Arg275. Whether the adjacent Lys277 residue contributes to tPA activation and fibrinolytic function remains unclear. Objectives To determine whether Lys277 can function as an alternative tPA cleavage site and whether Lys277-dependent processing is associated with altered fibrinolytic activity. Methods We combined phylogenetic analysis of PLAT sequences from 645 gnathostome species, engineered human tPA mutants affecting Arg275 and/or Lys277, plasmin- and plasma-cleavage assays, Edman sequencing, amidolytic and fibrin-plasminogen zymography, plasma clot-lysis assays, and rotational thromboelastometry. Results Arg275 was highly conserved across species, whereas species carrying Arg275 substitutions almost invariably retained Lys277; snakes contained only the Lys277 site. Plasmin cleaved wild-type tPA at Arg275-Ile276, whereas Arg275-mutant tPA was cleaved at Lys277-Gly278; mutation of both residues prevented two-chain formation. Compared with single-chain wild-type tPA, Arg275 single mutants shortened 50% lysis of fibrin-rich plasma clots by 1.9 to 2.4-fold and accelerated blood clot fibrinolysis. Human plasma generated tPA heavy-chain fragments from wild-type and Arg275-mutant tPA, but not from the Arg275/Lys277 double mutant, indicating cleavage at both sites under plasma conditions. Preparations enriched in Lys277-cleaved tPA, displayed greater blood clot lysis than Arg275-cleaved tPA, with corrected fibrinolysis speed up to 2.8-fold higher.