Nonlysine-analog plasminogen modulators promote autoproteolytic generation of plasmin(ogen) fragments with angiostatin-like activity.

We recently discovered several nonlysine-analog conformational modulators for plasminogen. These include SMTP-6, thioplabin B and complestatin that are low molecular mass compounds of microbial origin. Unlike lysine-analog modulators, which increase plasminogen activation but inhibit its binding to fibrin, the nonlysine-analog modulators enhance both activation and fibrin binding of plasminogen. Here we show that some nonlysine-analog modulators promote autoproteolytic generation of plasmin(ogen) derivatives with its catalytic domain undergoing extensive fragmentation (PMDs), which have angiostatin-like anti-endothelial activity. The enhancement of urokinase-catalyzed plasminogen activation by SMTP-6 was followed by rapid inactivation of plasmin due to its degradation mainly in the catalytic domain, yielding PMD with a molecular mass ranging from 68 to 77 kDa. PMD generation was observed when plasmin alone was treated with SMTP-6 and was inhibited by the plasmin inhibitor aprotinin, indicating an autoproteolytic mechanism in PMD generation. Thioplabin B and complestatin, two other nonlysine-analog modulators, were also active in producing similar PMDs, whereas the lysine analog 6-aminohexanoic acid was inactive while it enhanced plasminogen activation. Peptide sequencing and mass spectrometric analyses suggested that plasmin fragmentation was due to cleavage at Lys615-Val616, Lys651-Leu652, Lys661-Val662, Lys698-Glu699, Lys708-Val709 and several other sites mostly in the catalytic domain. PMD was inhibitory to proliferation, migration and tube formation of endothelial cells at concentrations of 0.3-10 microg.mL(-1). These results suggest a possible application of nonlysine-analog modulators in the treatment of cancer through the enhancement of endogenous plasmin(ogen) fragment formation.

Antibiotic A10255 (thioplabin) enhances fibrin binding and activation of plasminogen.

Three thiopeptide metabolites that enhance fibrin binding of plasminogen were isolated from a culture of Streptomyces sp. R1401. A combination of spectroscopic analyses revealed that these compounds were identical with the antibiotic A10255B, E and G. These agents enhanced fibrin binding of plasminogen and plasminogen/urokinase-mediated fibirinolysis at concentrations of 5 to approximately 20 microM. A10255B reversibily increased urokinase-catalyzed activation of plasminogen by lowering Km, while the agent did not enhance urokinase activity when substrates other than plasminogen were used, indicating that the agent affects plasminogen to increase its affinity to urokinase. A smaller but significant increase in activation was also observed when conformationally relaxed plasminogen derivatives such as Lys-plasminogen and mini-plasminogen were used. Two related thiopeptide antibiotics with a C-terminal amide had no effect on plasminogen activation, suggesting a role of the terminal carboxyl of the A10255 molecule in activity.