Non-lysine-analog plasminogen modulators: promotion of autocatalytic generation of plasmin(ogen) fragments with angiostatin-like activity and inhibition of tumor growth in vivo

Abstract number: P1359

Hasumi^* K., Ohyama^* S., Hu^* W., Harada^* T., Abe† S.

†Teikyo University, Japan ^*Tokyo Noko University, Japan;

We recently discovered several nonlysine-analog conformational modulators of plasminogen, including SMTP-6, thioplabin B and complestatin. Unlike lysine analog modulators, which increase plasminogen activation but inhibit its binding to fibrin, these agents enhance both activation and fibrin binding of plasminogen. Here we show that these nonlysine-analog modulators promote autocatalytic generation of plasmin(ogen) derivatives with its catalytic domain undergone extensive fragmentation (PMDs), which have angiostatin-like anti-endothelial activity. The enhancement of urokinase-catalyzed plasminogen activation by SMTP-6 followed 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. The PMD generation was observed when plasmin alone was treated with SMTP-6 and was inhibited by the plasmin inhibitor aprotinin, indicating autocatalytic generation of PMD. 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 the 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. PMDs of 68–77 kDa were inhibitory to proliferation, migration and tube formation of endothelial cells at concentrations of 0.2–20 mg mL^-1. The administration of orniplabin, a potent SMTP-6 analog, at an intraperitoneal dose of 1.5 mg kg^-1 in mice inhibited the growth of implanted Lewis lung carcinoma.