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

Abstract

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 lgAEmL )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.

Bacillolysin MA, a Novel Bacterial Metalloproteinase That Produces Angiostatin-like Fragments from Plasminogen and Activates Protease Zymogens in the Coagulation and Fibrinolysis Systems

“…After incubation for 30 min, 100 l of EGM containing 20 ng/ml epidermal growth factor was added and incubated for 3 days. The ability of cells to reduce formazan-forming reagent (WST-1; Dojindo Laboratories, Kumamoto, Japan) was determined as an index of cell number (26).…”

A new series of the SMTP plasminogen modulators with a phenylamine-based side chain

“…5 SMTPs, a family of triprenyl phenol metabolites produced by Stachybotrys microspora, enhance plasminogen activation by modulating plasminogen conformation. [5][6][7] SMTP-7, one of the most potent congeners, is effective in treating thrombotic stroke in animal models, [8][9][10][11] possibly involving a neuroprotective mechanism. [10][11][12] The SMTP molecule consists of a tricyclic g-lactam moiety, a geranylmethyl group, and an N-linked side chain.…”

Tumor shedding of laminin binding protein modulates angiostatin production in vitro and interferes with plasmin‐derived inhibition of angiogenesis in aortic ring cultures

“…PMDs have only been recently identified as the autoproteolytic products of plasmin(ogen) generated when incubation takes place in the presence of certain nonlysine analog plasminogen modulators. 65 The intense fragmentation of the catalytic domain that occurs during this type of plasmin processing suggests that these compounds bind to plasmin in a way that exposes the catalytic domain for cleavage, and suggests the occurrence of a major conformational change upon analog binding to plasmin. 65 According to this model, plasmin catalysis would be rapidly inhibited by the fragmentation leading to a significant change in the tumor environment, including a reduction in local proteolytic events.…”

“…65 The intense fragmentation of the catalytic domain that occurs during this type of plasmin processing suggests that these compounds bind to plasmin in a way that exposes the catalytic domain for cleavage, and suggests the occurrence of a major conformational change upon analog binding to plasmin. 65 According to this model, plasmin catalysis would be rapidly inhibited by the fragmentation leading to a significant change in the tumor environment, including a reduction in local proteolytic events. This expectation is supported by the overall reduction in proteolysis that is seen in our casein zymograms when plasmin is incubated in the presence of sLBP.…”