Fibrinolytic Activation Promoted by the Cyclopentapeptide Malformin: Involvement of Cytoskeletal Reorganization

Abstract

The fibrinolytic system contains an inactive proenzyme, plasminogen, which is converted to the active enzyme plasmin by two physiological plasminogen activators: tissue-type and urokinase-type plasminogen activators (t-PA and u-PA, respectively). Plasminogen activation mediated by t-PA is mainly involved in the dissolution of fibrin at the site of vascular injury, whereas u-PA binds to a specific cellular receptor (uPAR) and plays a role in pericellular proteolysis by activating cell-bound plasminogen. The fibrinolytic system is regulated at the level of plasminogen activators by specific inhibitors such as plasminogen activator inhibitor-1 (PAI-1), and at the level of plasmin by a 2 -antiplasmin.1) This system is implicated in several pathophysiological processes, including thrombosis, inflammation, vascular repair, tissue remodeling, tumor invasion, and angiogenesis. 2)While searching for a modulator of the fibrinolytic system, we identified several low-molecular-mass compounds that effectively enhance fibrinolytic activity.3) Malformin A 1 (Fig. 1A), a cyclic pentapeptide, is one of these compounds. 4) Malformin A 1 enhances the fibrinolytic activity of U937 human monocytoid cell line which can elaborate PA and PAI, 5) and which have been widely used to study the fibrinolytic system. The malformin effect is dependent on the existence of blood plasma, and its activity is abolished by a lysine analog that competes with plasminogen for the cell-surface receptor or fibrin, or by anti-u-PA serum. Thus, the plasminogen/u-PA system is involved in the action of malformin A 1 . However, malformin A 1 does not enhance fibrinolytic activity in cellfree systems. Therefore, malformin action requires both cellular and plasma-related functions. In this study, we attempted to elucidate the activities of plasma malformin cofactor and purified vitronectin after several activity-based fractionation steps. Malformin A 1 affects cytoskeletal reorganization, cell signaling, and the resultant cell-surface localization of plasminogen. This mechanism may account for the increase in the level of activation of cell-surface plasminogen. MATERIALS AND METHODS MaterialsMalformin A 1 was isolated from a culture of Aspergillus niger F7586 through ethyl acetate extraction, silica gel chromatography, and reverse-phase HPLC. Plasminogen was affinity purified from human plasma, 6) and the flowthrough fraction thus obtained was used as plasminogen-deficient plasma. The following materials were obtained from commercial sources: bovine vitronectin and bovine a 2 -macroglobulin from Yagai (Yagai, Yamagata, Japan); human fibrinogen, Arg-Gly-Asp (RGD) peptide, H-7, genistein, cytochalasin B, and nocodazole from Sigma-Aldrich (St. Louis, MO, U.S.A.); wortmannin and calphostin C from Kyowa Medex (Tokyo, Japan); LY294002, K252a, and PD98059 from Merck (Darmstadt, Germany); U-73122 from Wako (Osaka, Japan); Texas Red-X phalloidin from Invitrogen (Carlsbad, CA, U.S.A.); and t-butyloxycarbonyl-Val-LeuLys-4-methyl-coumaryl-7-amide (Boc-VLK-MCA) from Peptid...

Involvement of RSK1 activation in malformin-enhanced cellular fibrinolytic activity

“…Thereafter, the various biological activities of MA 1 and its analogs have been reported 6 – 18 . The mode of action of MA 1 in enhancing the fibrinolytic activity involves the uPA/plasminogen system on the cell surface and depends on the coordinated action of vitronectin as a plasma component 1 , 19 . However, the intracellular mechanism by which MA 1 enhances fibrinolytic activity remains unclear 19 .…”

Malformin A1 treatment alters invasive and oncogenic phenotypes of human colorectal cancer cells through stimulation of the p38 signaling pathway

“…Plant-associated microorganisms are known to produce a variety of metabolites with novel structures and interesting biological activities (4)(5)(6)(7)(8). MA1 isolated from the fungus A. niger has been found to possess a range of bioactive properties, which includes enhancing fibrinolytic activity, and acting as an antibacterial/viral/fungal agent (13)(14)(15)(16)(17)(18). Furthermore, MA1 exhibits a strong cytotoxic effect against various human cancer cell lines (9,(22)(23)(24).…”

Anti-TMV Activity of Malformin A1, a Cyclic Penta-Peptide Produced by an Endophytic Fungus Aspergillus tubingensis FJBJ11

“…Malformin A 1 ( 1 ), produced from Aspergillus niger , is a cyclic pentapeptide with a disulfide bond formed from two cysteine thiols. Malformin A 1 has been reported to possess a variety of biological activities including plant growth stimulation [ 12 ], phytochrome-mediated response modulation of Phaseolus vulgaris [ 13 ], antibacterial activity [ 14 , 15 ], prevention of interleukin-1 (IL-1) induced endothelial changes by inhibition of protein synthesis [ 16 ], as well as enhanced fibrinolytic activity [ 17 ]. Moreover, malformin A 1 was recently found to be strongly cytotoxic against the human cancer cell lines NCI-H460 (non-small cell lung carcinoma), MIA Pa Ca-2 (pancreatic cancer), MCF-7 (breast cancer), and SF-268 (CNS cancer; glioma) with slight selectivity towards the pancreatic cancer cell line (MIA Pa Ca-2) compared with the normal human primary fibroblast cells WI-38 [ 18 ].…”