title: Determination of FVIIa-sTF Inhibitors in Toxic Microcystis Cyanobacteria by LC-MS Technique.
abstract: The blood coagulation cascade involves the human coagulation factors thrombin and an activated factor VII (fVIIa). Thrombin and fVIIa are vitamin-K-dependent clotting factors associated with bleeding, bleeding complications and disorders. Thrombin and fVIIa cause excessive bleeding when treated with vitamin-K antagonists. In this research, we explored different strains of toxic Microcystis aeruginosa and cyanobacteria blooms for the probable fVIIa-soluble Tissue Factor (fVIIa-sTF) inhibitors. The algal cells were subjected to acidification, and reverse phase (ODS) chromatography-solid phase extraction eluted by water to 100% MeOH with 20%-MeOH increments except for M. aeruginosa NIES-89, from the National Institute for Environmental Studies (NIES), which was eluted with 5%-MeOH increments as an isolation procedure to separate aeruginosins 89A and B from co-eluting microcystins. The 40%-80% MeOH fractions of the cyanobacterial extract are active against fVIIa-sTF. The fVIIa-sTF active fractions from cultured cyanobacteria and cyanobacteria blooms were subjected to liquid chromatography-mass spectrometry (LC-MS). The 60% MeOH fraction of M. aeruginosa K139 exhibited an m/z 603 [M + H]⁺ attributed to aeruginosin K139, and the 40% MeOH fraction of M. aeruginosa NIES-89 displayed ions with m/z 617 [M - SO3 + H]⁺ and m/z [M + H]⁺ 717, which attributed to aeruginosin 89. Aeruginosins 102A/B and 298A/B were also observed from other toxic strains of M. aeruginosa with positive fVIIa-sTF inhibitory activity. The active fractions contained cyanobacterial peptides of the aeruginosin class as fVIIa-sTF inhibitors detected by LC-MS.
authors.affiliation: Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan. arojanas@riem.nagoya-u.ac.jp.|Department of Brain Functions, Division of Stress Adaptation and Protection, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan. arojanas@riem.nagoya-u.ac.jp.
authors.forename: Andrea Roxanne J
authors.initials: AR
authors.lastname: Anas
authors.affiliation: Graduate School of Environmental and Human Sciences, Meijo University, Tempaku, Nagoya 468-8503, Japan. g0973345@ccalumni.meijo-u.ac.jp.
authors.forename: Anna
authors.initials: A
authors.lastname: Nakajima
authors.affiliation: Graduate School of Environmental and Human Sciences, Meijo University, Tempaku, Nagoya 468-8503, Japan. 100973149@ccalumni.meijo-u.ac.jp.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Naruse
authors.affiliation: Graduate School of Environmental and Human Sciences, Meijo University, Tempaku, Nagoya 468-8503, Japan. 110973437@ccalumni.meijo-u.ac.jp.
authors.forename: Mineka
authors.initials: M
authors.lastname: Tone
authors.affiliation: Graduate School of Environmental and Human Sciences, Meijo University, Tempaku, Nagoya 468-8503, Japan. asukabe@bj8.so-net.ne.jp.
authors.forename: Hirohiko
authors.initials: H
authors.lastname: Asukabe
authors.affiliation: Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan. kiharada@meijo-u.ac.jp.
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Harada
mesh_terms: D000925:Anticoagulants / Q000494:pharmacology*; D002853:Chromatography, Liquid; D000458:Cyanobacteria / Q000737:chemistry*; D015942:Factor VIIa / Q000037:antagonists & inhibitors* / Q000187:drug effects; D005618:Fresh Water; D006801:Humans; D007564:Japan; D013058:Mass Spectrometry; D013329:Structure-Activity Relationship; D013925:Thromboplastin / Q000037:antagonists & inhibitors* / Q000187:drug effects
chemical_list: D000925:Anticoagulants; D013925:Thromboplastin; D015942:Factor VIIa
keywords: aeruginosins; anticoagulant; blood coagulation cascade; cyanobacteria; fVIIa-sTF inhibitors; peptides; toxic Microcystis