Ayadualin, a novel RGD peptide with dual antihemostatic activities from the sand fly Lutzomyia ayacuchensis, a vector of Andean-type cutaneous leishmaniasis.
Sequence analysis of the Lutzomyia (Lu.) ayacuchensis salivary gland cDNA library identified a short peptide containing an RGD (Arg-Gly-Asp) sequence flanked by two cysteine residues in the C-terminal end as the most abundant transcript. In the present study, a recombinant protein of the RGD-containing peptide, designated ayadualin, was expressed in Escherichia coli and its activity was characterized. Ayadualin inhibited both collagen and ADP-induced platelet aggregations by interfering with the binding of integrin αIIbβ3 to fibrinogen. The RGD sequence and cysteine residues located on both sides of the RGD sequence were essential for the inhibitory action. Moreover, ayadualin efficiently inhibited the intrinsic blood coagulation pathway irrespective of the RGD sequence. Measuring the enzymatic activity of coagulation factors using chromogenic substrates revealed that ayadualin efficiently inhibited factor XIIa (FXIIa) activity in a dose-dependent manner. In addition, pre-incubation of ayadualin with FXII inhibited FXIIa activity, while activated FXIIa was not affected by ayadualin, indicating that ayadualin inhibits the activation of FXII, but not enzymatic activity of FXIIa. These results indicated that ayadualin plays an important role in the blood feeding of Lu. ayacuchensis by inhibiting host hemostasis via dual mechanisms.
Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. Electronic address: hkato@vetmed.hokudai.ac.jp.
Hirotomo
H
Kato
Departamento de Medicina Tropical, Facultad de Medicina, Universidad Catolica de Guayaquil, Ecuador.
Eduardo A
EA
Gomez
Laboratory of Veterinary Hygiene, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan.
Megumi
M
Fujita
Laboratory of Veterinary Hygiene, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan.
Yuka
Y
Ishimaru
Department of Dermatology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan.
Hiroshi
H
Uezato
Department of Microbiology, Faculty of Life Sciences, Graduate School of Health Sciences, Kumamoto University, Kumamoto, Japan.
Tatsuyuki
T
Mimori
Laboratory of Veterinary Hygiene, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan.
Hiroyuki
H
Iwata
Department of Parasitology, Kochi Medical School, Kochi University, Kochi, Japan; Centro de Biomedicina, Universidad Central del Ecuador, Quito, Ecuador; Prometeo, Secretaría Nacional de Educacion Superior, Ciencia, Tecnologia e Innovacion (SENESCYT), Ecuador.
Yoshihisa
Y
Hashiguchi
D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D015956:Factor XIIa / Q000037:antagonists & inhibitors*; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry / Q000494:pharmacology*; D007303:Insect Vectors / Q000737:chemistry*; D016773:Leishmaniasis, Cutaneous; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D011576:Psychodidae / Q000737:chemistry*
D019476:Insect Proteins; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid; D015956:Factor XIIa
Anticoagulant; Antiplatelet; RDG peptide; Saliva; Sand fly