Ancylostoma ceylanicum anticoagulant peptide-1: role of the predicted reactive site amino acid in mediating inhibition of coagulation factors Xa and VIIa.
Hookworm infection is a leading cause of gastrointestinal blood loss and iron deficiency anemia in developing countries. Ancylostoma hookworms secrete potent anticoagulants, which have been shown to target coagulation factors Xa and the factor VIIa/Tissue Factor complex, respectively. The goal of these experiments was to determine the mechanism of action of three recombinant hookworm anticoagulants using in vitro assays. Three hookworm coagulation inhibitors were expressed and purified, along with site directed mutants targeting each of the predicted P1 inhibitory reactive site amino acid residues. Using chromogenic assays, it has been confirmed that Ancylostoma caninum Anticoagulant Peptide 5 (AcAP5) inhibits coagulation factor Xa (fXa) by a canonical, substrate-like mechanism. In contrast, Ancylostoma ceylanicum Anticoagulant Peptide-1 (AceAP1) binds to and inhibits fXa by both active site and non-active site mediated interactions. Data from in vitro studies also demonstrates that AceAP1 inhibits the factor VIIa/Tissue complex (fVIIa/TF) and displays a distinct pattern of fXa binding. Together, these data suggest that the human hookworm A. ceylanicum has evolved a single anticoagulant that targets multiple components of the mammalian coagulation response, effectively mimicking the concerted action of the two related inhibitors from A. caninum. Despite the amino acid sequence similarity, AceAP1 appears to interact with coagulation proteases fXa and fVIIa by a novel mechanism, perhaps explaining its spectrum of inhibitory activity.
Program in International Child Health, Department of Pediatrics, Yale School of Medicine, New Haven, CT 06520, USA.
Juliusz
J
Mieszczanek
Lisa M
LM
Harrison
Michael
M
Cappello
D019943:Amino Acid Substitution; D000722:Ancylostoma / Q000201:enzymology*; D000818:Animals; D001665:Binding Sites; D001777:Blood Coagulation; D005165:Factor V / Q000037:antagonists & inhibitors / Q000378:metabolism; D015942:Factor VIIa / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D065427:Factor Xa Inhibitors; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology
D065427:Factor Xa Inhibitors; D015801:Helminth Proteins; D011994:Recombinant Proteins; C094339:anticoagulant protein, Ancylostoma caninum; C022475:prothrombinase complex; D005165:Factor V; D015942:Factor VIIa; D015951:Factor Xa