Identification and localization of hookworm platelet inhibitor in Ancylostoma ceylanicum

Ancylostoma ceylanicum is a zoonotic hookworm, which mainly causes iron deficiency anemia (IDA) in humans and animals. Hookworm platelet inhibitor (HPI) has been isolated from adult Ancylostoma caninum and linked to the pathogenesis of hookworm associated intestinal hemorrhage and IDA. However, there is no available data about HPI from A. ceylanicum. To study the molecular characteristics of A. ceylanicum HPI (Ace-HPI), its corresponding cDNA was amplified from adult A. ceylanicum mRNA using the primers designed based on the Ac-HPI gene sequence, and its sequence homology and phylogenetic relationship were analyzed. The differential expression of Ace-hpi mRNA in the adult and third larval (L3) stages was compared using the quantitative real-time PCR. Ace-HPI reactivity and tissue localization were studied by Western blot and immunofluorescence, respectively. Platelet aggregation activity was monitored in a 96-well microplate reader. The results showed that the Ace-HPI encoding gene was 603 bp in length. Ace-HPI showed 91% homology to Ac-HPI, was closely related to Ac-ASP3, and belonged to the CAP superfamily. Ace-hpi transcripts were most abundant in the adult stage, followed by serum-stimulated infective larvae (ssL3), and finally in L3 stage, with a significant difference. Escherichia coli-expressed recombinant protein had good reactivity with the positive serum of A. ceylanicum-infected dogs. Immunolocalization indicated that Ace-HPI was located in the esophagus and cephalic glands of the adult. As well as, recombinant Ace-HPI inhibited the platelet aggregation in-vitro. HPI overexpression, anatomical location in adults, antigenicity and its in-vitro activity indicate its possible role in adult worm blood-feeding and as a valuable target for hookworm vaccine and drug development.