The molecular pathogenesis of hookworm anemia

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DESCRIPTION: (provided by the applicant): More than one billion people in developing countries are currently infected with blood feeding hookworms, intestinal nematodes that represent a leading cause of iron deficiency anemia in the world. The pathogenesis of hookworm anemia is a direct result of hemorrhage caused by the adult worm as it attaches to the intestinal mucosa. While it has been appreciated for nearly a century that adult hookworms produce potent inhibitors of thrombosis, only recently have the molecular mechanisms underlying the parasite blood feeding process been elucidated. Potent inhibitors of coagulation and platelet function have been identified in soluble protein extracts and secretory products of the human hookworm parasite Ancylostoma ceylanicum. The anticoagulant has been cloned from adult A. ceylanicum RNA, and the recombinant protein inhibits the activity of coagulation factor Xa by a novel mechanism. The platelet inhibitor blocks the function of two important platelet integrins, glycoprotein Jib/lila (GPIIb/IIIa) and GPIa/Iia, which mediate platelet binding to fibrinogen and collagen, respectively. We hypothesize that these anti-thrombotics play a central role in the pathogenesis of hookworm anemia by facilitating blood feeding and exacerbating gastrointestinal hemorrhage. The mechanism of action of the hookworm factor Xa inhibitor will be characterized using in vitro studies of factor Xa binding, protease mediated inhibitor cleavage, and site directed mutagenesis. The platelet inhibitor will be purified and cloned from A. ceylanicum, and its mechanism of action will be characterized using in vitro assays of GPIa/Iia and GPIJb/iIIa integrin binding. Using a reproducible animal model of A.ceylanicum infection, the role of the anticoagulant and platelet inhibitor in the pathogenesis of hookworm anemia will be characterized using a vaccine-based approach. Animals will be immunized with each recombinant inhibitor, followed by challenge with 50 infectious L3 hookworm larvae. The responses to immunization will be monitored by ELISA, and the degree to which antibodies directed at the anti-thrombotics from A. ceylanicum protect against hookworm anemia and weight loss will be assessed using clinical parameters and worm burden measurements. These studies will ultimately determine the role of blood feeding in the pathogenesis of hookworm disease, as well as identify potential targets for a human vaccine against this globally important parasite.