Anticoagulant repertoire of the hookworm Ancylostoma caninum.

126

123

Tissue factor (TF) is the physiological activator of the coagulation cascade that plays pathophysiological roles in metastasis, angiogenesis, and inflammation. Downstream in coagulation, thrombin is the central protease that signals through G protein-coupled, protease-activated receptors (PARs). However, the TF-VIIa-Xa complex upstream in coagulation also activates PAR1 and 2. Here, we address the question of whether signaling of the TF initiation complex is a relevant pathway that leads to TF cytoplasmic domain phosphorylation. In heterologous expression systems and primary endothelial cells, we demonstrate that the ternary TF-VIIa-Xa complex induces TF phosphorylation specifically by activating PAR2 but not through PAR1 signaling. In addition, TF cytoplasmic domain phosphorylation is induced only by TF-dependent signaling but not by other coagulation factors in endothelial cells. Phosphorylation of the Prodirected kinase target site Ser 258 is dependent on prior phosphorylation of Ser 253 by protein kinase C (PKC) ␣. TF phosphorylation is somewhat delayed and coincides with sustained PKC␣ activation downstream of PAR2 but not PAR1 signaling. Phosphatidylcholine-dependent phospholipase C is the major pathway that leads to prolonged PKC␣ recruitment downstream of PAR2. Thus, PAR2 signaling specifically phosphorylates TF in a receptor cross-talk that distinguishes upstream from downstream coagulation protease signaling.

Section: Materials-viia Inactive Sermentioning

confidence: 99%

Protease-activated Receptor 2-dependent Phosphorylation of the Tissue Factor Cytoplasmic Domain

Ahamed

Ruf

2004

126

123

“…For example, in a single village of ϳ800 people in South China, it was estimated that hookworms caused an annual blood loss of 200 liters (14). To obtain an unimpeded blood meal, hookworms secrete potent anticoagulants, the actions of which have been well characterized (15,16). Once blood is ingested by adult hookworms, they lyse erythrocytes using a pore-forming, membrane-bound hemolysin (17), releasing the red cell contents into the intestinal lumen for proteolytic degradation.…”

mentioning

confidence: 99%

A Multi-enzyme Cascade of Hemoglobin Proteolysis in the Intestine of Blood-feeding Hookworms

Williamson¹,

Lecchi

Turk

et al. 2004

157

115

Blood-feeding pathogens digest hemoglobin (Hb) as a source of nutrition, but little is known about this process in multicellular parasites. The intestinal brush border membrane of the canine hookworm, Ancylostoma caninum, contains aspartic proteases (APR-1), cysteine proteases (CP-2), and metalloproteases (MEP-1), the first of which is known to digest Hb. We now show that Hb is degraded by a multi-enzyme, synergistic cascade of proteolysis. Recombinant APR-1 and CP-2, but not MEP-1, digested native Hb and denatured globin. MEP-1, however, did cleave globin fragments that had undergone prior digestion by APR-1 and CP-2. Proteolytic cleavage sites within the Hb ␣ and ␤ chains were determined for the three enzymes, identifying a total of 131 cleavage sites. By scanning synthetic combinatorial peptide libraries with each enzyme, we compared the preferred residues cleaved in the libraries with the known cleavage sites within Hb. The semi-ordered pathway of Hb digestion described here is surprisingly similar to that used by Plasmodium to digest Hb and provides a potential mechanism by which these hemoglobinases are efficacious vaccines in animal models of hookworm infection.

“…This doubling time concentration has been used previously to measure the potency of recombinant hookworm anticoagulants (5,6,17 …”

Section: Hookworm Life Cycle and Preparation Of Hex And Es-allmentioning

confidence: 99%

“…To date, the two predominant anticoagulant serine protease inhibitors have been cloned from the dog hookworm Ancylostoma caninum. One of these, A. caninum anticoagulant peptide 5 (AcAP5), 1 is a potent and specific inhibitor of coagulation factor Xa, whereas the second, AcAPc2, blocks the activity of factor VIIa in complex with membrane-bound tissue factor (5,6). Both of these hookworm proteins are members of a family of related serine protease inhibitors first identified in the non-bloodfeeding intestinal nematode Ascaris suum (7)(8)(9).…”

mentioning

confidence: 99%

Molecular Characterization of Ancylostoma Inhibitors of Coagulation Factor Xa

Harrison

Nerlinger

Bungiro

et al. 2002

Bloodfeeding hookworms, which currently infect over a billion people in the developing world, are a leading cause of gastrointestinal hemorrhage and iron deficiency anemia. The major anticoagulant inhibitor of coagulation factor Xa has been identified from the hookworm parasite Ancylostoma ceylanicum using reverse transcription PCR and 3-rapid amplification of cDNA ends. This is the first anticoagulant cloned from a hookworm species for which humans are recognized permissive hosts. Despite ϳ50% amino acid similarity, A. ceylanicum anticoagulant peptide 1 (AceAP1) is both immunologically and mechanistically distinct from AcAP5, its homologue isolated from the dog hookworm Ancylostoma caninum. Studies using plasma clotting times and single stage chromogenic assays of factor Xa activity have demonstrated that the recombinant AceAP1 protein is substantially less potent than AcAP5 and that soluble whole worm protein extracts of adult A. ceylanicum possess less anticoagulant activity than extracts of A. caninum. These values correlate with previously reported differences in bloodfeeding capabilities between these two species of hookworm, suggesting that factor Xa inhibitory activity is predictive of hookworm bloodfeeding capabilities in vivo. These fundamental differences in the mechanism of action and immunoreactivity of the major anticoagulant virulence factors from related Ancylostoma hookworm species may have significant implications for human vaccine development.