Dirofilaria immitis possesses molecules with anticoagulant properties in its excretory/secretory antigens

Abstract

Dirofilaria immitis is a parasitic nematode that survives in the circulatory system of suitable hosts for many years, causing the most severe thromboembolisms when simultaneous death of adult worms occurs. The two main mechanisms responsible for thrombus formation in mammals are the activation and aggregation of platelets and the generation of fibrin through the coagulation cascade. The aim of this work was to study the anticoagulant potential of excretory/secretory antigens from D. immitis adult worms (DiES) on the coagulation cascade of the host. Anticoagulant and inhibition assays respectively showed that DiES partially alter the coagulation cascade of the host and reduce the activity of the coagulation factor Xa, a key enzyme in the coagulation process. In addition, a D. immitis protein was identified by its similarity to the homologous serpin 6 from Brugia malayi as a possible candidate to form an inhibitory complex with FXa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry. These results indicate that D. immitis could use the anticoagulant properties of its excretory/secretory antigens to control the formation of blood clots in its immediate intravascular habitat as a survival mechanism.

Keywords: Anticoagulant; Dirofilaria immitis; coagulation cascade; excretory/secretory antigens; factor Xa; serpin.

Fig. 1.

Anticoagulant activity of DiES evaluated by measuring the APTT, the PT and the TT. Plasma from healthy dogs was incubated with 0.5 μg of DiES (■) or with PBS as negative control (●), and the corresponding reagent (APTT, PT or TT). Each point is the mean of three replicates ± s.d.. The asterisk (*) indicates significant differences (P < 0.05).

Fig. 2.

Inhibition of FXa by DiES. (■) 0.1 μg of DiES were incubated with 4 nm FXa and 200 μm S-2765 in a total volume of 100 μL. (●) The presence of DiES was omitted and substituted by PBS as negative control in the previous reaction mixtures. Each point is the mean of three replicates ± s.d. The asterisk (*) indicates significant differences (P < 0.05).

Fig. 3.

Binding of DiES to FXa. SDS-PAGE with 20 μg of DiES (1), 1 μg of FXa (2) and 20 μg of DiES + 1 μg of FXa (3) incubated in 5 mm Hepes buffer, pH 7.4. The two arrows mark the bands belonging to FXa in the sample incubated with DiES. The reference of the molecular weight pattern is indicated on the left.

Fig. 4.

Alignment of the serpin protein 6 of B. malayi (XP_001900434.1) with the antithrombin III of C. lupus familiaris (XP_537187.4) and H. sapiens (AAB40025.1). The amino acids conserved in all the sequences are labelled with asterisks, and conservative and semiconservative substitutions are labelled with two and one point, respectively. The percentage of identity of the whole sequences, as well as the serpin signatures between B. malayi sequence and the others is indicated. The predicted signal peptide is shaded in grey, and the serpin signatures highlighted in yellow.