The blood fluke Schistosoma mansoni cleaves the coagulation protein high molecular weight kininogen (HK) but does not generate the vasodilator bradykinin

Abstract

Background: Schistosomes are blood dwelling parasitic worms that cause the debilitating disease schistosomiasis. Here we examined the influence of the parasites on their external environment by monitoring the impact of adult Schistosoma mansoni worms on the murine plasma proteome in vitro and, in particular, on how the worms affect the blood coagulation protein high molecular weight kininogen (HK).

Methods: Following the incubation of adult schistosomes in murine plasma, two-dimensional differential in-gel electrophoresis (2D-DIGE) was conducted to look for changes in the plasma proteome compared with control plasma. A major change to the blood protein kininogen (HK) was observed, and the interaction of Schistosoma mansoni parasite with this protein alone was then investigated by western blot analysis and activity assays. Finally, the generation of bradykinin from HK was monitored using a bradykinin detection kit.

Results: The most striking change to the plasma proteome concerned HK; while the full-length protein was more abundant in control plasma, carboxyl-terminal truncated forms were more abundant in plasma that contained schistosomes. Incubating parasites in buffer with pure HK followed by Western blot analysis confirmed that human HK is degraded by the worms. The resulting digestion pattern differed from that brought about by kallikrein, a host serine protease that normally acts on HK to release the vasodilator bradykinin. We found that live schistosomes, while digesting HK, do not generate bradykinin nor do they cleave a chromogenic kallikrein substrate. Since the cleavage of HK by the worms is not impeded by the serine protease inhibitor PMSF but is blocked by the cysteine protease inhibitor E64c, we hypothesize that schistosome tegumental cysteine proteases are responsible for HK cleavage.

Conclusions: Since proteomic and biochemical studies have revealed that the schistosome tegument contains two cysteine proteases belonging to the calpain family (SmCalp1 and SmCalp2) we conclude that these are likely responsible for the HK cleavage reported here. Schistosome cleavage of HK should help impede blood clotting and inflammation around the worms in vivo and so promote their ease of movement within the vasculature of their hosts.

Keywords: Bradykinin; Calpain; Coagulation; High molecular weight kininogen; Schistosoma mansoni.

Fig. 1

Impact of schistosomes on the mouse plasma proteome. a Image (right) showing 2D gel resolution of samples obtained following normal heparinized mouse plasma incubation for 1 h at 37 °C either in the presence of schistosome parasites (red) or without parasites (green). Most proteins are the same in both samples and appear yellow. The numbers on the sides of the images represent molecular mass markers (kDa) and the numbers at the bottom represent pH values. The section containing protein spots of interest is bounded by a green box at the top and this section is magnified and shown on the left. Four spots are circled and numbered. Two exhibits greater relative abundance in the absence of parasites (green spots 1, 2); two are more abundant in the presence of parasites (red spots 3 and 4). b Gel spot analysis by DeCyder software. The left panel depicts an analysis of gel spot 2. Here the sizable protein peak (bounded by a yellow line in the figure) that is seen in the control sample (- parasites) is greatly diminished in the sample containing parasites (+ parasites). Spot 4, right panel, shows the opposite effect in which the identified spot is barely detectable in the control sample (-) but becomes prominent in the presence of the worms (+). c Diagrammatic representation of the HK protein with its 6 domains (D1-D6). The 9-amino acid vasodilator bradykinin is contained within domain 4 and is indicated by an orange line. The positions of peptides that were identified by mass spectroscopy following trypsin digestion of 2D-DIGE spots 1 and 2 are indicated by thin green lines protruding above the HK protein. Positions of peptides identified by mass spectroscopy following trypsin digestion of 2D-DIGE spots 3 and 4 are indicated by red lines protruding below the protein

Fig. 2

Analysis of HK cleavage by schistosomes using western blotting. a High molecular weight kininogen (HK) was incubated in the absence (-) or presence of parasites (S, schistosomula; M, males) for different time periods (6 or 24 h, as indicated). Samples were resolved by SDS-PAGE, blotted to membrane and probed by western blotting. A number of prominent HK degradation products are detected (at ~40 and 16 kDa, arrows) only in the presence of parasites. The numbers on the right indicate molecular mass markers (kDa). b Parasites or kallikrein, as indicated, were incubated with HK for 24 h and cleavage products were resolved by SDS-PAGE, blotted to membrane and probed for HK digestion by western blotting. Numbers represent molecular mass markers (kDa, right). c HK was incubated for 6 h with parasites either in the presence or absence of serine protease inhibitor PMSF (0.5 mM), as indicated (left panel); or in the presence or absence of cysteine protease inhibitor E64c (0.1mM) (right panel). The presence of PMSF (left panel) does not impede parasite-mediated cleavage of HK. The characteristic ~40 kDa product is indicated by the arrow. In contrast, the presence of E64c (right panel) does impede parasite-mediated cleavage of HK

Fig. 3

Kallikrein activity assay. Cleavage of kallikrein substrate (H-D-Pro-Phe-Arg-pNa) by living schistosomes (triangle) and human kallikrein (0.02 μg) in the presence (square) or absence (circle) of PMSF (2 mM) (Mean +/- SD, n = 3). Released pNa was measured every 5 minutes at OD₄₀₅. All conditions differ significantly from “Kallikrein”; two-way ANOVA: F_(24,52) = 94.76, F_(2,52) = 2318.22, F_(12,52) = 124.82, P < 0.0001

Fig. 4

Bradykinin detection. Bradykinin generated following incubation of HK with either male schistosomes (HK + males) or with pure human kallikrein (HK + Kallikrein) or alone (HK) for 24 h at 37^oC. Positive (+) and negative (-) controls used in this ELISA assay are bradykinin, 2.8 nM and 0 nM. (Mean +/- SD, n = 3)

Fig. 5

Depiction of biochemical pathways involving high molecular weight kininogen (HK, blue circles) that schistosomes may disrupt (red). HK is a cofactor in the conversion of coagulation factor XII to its active form (Factor XIIa) and the conversion factor XI to its active form (Factor XIa). HK is also involved in the Factor XIIa cleavage of prekallikrein to its active form, kallikrein. Finally, HK is cleaved by kallikrein to generate bradykinin. Cleavage of HK by parasites has the potential to disrupt all of these pathways