C4a/C4a-desArg, and C5a/C5a-desArg were detected using BD Cytometric Bead Array Human. Anaphylatoxin Kit. Cytometric measurements were performed in ...
Complement cascade activation by Bothrops lanceolatus venom: a “pathway” to improving patient management in case of envenomation?
Clinical aspect of a Bothrops lanceolatus bite.
Marie Delafontaine1, 2; Danielle Paixão-Cavalcante1; Fernanda C. V. Portaro1; Laurence Mathieu2; Joël Blomet2; Denise V. Tambourgi1 (Résière et al, 2008)
Aim: To investigate the complement cascade activation potential of Bothrops lanceolatus venom (BlV), in order to understand the mechanism of local lesion and maybe establish the basis for improving the treatment of local symptoms in case of envenomation.
Why could B. lanceolatus venom activate the complement system ? A
B
A
B
Action of Bothropic South American venoms on complement cascade: -
Activation on the 3 pathways Cleavage of the soluble serine protease inhibitor C1-Inh Direct cleavage of C3, C4 and C5 No action on the surface complement regulators Type I metallo proteases involved
(Pidde-queiroz et al, 2010, 2013; Farsky et al, 2000)
WGA
Con A
Presence of glycosylated proteins Cross-reaction in BlV Brazilian Venom samples (15 µg) were separated antivenom by SDS-PAGE on 12% acrylamide gel, in non-reducing [A] or reducing [B] conditions, and submitted to Westernblotting using the peroxidaseconjugated lectins, concanavalina A (Con A) or wheat germ agglutinin Triticum vulgaris lectin (WGA).
with the bothropic
Samples (5 µg) of B. jararaca (Bj) and B. lanceolatus (Bl) venoms submitted to SDS-PAGE were submitted to Western-blotting using anti-bothropic serum.
Inhibition of flourescent substrates hydrolysis by antibothropic serum BlV was incubated 30 min at room temperature with buffer in the presence of antibothropic serum (20 µL). Assays were carried out, at 37°C, in 100 µL of buffer, containing 5 µM of Abz-peptides.
B. lanceolatus venom activates complement cascade by the three pathways and induces production of anaphylatoxins 50% Inhibition Concentration (µg of venom/mL of diluted NHS)
95% Confidence Interval (µg of venom/mL of diluted NHS)
Classical
156.6
147.3 - 166.6
Alternative
294.5
273.2 - 317.4
Lectin
396.3
375.7 - 418.0
15 SN H S C 5 b -9 c o n c e n t r a t io n ( µ g /m L )
Complement Pathway
*
B .la n c e o la tu s B .la n c e o la tu s + P h e n a n t r o lin e
10
5
0 TCC
Action of BlV on complement pathways. Samples of diluted (1:2) normal human serum (NHS) were incubated with BlV for 30 min (alternative and lectin pathways) or 1 hour (classical pathway) at 37°C. The residual lytic activities of classical and alternative complement pathways were assessed using antibody-sensitised sheep erythrocytes or rabbit erythrocytes, respectively. Mannan-coated plates were used to measure the residual lectin complement pathway activity by ELISA. Data are representative for 3 different experiments. Analysis was performed using the software GraphPad Prism6.
Action of BlV on complement terminal pathway Samples of NHS were incubated with BlV (0,5 mg/mL of diluted NHS (1:2)) for 30 min at 37°C, with or without 1,10phenantroline (10 mM). Concentration of the soluble complement terminal complex (TCC) was assesed using the MicroVue SC5b-9 Plus Enzyme Immunoassay Kit (Quidel, USA). Statistic analysis was performed using the software GraphPad Prism6.
Production of anaphylatoxins induced by BlV. Samples of normal human serum (NHS) were incubated with BlV (0,5 mg/mL of diluted NHS (1:2)) for 30 min at 37°C, with or without 1,10-phenantroline (10 mM). The anaphylatoxins C3a/C3a-desArg, C4a/C4a-desArg, and C5a/C5a-desArg were detected using BD Cytometric Bead Array Human Anaphylatoxin Kit. Cytometric measurements were performed in a FACScalibur (Becton Dickinson, California, USA). Statistic analysis was performed using the software GraphPad Prism6.
B. lanceolatus venom directly cleaves complement cascade components C3, C4 and C5, as well as the regulator C1Inh Cleavage of human purified complement proteins by BlV BlV (2 µg) C (2 µg) Inhibitors
30’ – 37°C
SDS-PAGE in reducing conditions
Silver staining
BlV potently activates the complement cascade via classical, alternative and lectin pathways, cleaving directly the central components of the cascade by the action of metallo and serine proteinases. This fact could play an important role in the local inflammatory reaction and haemostasis disturbance caused by BlV, via a strong release of anaphylatoxins. More investigations are still needed to identify the proteases involved and their mechanisms of action on complement, however the present data suggest that the mechanism could be similar to what was described with other bothropic venoms.
1
Immunochemistry Laboratory, Butantan Institute, Av. Prof. Vital Brazil, 1500, CEP 05503-900, São Paulo, Brazil 2 Prevor Laboratory, Moulin de Verville, 95760 Valmondois CEDEX, France
