PubMed · 3615437
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M Heideman, A Bengtson. 1987. Complement activity in shock.. https://pubmed.ncbi.nlm.nih.gov/3615437/
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We have shown previously that the extracellular cysteine proteinase of Entamoeba histolytica trophozoites activates the alternative pathway of complement by specifically cleaving C3. This unique mechanism of complement activation leads to passive lysis of nonpathogenic, but not of pathogenic strains. In an attempt to investigate the relationship between the cleavage of complement components C3 and C5 and the pathogenesis of amebiasis, we investigated the production of the anaphylatoxins C3a and C5a, which have diverse effects on the host immune response. The concentration of proteinase required to cleave purified C5 was at least 5 to 10 times that needed for C3 cleavage, but these levels are easily obtainable as demonstrated by cleavage of 125I-labeled C5 during incubation with purified trophozoites. When the C3a-like cleavage fragments were purified by gel filtration, they were found to be extensively degraded during a 1-h incubation of C3 with the proteinase. Subsequent evaluations of the C3a- and C5a-like cleavage products generated earlier in the reaction using immunoblots and cellulose acetate electrophoresis revealed rapid degradation, even during incubation periods as short as 5 min. Because C-terminal fragments as small as 20 amino acid residues can mimic the biologic functions of C3a or C5a, we tested cleavage products for activity. In sensitive bioassays, including guinea pig platelet aggregation for C3a activity and chemotaxis for C5a activity, we demonstrated that proteolysis renders these molecules inactive. These studies suggest that the extracellular cysteine proteinase of E. histolytica, which is capable of activating the complement system, may also provide a mechanism to circumvent normal host immunity by inactivating the proinflammatory factors C3a and C5a.
EDTA-blood samples derived either from healthy staff or septic patients were investigated for in vitro complement activation during the first 48 h after blood drawing at 4 degrees C and 20 degrees C. For this purpose C3a/C3a desArg plasma levels were determined by the ABICAP C3a assay. Within the septic group no complement activation was detectable during the whole observation period. However, if blood from healthy persons was stored for longer than 6 h at 20 degrees C complement activation occurred. The most profound activation was found in EDTA-blood stored for 48 h at 20 degrees C. C3a values in this sample increased four-fold from 56 +/- 7 ng/ml to 222 +/- 38 ng/ml. From these data we conclude that both immediate cooling of EDTA-blood to 4 degrees C, as well as the immediate separation of plasma as proposed by Mollnes et al. (Clin. Exp. Immunol. (1988) 73, 484), is not necessary for determination of anaphylatoxin plasma values. Storage of EDTA-blood samples for up to 6 h without the need to perform centrifugation should allow anaphylatoxin measurement to become a routine parameter for diagnosis of inflammatory diseases.