Figure 4.

C5a is the key effector molecule for platelet/granulocyte aggregate (PGA) formation in the presence of dysregulated Factor H (FH) cell surface protection. 20 µl of lepirudin anticoagulated whole blood was incubated with thrombin receptor-activating peptide (TRAP) (10 µM) without or with rH19–20 (10 µM) ± anticomplement reagents (Cp20: 50 µM, PMX53: 16.7 µM, OmCI: 0.5 µM, and Eculizumab and IgG4 isotype control: 100 µg/ml), in a total 80 µl volume. Assuming 3 µM FH in each donors’ plasma and 60% plasma per blood sample, the molar ratio of rH:FH was ~22:1. A sample that did not receive TRAP [non-activated (NA)] or that was preincubated with Cp20 (NA + Cp20) was included for A and B, respectively. (A) PGA formation and (B) C5a generation were determined as described in Section “Materials and Methods.” Graphs are representative of (A) three and (B) two independent experiments done with blood from different human volunteer donors. Results are shown as mean and SD of duplicate observations. The data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. p < 0.001 (***) and p > 0.05 (n.s.).