Figure 1.

Factor H (FH) C-terminal domains are most critical for control of the alternative pathway on platelets and neutrophils. (A) Thrombin-activated platelets preincubated with 25 µg/ml P₂–P₄ (Thr + P; 2 × 10⁷/ml) were incubated without or with rH fragments (18 µM) in the presence of 5 mM MgEGTA and 60% properdin-depleted serum. Assuming 3 µM FH in serum, the molar ratio of rH:FH was ~10:1. Non-activated (NA) platelets were included as negative controls. C3 fragment deposition on platelets was determined as described in Section “Materials and Methods.” Results are representative of independent experiments done with platelets from different human volunteer donors that tested the entire rH fragment panel and are graphed as mean and SD of triplicate observations, n = 2. The data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test against Thr + P. p < 0.001 (***). (B) Neutrophils (2 × 10⁶/ml) were incubated without or with rH fragments (20 µM) and 33% C8-depleted serum in the presence of 2.5 mM MgEGTA. Assuming 3 µM FH in serum, the molar ratio of rH:FH was ~20:1. Samples that received 10 mM EDTA were included as negative controls. C3 fragment deposition on neutrophils was determined as described in Section “Materials and Methods.” Results are representative of independent experiments done with neutrophils from different human volunteer donors that tested the entire rH fragment panel (indicated in each graph) and are graphed as mean and SD of duplicate observations, n = 2. The data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test against MgEGTA alone. p < 0.05 (*) and p < 0.001 (***).