Cross-Talk between the Complement Pathway and the Contact Activation System of Coagulation: Activated Factor XI Neutralizes Complement Factor H

Abstract

Complement factor H (CFH) is the major inhibitor of the alternative pathway of the complement system and is structurally related to beta2-glycoprotein I, which itself is known to bind to ligands, including coagulation factor XI (FXI). We observed reduced complement activation when FXI activation was inhibited in a baboon model of lethal systemic inflammation, suggesting cross-talk between FXI and the complement cascade. It is unknown whether FXI or its activated form, activated FXI (FXIa), directly interacts with the complement system. We explored whether FXI could interact with and inhibit the activity of CFH. We found that FXIa neutralized CFH by cleavage of the R341/R342 bonds. FXIa reduced the capacity of CFH to enhance the cleavage of C3b by factor I and the decay of C3bBb. The binding of CFH to human endothelial cells was also reduced after incubating CFH with FXIa. The addition of either short- or long-chain polyphosphate enhanced the capacity of FXIa to cleave CFH. FXIa also cleaved CFH that was present on endothelial cells and in the secretome from blood platelets. The generation of FXIa in plasma induced the cleavage of CFH. Moreover, FXIa reduced the cleavage of C3b by factor I in serum. Conversely, we observed that CFH inhibited FXI activation by either thrombin or FXIIa. Our study provides, to our knowledge, a novel molecular link between the contact pathway of coagulation and the complement system. These results suggest that FXIa generation enhances the activity of the complement system and thus may potentiate the immune response.

Figure 1:

Characterization of the interaction between FXIa and CFH. (A) CFH (400 nM) was incubated with FXIa (10 nM) for selected times (0-2 hrs) at 37°C or (B) with increasing concentrations of FXIa (0-30 nM) for 2 hrs before being separated by SDS-PAGE under reduced conditions and analyzed by Coomassie blue staining. Data are mean ± SD (n = 3). (C) CFH (400 nM) was incubated with FXIa (10 nM) for selected times (0-2 hrs) at 37°C in the presence or absence of 1A6, 14E11, 10C9 (20 μg/ml) or aprotinin (50μM). In selected experiments CFH was incubated with rFXIa

Figure 2:

Characterization of complement factor H proteolysis by FXIa. (A) Complement factor H (200 nM) was incubated with FXIa (10 nM) for selected times (0-2 hrs) at 37°C before being analyzed with two different antibodies: anti-CFH N-terminal domain antibody or (B) anti-CFH C-terminal domain antibody. Protein bands were subjected to aminoterminal sequencing, using an automated Edman sequencer. Numbers refer to position of amino acid in full-length CFH protein (C). The identity of the fragments formed upon cleavage of CFH by FXIa are shown in a schematic overview. (D) Complement factor H (400 nM) was incubated with kallikrein, FXa, XIIa or thrombin (30 nM) for selected times (0-2 hrs) at 37°C before being separated by SDS-PAGE under reducing conditions and analyzed by Coomassie blue staining.

Figure 3:

Effect of polyphosphates on factor H proteolysis by FXIa. (A) Complement factor H (400 nM) was incubated with FXIa (10 nM) for selected times (0-2 hours) at 37°C in the presence or absence of 10 μM or 50 μM short or long polyP. (B) CFH cleavage in the absence or presence of short or long polyP (50 μM) was quantified by densitometry. * indicates between-groups differences with P < 0.01. Two-way ANOVA was used for statistical comparisons. Data are mean ± SD (n = 3).

Figure 4:

Evaluation of CFH cofactor activity assay, decay acceleration assay and binding to endothelial cells. (A) C3b (300 nM), FI (20 nM) and CFH (50 nM) or (B) CFH (25 nM) were incubated in solution and the generation of iC3b at specific time points (10, 20, 40 and 80 min) was monitored by SDS-PAGE analysis. In selected experiments CFH was preincubated with FXIa for 2 hrs. Aprotinin was added to all samples to stop the reaction. The disappearance of the αC3b-chain (114 kDa) and the appearance of three new bands (iC3bα) indicate proteolytic cleavage of C3b. (C) 96 well plates were coated with 5 μg/ml C3b and then incubated at 37°C for 2 h with 50 ng/ml factor B, 25 ng/ml factor D, and 1000 ng/ml properdin. The wells were then rinsed and incubated with increasing concentrations of CFH (0.25-2 ug/ml) for 30 min. In selected experiments CFH was incubated with FXIa for 2 hrs. Aprotinin was added to all samples to stop the reaction. The C3bB and C3bBb complexes were detected by ELISA using polyclonal goat anti-human FB antibody. (D) CFH was incubated with FXIa for 2 hrs. Aprotinin was added to all samples to stop the reaction and the decay acceleration assay was measure using sheep red blood cells coated with rabbit anti-sheep erythrocyte antiserum. CFH-depleted human serum in the presence of increasing concentrations of CFH were added and the erythrocyte lysis was measure at 541 nm. (E) CFH (200 nM) was incubated with FXIa for 2 hrs, aprotinin was added to all samples to stop the reaction, and the binding of CFH to HUVECs was measured by cell surface detection of CFH by using an anti-CFH C-terminal domain antibody. * indicates between-groups differences with P < 0.01. Two-way ANOVA was used for statistical comparisons. Data are mean ± SD (n = 3).

Figure 5:

Characterization of CFH proteolysis by FXIa in serum. (A) HUVECs incubated with CFH (200 nM) for 1 hr at 37°C, washed and treated with FXIa (0-30 nM) for 30 min at 37°C. Aprotinin was added to all samples to stop the reaction. Cells lysates were analyzed by Western blotting with an anti-CFH C-terminal domain antibody. (B) Supernatant from activated platelets (2.5 x 10⁸) was incubated with FXIa (30 nM) for selected times (0-180 min). CFH was analyzed by Western blotting with an anti-CFH C-terminal domain antibody. (C) Human plasma or FXI-depleted plasma (FXI−/− plasma) was incubated with aPTT reagent or FXIa (30 nM) for 1 hr at 3 °C. CFH was analyzed by Western blotting with an anti-CFH C-terminal domain antibody. (D) FI-depleted serum was incubated with FXIa (30 nM) for selected times (0-180 min). CFH was analyzed by Western blotting with an anti-CFH C-terminal domain antibody. (E) C3b (300 nM) and FI (5 nM) were incubated in FI-depleted serum (1/5) at 37°C for selected times (10, 20, 40 and 80 min). Samples were separated by SDS-PAGE under reducing conditions and the generation of iC3b was analyzed by Western blotting using an anti-human C3b antibody. In selected experiments FI-depleted serum was incubated with FXIa (30 nM) for 3 hrs. Aprotinin was added to all samples to stop the reaction.

Figure 6:

Inhibition of FXI activation by CFH. (A) FXI (30 nM) was incubated with either thrombin (5 nM) or (B) FXIIa (5 nM) in the presence of dextran sulfate (DXS, 2 ug/ml) for 15 min at 37°C in the presence or absence of increasing concentrations of factor H (0-800 nM) or β2GPI. FXIa generation was measured with a chromogenic substrate. (C) FXI (100 nM) was incubated with either thrombin (10 nM) or FXIIa (10 nM) in the absence of DXS for 30 min at 37°C in the presence or absence of factor H (800 nM) or β2GPI (800 nM). FXIa generation was measured with a chromogenic substrate. (D) FXII activation was measured following the addition of 200 nM FXII, 50 nM PK and 50 nM HK in the presence of aPTT reagent, 10 μM long polyP or DXS (2 μg/ml) in the presence or absence of factor H or β2GPI (800 nM). FXIIa generation was measured with a chromogenic substrate. (E) 96-well plates were coated with 5 μg/ml streptavidin prior to addition of vehicle, biotinylated CFH (bio-CFH) or β2GPI (bio- β2GPI) (100 nM). Increasing concentrations of FXI was added and binding was detected with a specific antibody against FXI. (F) 96 well plates were coated with 5 μg/ml streptavidin prior to addition of vehicle or biotinylated CFH (bio-CFH). FH-depleted plasma was added and binding was detected with a specific antibody against FXI or a mouse IgG control. * P < 0.05 with respect to vehicle. Mann-Whitney U test was used for statistical comparisons. Data are mean ± SD (n = 3).