The interaction between factor H and VWF increases factor H cofactor activity and regulates VWF prothrombotic status

Endothelial cells (ECs) represent an important interface between primary hemostasis, coagulation, and complement activation. During inflammation and endothelial injury, activated ECs release procoagulant and proinflammatory molecules, including multimeric von Willebrand factor (VWF) that is stored in the Weibel-Palade bodies (WPBs). In addition, ECs secrete complement molecules including factor H (FH).^1-4  We investigated the colocalization of FH and VWF in the WPBs of human ECs. We further demonstrated the interaction between the 2 molecules and the relevance of this interaction for their respective functions.

Blood was collected, with informed consent provided according to the Declaration of Helsinki, during the normal follow-up of a type 3 von Willebrand disease (VWD) patient with combined heterozygous p.Gln1311X and p.Ser2079X mutations, 5 days after replacement therapy. Because the study did not require additional blood sampling, an approval from an ethics committee was not required under French law according to article L.1121-1 of the public health code.

VWF (Wilfactin, LFB, or recombinant VWF; Baxter) or polyclonal anti-VWF antibodies were coated on enzyme-linked immunosorbent assay (ELISA) plates (Nunc) and incubated with normal plasma, FH (Comptech), or recombinant FH fragments consisting of domains 1 to 4, 6 to 8, and 19 to 20.^5,6  Binding of FH or fragments was revealed using a goat anti-human FH antiserum (Quidel).

VWF was coupled to the CM5 biosensor chip using a Biacore 2000 (GE Healthcare). FH binding to both VWF-coated and uncoated flow cells was monitored. Data were analyzed using the BIAevaluation software after subtraction of the signal generated by the uncoated flow cell.

Immunoglobulin G (IgG)-depleted plasma (by protein G adsorption) was incubated with CNBr-sepharose beads (Sigma-Aldrich) coated with anti-VWF IgG (Dako). Immunoprecipitated FH was detected by immunoblotting.

Fixed and permeabilized human umbilical vein ECs (HUVECs) grown on coverslips were incubated with a pool of 3 biotinylated anti-FH antibodies: FH1 and FH2 (Quidel) and OX24 (AbDSerotec) and with a polyclonal anti-VWF antibody (Dako). Cells were washed and mounted in fluorescent mounting medium Fluoprep (bioMérieux). Images were acquired using an Axiovert 200M microscope (Zeiss) equipped with Apotome.

FH cofactor activity was assessed using purified C3b, factor I (FI), and iC3b (Comptech). The reaction was stopped by adding reducing sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis sample buffer and analyzed by immunoblotting with a goat polyclonal anti-human C3 antibody (Calbiochem) and a rabbit anti-goat IgG–horseradish peroxidase (Southern Biotech).

VWF (10 nM) was incubated with FH (645 nM) for 30 minutes at 37°C. BaCl₂-activated recombinant ADAMTS13 (10 nM) was added. The reaction was stopped by adding 10 mM EDTA. Residual VWF:antigen was measured by ELISA^7,8  and multimer distribution by a 2% SDS agarose gel.

In the case of endothelial injury, complement and coagulation are simultaneously activated to assure wound healing and elimination of the aggressing agent. These cascades have to be tightly regulated to avoid overactivation, which would otherwise lead to tissue injury aggravation rather than healing. We investigated the respective localizations of complement regulator FH and VWF in resting HUVECs. When HUVECs were stained for FH and VWF, both molecules appeared colocalized in the WPB (Figure 1A). The detection of FH in the WPBs is in contrast with earlier work that used different detecting antibodies.⁴  It also contrasts with the absence of FH localization with VWF in the α-granules of platelets.⁹  Stimulation of HUVECs with heme¹⁰  resulted in 2.3-fold and 3.3-fold increases in VWF and FH concentration in culture supernatant, respectively (Figure 1B), suggesting that some of the FH found in the circulation originates from ECs. Both purified FH and FH in plasma interacted in a dose-dependent manner with immobilized recombinant (not shown) and plasma-derived VWF (Figure 1C-D), at physiological pH and ionic strength. VWF-FH complexes were detected in normal plasma by ELISA (Figure 1F) and by immunoprecipitation (Figure 1F inset). No complex was detected in the plasma of a type 3 VWD patient or in FH-depleted serum containing VWF, suggesting the specificity of the interaction. Using a polyclonal antibody that equally recognizes FH and all recombinant FH fragments (supplemental Figure 1), VWF was found to bind to both the recombinant FH fragments consisting of the short consensus repeat (SCR) domains 6 to 8 (SCR6-8) and domains 19 to 20 (SCR19-20) that are responsible for FH anchoring to the cell surface. No binding was observed to regulatory N-terminal domains 1-4 (SCR1-4) of FH that are responsible for FH binding to C3b (Figure 1D). Accordingly, SCR6-8 and SCR19-20, but not SCR1-4, bound VWF by surface plasmon resonance (SPR) (supplemental Figure 2), suggesting that VWF immobilization does not inactivate putative binding sites for FH. The identification of the FH domains 6 to 8 and 19 to 20 as the interacting partners does not exclude that other FH domains are implicated in complex formation.

We further studied FH-VWF binding in real-time using SPR. The injection of FH on a VWF-immobilized chip showed dose-dependent binding with an apparent K_D of 180nM ± 20nM (Figure 1E). At physiological ionic strength, FH-VWF association and dissociation constants were 2.10 × 10⁴ ± 0.39 × 10⁴ (nM × s)⁻¹ and 3.65 × 10⁻³ ± 0.35 × 10⁻³s⁻¹, respectively. The FH-VWF interaction was pH and NaCl-dependent (supplemental Figures 3 and 4), suggesting that it is driven by electrostatic forces, as most of the interactions between complement proteins. Whether the interaction occurs in the WPB, at the time of secretion and/or on the surface of activated ECs and platelets remains to be investigated.

FH controls inappropriate complement activation by acting as a cofactor for FI in the cleavage and inactivation of C3b into iC3b, as shown by the dose-dependent appearance of the 64- and 43-kDa proteolysis bands of the α′ chain of C3b (Figure 2A). The presence of VWF was associated with enhanced FI-mediated inactivation of C3b at all FH concentrations tested, while the presence of HSA had no effect (Figure 2A). Accordingly, incubation of fixed low amounts of FH in the presence of increasing concentrations of VWF led to a dose-dependent increase in FH cofactor activity (Figure 2B). Incubation of VWF with C3b and FI, in the absence of FH, did not yield iC3b generation, indicating that VWF has no effect by itself on C3b degradation by FI but enhances FH cofactor activity.

Upon secretion by ECs, ultralarge VWF multimers are rapidly hydrolyzed by ADAMTS13.^11,12  Addition of FH to an ADAMTS13-dependent VWF proteolysis assay resulted in a drastic reduction in VWF proteolysis (Figure 2C-D), in contrast to factor VIII (FVIII) which enhances VWF cleavage.¹³  The time to reach 50% hydrolysis of the initial VWF level was 10 hours ± 1 hour in the absence of FH vs 47 hours ± 2 hours in the presence of FH (Figure 2C). Accordingly, as depicted in Figure 2D upon VWF multimer analysis, the addition of FH was associated with increased amounts of high-molecular-weight multimers and reduced amounts of low-molecular-weight multimers. Moreover, addition of FH to VWF, at a physiological concentration ratio, was associated with a consistent 1.7-fold increase in VWF-mediated platelet aggregation (19.2% ± 6.7% vs 32.8% ± 8.4% in the absence or presence of FH, respectively, P = .044; Figure 2E). FH alone was devoid of an aggregating effect. Likewise, the presence of an excess of HSA (4 mg/mL) either with or without FH had no effect on VWF-mediated platelet aggregation (not shown). Of note, the presence of FH did not alter the capacity of VWF to bind collagen (supplemental Figure 5) or FVIII (not shown). Together, these results suggest that VWF-FH interaction facilitates VWF-mediated maintenance of hemostasis after injury.

In conclusion, we describe a novel type of interaction that implicates VWF and FH, which may participate in blood clotting at the site of injury by strengthening platelet aggregation and protects VWF from excessive proteolysis by ADAMTS13. In parallel, this interaction enhances the anti-inflammatory role of FH in regulating complement activation on activated or damaged endothelium. Our data provide a further association of VWF with the innate immune system.

The authors thank Paulette Legendre for technical assistance (Institut National de la Santé et de la Recherche Médicale [INSERM] U770).

This work was supported by INSERM, Centre National de la Recherche Scientifique, and Université Pierre et Marie Curie Paris 6. Recombinant ADAMTS13 and recombinant VWF were gifts from Baxter Bioscience (Vienna, Austria) and recombinant FVIII (Helixate) from CSL-Behring (Marburg, Germany).

Contribution: J.R., L.T.R., and S.L.-D. planned the work; J.R., L.T.R., J.D.D., Y.R., and M.I. performed experiments; L.H.-M., O.C., T.S.J., A.B.-D., and V.F.-B. contributed essential material; J.R., L.T.R., J.D.D., Y.R., S.V.K., V.F.-B., and S.L.-D. analyzed the data; and J.R., L.T.R., and S.L.-D. wrote the report.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Sébastien Lacroix-Desmazes, INSERM UMR S 872 Equipe 16, Centre de Recherche des Cordeliers, Paris, F-75006 France; e-mail: sebastien.lacroix-desmazes@crc.jussieu.fr; and Véronique Frémeaux-Bacchi, Service d’Immunologie Biologique, Hôpital Européen Georges Pompidou, 20-40 rue Leblanc, 75908 Paris cedex 15, France; e-mail: veronique.fremeaux-bacchi@egp.aphp.fr.