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M. Rijkers et al.
pre-incubated with a blocking antibody directed to the tail of C1q (anti-C1q), preventing binding of C1q to Fc-tails of immunoglobulins. Complete blockage of WIM8E5 induced C3b and C4b deposition on platelet surfaces was achieved by anti-C1q at a concentration of 50 mg/ml (Figure 3A-B). The blocking effect of anti-C1q was dose dependent (Figure 3C). These results indicate a crucial role for C1q binding in WIM8E5 induced C3b and C4b depo- sition, suggesting that complement activation occurs via the classical pathway.
It has been described that IgG molecules can form hexa- mers to which C1q can efficiently bind.23 Employing a synthetic peptide shown to interfere with Fc-dependent hexamer formation and subsequent complement deposi- tion,23,24 we studied whether anti-HLA antibody-induced complement deposition was blocked by this peptide. IgG- Fc:Fc blocking peptide inhibited C3b and C4b deposition by WIM8E5 or by the combination of SN607D8 and SN230G6 in a dose dependent manner (Figure 3D-E). No effects were observed with an irrelevant control peptide (Figure 3F-G). Together, these results suggest that anti- HLA antibody-induced complement deposition on platelets involves Fc tail-mediated assembly of IgG hexa- mers, creating a suitable binding platform for C1q.
Anti-HLA antibodies induce complement- and FcγRIIa-dependent platelet activation
We have recently shown that a subset of anti-HLA mAbs (including WIM8E5) can activate platelets directly through FcγRIIa in the absence of a complement source.8 Under these conditions, WIM8E5-induced CD62P expo- sure was completely blocked with either anti FcγRIIa- blocking antibody IV.3 or through Syk inhibitor IV (which blocks downstream signaling of FcγRIIa25) (Figure 4A). Incubation of platelets with WIM8E5 in the presence of
serum as a complement source resulted in a marked increase in CD62P exposure on platelets (Figure 4A). Under these conditions, CD62P exposure was only slight- ly inhibited by blocking FcγRIIa or with Syk inhibitor IV (Figure 4A). These results suggest that in the presence of complement activation, platelet activation is only partially FcγRIIa-dependent. To study the effect of complement activation on α-granule release, complement activation was blocked with anti-C1q. This significantly inhibited C3b deposition, but also only partly blocked α-granule release as measured by CD62P exposure (Figure 4B). However, a combination of Syk inhibitor and anti-C1q blocked WIM8E5-induced CD62P exposure as well as C3b deposition completely (Figure 4B).
Previously, Wiedmer et al. described a mechanism where the formation of a MAC on platelet surfaces induces platelet activation and α-granule release caused by calcium influx through the MAC.26 We used a C5 blocking antibody (Eculizumab) to prevent cleavage of C5 and thereby the formation of a MAC. Like anti-C1q, Eculizumab partly inhibited CD62P surface exposure (Figure 4C). WIM8E5-induced CD62P exposure was com- pletely inhibited when Eculizumab was combined with either Syk inhibitor IV or FcγRIIa blocking antibody IV.3 (Figure 4C). Similar results were obtained employing com- binations of HLA mAbs (Online Supplementary Figure S1). Together, these results suggest that in presence of a com- plement source, platelet activation by anti-HLA antibodies is dependent on formation of a MAC as well as FcγRIIa- dependent signaling.
MAC formation leads to calcium influx
To confirm MAC formation, platelets activated with a combination of SN607D8 and SN230G6 were stained with an anti-C5b-9 antibody. As expected,
AB
C
DE
Figure 6. IVIg and C1 esterase inhibitor inhibit complement deposition on platelets. (A-B) Platelets were incubated with increasing concentrations of IVIg (0-10 mg/ml), after which WIM8E5 (20 mg/ml) (A) or SN607D8 and SN230G6 (0.2 mg/ml) (B) were added. C3b deposition was measured on the platelet surfaces. (n=3). (C-E) Platelets were pre-incubated with 0-600 μg/ml C1 esterase inhibitor. C3b deposition upon incubation with (C) 20 mg/ml WIM8E5 or (D) 0.2 mg/ml SN607D8 and SN230G6 was measured. (E) Inhibitory effect of C1 esterase inhibitor on C4b deposition was measured upon incubation with WIM8E5 (20 mg/ml) or SN607D8 and SN230G6 (0.2 mg/ml) (n=3).
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haematologica | 2019; 104(2)