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M. Rijkers et al.
tion, probably largely affected by the number of available epitopes for WIM8E5, is correlated with the level of platelet complement activation.
Combinations of anti-HLA mAbs induce complement deposition on platelets
Next, less broadly reacting anti-HLA human IgG1 mAbs were tested for their ability to induce C3b deposition on platelet surfaces. No C3b deposition was observed when
A
B
SN607D8, SN230G6, HDG8D9 and BRO11F6 were incu- bated separately (Figure 2A; upper panel). However, by combining these HLA mAbs, thereby mimicking the poly- clonal nature of HLA alloimmunized patient sera, several combinations promoted C3b deposition on platelets (Figure 2A; lower panel and Figure 2B). This effect was very strong when a combination of anti- HLA-A2 mAbs, SN607D8 and SN230G6, were used (Figure 2B). Also, combinations of monoclonal antibodies anti-HLA-A2
C
Figure 4. Platelet activation occurs via complement activation and FcγRIIa- dependent activation. (A) C3b deposition and CD62P exposure were meas- ured in the presence and absence of serum upon incubation with WIM8E5, with or without pre-incubation of the platelets with Syk inhibitor (5 mM) or FcγRIIa blocking antibody IV.3 (10 mg/ml). (B) In the presence of serum, platelets were pre-incubated with anti-C1q (50 mg/ml), Syk inhibitor IV (5 μM) or both and CD62P and C3b deposition were measured upon addition of WIM8E5 (20 mg/ml). Representative flow cytometry graph, bar graph is mean ± SD (n=8). (C) Platelets were pre-incubated with IV.3 (10 mg/ml), Syk inhibitor IV (5 mM) and/or the C5 inhibitor Eculizumab (10 mg/ml) in the pres- ence of serum. C3b and CD62P exposure were measured upon incubation with WIM8E5 (20 mg/ml). Representative flow cytometry graphs, bar graph represents mean ± SD (n=8). Syk inh: Syk inhibitor; *P<0.05; **P<0.01; ***: P<0.005; ****P<0.001; ns: not significant.
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haematologica | 2019; 104(2)