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M. Rijkers et al.
WIM8E5 and SN607D8 was significantly decreased in the presence of Syk inhibitor IV (Figure 6D). The results obtained were derived from three independent experi- ments employing platelets from different donors (Online Supplementary Figure S7). Together, these results show that FcγRIIa-dependent activation of a subset of HLA antibod- ies promotes uptake of platelets by macrophages.
Discussion
The presence of HLA antibodies in patients receiving platelet transfusions is in some cases associated with rapid platelet clearance, which may be accompanied by transfu- sion reactions, including chills and fever.2,7 Here, we stud- ied whether HLA antibodies can activate platelets. We showed that a subset of human HLA monoclonal antibod- ies induced platelet α-granule release, integrin αIIbb3 activa- tion and platelet aggregation. All these effects could be fully inhibited by blocking FcγRIIa-dependent signaling in platelets. This indicates that activation by HLA antibodies is induced upon crosslinking of HLA molecules with the platelet Fc receptor FcγRIIa. Employing sera from HLA- antibody positive and refractory patients we confirmed that FcγRIIa-dependent platelet activation can be induced on donor platelets. We also showed that FcγRIIa-depen- dent activation enhances phagocytosis of platelets by macrophages.
Although all the monoclonal antibodies tested in this study bound efficiently to HLA-matched platelets, only three of them significantly induced CD62P surface expo- sure. It has been reported that high levels of HLA antibod- ies in patients correlate with a higher risk of refractoriness,10 however, we did not observe a correlation between the level of IgG bound to platelets and the ability of HLA monoclonal antibodies or patients’ sera to induce platelet activation. No correlation between platelet activa- tion and binding of HLA monoclonal antibodies to specific HLA alleles was found. Antibodies WIM8E5, GV5D1 and GV2D5 all bind to HLA-A1, but only WIM8E5 and GV5D1 induce significantly increased CD62P membrane exposure on platelets. Similarly, whereas SN607D8 and SN230G6 both bind to HLA-A2, only SN607D8 induces platelet activation. Recently the affinities of SN607D8 and SN230G6 for HLA-A2 were reported: SN607D8 has a KD of 1.2x10-8 M and SN230G6 has a KD of 5.9x10-10 M.33 It is likely that the affinity of HLA antibodies affects their abil- ity to activate platelets to some extent. However, as the non-activating antibody SN230G6 has a significantly higher affinity than that of the activating antibody SN607D8, the propensity of HLA monoclonal antibodies to induce FcγRIIa-dependent platelet activation is not exclusively dependent on their affinity. For SN607D8 and SN230G6, residues critical for their binding to HLA have been determined (149A and 152V25, and 62GE34 respective- ly). These residues are located on opposite sides of the peptide-binding groove. We, therefore, hypothesize that the location of the binding site on HLA determines whether the Fc tail of an HLA antibody can bind and crosslink with FcγRIIa, causing activation through Syk.
Supplementary Figure S7). Similarly, differences in platelet clearance via activating and non-activating antibodies have been described in human FcγRIIa transgenic mice, in which an anti-CD9 antibody inducing FcγRIIa-platelet activation led to more rapid development of thrombocy- topenia compared to a non-activating platelet specific antibody.35 Besides, shock and thrombosis were observed only in the presence of platelet-activating antibodies and not with non-activating antibodies.35 The activating poten- tial of HLA antibodies has not been correlated with a risk of adverse effects, possibly because HLA-incompatible platelets are usually given to thrombocytopenic patients with low platelet counts, reducing the risk of thrombosis.
Previous studies on the effect of HLA antibodies on platelets have suggested that they are either inert36 or induce platelet activation through complement activation.20 Our experiments were performed with washed platelets in the absence of complement, so we cannot rule out that complement activation plays a role as well. Rubinstein et al. showed that crosslinking of a murine pan-anti-HLA antibody induced platelet activa- tion.19 However, the effect of the anti-HLA mouse mono- clonal antibody that they used was only observed by arti- ficial crosslinking of the HLA antibodies with goat anti- mouse IgG. Employing a mouse model, Waterman et al. showed that Fcγ receptors were critical for mice to devel- op platelet refractoriness induced by major histocompati- bility complex alloantibodies.37 Previous studies have shown that both murine and human HLA antibodies can induce release of Weibel-Palade bodies and subsequent CD62P exposure on endothelial cells, inducing enhanced monocyte adherence to endothelial cells via both CD62P binding and crosslinking of endothelial HLA with mono- cyte Fc receptors.38 It has been described previously that platelets can be activated by monoclonal antibodies directed to b2-microglobulin in an FcγRIIa-dependent manner.32 Similar to our observations, Rubinstein et al. reported differences in the platelet-activating abilities of several monoclonal antibodies directed towards different epitopes on b2-microglobulin.32 However, activation by anti-b2-microglobulin monoclonal antibodies was pro- posed to occur in an inter-platelet fashion, while our data provide evidence for intra-platelet activation by HLA monoclonal antibodies. The mechanism of platelet activa- tion by HLA monoclonal antibodies is similar to that of the platelet activation described in heparin-induced thrombocytopenia, in which antibodies directed to the platelet factor 4/ heparin complexes develop and induce FcγRIIa-dependent platelet activation.17 Intravenous immunoglobulin has been shown to have a beneficial effect in patients with heparin-induced thrombocytope- nia;39,40 here we have shown that intravenous immunoglobulin inhibits HLA monoclonal antibody- induced FcγRIIa-dependent platelet activation in vitro. Based on our observations it may be worth exploring whether intravenous immunoglobulin can be used to reduce the rapid clearance of transfused platelets in a sub- set of refractory patients with anti-platelet antibodies.
Li et al. described a mechanism in which platelets are activated by GPIbα antibodies in an FcγRIIa-independent way.16 Recently, Quach et al. showed that this activation was dependent on mechanomolecular signaling.41 Similar to our data, Li et al. and Quach et al. documented enhanced CD62P exposure on platelets. In addition, their data suggested that activation of platelets coincides with
We showed that FcγRIIa-dependent signaling enhanced platelet phagocytosis by macrophages. The degree of Syk- dependent phagocytosis correlated with the ability of the HLA monoclonal antibodies to activate and induce phos- phatidylserine exposure on the platelets (Online
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haematologica | 2018; 103(10)