A subset of anti-HLA antibodies induces FcγRIIa-dependent platelet activation

Abstract

HLA antibodies are associated with refractoriness to platelet transfusion, leading to rapid platelet clearance, sometimes coinciding with clinical side effects such as fever and chills. The presence of HLA antibodies is not always manifested by clinical symptoms. It is currently unclear why refractoriness to platelet transfusion is only observed in a subset of patients. Here, we utilized the availability of a unique panel of human monoclonal antibodies to study whether these were capable of activating platelets. Three out of eight human HLA-specific monoclonal antibodies induced activation of HLA-matched platelets from healthy donors as evidenced by enhanced α-granule release, aggregation, and αIIbb3 activation. The propensity of HLA monoclonal antibodies to activate platelets was independent of the HLA subtype to which they were directed, but was dependent on the recognized epitope. Activation was fully inhibited either by blocking FcγRIIa, or by blocking FcγRIIa-dependent signaling with Syk inhibitor IV. Furthermore, activation required the presence of the IgG-Fc part, as F(ab’)2 fragments of HLA monoclonal antibodies were unable to induce platelet activation. Mixing experiments revealed that activation of platelets occurred in an intra-platelet dependent manner. Accordingly, a proportion of sera from refractory patients with HLA antibodies induced FcγRIIa-dependent platelet activation. Our data show that a subset of HLA antibodies is capable of crosslinking HLA and FcγRIIa thereby promoting platelet activation and enhancing these cells’ phagocytosis by macrophages. Based on these findings we suggest that FcγRIIa-dependent platelet activation may contribute to the decreased platelet survival in platelet-transfusion-dependent patients with HLA antibodies.

Anti-HLA antibodies with complementary and synergistic interaction geometries promote classical complement activation on platelets

“…IVIg can inhibit FcγRIIa-dependent platelet activation by anti-HLA antibodies. 8 Similar effects have been observed in the context of heparin-induced thrombocytopenia. 28,29 Here, we also tested if IVIg affects complement activation and observed a dose-dependent inhibition of C3b deposition by IVIg (Figure 6A–B).…”

“…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 exposure was completely blocked with either anti FcγRIIa-blocking antibody IV.3 or through Syk inhibitor IV (which blocks downstream signaling of FcγRIIa 25 ) (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).…”

“…We have previously shown that HLA antibodies can induce FcγRIIa-dependent platelet activation, leading to CD62P surface exposure. 8 Therefore, we tested whether the complement activation by HLA antibodies occurs via the classical pathway, or whether CD62P surface exposure directly leads to C3b binding followed by activation of the alternative complement pathway. Since a blocking antibody directed to C1q completely blocked C3b and C4b deposition, complement activation by HLA antibodies appears to be fully dependent on the classical pathway.…”

“…Ideally, the ability of HLA antibodies of individual patients to induce platelet activation 8 and/or complement deposition on donor platelets should be assessed before transfusion. Previous studies have shown that platelet cross match does increase platelet recovery in patients, but data on occurrence of hemorrhage and mortality are lacking.…”

“…Previously, we showed that a subset of human monoclonal HLA antibodies and patient sera containing HLA antibodies induce FcγRIIa-dependent platelet activation and enhanced phagocytosis by macrophages. 8 However, it remains unclear to which extent this HLA antibody-mediated activation of platelets contributes to platelet clearance and which other mechanisms contribute to platelet clearance in refractory patients. In the current study we have focused on the role of complement activation by HLA antibodies.…”