Anti-Platelet IgG-Fc Glycosylation As a Novel Regulator in Platelet Transfusion Refractoriness
Authors: Mons Dirk-Jan , van Osch Thijs , Rayadoost Farahnaz , van der Velden Saskia , Wuhrer Manfred , Hazenberg Mette D. , Vidarsson Gestur , Kapur Rick 
Published: December 2024

Platelet transfusion refractoriness (PTR) is a major clinical burden that remains challenging and very costly to manage. While platelet transfusions significantly reduce mortality in patients with thrombocytopenia, in approximately 5-15% of patients with chronic platelet support PTR occurs, characterized by a rapid clearance of transfused platelets. HLA-antibodies directed against the transfused platelets are frequently responsible for this process.

It is presumed that HLA-antibody mediated PTR occurs via IgG-Fc Receptor (FcγR)-mediated phagocytosis of platelets and IgG-Fc induced complement activation. However, these mechanisms have not been systematically elucidated in detail. Remarkably, not all patients with HLA-antibodies develop PTR to unmatched platelet transfusions. Antibody effector functions are dependent on several factors, including antibody Fc-glycosylation. We have previously shown that increased galactosylation of platelet antibodies (and to some degree Fc sialylation) enhances complement activation on platelets (Van Osch, Haematologica 2022). Furthermore, IgG-Fc afucosylation is known to enhance binding to FcγRIIIa/b, resulting in increased phagocytosis of platelets (Kapur et al, Blood, 2014). Notably, the regulatory role of platelet IgG Fc-glycosylation, i.e. the specific contribution of platelet IgG Fc-fucosylation, -galactosylation and -sialylation, has not been investigated in the context of PTR. We investigated how IgG-Fc glycosylation patterns determine the functionality of HLA-antibodies in clearing transfused platelets in an in vivo mouse model of PTR.

Monoclonal thrombocytopenia-inducing antibodies targeting mouse major histocompatibility complex (MHC)-I subclass H-2Kd (clone 34-1-2S) were recombinantly cloned (van der Velden et al, Blood 2024). During transfection the antibodies were glyco-engineered with enhanced Fc-galactosylation, Fc-sialylation or decreased Fc-fucosylation as a result. Low fucosylated antibodies were produced using the decoy substrate 2-fluorofucose (2FF). Highly galactosylated and highly sialylated antibodies were produced by overexpressing enzymes B4GALT1 and ST6GALT, respectively. The glycosylation profiles were confirmed using mass spectrometry.

A mouse model was set up mimicking a passive form of PTR. Male C57BL/6 mice were transfused with 200 x106 platelets consisting of unmatched (H2-Kd) platelets from donor Balb/c mice and matched (H-2Kb) platelets. Platelets were labeled with CFSE or Celltrace Yellow before transfusion. The main read-out was the percentage of unmatched compared to matched transfused platelets corrected for the pre-treatment ratio. Directly after transfusion the mice were intravenously injected with either 4.5 mg/kg 34-1-2S glyco-engineered antibody or an antibody isotype control. 10 minutes, 1 hour and 24 hours after antibody administration blood was drawn, consistent with the clinical practice regarding PTR diagnosis.

Survival of unmatched platelets 24 hours after transfusion was 73.0% (SD±25.2, N=2) for the group receiving wild-type 34-1-2S antibody and 44.4% (SD±19, N=4), 42.1 % (SD±4.2, N=2) and 37.9% (SD±9.4, N=4) for the groups receiving low fucosylated, highly galactosylated and highly sialylated versions of the 34-1-2S antibody, respectively. Mice receiving the antibody isotype control showed a platelet survival of 67.1% (SD±19.0, N=3). There were no clear differences in platelet survival 10 minutes or 1 hour after antibody injection. No adverse events were observed.

Our preliminary data reveal that low fucosylated, highly galactosylated and highly sialylated MHC-I targeting antibodies are more potent in clearing mismatched transfused platelets in vivo. This suggests that FcγRIII dependent phagocytosis and complement dependent clearance (CDC) of platelets play a role in PTR. We are currently expanding the sample sizes in all groups, and are in parallel conducting experiments in an in vivo mouse model of active PTR (based on in vivo antibody production due to immunizations). In both models, we will investigate the role of CDC by using a recombinant mouse C5 antibody (clone BB5.1.PGLALA) which we have generated. These data improve our understanding of the pathophysiology of PTR and contribute to the development of more personalized approaches to solve this major clinical problem.