PB1336 - Novel Insight into Platelet Refractoriness: Desialylation, Apoptosis and Treatment

Background: Platelet refractoriness is a significant burden in patients with solid and haematological malignancies. Alloantibodies against human leucocyte antigen (HLA) are the commonest immune cause. Despite leucodepletion, the incidence remains high, and the mechanistic effects of anti-HLA antibodies on platelets are yet to be fully elucidated.

Aims: (1) Ascertain if anti-HLA antibodies lead to platelet apoptosis or desialylation.
(2) Establish a mouse model for platelet refractoriness.
(3) Determine if blockage of anti-HLA antibodies binding protects platelets from destruction in mice.

Methods: The study was approved by the institution’s Ethics Committees. Sera were obtained from 5 patients with immune platelet refractoriness. Firstly, the presence of anti-platelet antibodies was confirmed. Washed platelets were treated with patients' or controls' sera followed by incubation with anti-human IgG. For determination of desialylation and apoptosis, platelets were incubated with RCA-1, anti-Neu1 antibody, and DiOC6 respectively, and analysed by flow cytometry. Purified anti-HLA monoclonal antibody W6/32 was incubated with platelets in some experiments to determine its role in blockade of patient antibody binding to platelets. Finally, a nonobese diabetic/severe combined immunodeficient murine model of platelet refractoriness was established to examine the site of platelet uptake and the therapeutic effect of W6/32 fragment antigen-binding (FAB) portion in platelet preservation.

Results: HLA antibodies did not induce detectable desialylation but caused apoptosis (P=0.548, P=0.310, P=0.039 for RCA-1, NEU1 and DiOC6, respectively). W6/32 reduced HLA antibody binding to donor platelets in vitro (P=0.029). Anti-HLA antibodies lead to splenic platelet sequestration. Lack of hepatic uptake further supports the absence of desialylation observed in vitro. Pre-treating platelets with W6/32 FAB reduced patient’s antibody mediated platelet clearance (P=2.41e-12).

Conclusion(s): Anti-HLA antibodies do not lead to platelet desialylation but induce platelet apoptosis. W6/32 reduces antibody binding in vitro and its FAB showed efficacy in preventing anti-HLA antibody mediated platelet destruction in vivo. For further confirmation, human studies may be warranted.