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Posttransfusion purpura (PTP) is an uncommon but life-threatening condition characterized by profound thrombocytopenia occurring ∼1 week after a blood transfusion. The hallmark of PTP is a potent immunoglobulin G antibody specific for a transfused platelet-specific alloantigen, usually located on glycoprotein IIb/IIIa (GPIIb/IIIa; αIIb/β3 integrin). It is widely thought that this alloantibody somehow causes the thrombocytopenia, despite absence from host platelets of the alloantigen for which it is specific. In studies described here, we found that cross-strain platelet immunization in mice commonly induces GPIIb/IIIa-specific alloantibodies combined with platelet-specific autoantibodies and varying degrees of thrombocytopenia, and we identified 1 strain combination (129S1Svlm/PWKPhJ) in which 95% of immunized mice made both types of antibody and developed severe thrombocytopenia. There was a strong inverse correlation between autoantibody strength and platelet decline (P < .0001) and plasma from mice that produced autoantibodies caused thrombocytopenia when transfused to syngeneic animals, arguing that autoantibodies were the cause of thrombocytopenia. The findings define a model in which a routine alloimmune response to platelets regularly transitions to an autoimmune reaction capable of causing severe thrombocytopenia and support the hypothesis that PTP is an autoimmune disorder.

PTP is characterized by profound thrombocytopenia (TP) associated with a strong allo-response against a transfused human platelet antigen (HPA), usually HPA-1a. It is unclear why individuals with PTP develop TP, since their alloantibodies are non-reactive with autologous platelets. We recently studied a patient with PTP in whom, using autologous platelets obtained after recovery, convincing evidence was obtained that TP was caused by GPIIb/IIIa-reactive autoantibodies produced concomitantly with potent alloantibodies specific for platelet antigens HPA-1a and HPA-2a (Am J Hematol 91;848-51, 2016). Sporadic reports by others suggest that production of an autoantibody by an individual mounting an allo-response against a blood cell alloantigen is not rare. We undertook studies to determine whether this phenomenon can be recapitulated in a mouse model that will lend itself to the study of underlying mechanisms. The two most immunogenic platelet glycoproteins (GP), αIIb/β3 integrin (GPIIb/IIIa) and GPIb/IX are closely homologous in various mouse strains. We used available data bases to select strains C57BL/6J, 129S1 and SPRET in which the composition of these glycoproteins (GP) differed maximally between strains. Mice were immunized weekly for 5 weeks with autologous or allogeneic platelets. After 2-3 weeks, 93% of 28 animals produced IgG antibodies (abs) specific for platelets of the donor strain. At 3-4 weeks, 36% also produced abs specific for autologous platelets as shown by an increase in platelet-associated IgG and a significant drop in platelet levels; 70% of these mice had detectable plasma abs that bound to platelets from the recipient strain. Immunoprecipitation studies showed that all abs detected were specific for GPIIb/IIIa despite disparities of only 2-6 amino acids in these GPs between strains. No abs were produced following immunization with autologous platelets. To enhance the observed immune responses, C57BL/6 PKCd KO mice that are defective in development of B cell tolerance were studied. Each of two KO mice immunized with 129S1 platelets produced both allo- and autoantibodies at 3 weeks and became profoundly thrombocytopenic (~10% of normal). These findings show that mice mounting an allo-response against GPIIb/IIIa commonly produce autoantibodies against the same protein, leading to thrombocytopenia, as in PTP experienced by transfused humans. An alloimmune response is usually triggered by a single amino acid disparity between donor and recipient. We propose that in this process, random somatic hypermutation occasionally modifies one or more amino acids in the antigen-combining site of a B cell receptor (BCR) in such a way that recognition of the amino acid that governs allo-antigenicity is lost and specificity for a corresponding autoepitope is gained (Figure 1). Sequencing of allo- and autoantibodies produced following murine cross-species platelet immunization will enable this hypothesis to be formally tested. Molecular characterization of epitopes recognized by alloantibodies in this model are expected to define for the first time in an animal, platelet-specific alloantigen systems comparable to human HPA that can be used to study pathogenesis of PTP and the alloimmune disorder neonatal alloimmune thrombocytopenia (NAIT) under "natural" conditions. Figure 1 Figure 1. Disclosures Aster: Bloodcenter of Wisconsin: Patents & Royalties: A patent application has been filed on a Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591.

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