An Authentic Murine Model of Fetal/Neonatal Alloimmune Thrombocytopenia

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies directed against paternally-inherited antigens present on the surface of fetal platelets. The human platelet alloantigen, HPA-1a (also known as the Pl^A1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Caucasians. A single Leu33Pro amino acid polymorphism residing within the ~50 amino acid plexin/semaphorin/integrin (PSI) domain near the N-terminus of the integrin β3 subunit (platelet membrane glycoprotein (GP)IIIa), is responsible for generating the HPA-1a and HPA-1b epitopes in human GPIIIa. Neither human polyclonal nor mouse monoclonal anti-HPA-1a antibodies, however, recognize murine GPIIIa due to amino acid differences both within and surrounding the HPA-1a alloepitope. As a result, there are currently no authentic mouse models of FNAIT capable of recapitulating the human alloimmune response to this clinically important platelet alloantigen. We have recently shown that humanizing residues 30, 32, 33, and 39 within the PSI domain, as well as amino acid 470 within the linearly distant but conformationally close Epidermal Growth Factor (EGF) 1 domain, of murine GPIIIa, is sufficient to re-create the epitope recognized by most maternal anti-HPA-1a alloantibodies. We therefore used CRISPR/Cas9 gene editing technology to generate transgenic mice whose platelets express human residues A₃₀P₃₂L₃₃D₃₉Q₄₇₀ on a murine GPIIIa backbone - hereafter referred to as APLDQ mice. Intraperitoneal injection of an anti-HPA-1a mAb induced severe thrombocytopenia in these APLDQ, but not wild-type, mice. Furthermore, platelets from APLDQ mice, when introduced into wild-type mice, elicited a strong polyclonal immune response that was specific for, and importantly restricted to, the epitopes created by these humanized residues, demonstrating that the APLDQ humanized form of murine GPIIIa is immunogenic in mice. Wild-type female mice pre-immunized with APLDQ platelets, when bred with APLDQ male mice, gave birth to severely thrombocytopenic pups, many of whom exhibited an accompanying bleeding phenotype. Notably, administration of intravenous immunoglobulin G (IVIG) into pregnant female mice at days 10 and 17 lowered the concentration of anti-APLDQ alloantibodies in both the maternal and fetal circulation, and importantly normalized the platelet count in the pups. Taken together, these data establish a novel murine model of FNAIT that recapitulates many of the clinically important features of FNAIT. Availability of APLDQ humanized transgenic mice should pave the way for the pre-clinical development and testing of novel therapeutic and prophylactic modalities to treat or prevent FNAIT in humans.