Maternal anti-αIIb antibodies target fetal hematopoietic stem cells disrupting hematopoiesis and causing pregnancy loss in a murine model of fnait

Abstract Fetal and Neonatal Alloimmune Thrombocytopenia (FNAIT) is a life-threatening complication of pregnancy (~1 in 1000 live births) in which maternal IgG antibodies target paternally inherited fetal platelet antigens, leading to thrombocytopenia, hemorrhage, and pregnancy loss. Approximately 80–90% of reported FNAIT cases involve antibodies against integrin β3 subunit (e.g., HPA-1a), whereas anti-αIIb antibodies (e.g., HPA-3a or HPA-9b) account for only ~2–3%. This disparity is unlikely the result of polymorphism frequencies, as αIIb and β3 variants occur at similar rates in the population. Despite their rarity, anti-αIIb-mediated cases are particularly severe, frequently associated with thrombocytopenia, major bleeding, and pregnancy loss. This severity, paired with rarity, raises the possibility that anti-αIIb antibodies may cause early fetal loss before diagnosis, leading to their underreporting. Since FNAIT is usually detected only after live birth with thrombocytopenia after other causes are ruled out, pregnancies affected by anti-αIIb may be missed entirely. Notably, integrin αIIb is also expressed on fetal hematopoietic stem cells (HSCs) during early hematopoiesis, suggesting that maternal anti-αIIb antibodies may directly impair fetal blood development and viability. We developed a murine model of anti-αIIb–mediated FNAIT by immunizing αIIb-knockout (αIIb−/−) females with wild-type (WT) platelets to generate anti-αIIb antibodies. Immunized females were then bred with WT males to generate uniformly αIIb-positive fetuses. Anti-αIIb FNAIT led to miscarriage in ~66% of pregnancies, twice the rate seen in anti-β3 FNAIT (p<0.02). Surviving fetuses at E12.5 and E14.5 had significantly smaller livers, reduced liver-to-body weight ratios, and markedly decreased hematopoietic output. Fetal liver HSCs were identified using Lin−Sca-1+c-Kit+ (LSK) markers, with multipotent progenitors (MPP2, MPP3, MPP4) defined by SLAM markers (CD48, CD150, CD135). Total LSK cells and MPP2/MPP3 subsets were significantly reduced in FNAIT fetal livers, while MPP4 frequencies remained unchanged. This significantly affected the downstream progeny of these progenitors, noted by a significant depletion of myeloid cells, macrophages, and megakaryocytes, whereas the lymphoid and erythroid lineages remained unchanged. To confirm antigen-specificity, we used a heterozygous breeding strategy where immunized αIIb−/− females were mated with αIIb+/− males to generate litters containing both αIIb-positive and αIIb-negative fetuses, enabling within-litter comparisons under identical maternal and environmental conditions to determine whether only antigen-expressing fetuses were selectively affected. Anti-αIIb antibodies significantly skewed offspring genotypes, with far fewer αIIb-positive fetuses than expected. These αIIb-positive fetuses had markedly reduced HSPCs, whereas αIIb-negative littermates developed normally with intact hematopoietic profiles. This genotype-specific loss confirms that the hematopoietic impairments and pregnancy loss were driven by direct targeting of αIIb-expressing fetal cells. Our findings demonstrate that maternal anti-αIIb antibodies induce a severe, previously unrecognized form of FNAIT in which fetal HSCs are directly targeted, leading to impaired fetal liver hematopoiesis, disrupted liver development, and early pregnancy loss. Unlike anti-β3-mediated FNAIT, which typically presents postnatally through thrombocytopenia and/or bleeding diatheses, anti-αIIb antibodies act earlier on αIIb-expressing progenitors, resulting in fetal demise. Our findings suggest FNAIT-related miscarriage is likely underdiagnosed, as current prenatal care does not include routine anti-platelet antibody screening, and alloimmune causes of miscarriage are rarely considered. Moreover, the diagnosis is typically made only after a thrombocytopenic live birth, excluding early losses from recognition. These factors may obscure the true prevalence of severe cases like those caused by Maternal anti-αIIb antibodies. Thus, our findings support further investigation into anti-platelet antibodies in women with unexplained prenatal loss, where early diagnosis and intervention using IVIG or FcRn blockade could be employed to alleviate fetal complications.

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