Abstract

Background

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe bleeding disorder that often results in intracranial hemorrhage (ICH), leading to neurological impairments or death. FNAIT has also been linked with miscarriage although the incidence of fetal loss has not been adequately studied. It is the most common cause of severe thrombocytopenia in live born neonates and accounts for up to 35% of all neonates admitted into the intensive care unit. In FNAIT, maternal alloantibodies cross the placenta and target paternally-derived fetal platelet antigens, most commonly platelet GPIIbIIIa (αIIbβ3 integrin). Polymorphisms in human platelet antigen-1a (HPA-1a), located on the integrin β3 subunit are the most common cause of FNAIT and are most widely studied, however the reported incidence of anti-αIIb-mediated FNAIT has recently increased; but the pathogenesis of this disease is unclear.

Methods and Results

To establish mouse models of anti-αIIb antibody mediated FNAIT, αIIb deficient (αIIb-/-) and human αIIb transgenic mice were employed in this study. We first immunized female αIIb-/- mice by weekly transfusions of wild type (WT) mouse platelets 2 times or 4 times. We demonstrated that the αIIb-/- mice were immunoresponsive to the αIIb antigen and both IgG1 and IgG2 antibodies against αIIb (i.e.TH2-like and TH1-like immune response, respectively) were detected two weeks after platelet transfusion. To test whether αIIb antisera can cause platelet destruction, we injected the antisera into WT mice and found severe thrombocytopenia in the recipient mice (P<0.001). Subsequently, immunized female αIIb-/- mice were bred with WT males to generate heterozygous fetuses and to recapitulate the features of FNAIT. Fetuses and neonates generated from naïve αIIb-/- females bred with WT males were used as a control.

We found the severity of symptoms was correlated with the level of antibody titers in the maternal circulation. In 2 times and 4 times immunized females, miscarriage was observed (n= 2/9; and n =3/3, respectively). Upon post mortem dissection of miscarrying mothers at embryonic day 15.5, fetuses possessed ICH in 2 times and 4 times immunized females (n=2/14; and 5/12, respectively). Live born neonates from 2 times immunized mothers showed severe thrombocytopenia (P<0.001), purpura (n=9/22) and ICH (n=4/22). In addition, neonate body weight decreased compared to naïve controls (P<0.05), suggesting intrauterine growth restriction. There were no live pups delivered from 4 times immunized mothers. We also tested the effect of antisera from 2 times immunized αIIb-/- mice on platelet function and found the antisera enhanced ADP-induced platelet aggregation. This is different from the anti-β3 sera that inhibited platelet aggregation in our previous FNAIT model. Whether the different effects on platelets may lead to different pathology in FNAIT is under investigation in the laboratory.

In addition to the knockout mouse model, we also used human αIIb transgenic mice, which do not express mouse αIIb on their platelets, in order to generate an alloimmune model of FNAIT. This model may better mimic the conditions of human FNAIT caused by the polymorphisms in αIIb genes. Following the same protocol as we used in the αIIb-/- mice, we found anti-murine αIIb antibodies were induced although the antibody titer was slightly lower. After breeding with male WT mice, immunized female human αIIb transgenic mice deliver pups with thrombocytopenia and ICH. Miscarriage was also observed, however the severity and symptoms of the disease were less. Furthermore, we also established another model of mouse-anti-human FNAIT by breeding humanized αIIb male mice with immunized female WT mice. We will compare the differences in these iso- and alloimmune models to elucidate the pathogenesis of αIIb mediated FNAIT.

Conclusions and Significance

We established the first anti-αIIb model of FNAIT and the first alloimmune animal models of FNAIT. Our results indicate that these models recapitulate human disease symptoms such as; lower platelet counts, bleeding diasthesis, and decreased birth weight compared to controls. Both neonates and fetuses possess ICH, and some immunized mothers underwent miscarriage. These models give us the opportunity to compare iso- vs. alloimmune FNAIT, and to compare the pathology between anti- αIIb and our anti-β3 mediated FNAIT.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.