Seminars in Fetal & Neonatal Medicine 21 (2016) 19e27

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Review

Fetal and neonatal alloimmune thrombocytopenia Darko Zdravic a, b, c, d, Issaka Yougbare b, c, d, Brian Vadasz a, b, c, Conglei Li a, b, c, Alexandra H. Marshall b, c, Pingguo Chen b, c, d, Jens Kjeldsen-Kragh b, e, Heyu Ni a, b, c, d, e, f, g, * a

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Toronto Platelet Immunobiology Group, Toronto, ON, Canada c Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St Michael's Hospital, Toronto, ON, Canada d Canadian Blood Services, Toronto, ON, Canada e Department of Clinical Immunology and Transfusion Medicine, University and Regional Laboratories Region Skåne, Lund, Sweden f Department of Medicine, University of Toronto, Toronto, ON, Canada g Department of Physiology, University of Toronto, Toronto, ON, Canada b

s u m m a r y Keywords: Platelets Fetal and neonatal alloimmune thrombocytopenia b3 integrin and GPIba Intraveneous immunoglobulin G Antibody-induced immune suppression

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is an alloimmune disorder resulting from platelet opsonization by maternal antibodies that destroy fetal platelets. The major risk of FNAIT is severe bleeding, particularly intracranial hemorrhage. Miscarriage has also been reported but the incidence requires further study. Analogous to adult autoimmune thrombocytopenia (ITP), the major target antigen in FNAIT is the platelet membrane glycoprotein (GP)IIbIIIa. FNAIT caused by antibodies against platelet GPIba or other antigens has also been reported, but the reported incidence of the anti-GPIba-mediated FNAIT is far lower than in ITP. To date, the maternal immune response to fetal platelet antigens is still not well understood and it is unclear why bleeding is more severe in FNAIT than in ITP. In this review, we introduce the pathogenesis of FNAIT, particularly those new discoveries from animal models, and discuss possible improvements for the diagnosis, therapy, and prevention of this devastating disease. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction 1.1. Roles of platelets, GPIIbIIIa, and GPIba in hemostasis Platelets are anucleate cells generated from megakaryocytes in the bone marrow [1]. After release into the blood, these small cells surveil the circulation, playing key roles in vascular repair and hemostasis [2,3]. Over the last few decades, our understanding of platelets and their functions has been significantly advanced. Recent studies demonstrate that platelets are versatile and involved in inflammation, immune responses, cancer, angiogenesis, lymphatic vessel development, atherosclerosis, and more [4,5]. However, hemostasis and thrombosis are likely still the major physiological and pathological functions of platelets.

* Corresponding author. Address: St Michael's Hospital, Room 420, LKSKI e Keenan Research Centre for Biomedical Science, 209 Victoria Street, Toronto, Ontario M5B 1W8, Canada. Tel.: þ1 416 847 1738. E-mail address: [email protected] (H. Ni). http://dx.doi.org/10.1016/j.siny.2015.12.004 1744-165X/© 2015 Elsevier Ltd. All rights reserved.

Platelet membrane glycoprotein (GP) GPIba and GPIIbIIIa (aIIbb3 integrin) are the two major receptors on the platelet surface. GPIba is the receptor for von Willebrand factor (vWF), which is essential for initiating platelet tethering and adhesion to the site of injury, particularly at high shear. Subsequent firm platelet adhesion is mediated by binding of several integrins to their ligands on the vessel wall (e.g. integrin aIIbb3 to fibronectin and fibrinogen/fibrin, and a2b1 to collagen) [2,6]. At low shear, interactions between platelet integrins and their ligands may directly initiate platelet adhesion [7]. Following the first layer of platelet adhesion and activation, platelet aIIbb3 integrin mediates platelet aggregation and hemostatic plug formation by interacting with its ligands, including fibrinogen and other ligands (i.e. fibrinogen-dependent and -independent platelet aggregation) [8,9]. Platelet accumulation (adhesion and aggregation) at the site of injury has been considered the first wave of hemostasis [2,3,10]. Platelets also contribute to the second wave of hemostasis, blood coagulation. Following platelet activation, platelets can express phosphatidylserine and generate a negatively charged surface, which harbors the coagulation factors and markedly potentiates

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thrombin generation (i.e. cell-based thrombin generation) [11]. Furthermore, platelets may release their a-granule components at the site of injury and contribute to the newly identified fibronectinmediated “protein wave” of hemostasis [12e14]. Given the crucial role of platelets in hemostasis, increased platelet destruction and clearance may lead to bleeding disorders (thrombocytopenias).

contrast to hemolytic disease of the newborn (the red blood cell analog of FNAIT), FNAIT cases may occur during the first pregnancy [32], thus challenging diagnostic and therapeutic armamentaria. The rate of recurrence among subsequent platelet antigen-positive siblings is close to 100% [34], with the degree of thrombocytopenia being either the same or more severe. Collectively, FNAIT is a severe and potentially life-threatening disease.

1.2. Roles of GPIIbIIIa and GPIba in thrombocytopenias 2.1. Platelet alloantigens Both GPIba and GPIIb (the aIIb subunit of the aIIbb3 integrin) are almost exclusively expressed on platelets and their precursor megakaryocytes. GPIIIa (the b3 subunit), however, may also be expressed with the aV subunit (i.e. aVb3 integrin) on other cells, including trophoblast cells [15], endothelial cells, and endothelial progenitor cells, which may mediate new vessel development (angiogenesis) [16,17] and play important roles in placental development. In addition to their physiological functions listed above, GPIba and aIIbb3 integrin are also the major antigenic targets of autoimmune and alloimmune antibodies that cause platelet destruction. In autoimmune thrombocytopenia (ITP) patients, >70% of platelet autoantibodies target aIIbb3 integrin, whereas the remaining 20e40% have specificity for the GPIba complex or both [18]. Alloantibodies targeting GPIba or aIIbb3 can be generated after transfusion with “foreign” or allogeneic platelets, or during pregnancy after maternal exposure to fetal platelets (containing paternally derived alloantigens). Alloantibody generation may result in alloimmune diseases, such as post-transfusion purpura and fetal and neonatal alloimmune thrombocytopenia (FNAIT). Analogous to ITP, >75% of platelet-specific antibodies in FNAIT patients have specificity against aIIbb3 integrin [19,20]. In contrast, reported cases of anti-GPIba antibodies are rare, although severe cases have been reported. Whereas steroid therapy and intraveneous immunoglobulin G (IVIG) have been shown to increase platelet count and prevent bleeding in ITP [21,22] and in some cases of FNAIT [20,23], whether these therapies are equally efficient in treating anti-GPIba versus anti-aIIbb3-mediated thrombocytopenias is unclear, since the former may cause platelet destruction through an Fc-independent pathway [24e26]. Recent studies demonstrated that thrombocytopenia caused by anti-GPIba may differ from that caused by antib3 integrin antibodies. Anti-GPIba antibodies may deliver signals and activate platelets, leading to thrombocytopenias that are resistant to IVIG therapy in both murine models and human patients [25e27]. Most recently, anti-GPIba antibodies have been found to induce platelet sialidase neuraminidase-1 translocation and platelet desialylation [28,29]. These desialylated platelets are likely cleared in the liver via hepatocyte AshwelleMorell receptors, a significantly different process from the classical Fc-FcgR-dependent macrophage phagocytosis in the spleen. However, whether this mechanism also occurs in the fetal reticuloendothelial system (RES) and whether it affects FNAIT is currently unknown. 2. Pathogenesis of FNAIT FNAIT is the most frequent cause of severe thrombocytopenia in liveborn neonates [30,31], with an estimated frequency of 0.5e1.5 per 1000. This number, however, does not include miscarriages, since the incidence of mortality of FNAIT fetuses has not been adequately studied [32]. Intrauterine death or intracranial hemorrhage (ICH) may occur as early as 14e16 weeks of gestation [30], and up to 10% of liveborn neonates with FNAIT had ICH in utero before the 30th week. Postnatal ICH is also frequent (10e20%) in FNAIT neonates and may be fatal in up to 5% of cases [20]. In those who survive ICH, neurological impairment is frequent [33]. In

FNAIT is initiated by antigens on fetal platelets inherited from the father and are lacking in the mother [31,35]. The maternal immune system regards the fetal platelets as foreign and generates alloantibodies to target them. Each unique antibody specificity is assigned a human platelet alloantigen (HPA) number [36,37]. The “a” is designated for high frequency allelic forms and the “b” for low frequency forms. Thirty-six different HPAs located on six separate platelet surface proteins (integrins b3, aIIb, a2; glycoproteins GPIba, GPIbb, and CD109) have been reported and attributed to causing FNAIT [38]. The major platelet alloantigens are illustrated in Figure 1. Approximately half of all reported platelet surface antigens are located on the integrin b3 subunit, covering a wide range of the extracellular portion of the b3 subunit, from the N-terminal PSI domain to the Cterminus of the extracellular region. This suggests that the entire b3 subunit could contain alloantigens and cause FNAIT. In the Caucasian population, 75e85% of maternally derived antibodies reported target HPA-1a on integrin b3 (residue 33 Leu and Pro on the b3 integrin subunit) [19,31]. The HPA-1 leucine/ proline polymorphism is relatively rare in the African population, and even more rare in the Asian population [37]. HPA-5a (residue 505 Lys and Glu on the a2 subunit of a2b1 integrin e the collagen receptor) alloimmunization is the second leading cause of FNAIT in the Caucasian population, but seems to cause a less severe form of FNAIT. In Japan, the most frequent antibodies involved in FNAIT are anti-HPA5b followed by anti-HPA4b (residue 143 Gln and Arg of the integrin b3 subunit) [39,40]. The frequency of reported cases caused by antibodies targeting other antigens, such as HPA-2a (residue 145 Thr and Met on GPIba) and HPA-3a (residue 843 Ile and Ser on aIIb), are quite low. Currently it is unclear why the reported cases of anti-HPA-2a (GPIba)-mediated FNAIT are rare but studies from animal models suggest that anti-GPIba antibodies can induce a non-classical FNAIT (i.e. miscarriage but no bleeding in neonates) [41], which may not be easily identified and reported by clinicians. Anti-HPA-3a (aIIb)mediated FNAIT was first reported in 1980. Although cases of antiHPA-3a-mediated FNAIT are relatively rare (3e5% of cases), they are clinically severe and can cause miscarriages [39]. Anti-aIIb HPAs are reportedly difficult to detect [39]; thus, both anti-GPIba-and antiaIIb-mediated FNAIT may be underreported. In addition to HPAs, platelet GPIV (CD36) deficiency has been found in 4e8% of Asians and anti-CD36-mediated FNAIT has been reported in both Asian and African populations [42e44]. Further, anti-HLA antigens may also be involved in FNAIT. However, since HLA antigens are broadly expressed on many fetal cells and tissues and are less specific to platelets, the importance of anti-HLA antibodies in FNAIT is debatable. 2.2. Maternal and fetal immune responses The maternal immune response to fetal platelet antigens is still not well understood. Currently it is unclear how fetal platelet antigens can breakthrough the immunoprivileged site (placenta) and enter the maternal immune system. In addition to the fetal platelets that may enter maternal blood, it has been speculated that interaction between maternal blood cells and antigen-positive

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HPA-19w K137Q HPA-16w T140I HPA-4 R143Q HPA-17w T195M

HPA-2 T145M

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HPA-5 E505K HPA-18W TQ716H HPA-13W T799M

HPA-7w P407A HPA-10w R62Q

HPA-1 L33P

HPA-12 G15Q

HPA-6w R489Q

HPA-20w T619M

HPA-8w R636c HPA-11w R633H HPA-14w Del K611 HPA-21w E628K

HPA-9w V837M HPA-3 1843S

GPIIb GPIa

GPIbα

GPIbβ

GPIbβ

GPIX

GPIIIa PIIa GPIIa

Fig. 1. Diagram of some major human platelet antigens (HPAs) on various glycoprotein complexes that are responsible for alloimmunization of pregnant women leading to fetal and neonatal alloimmune thrombocytopenia (FNAIT). All HPAs result in a single amino acid substitution except for HPA-14w where the antigen is a result of a one amino acid deletion. Thirty-six HPAs on six platelet surface proteins have been discovered so far, 26 of which are caused by bi-allelic polymorphisms, whereas two are caused by tri-allelic polymorphisms. Approximately half of all reported platelet surface antigens are located on the integrin b3 subunit, covering a wide range of the extracellular portion of the b3 subunit, from the N-terminus to the C-terminus of the extracellular region.

trophoblasts or trophoblast microparticles may play a role. After digestion of fetal antigens, the antigen-presenting cells (APCs), such as dendritic cells and macrophages, load the antigen peptides on to their MHC class II molecules and present the MHCepeptide complex to T cell receptors on CD4þ T helper (Th) cells. These Th cells then proliferate and release cytokines that promote B cell differentiation, proliferation, and antibody production including immunoglobulin class switching and somatic hypermutation that may increase the antibody affinity. HLA-DRB3*01:01 (MHC class II) has been linked with anti-HPA1a generation and FNAIT. The largest prospective FNAIT study so far has reported that women who are HLA DRB3*01:01 positive have an approximately 25 times higher risk of HPA-1a-immunization when compared to women who lack this allele [45]. Of the women who are already HPA-1a-immunized, ~90% are HLA DRB3*01:01 positive [45]; and the HPA-1a-immunized women who are HLA DRB3*01:01 positive [45] have significantly higher levels of anti-HPA-1a antibodies [46]. The reason for the increased immunization risk is related to the ability of the peptide carrying the HPA-1a epitope to be presented efficiently to antigen-specific T cells. Several studies have shown that the antigenic peptide harboring the HPA-1a epitope is efficiently presented to Th cells by APCs carrying the HLA molecule encoded by the HLA DRB3*01:01 allele [47]. Hence, in women lacking this HLA molecule, the antigenic peptide will bind to other less suitable HLA class II molecules and will not efficiently trigger Th cells. Notably, CD4þ and CD8þ T regulatory cells may play important roles in suppressing immune responses and maintaining immune tolerance against anti-platelet responses [48e51]. However, the roles of these different subsets of CD4þ and CD8þ T regulatory cells in the maternal immune response

to fetal platelet antigens during pregnancy (a stage of “physiological immunotolerance”) are still largely unknown. It was recently demonstrated that an MHC class I-related receptor, neonatal Fc receptor (FcRn), plays a key role in the maternofetal transfer of IgG antibody. Interestingly, it seems that the fetal, rather than maternal FcRn is indispensable for transplacental transportation of different isotypes of IgGs [52], although the affinity between FcRn and different IgG isotypes may affect this process [53]. However, it is still not entirely clear where and how the IgG-FcRn interaction occurs in the placenta and how IgGs are transported to the fetal side. The mechanisms of platelet destruction and clearance in FNAIT are thought to be similar to ITP: the antibody binds to a platelet via the Fab portion and bridges the opsonized platelet to a macrophage through interaction between Ig-Fc and the macrophage Fc receptor, resulting in clearance of opsonized platelets [54]. Interaction of the Fc portions of different IgG isotypes (e.g. IgG1 vs. IgG2a) and various Fc receptors (e.g. FcRIIIA vs FcRIIB) on macrophages may vary and, thus, may initiate or prevent phagocytosis [55] and regulate platelet clearance in the reticuloendothelial system (RES, e.g. the spleen). Recently Kapur et al. showed that anti-HPA-1a antibodies causing FNAIT possess oligosaccharides that are deficient in core fucose residues [56]. This feature has also been associated with increased affinity to FcgRIIIa/b and enhanced platelet phagocytosis. Thus, these a-fucosylated antibodies may be more efficient at promoting antibody-mediated phagocytosis of sensitized platelets. Further investigation will be important to determine whether differences exist between the adult and fetal/neonatal RES and also whether Fc-independent platelet destruction occurs in fetuses, which would cause them to respond to therapies differently.

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Although platelet destruction is thought to be the major contributor to bleeding in affected patients, there has been little study of the effect of anti-platelet antibodies on vascular injury and platelet function, which may contribute to bleeding. Emerging information suggests that impairment of angiogenesis [57], rather than platelet or fibrin clots [58], may be the major cause of fetal/ embryonic hemorrhage [59]. Among the questions which remain unanswered: (i) whether the HPA-1a antigen induces antibody generation more readily than other HPA antigens (e.g. HPA-2a on GPIba), and whether the anti-HPA-1a antibody can more easily cross the placenta (via FcRn) or cause platelet phagocytosis based on the prevalence of their IgG isotypes; (ii) whether platelet clearance in FNAIT caused by anti-GPIba is also mediated by an Fc-independent pathway [24e26] in the fetal/ neonatal RES, or whether IVIG may be less effective for patients with anti-GPIba-mediated thrombocytopenia [25]; (iii) whether antiGPIba-mediated FNAIT is less frequently reported because GPIba is either less immunogenic/exhibits less severe symptoms (i.e. not reported) or is too severe (due to miscarriage) to be identified by clinicians; (iv) whether the more severe bleeding found in FNAIT (versus ITP) is due to maternal anti-b3 antibodies that target fetal/ neonatal angiogenic vessels, which are particularly abundant in fetal and neonatal brains. Given the ethical barriers in performing basic research on human fetuses and neonates, it would be very useful to have an animal model of FNAIT to address these important questions. 3. Lessons learned from animal models of FNAIT The first animal model of FNAIT was reported in 2006 [60]. Currently, few other animal models are available to study the maternal immune response to fetal platelet antigens [61,62]. In contrast, several animal models of ITP have been reported [4,49,51,63]. Although these ITP models provide important information for understanding antibody-mediated macrophage phagocytosis and the role of IVIG, they have not been instructive in the understanding of the maternal immune responses against fetal platelet antigens that cause FNAIT. 3.1. The first animal model of FNAIT and examination of IVIG therapy Since the entire extracellular domain of the b3 subunit can contain alloantigens and cause FNAIT in humans, a study of the immune response to the entire b3 integrin subunit is important. The first animal model of FNAIT was, therefore, established using b3 integrin knockout (b3e/e) mice [60]. This model clarified that maternal antib3 integrin antibody levels correlate with severity of disease. Importantly, this model demonstrated that maternal IVIG administration can decrease pathogenic antibody titer in both the maternal and fetal circulation, and significantly ameliorates the symptoms of FNAIT, although it was previously debated whether IVIG is an efficient therapy to control this disorder. These results suggest that fetal IVIG transfusion may not only be more complicated and risky [64], but may also be less efficient than maternal administration. 3.2. Role of neonatal Fc receptor (FcRn): mechanisms of IVIG and anti-FcRn therapy for FNAIT and other fetal and neonatal disorders Studies have shown that FcRn protects IgG from clearance, regulates IgG homeostasis [65], and plays an important role in transplacental IgG transport. However, the role of FcRn in the pathogenesis and therapy of FNAIT has not been adequately investigated. Chen et al. developed an animal model of FNAIT using combined b3 integrin- and FcRn-deficient (b3e/eFcRne/e) mice and demonstrated that FcRn is indispensable for the induction of FNAIT.

By breeding b3e/eFcRne/þ or b3e/eFcRne/e females with b3þ/ FcRne/e or b3þ/þFcRne/þ males, Chen et al. clearly demonstrated that fetal rather than maternal FcRn is responsible for the transplacental transport of various IgG isotypes including IgG1, IgG2b, and IgG3. This study further showed that anti-FcRn and IVIG therapy prevented FNAIT and IVIG-ameliorated FNAIT through both FcRn-dependent and FcRn-independent pathways. These data suggest that targeting FcRn may be a potential therapy for human FNAIT [52]. In addition, anti-FcRn therapy may be useful for prevention of transplacental transport of pathogenic antibodies to the fetus in other alloimmune diseases, such as hemolytic disease of the fetus and newborn (HDFN) and alloimmune neonatal neutropenia, or pathogenic antibody transfer from mothers with autoimmune disorders, including ITP, Grave's dis€ gren's syndrome, autoimmune hemolytic anemia, and ease, Sjo systemic lupus erythematosus.

þ

3.3. Anti-GPIba-mediated FNAIT: non-classical FNAIT and the role of infection Multiple groups worldwide have reported FNAIT-related miscarriage, but the incidence is currently unknown. The mechanism of fetal loss has not been previously examined; notwithstanding, fetal bleeding was assumed to be the cause of fetal death. As discussed, aIIbb3 integrin and GPIba are the primary autoantigens in ITP, but the reported incidence of anti-GPIba-mediated FNAIT is rare. Li et al. established an FNAIT model using GPIbae/e mice [41], similar to the anti-b3 model [60]. After comparing the pathogenesis of anti-GPIba- and anti-b3-mediated FNAIT, Li et al. unexpectedly found miscarriage in most of the anti-GPIba-mediated FNAIT cases and >83% of female mice did not deliver any pups (usually 6 to 12 pups per litter). This frequency is far higher than that observed in the anti-b3-mediated FNAIT model. Mothers with antiGPIba antibodies exhibited extensive fibrin deposition in their placentas, which severely impaired placental function and blood perfusion. Li et al. further demonstrated, for the first time, that antiGPIba (but not anti-b3) sera activated platelets and induced phosphatidylserine expression, which likely enhanced the cell-based thrombin generation and fibrin formation in vitro and thrombus formation in vivo. Therefore, the maternal immune response to fetal GPIba may cause a previously unidentified, non-classical FNAIT characterized by spontaneous miscarriage without neonatal bleeding. The high frequency of miscarriage may have masked the severity and frequency of anti-GPIba-mediated FNAIT in patients. Importantly, both IVIG and anti-FcRn therapy efficiently prevented fetal death in this non-classical form of FNAIT [41], possibly through prevention of transplacental transport of pathogenic antibodies. Viral and bacterial infections have been frequently linked with the pathogenesis of ITP; however, it was previously unclear whether these infections contribute to the severity of FNAIT. In the next study, Li et al. examined immune responses against platelet antigens by transfusing WT mouse platelets into b3e/e or GPIbae/e mice. To mimic viral and bacterial infections, polyinosinic:polycytidylic acid (Poly I:C) and lipopolysaccharide (LPS) were injected intraperitoneally immediately after platelet transfusions. FNAIT was established by breeding the immunized female mice with male WT mice. Li et al. demonstrated that the immunogenicity of platelet GPIba was lower compared to b3 integrin, which may provide an additional explanation as to why the reported incidence of anti-GPIba-mediated FNAIT is far lower than ITP. Interestingly, co-stimulation with Poly I:C or LPS markedly enhanced the immune response against platelet GPIba and caused severe FNAIT-associated pathologies (i.e. miscarriage). Poly I:C or LPS also enhanced the immune response against b3 integrin. These data suggest that viral and bacterial infections aggravate the anti-

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platelet GPIba response [66], which may lead to a non-classical form of FNAIT (i.e. miscarriage). 3.4. Role of anti-angiogenic effect in intracranial hemorrhage (ICH) and intrauterine growth restriction (IUGR) ICH is the major risk factor for FNAIT. Although thrombocytopenia resulting from maternal antibodies targeting b3 integrin or other platelet antigens (e.g. GPIba) has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately  et al. found explored. Utilizing murine models of FNAIT, Yougbare that ICH occurred in fetuses and neonates with anti-b3 integrinmediated FNAIT. Interestingly, ICH was not found in any of the several hundred anti-GPIba-mediated FNAIT fetuses and neonates studied, despite similar thrombocytopenia in anti-b3- and antiGPIba-mediated FNAIT mice. Reduced brain and retinal vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis were observed only in anti-b3-mediated FNAIT, not in anti-GPIba-mediated FNAIT, and were abrogated by maternal IVIG  et al. treatment. In cultured human endothelial cells, Yougbare further demonstrated that cell proliferation, network formation, and Akt phosphorylation were inhibited only by murine anti-b3 sera and human anti-HPA-1a IgG purified from mothers with FNAIT children [59]. These data suggest that fetal hemostasis may be unique in that impairment of angiogenesis rather than thrombocytopenia is likely the major cause of ICH. Since the fetal brain is one of the most angiogenic organs during development and may have far more angiogenic vessels than adult brains, this may explain why the frequency of ICH in FNAIT is 10e100 times higher than in ITP [67]. Importantly, maternal IVIG therapy can effectively prevent this devastating disorder in the murine model of FNAIT. Because thrombocytopenia may not be the major cause of ICH, increasing fetal platelet counts via weekly fetal platelet transfusion may not be an efficient therapy. Given that fetal platelet transfusion is an invasive therapy and may cause fetal loss, this prenatal therapeutic method must be used with caution. Since anti-b3 integrin antibodies impair angiogenesis, they may also attack angiogenic vessels in the placenta and fetus, potentially leading to intrauterine growth restriction (IUGR). Consistent with this hypothesis, a Norwegian FNAIT study showed a clear association between the levels of maternal HPA-1a antibodies and reduced birth weight in boys [68]. Studies investigating the mechanisms of IUGR and placental pathology are currently underway, with some preliminary data reported [69]. 3.5. Possible prophylaxis for FNAIT by antibody-mediated immune suppression (AMIS) FNAIT is a severe disorder and currently no effective therapy is available and no prophylactic strategies exist. Results from several large screening studies suggest that the pathophysiology of FNAIT may be more similar to hemolytic disease of the fetus and newborn (HDFN) than previously thought. Immunization against HPA-1a might, therefore, be preventable by a prophylactic regimen of AMIS, which has been documented to be useful prophylaxis against HDFN. To test whether passive administration of anti-b3 integrin antibodies could induce AMIS and thereby prevent clinical complications of FNAIT, Tiller et al. induced AMIS in b3e/e mice by intravenous administration of human anti-HPA-1a IgG (both polyclonal and monoclonal antibodies) or murine anti-b3 sera, which were given as prophylaxis following transfusion of HPA-1a-positive human platelets or murine wild-type platelets, respectively. Tiller et al. found that AMIS against both human and murine platelet antigens was induced in b3e/e mice and that the amount of

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maternal anti-platelet antibodies was reduced by up to 90%. Importantly, neonatal platelet counts were increased and pregnancy outcome was improved dose-dependently. Tiller et al. also observed that the severity of thrombocytopenia inversely correlated with birth weight [70]. Interestingly, preliminary data showed that anti-b3 integrin sera used to induce AMIS also downregulated anti-GPIba antibodies, suggesting that AMIS is not necessarily restricted to the specificity of the antibody used to induce the immune suppression. This work indicates that prophylactic administration of antiplatelet antibodies induces AMIS and prevents poor pregnancy outcome in FNAIT mice. It further suggests that both poly- and monoclonal anti-HPA-1a antibodies should be tested as prophylactic reagents against human FNAIT. 4. Management and prevention strategies for FNAIT 4.1. The role of the diagnostic laboratory 4.1.1. Platelet count Most cases of FNAIT are discovered when a child is born at term with petechiae or other signs of bleeding in the absence of any other condition known to be associated with neonatal thrombocytopenia. Thrombocytopenic neonates usually present with low platelet counts during the first 72 h after birth [19,30,31,33,34]. However, in the absence of petechiae many FNAIT cases with mild to moderate thrombocytopenia (>50106/mL) are overlooked [71]. 4.1.2. Antibody detection If at any time FNAIT cannot be excluded, then a clinical laboratory diagnosis should be used to confirm the presence of platelet antibodies in the maternal circulation [31,33]. Although there are several commercially available laboratory assays for detection of maternally derived anti-platelet antibodies, the monoclonal antibody immobilization of platelet antigen (MAIPA) assay has long been considered as the gold standard for detection of platelet antibodies [31]. The MAIPA assay is versatile in that it has a multitude of important uses in the clinical diagnostic laboratory, which include antibody differentiation, antibody screening, and serumeplatelet cross-matching assays. Furthermore, MAIPA allows for the detection of rare FNAIT antigens beyond the scope of the widely reported HPA1, HPA-3, HPA-5, and HPA-15 [72]. Given that MAIPA assays usually take 2e3 days, which is too long for neonates to wait for treatment, they are best used to confirm the diagnosis and to monitor the maternal antibody level in the subsequent pregnancies. In rare FNAIT cases, antibodies may not be detectable in samples taken at the time of childbirth [72]. In such cases, where the most likely clinical diagnosis is FNAIT, follow-up serology tests must be performed, especially in cases of subsequent pregnancies. There have been reports of severe thrombocytopenia caused by anti-platelet antibodies, yielding negative serology results due to the low avidity of the aforementioned antibodies. In these cases the antibodies will not remain bound to their specific antigens during the washing steps of laboratory procedures. In many such FNAIT cases antibodies can be detected by surface plasmon resonance technology [73]. 4.1.3. Genetic testing The familial recurrence rate in subsequent pregnancies is dependent upon the genotype of the father. Families with homozygous dominant fathers have a nearly 100% recurrence rate in subsequent pregnancies, whereas those with heterozygous fathers have an approximately 50% recurrence rate [72]. Therefore, there is a significant risk to the offspring of multigravidous women with a history of FNAIT. In all cases, whether the father is homozygous or heterozygous for the causative HPA, genetic testing should be

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performed on the fetus. Today, fetal HPA-1a typing can easily be carried out based on cell-free fetal DNA in the mother's plasma [74]. 4.2. Management of FNAIT 4.2.1. Prenatal management 4.2.1.1. Intravenous immunoglobulin. Though there is a lack of approved drugs for this condition, many centers use high-dose intravenous immunoglobulin (IVIG) off-label to treat HPA-1aimmunized pregnant women [31,75]. IVIG is a blood product prepared from the plasma pooled from more than 1000 healthy donors. It has been used to treat several auto- and alloimmune diseases, particularly for ITP and other antibody-mediated thrombocytopenias [21,76]. Proposed mechanisms of action of IVIG include RES blockade, occupancy of FcRn and accelerated antibody clearance [77], anti-idiotypic antibody activity, induction of T/B cell tolerance [76], and inhibition of dendritic cell function [78]. Recently, inhibition of macrophage phagocytosis via IVIGeFcgRIIB interaction, for example, sialylated IgGs, has been highlighted by Ravetch et al. [79], but the mechanisms of action of IVIG in these diseases remain poorly understood. Unfortunately, IVIG is expensive and associated with significant side-effects, and its efficacy in preventing ICH is controversial [75,80,81]. The dosage used for treatment of HPA-1a-immunized women varies from 0.5 to 1.0 g/kg/week in Sweden and The Netherlands [82] to 2 g/kg/week starting from gestational week 12 in extremely high-risk pregnancies in the USA [83]. As the price of IVIG is approximately US$75 per gram, IVIG treatment of one HPA1a-immunized woman with a high risk of FNAIT can add up to $300,000 in the USA. Some centers stratify the treatment according to the presence or absence of ICH in the previous child and the timing of when ICH occurred, i.e. early in pregnancy, late in pregnancy, or perinatally [83]. Although it has been reported by Bussel et al. [84] and in animal models [52,60] that IVIG is a useful therapy for FNAIT [30], the efficacy of this therapy in FNAIT is still debatable [64,81,84e87]. 4.2.1.2. Steroid therapy. Many treatment protocols using IVIG also include glucocorticosteroids and varying degrees of intrauterine monitoring during pregnancy. Recent studies have reported that IVIG with steroids was a more effective therapy than IVIG alone at preventing severe thrombocytopenia [30]. Bussel et al. suggest stratifying based on whether the first child had ICH [83]. For cases with a sibling without ICH, they recommend the mother be given IVIG (1 g/kg/week) from 20e22 weeks along with prednisone (0.5 mg/kg/day) from 30e32 weeks of gestation until delivery. For high-risk cases with a sibling with ICH, therapy would start at 16e18 weeks. Vinograd and Bussel recommend two infusions of IVIG per week (1 g/kg/week) with prednisone (0.5 to 1 mg/kg/day) for the most severely affected fetuses [88]. 4.2.1.3. Cesarean section. Elective cesarean section may help prevent ICH from occurring at delivery. Bussel et al. recommended cesarean section at 39 weeks for both low- and high-risk cases (i.e. whether or not the sibling had an ICH) [83]. However, studies from Bertrand et al. suggest assigning risk based on antibody concentration [30,89]. Maternal anti-HPA-1a alloantibody concentrations were measured by MAIPA. Importantly, they found a negative correlation between the area under the curve (AUC) of the antibody concentration data and newborn platelet count at delivery; low AUC correlated with safe newborn platelet count, whereas high AUC correlated with severe thrombocytopenia. Based on these findings, Bertrand et al. found a threshold and recommended vaginal delivery when the AUC is 23 IU/mL.

4.2.1.4. Platelet transfusion. In some cases, the efficacy of IVIG is unpredictable and therefore weekly in-utero platelet transfusions have been performed [81,85,90,91]. However, in-utero platelet transfusion is an invasive procedure that carries a risk of fetal loss, especially for fetuses with an existing low platelet count [92]. Moreover, if the bleeding disorders (e.g. ICH) are caused by impaired angiogenesis rather than thrombocytopenia, as observed in mice [58,59], platelet transfusions may not be an effective treatment. Fetal platelet transfusion may need re-evaluation in order to better balance the risk and efficacy of this therapy. 4.2.1.5. Anti-FcRn therapy. Anti-FcRn monoclonal antibody (mAb), which blocks transplacental transport of maternally derived pathogenic IgGs, has been studied in several animal models. The data are encouraging: miscarriage, platelet counts, bleeding diatheses, and impaired angiogenesis are all markedly improved [41,52,59]. However, the mechanisms of this therapy are not well understood and detailed information as to how FcRn transports IgG across the placenta is almost completely unknown. In addition, the safety and efficacy of anti-FcRn therapy need to be examined in clinical trials. 4.2.2. Postnatal management 4.2.2.1. Platelet transfusion. Many neonates exhibit signs of bleeding unexpectedly during or immediately after parturition, and appropriate therapies are not always readily available. Currently, transfusion of donor-matched, antigen-negative platelets is the best treatment of choice for neonates born with FNAIT who exhibit signs of bleeding. In up to 95% of cases of newborns with FNAIT, platelets from HPA-1a- and HPA-5b-negative individuals are compatible and effectively increase platelet counts with improved survival [93,94]. Unfortunately, few hospitals and blood transfusion centers keep genotyped platelets stored for urgent transfusions [95]. If compatible platelets are not available, and if the mother is suitable for platelet apheresis, it is possible to transfuse the newborn with washed, irradiated and leukoreduced platelets harvested from the mother. Since this procedure takes several hours, this approach can only be used in cases where FNAIT is expected, for instance due to the obstetric history. In such cases, platelet apheresis can be performed before delivery. Another treatment option is the immediate transfusion of random donor platelets [96]. Although this treatment has been reported to be of great benefit in some cases, it is primarily used as an alternative strategy to temporarily increase platelet counts until compatible platelets become available [97,98]. Furthermore, IVIG may also improve the neonatal platelet count and enhance hemostasis, particularly when antigen-negative platelet transfusions are not available. 4.2.2.2. IVIG therapy. Since neonatal alloimmune thrombocytopenia can be life-threatening and ICH may lead to neurological impairment, rapid and effective therapies are required. Compatible (antigen-negative) platelets for transfusion may be difficult to obtain on short notice, and maternal platelets cannot be readily prepared in all circumstances or at all hospitals. However, IVIG can be quickly and readily made available. Although some benefit has been observed, no random clinical trial data are available for neonatal IVIG therapy and it is currently unknown whether the neonatal RES response to IVIG is similar to that of adults in ITP. Further research is required to improve our understanding of the therapeutic role of IVIG in this disease, to understand its efficacy, its mechanisms of action, and to elucidate which patients might best respond to IVIG therapy.

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4.3. Prevention of FNAIT: genetic screening and AMIS prophylaxis

Acknowledgements

During the last decade there has been discussion in many European countries as to whether it is time for inclusion of HPA-1a typing in the healthcare program for pregnant women in order to identify pregnancies at risk of FNAIT. No countries have so far introduced general HPA-1a typing. One of the main arguments against general HPA-1a typing is that there is no consensus regarding the optimal treatment of risk pregnancies. The PROFNAIT Consortium (www.profnait.eu) was established in 2011 on the initiative of Prophylix Pharma AS (www. prophylixpharma.com) e a small Norwegian biotech company e based on clinical observations and animal studies [70,99]. The drug under development is purified hyperimmune anti-HPA-1a IgG from plasma collected from HPA-1a-immunized women who previously have had a pregnancy complicated by FNAIT. The hyperimmune anti-HPA-1a IgG will be used for the prevention of FNAIT by antibody-mediated immune suppression. The idea was to apply the same prophylactic principle by which hyperimmune anti-D IgG had been used successfully during the last four decades for the prevention of hemolytic disease of the fetus and newborn. It is anticipated that the drug (hyperimmune anti-HPA-1a IgG) will be produced in 2016, that the clinical trial will begin in early 2017, and that the drug will be licensed for clinical use in 2020.

We thank Dr John Freedman, Dr Richard O. Hynes, and Dr Zaverio M. Ruggeri for their long-term support for these research projects; also Mr Sean Lang and Miss Xun Fu and Dr Xiaohong Xu for their help during the manuscript preparation.

Practice points  Maternal antibodies against fetal platelet antigens may cause miscarriage, and the maternal immune response against GPIba may mainly lead to a non-classical FNAIT (i.e. miscarriage but not neonatal bleeding).  Some anti-platelet antibodies cross-react with aVb3 integrin on endothelial cells and impair angiogenesis, which may be the key mechanism to cause the ICH in FNAIT. Detection of maternal anti-aVb3 antibodies may be important for diagnosis and for ICH prevention.  Intravenous immunoglobulin G is a useful therapy, and a monoclonal antibody targeting the neonatal Fc receptor may prevent pathogenic IgG from crossing the placenta and may, therefore, be an efficient therapy to control this type of disease.  Similar to anti-RhD therapy, AMIS may be useful to prevent FNAIT.

Research directions  Study the relationship between miscarriage and antiplatelet antibodies in affected pregnant women.  Study whether the cross-reaction of anti-platelet antibodies with aVb3 integrin on endothelial cells is the cause of intracranial hemorrhage and intrauterine growth restriction.  Study whether antibody-mediated immune suppression may be a useful prophylaxis against fetal and neonatal alloimmune thrombocytopenia.  Re-evaluate the efficacy of prenatal fetal platelet transfusion for control of fetal bleeding disorders.  Study the therapeutic potential of anti-FcRn antibodies for treatment of fetal and neonatal alloimmune thrombocytopenia.

Conflict of interest statement Jens Kjeldsen-Kragh is one of the founders and owners of Prophylix Pharma AS, which leads an EU-founded consortium developing a hyperimmune anti-HPA-1a IgG for the prevention of fetal and neonatal alloimmune thrombocytopenia. Funding sources This work was supported by the Canadian Institutes of Health Research (MOP 68986, MOP 119540 and MOP 119551), Equipment Funds from the Canada Foundation for Innovation, St Michael's Hospital, and Canadian Blood Services. D. Zdravic is a recipient of a Canadian Blood Services graduate fellowship and a University of Toronto, Department of Laboratory Medicine and Pathobiology  is a recipient of a Canadian departmental fellowship. I. Yougbare Blood Services postdoctoral fellowship. B. Vadasz is a recipient of graduate fellowships from the Department of Laboratory Medicine and Pathobiology, University of Toronto and has received a Queen Elizabeth II Ontario Graduate Scholarship. C. Li is a recipient of the Connaught Scholarship, and Laboratory Medicine and Pathobiology Departmental Fellowships, University of Toronto. References [1] Junt T, Schulze H, Chen Z, Massberg S, Goerge T, Krueger A, et al. Dynamic visualization of thrombopoiesis within bone marrow. Science 2007;317(5845): 1767e70. [2] Ruggeri ZM. Mechanisms initiating platelet thrombus formation. Thromb Haemost 1997;78:611e6. [3] Hou Y, Carrim N, Wang Y, Gallant RC, Marshall A, Ni H. Platelets in hemostasis and thrombosis: novel mechanisms of fibrinogen-independent platelet aggregation and fibronectin-mediated protein wave of hemostasis. J Biomed Res 2015;29:437e44. [4] Li C, Li J, Li Y, Lang S, Yougbare I, Zhu G, et al. Crosstalk between platelets and the immune system: old systems with new discoveries. Adv Hematol 2012;2012:384685. [5] Murphy AJ, Bijl N, Yvan-Charvet L, Welch CB, Bhagwat N, Reheman A, et al. Cholesterol efflux in megakaryocyte progenitors suppresses platelet production and thrombocytosis. Nat Med 2013;19(5):586e94. [6] Wang Y, Andrews M, Yang Y, Lang S, Jin JW, Cameron-Vendrig A, et al. Platelets in thrombosis and hemostasis: old topic with new mechanisms. Cardiovasc Hematol Disord Drug Targets 2012;12(2):126e32. [7] Ni H, Freedman J. Platelets in hemostasis and thrombosis: role of integrins and their ligands. Transfus Apher Sci 2003;28:257e64. [8] Yang H, Reheman A, Chen P, Zhu G, Hynes RO, Freedman J, et al. Fibrinogen and von Willebrand factor-independent platelet aggregation in vitro and in vivo. J Thromb Haemost 2006;4(10):2230e7. [9] Jackson SP. The growing complexity of platelet aggregation. Blood 2007;109: 5087e95. [10] Ni H, Denis CV, Subbarao S, Degen JL, Sato TN, Hynes RO, et al. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. J Clin Invest 2000;106(3):385e92. [11] Roberts HR, Hoffman M, Monroe DM. A cell-based model of thrombin generation. Semin Thromb Hemost 2006;32(Suppl 1):32e8. [12] Ni H, Papalia JM, Degen JL, Wagner DD. Control of thrombus embolization and fibronectin internalization by integrin alpha IIb beta 3 engagement of the fibrinogen gamma chain. Blood 2003;102:3609e14. [13] Wang Y, Reheman A, Spring CM, Kalantari J, Marshall AH, Wolberg AS, et al. Plasma fibronectin supports hemostasis and regulates thrombosis. J Clin Invest 2014;124(10):4281e93. [14] Wang Y, Carrim N, Ni H. Fibronectin orchestrates thrombosis and hemostasis. Oncotarget 2015;6:19350e1. [15] Kumpel BM, Sibley K, Jackson DJ, White G, Soothill PW. Ultrastructural localization of glycoprotein IIIa (GPIIIa, beta 3 integrin) on placental syncytiotrophoblast microvilli: implications for platelet alloimmunization during pregnancy. Transfusion 2008;48:2077e86.

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