Alloimmune Thrombocytopenia Research Articles

Congenital thrombotic thrombocytopenic purpura simulating alloimmune thrombocytopenia

Congenital thrombotic thrombocytopenic purpura (cTTP) is a thrombotic microangiopathic caused by severely reduced activity of the von Willebrand factor-cleaving protease A Disintegrin And Metalloproteinase with a Thrombospondin type 1 Motif, member 13 (ADAMTS-13), which leads to small-vessel platelet-rich thrombi, thrombocytopenia, and microangiopathic hemolytic anemia (MHA) in the absence of neutralizing antibodies. We describe a case of cTTP in a female neonate presenting initially on the 1st day of life with asymptomatic thrombocytopenia thought to be alloimmune thrombocytopenia, then progressed to severe neonatal jaundice, pallor, and MHA, in whom ADAMTS-13 levels <5%, and the anti-ADAMTS-13 antibody titer was negative confirming the diagnosis of cTTP. The patient initially received intravenous immunoglobulin infusion before documenting MHA. She received fresh frozen plasma infusions which successfully reversed the MHA (by supplying ADAMTS-13) and prevented organ damage in this patient.

Prevention of Fetal/Neonatal Alloimmune Thrombocytopenia in Mice: Biochemical and Cell Biological Characterization of Isoforms of a Human Monoclonal Antibody.

Maternal alloantibodies toward paternally inherited Ags on fetal platelets can cause thrombocytopenia and bleeding complications in the fetus or neonate, referred to as fetal and neonatal alloimmune thrombocytopenia (FNAIT). This is most commonly caused by Abs against the human platelet Ag (HPA)-1a in Caucasians, and a prophylactic regimen to reduce the risk for alloimmunization to women at risk would be beneficial. We therefore aimed to examine the prophylactic potential of a fully human anti-HPA-1a IgG1 (mAb 26.4) with modified Fc region or altered N-glycan structures. The mAb 26.4 wild-type (WT) variants all showed efficient platelet clearance capacity and ability to mediate phagocytosis independent of their N-glycan structure, compared with an effector silent variant (26.4.AAAG), although the modified N-glycan variants showed differential binding to FcγRs measured in vitro. In an in vivo model, female mice were transfused with platelets from transgenic mice harboring an engineered integrin β3 containing the HPA-1a epitope. When these preimmunized mice were bred with transgenic males, Abs against the introduced epitope induced thrombocytopenia in the offspring, mimicking FNAIT. Prophylactic administration of the mAb 26.4.WT, and to some extent the mAb 26.4.AAAG, prior to platelet transfusion resulted in reduced alloimmunization in challenged mice and normal platelet counts in neonates. The notion that the effector silent variant hampered alloimmunization demonstrates that rapid platelet clearance, as seen with mAb 26.4.WT, is not the sole mechanism in action. Our data thus successfully demonstrate efficient Ab-mediated immunosuppression and prevention of FNAIT by anti-HPA-1a monoclonal variants, providing support for potential use in humans.

Neonatal alloimmune thrombocytopenia: difficulties in diagnosis and therapy

Neonatal alloimmune thrombocytopenia: difficulties in diagnosis and therapy

Neonatal alloimmune thrombocytopenia as a cause of fetal intracranial hemorrhage: Case report

Neonatal alloimmune thrombocytopenia as a cause of fetal intracranial hemorrhage: Case report

Контраверсійні питання менеджменту вагітності при імунній тромбоцитопенії

Among autoimmune cytopenias during pregnancy, immune thrombocytopenia (ITP) occurs most often. Thrombocytopenia (TP), a decrease in platelet count less than 150x109/l, occurs in 5 to 12% of pregnancies. Immune thrombocytopenia is a diagnosis of exclusion and requires differential diagnosis primarily with gestational TP. Preconception counseling is extremely important in ITP to develop an individual pregnancy management plan. Prediction and calculation of antero-, perinatal and obstetric risks for the fetus and pregnant woman should be performed repeatedly during pregnancy. The choice of the time of initiation of therapy and the type of medication is not clear due to the specificity of the drugs and the possible impact on the fetus. The issue of neonatal severe thrombocytopenia and hemorrhagic complications that may occur at the antenatal stage in some cases is not explained by maternal immune thrombocytopenia, and fetal3neonatal alloimmune thrombocytopenia (FNAT). An unambiguous guide to the prevention and management of cases with FNAT has not been developed at present. We have highlighted current global trends on this issue. Pregnant women with severe TP are a group of high perinatal risk, pregnancy and childbirth should take place in a maternity hospital of III level with the involvement of a multidisciplinary team and an individual plan of pregnancy management, development of delivery and management of postpartum and neonatal periods. The research was carried out in accordance with the principles of the Helsinki declaration. The study protocol was approved by the Local Ethics Committee of the participating institution. The informed consent of the patient was obtained for conducting the studies. No conflict of interest was declared by the authors. Key words: immune thrombocytopenia, pregnancy, fetal-neonatal alloimmune thrombocytopenia, gestational thrombocytopenia.

Prevention of Fetal/Neonatal Alloimmune Thrombocytopenia in a Preclinical Model By Monoclonal Anti-HPA-1a Antibody Prophylaxis: Structural and Functional Properties of Antibody Variant Isoforms

Maternal alloantibodies to paternally inherited platelet antigens can cause fetal/neonatal alloimmune thrombocytopenia (FNAIT), rendering the fetus or newborn prone to bleeding and intracranial hemorrhage (ICH) with risk of lifelong disabilities or death. In Caucasians, the vast majority of cases are due to antibodies to the Human Platelet Antigen (HPA)-1a epitope on glycoprotein (GP)IIIa. To date, there are neither any means to prevent or reduce the risk of alloimmunization and subsequent FNAIT, nor safe and efficient treatment during affected pregnancies. Thus, a prophylactic regimen by administration of monoclonal antibodies to women at risk would be highly beneficial. Knowledge about the optimal functional design of prophylactic monoclonal antibodies for antenatal or post-natal use is however still limited. We have previously isolated and characterized a fully human anti-HPA-1a monoclonal antibody, mAb26.4. In the current study we have explored the prophylactic potential of this antibody by testing a panel of different IgG1 designs, including the wild-type mAb26.4, variants with modified Fc-region N-glycans, as well as an effector silent variant. We performed analyses of properties relevant for immunosuppression: in vitro Fc-receptor binding and capacity to induce phagocytosis, in vivo half-life measurements in humanized FcRn mice and platelet clearance in a recently developed transgenic mouse strain with a recreated HPA-1a epitope on murine GPIIIa. The prophylactic capacity by antibody-mediated immune suppression in vivo, were further tested in the FNAIT pre-clinical murine model, in which BALB/c females can be immunized by transfusion of HPA-1a-expressing platelets from the transgenic mice and where subsequent breeding of pre-immunized mice with transgenic males cause thrombocytopenia in off-springs mimicking FNAIT. By intravenous administrations of mAb26.4 variants prior to platelet transfusions, the mice generated no or low anti-platelet responses compared to control mice, and normal platelet counts in pups upon subsequent breeding. Our data thus successfully demonstrates efficient immunosuppression and prevention of FNAIT by anti-HPA-1a human monoclonal variants, providing further support for potential use in humans. DisclosuresSkogen: Prophylix Pharma AS: Current holder of individual stocks in a privately-held company. Newman: Rallybio: Consultancy, Research Funding.

Prophylactic Administration of Antibodies Specific for the HPA-1a Epitope Prevents Alloimmunization and Platelet Destruction in a Murine Model of Fetal Neonatal Alloimmune Thrombocytopenia

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal alloantibodies directed against paternally inherited antigens present on the surface of fetal and neonatal platelets. The human platelet alloantigen HPA-1a (formerly known as the Pl A1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Caucasians. A single Leu33Pro amino acid polymorphism near the amino terminus of the integrin b3 subunit (known as GPIIIa in the platelet literature) serves as the central target for alloantibody binding, leading to clearance of both fetal and neonatal platelets, thrombocytopenia, and in the most severe cases, spontaneous- or trauma-induced intracranial hemorrhage. Unlike hemolytic disease of the newborn, which occurs in pregnancies subsequent to parturition-induced alloimmunization, an estimated 25% to 50% of FNAIT cases occur without warning during gestation of the first pregnancy. Though long proposed, there are currently no approved therapies for the prevention of FNAIT. We recently described the development of transgenic mice expressing the human HPA-1a allogeneic epitope on a murine GPIIIa backbone. Transfusion of such platelets into wild-type female mice induced the generation of high-titer anti-HPA-1a alloantibodies that can cross the placenta and recapitulate many of the relevant clinical features of FNAIT. To test the hypothesis that rapid elimination of fetal HPA-1a positive platelets from the circulation of a mother who is HPA-1a negative might prevent maternal alloimmunization and the development of FNAIT, we administered either a hyperimmune plasma-derived polyclonal anti-HPA-1a antibody derived from females having previous cases of FNAIT (termed RLYB211), or a novel human monoclonal antibody directed against the HPA-1a epitope (termed RLYB212), to wild-type female mice prior to challenging them with HPA-1a-positive murine platelets. RLYB211 and RLYB212 were each able to effect the rapid removal of HPA-1a-positive platelets from murine circulation and prevent the development of anti-HPA-1a alloantibodies. Importantly, wild-type female mice pretreated with RLYB211 prior to exposure to HPA-1a-positive platelets, and then impregnated by HPA-1a-positive males, gave birth to HPA-1a-positive pups with significantly improved platelet counts and no bleeding symptoms. These preclinical data establish the potential for prophylactic polyclonal and monoclonal anti-HPA-1a antibody therapy for the prevention of FNAIT in humans. DisclosuresSheridan: Rallybio: Current Employment, Current equity holder in publicly-traded company. Newman: Rallybio: Consultancy, Research Funding.

Genetic Removal of Terminal Sialic Acid Residues from the O-Linked Glycans Adjacent to the HPA-9b Polymorphism of Platelet Membrane Glycoprotein IIb Improves the Binding and Detection of HPA-9b Patient Alloantibodies

Sialic acids occupy the terminal position of glycan chains, and have the potential to influence the antigenicity of glycoproteins. Antibody binding sites on a glycoprotein can be solely protein in nature, or can include or be affected by nearby glycan chains, which may either mask the epitope, or conversely comprise part of the antibody binding site. The polymorphisms responsible for formation of the Human Platelet Alloantigens (HPA)-3 (Ile843Ser) and HPA-9 (Val837Met) are next to each other near the C-terminus of the extracellular domain of platelet membrane glycoprotein (GP)IIb, and are adjacent to sialyl-Core 1 O-glycans that emanate from serines 845 and 847. Previous studies have shown that these O-linked glycans are required to support the binding of some of HPA-3a alloantibodies. Loss of these glycans, especially terminal sialic acid residues, during platelet storage or preparation, can present major difficulties in detecting clinically important anti-HPA-3a alloantibodies in suspected cases of fetal/neonatal alloimmune thrombocytopenia (FNAIT). Similarly, detection and identification of anti-HPA-9b alloantibodies from FNAIT patient sera can also be extremely challenging, resulting in the inability to resolve clinical cases of this bleeding disorder. Whether the nearby O-glycans on serines 845 and 847 of GPIIb affect the antigenicity of HPA-9b, and/or influence the binding of anti-HPA-9b alloantibodies in clinically significant cases of FNAIT is unknown.We previously reported the generation of bioengineered, HLA class I-negative, HPA-9a or -9b allele-specific megakaryocytes (MKs) from human induced pluripotent stem cells (iPSCs) that are suitable for whole-cell flow cytometric detection of anti-HPA-9b alloantibodies (Blood 2019;134(22):e1-e8). Unexpectedly, treatment of these allele-specific MKs with neuraminidase actually enhanced the binding of anti-HPA-9b alloantibodies, suggesting that terminal sialic acids on GPIIb partially mask the HPA-9b epitope. To test the hypothesis that removal of terminal sialic acids on nearby O-glycans, or removal of the entire O-glycan chains emanating from Ser845/847 of GPIIb, might enhance the detection of anti-HPA-9b patient alloantibodies, we created a series of deletion mutants in two major sialidases, ST3GAL1 and ST3GAL2, known to be responsible for transferring terminal sialic acid residues to Core 1 O-glycans, in our HPA-9a and -9b allele-specific iPSCs. Immunoprecipitation/western blot analysis confirmed the complete removal of terminal sialic acids on the O-glycan chains of GPIIb in ST3GAL1/2 knockout (KO) iPSC-derived MKs, as reported by the binding of the lectin PNA to the exposed Core 1 structure on GPIIb. These sialylation-deficient ST3GAL1/2 KO HPA-9b MKs exhibited dramatically increased anti-HPA-9b alloantibody binding, further confirming the notion that HPA-9b epitopes are partially masked by terminal sialic acids on nearby GPIIb O-glycan chains. Finally, allele-specific iPSCs lacking the complete O-glycan chains attached to serines 845 and 847 of GPIIb were generated by mutating those residues to alanines using a similar CRISPR/Cas9 gene editing approach. Interestingly, O-glycan chain-deficient Ala845/847 mutant MKs carrying the HPA-9b polymorphism exhibited slightly to moderately reduced binding of anti-HPA-9b alloantibodies, indicating that the presence of the Core 1 O-glycan chains attached to GPIIb serine residues 845 and 847 contribute to the presentation of the HPA-9b epitope - perhaps by stabilizing the conformation of the glycoprotein in this region. Taken together, these data suggest that detection of anti-HPA-9b alloantibodies may be enhanced through the use of iPSC-derived HPA-9b-specific MKs that have been genetically altered to lack nearby terminal sialic acid residues, but retain the glycan chains to which they are attached. DisclosuresCurtis: Rallybio: Consultancy. Newman: Rallybio: Consultancy, Research Funding.

Surveillance Program of Romiplostim Use Connected to Pregnancy

Surveillance Program of Romiplostim Use Connected to Pregnancy

Management of high-risk pregnancies in a context of maternal antiplatelet alloimmunization: Expert opinion of the French-speaking working group

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a potentially devastating disease, seen in 1/800-1000 neonates. FNAIT is the most common cause of early-onset isolated severe neonatal thrombocytopenia in maternity wards. A working group on fetomaternal platelet alloimmunization was created in 2017, under the auspices on the French Group of Thrombosis and Hemostasis (GFHT). The objective was to survey clinical practices for management of high-risk pregnancies in a context of suspected or confirmed FNAIT. Recommendations published by the ICTMG were translated in French, and discussed (Fetal and neonatal alloimmune thrombocytopenia: recommendations for evidence-based practice, an international approach. British J of Haematology, 2019, 185, 549-562). The study involved centers from France, Switzerland and Belgium: Angers, Besançon, Bordeaux, Brest, Créteil/Clamart, Genève, Grenoble, Liège, Lille, Lyon, Marseille, Nantes, Nîmes, Paris (hôpitaux Necker, Robert Debré et Trousseau), Poitiers, Rennes, Saint-Etienne, Strasbourg, Toulouse, Tours. Expert opinion was validated on September 23, 2020 (consensus≥90%).

Case Of NAIT Causing Severe Thrombocytopenia due to Anti-HLA Class I

Neonatal alloimmune thrombocytopenia (NAIT) is the leading cause of thrombocytopenia in otherwise healthy new-born. (1,2) Maternal antibodies raised against paternally inherited alloantigen carried on fetal platelet causing NAIT. Maternal IgG antibodies passed through to the fetal via the placenta, attack and cause the destruction of the fetal platelet. (3) We present a case of NAIT without any complications in a premature baby (35 weeks) with VACREL association, G6PD deficiency, left calcified cephalohaematoma, cardiomegaly and hypospadias with severe thrombocytopenia (platelet counts is 23 109/L) at day two of life and received twice platelet transfusion. Platelet count initially 123 109/L at birth but significantly drop and persistently less than 50 109/L until day 10 of life before it normalized. Maternal serum antibody screening was negative, but platelet immunology test detected maternal platelet-reactive antibody Anti-HLA Class I and correlates with incompatible parental crossmatch indicating that parent had “platelet-antigen incompatibility”. The goal of obstetric management is to identify pregnancies at risk and prevent intracranial haemorrhage. (4) There is no evidence to support routine screening for pregnancies as per current practice. (2, 5) The latest treatments include maternal administration of intravenous immunoglobulin to suppress maternal antibody production and or to reduce placental transfer of antibodies; with or without steroids during antepartum period besides planning of mode, timing and method of delivery. (2, 5, 6, 7) This is a rare and unique case of NAIT secondary to Anti-HLA Class I antibody and hence clinician should be au fait with the diagnosis and management as it is infrequent among Malaysian.International Journal of Human and Health Sciences Supplementary Issue-2: 2021 Page: S21

Preclinical evaluation of immunotherapeutic regimens for 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 (formerly known as the PlA1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Whites. A single Leu33Pro amino acid polymorphism residing within the ∼50-amino-acid plexin-semaphorin-integrin domain near the N-terminus of the integrin β3 subunit (platelet membrane glycoprotein IIIa [GPIIIa]) is responsible for generating the HPA-1a and HPA-1b epitopes in human GPIIIa and serves as the central target for alloantibody-mediated platelet destruction. To simulate the etiology of human FNAIT, wild-type female mice were pre-immunized with platelets derived from transgenic mice engineered to express the human HPA-1a epitope on a murine GPIIIa backbone. These mice developed a strong alloimmune response specific for HPA-1a, and when bred with HPA-1a+ males, gave birth to severely thrombocytopenic pups that exhibited an accompanying bleeding phenotype. Administering either polyclonal intravenous immunoglobulin G or a human monoclonal blocking antibody specific for the HPA-1a epitope into pregnant female mice resulted in significant elevation of the neonatal platelet count, normalized hemostasis, and prevented bleeding. The establishment of an alloantigen-specific murine model that recapitulates many of the clinically important features of FNAIT should pave the way for the preclinical development and testing of novel therapeutic and prophylactic modalities to treat or prevent FNAIT in humans.

Deteksi antibodi trombosit spesifik anti-hpa-3 pada neonatus dengan trombositopenia

Background: The immune factor causing thrombocytopenia in neonates can occur even during pregnancy. Antigen discrepancies in the platelet membrane expressed by the fetus and maternal can be the cause of thrombocytopenia. This is due to the presence of anti-platelet antibodies produced by the maternal body as a form of immune response after exposure to antigens on the platelet membrane expressed by the fetus. Human platelet antigen detection has not been carried out in Indonesia, there is no known antigen on the platelets. This allows the occurance of alloimmune thrombocytopenia in neonates, one of sign that can be recognized through clinical symptoms was neonates with thrombocytopenia. Screening anti-platelet antibodies in neonates with thrombocytopenia is an effort to find the possibility of anti-human platelet antigen antibodies. &#x0D; Objective: The aim of this study is to find out the presence of anti-platelet antibodies in neonates with thrombocytopenia.&#x0D; Methods: The study is descriptive observational with cross sectional design. The subject in this study were neonates with thrombocytopenia according to the study criteria. The collected sample is screened for the presence of anti-platelet antibodies, then identification of anti-HPA antibodies.&#x0D; Results: Screening in 30 neonates showed that 3 neonatal positive anti-platelet antibodies, 2 with borderline and 25 negative anti-platelet antibodies. Identification of anti-platelet antibodies was performed in five samples (result of screening, three with positive screening and two borderline), indicating that all were negative for the anti-human platelet antigen antibody GPIIb/IIIa specific HPA-3. &#x0D; Conclusion: Based on the screening result, five samples were found to have anti-platelet antibodies in neonates with thrombocytopenia, but after identification none of them were found to be specific anti-HPA antibodies to GPIIb/IIIa.

Are HPA-5b antibodies a significant cause of FNAIT and associated bleeding or merely an incidental finding?

Are HPA-5b antibodies a significant cause of FNAIT and associated bleeding or merely an incidental finding?

Clinical characteristics of human platelet antigen (HPA)-1a and HPA-5b alloimmunised pregnancies and the association between platelet HPA-5b antibodies and symptomatic fetal neonatal alloimmune thrombocytopenia.

SummaryFetal neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies directed against the human platelet antigens (mostly HPA‐1a or HPA‐5b) of the (unborn) child and can lead to severe bleeding. Anti‐HPA‐1a‐mediated FNAIT shows a severe clinical outcome more often than anti‐HPA‐5b‐mediated FNAIT. Given the relatively high prevalence of anti‐HPA‐5b in pregnant women, the detection of anti‐HPA‐5b in FNAIT‐suspected cases may in some cases be an incidental finding. Therefore we investigated the frequency of anti‐HPA‐5b‐associated severe bleeding in FNAIT. We performed a retrospective nationwide cohort study in cases with clinical suspicion of FNAIT. HPA antibody screening was performed using monoclonal antibody‐specific immobilisation of platelet antigens. Parents and neonates were typed for the cognate antigen. Clinical data were collected by a structured questionnaire. In 1 864 suspected FNAIT cases, 161 cases (8·6%) had anti‐HPA‐1a and 60 (3·2%) had anti‐HPA‐5b. The proportion of cases with severe bleeding did not differ between the cases with anti‐HPA‐1a (14/129; 11%) and anti‐HPA‐5b (4/40; 10%). In multigravida pregnant women with a FNAIT‐suspected child, 100% (81/81) of anti‐HPA‐1a cases and 79% (38/48) of anti‐HPA‐5b cases were HPA‐incompatible, whereas 86% and 52% respectively were expected, based on the HPA allele distribution. We conclude that anti‐HPA‐5b can be associated with severe neonatal bleeding symptoms. A prospective study is needed for true assessment of the natural history of anti‐HPA‐5b mediated FNAIT.

Structure of an Extended β Integrin

Integrins are a family of 24 α/β heterodimeric adhesion receptors that transmit bidirectional signals across the cell membrane. Each integrin subunit contains a large extracellular domain composed of headpiece and leg piece, a transmembrane domain and usually a short cytoplasmic tail. The α headpiece consists of the common β-propeller and thigh domains, and an extra αI domain inserted into the β-propeller domain in a subset of α subunits such as αL. The α leg piece includes calf-1 and -2 domains. The β headpiece contains βI, which is a structural homolog of αI domain, hybrid, PSI, and I-EGF1 domains. The β leg piece contains I-EGF2-4 and β-tail domains. Structural studies in the last 15 years, largely based on the two β3 integrins, αIIbβ3 and αVβ3, greatly advanced our understanding of integrin conformational regulation upon activation. The platelet specific αIIbβ3 plays an essential role in hemostasis and thrombosis, while the αVβ3 is important for the growth and survival of many cell types. In the resting state, integrin adopts a bent conformation with the headpiece folded onto the leg piece. Crystallographic and EM studies of αIIbβ3 and αVβ3 revealed two major conformational rearrangements, the swing-out motion of the hybrid domain, resulting in headpiece opening, and the switchblade-like movement of the headpiece from the leg domains, leading to integrin extension and leg separation. Despite the visualization of β3 extension by EM at very low resolution, a high-resolution extended β3 structure has been missing.A Leu33Pro polymorphism at the β3 PSI domain defines the human platelet alloantigen 1 (HPA-1) system. The mismatched HPA-1a/b (Leu33/Pro33) is a major cause of two severe bleeding disorders, fetal/neonatal alloimmune thrombocytopenia (FNAIT) and post-transfusion purpura (PTP) due to the generation of anti-HPA-1 alloantibodies. In addition, it has been controversial whether the Pro33 variant can enhance β3 activity, being a risk factor for thrombosis. It is also unknown whether the Leu33Pro substitution affects the conformation of PSI domain, which undergoes outward movement along with the headpiece opening and extension.It has been difficult to obtain the crystal structure of a full-length extended integrin largely due to the conformational heterogeneity and flexibility, especially in the lower leg domains. In this study, we engineered a chimeric β3 fragment, denoted as cβ3-E2, including the domains of PSI, hybrid, I-EGF1, I-EGF2, and the βI replaced with the αL αI domain, which enables the expression of β3 in the absence of α subunit. We determined the crystal structures of cβ3-E2 in both Leu33 and Pro33 forms. Strikingly, two conformations of cβ3-E2, an intermediate state and a fully extended state are simultaneously present in one crystal. The β3 extension occurs at the I-EGF1 and I-EGF2 interface, denoted as β-knee. Our structure revealed the structural changes of the flexible loop, C473-C486 or C1-C2 loop of I-EGF2 in the β-knee, which has been shown to modulate integrin activation by mutagenesis studies. No significant structural differences were observed between the PSI domains having Leu33 and Pro33. We next compared the ligand binding of β3-Leu33 and β3-Pro33 induced by Mn2+ or talin head domain. No significant differences were observed either in αIIbβ3 or αVβ3, indicating that the Leu33Pro substitution may not affect β3 integrin activation. However, the PSI C26-C38 loop harboring the Leu33Pro polymorphism moves closer to the I-EGF1 domain in the extended conformation. In addition, we found that a completely conserved arginine (Arg93 in β3) at the PSI/hybrid domain interface is important for maintaining the resting integrin state since the alanine or glutamine substitution renders β1, β2 and β3 integrin hyperactive. Interestingly, a naturally occurring β3-Arg93Gln substitution has been shown to disrupt the HPA-1a epitope. Finally, based on our cβ3-E2 structure, we built the extended full-length β3 structures with either closed or open headpiece, which can perfectly fit into the EM structures of extended αIIbβ3 with either closed or open headpiece, respectively. In summary, our study provides structural details of an extended β3 integrin, showing the conformational changes at the PSI, I-EGF1 and I-EGF2 domains, demonstrating their importance in regulating integrin activation and alloimmune response. DisclosuresNo relevant conflicts of interest to declare.

Structural and Functional Characterization of a Single Chain β3 Integrin in Solution and on the Cell Surface and the Implications on Integrin Activation and Pathogenesis of Anti-HPA-1a Alloantibodies

The β3 integrins, including αIIbβ3 and αVβ3, are among the most well studied integrins. αIIbβ3 is specific to platelets and essential for hemostasis and thrombosis. αVβ3 is ubiquitously expressed in many cell types, including platelets and endothelial cells, and is important for cell growth and survival. The β3 subunit contains the headpiece formed by PSI, hybrid, βI, and I-EGF1 domains, the leg piece formed by the I-EGF 2-4 and β-tail domains, a transmembrane (TM) domain and a short cytoplasmic tail (CT). Structural studies revealed both αIIbβ3 and αVβ3 complexes as a bent conformation with the headpiece folded onto the leg piece, representing a resting state. Upon activating by ligand binding to the headpiece or talin/kindlin binding to the CT, β3 integrin undergoes a large conformational rearrangement, showing the headpiece extension from the leg piece and the separation of the leg from the α subunit. Only the interface at the headpiece between the β3 βI and the α β-propeller domains is maintained during the bent to extended transition. The interactions at the α/β TM, CT and leg domains are broken in the active conformation. It remains unknown how the extended state of β3 is triggered and maintained, especially whether the α subunit contributes to regulating the β3 conformation.A Leu33Pro polymorphism at the β3 PSI domain, forming human platelet antigen-1a/1b (HPA-1a/1b), can induce alloantibodies against incompatible HPA-1a during pregnancy or after blood transfusion, leading to two severe bleeding disorders, fetal/neonatal alloimmune thrombocytopenia (FNAIT) and post-transfusion purpura (PTP). The severe risk of FNAIT is intracranial hemorrhage (ICH), often leading to death or life-long neurological disability. The direct cause of ICH remains unknown, but recent studies suggest a strong connection between the ICH and the presence of a subtype of anti-HPA-1a that is specific to the αVβ3 complex on endothelial cells. The other anti-HPA-1a subtypes are αIIbβ3 specific and complex independent. The αVβ3-specific anti-HPA-1a can interfere with endothelial cell function by inhibiting the αVβ3-mediated cell adhesion and angiogenesis, which may be responsible for the development of ICH. Certain anti-HPA-1a antibodies were also found to affect αIIbβ3-mediated platelet function. It is unknown how the anti-HPA-1a antibodies, which bind to the β3 PSI domain, can distinguish the β3 between αVβ3 and αIIbβ3, and how they block β3 integrin function.To address the above questions, we engineered a chimeric β3 integrin (cβ3) by replacing the βI domain with the αI domain of αL integrin, enabling to express the full-length β3 as a monomer in the absence of α subunit. With this strategy, the conformation of β3 can be studied without α subunit. On the cell surface, the cβ3 constitutively adopts an extended active conformation, evidenced by the binding of the αL ligand ICAM-1 in a metal ion-dependent manner, and the binding of active conformation-dependent anti-β3 mAbs. Very surprisingly, the cβ3 ectodomain exists in a compact conformation in solution as indicated by size exclusion chromatography and dynamic light scattering. This was confirmed by a high-resolution crystal structure of cβ3, which shows a bent shape that is comparable with the β3 conformation in the bent structures of αVβ3 and αIIbβ3. Our cβ3 structure revealed a rotational movement at the ankle region connecting the I-EGF4 and the β-tail domains. Mutagenesis study demonstrated that the plasticity of the ankle region regulates the activation of β3 integrin. These data demonstrate that the β3 integrin can adopt either a bent or extended conformation even without α subunit. Our data suggest that the TM, CT, cell membrane, and/or other interacting molecules are responsible for the active form of β3 on the cell surface. We next used our monomeric cβ3 to detect the anti-HPA-1a antibodies in FNAIT and PTP, aiming to distinguish the complex-dependent and independent subtypes. Remarkably, we found that anti-αIIbβ3 alloantibodies in FNAIT only recognize the bent conformation of either αIIbβ3 or cβ3 but not the extended form. Structural analysis of the PSI domains of the available β3 crystal structures revealed conformational diversity and flexibility, which may account for the heterogeneity of the anti-HPA-1a antibodies. More importantly, the bent conformation specific anti-HPA-1a antibodies may stabilize the resting integrin and block its cellular function. DisclosuresCurtis:Ionis Pharmaceuticals: Consultancy.

Maternal Anti-αIIb Antibodies Target Fetal Hematopoietic Stem Cells and May Lead to Pancytopenia and Miscarriage in Fnait

Background: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a devastating disorder characterized by an aberrant maternal immune response against paternally inherited polymorphisms on fetal platelet antigens. Affected fetuses can experience severe bleeding (e.g. intracranial hemorrhage; ICH), neurological sequelae and death. The targeted platelet antigens are located predominantly on platelet surface receptor αIIbβ3 integrin. It is estimated that 0.5-1.5/1000 live neonates are born with FNAIT although this incidence may largely underestimate the prevalence of the disease due to miscarriage. We have previously demonstrated that anti-β3 alloantibodies can target not only fetal platelets but also the β3 subunit on endothelial cells leading to ICH and placental vascular pathologies (J Clin Invest. 2015; Nat Commun. 2017). Although the number of polymorphisms in the αIIb subunit is similar to the β3 subunit, reported incidences of anti-β3 mediated FNAIT are far greater. It is currently unknown whether this due to miscarriage in anti-αIIb mediated FNAIT. αIIb was previously considered to be exclusively expressed on platelets, however hematopoietic stem cells (HSCs) and their early progenitors can express αIIbβ3 during embryonic development and αIIb is one of the earliest markers of hematopoietic commitment. Throughout gestation, HSCs migrate from yolk sac to the aorta-gonadal-mesonephros, placenta, fetal liver and finally bone marrow. We hypothesize that miscarriage is prevalent in anti-αIIb mediated FNAIT and that maternal anti-αIIb antibodies may target HSCs resulting in decreased blood cell counts, increased bleeding and fetal death.Methods: We established an animal model of anti-αIIb mediated FNAIT by immunizing αIIb-/- mice with 108 wild-type (WT, αIIb+/+) platelets. Immunized αIIb-/- mice were then bred with WT males. We used non-immunized (naïve) αIIb-/- x WT breeding pairs as a control. Sera of immunized and naïve mice were collected prior to breeding and ultrasound was used to detect fetal death. Mice were sacrificed at E14.5 and fetuses, placentas and fetal livers were weighed. Cell suspensions were prepared from these tissues as well as fetal blood and analyzed with flow cytometry. Fluorescent markers were used to identify HSCs (CD34+/CD117+/Lin- or CD34+/CD45+) and blood cell counts for T cells (CD3+,CD4+,CD8+), B cells (CD19+), myeloid lineage cells (CD11b+) and megakaryocytes (GPIbα+).Results: Sera of immunized mice showed a significant anti-αIIb immune response. Pups of immunized mice had low platelet counts, reduced body weight and bleeding diathesis. Ultrasound revealed miscarriage occurred mainly at E14.5 of immunized αIIb+/- fetuses. The miscarriage rate in our anti-αIIb mediated FNAIT model appeared significantly higher than the anti-β3 model. At E14.5, our results showed live fetuses of immunized mice had significantly lower body and liver weight although no significant difference in placental weight was observed. We found fetal liver HSCs have increased maternal antibody binding when incubated with sera from immunized αIIb-/- mice versus naïve αIIb-/- mice in vitro, suggesting that maternal anti-αIIb antibodies target fetal HSCs in vivo . Preliminary data also demonstrated that fetuses in the immunized group had reduced overall HSCs as well as αIIb+ HSC subsets in the placenta, liver and yolk sac. Fetal blood analysis showed reduced hematopoietic lineage cells in the immunized group, particularly T cells (CD3+), B cells and megakaryocytes.Conclusions: We established an anti-αIIb mouse model of FNAIT and revealed a high rate of miscarriage, which may explain the paucity of reported human cases. Our results indicate maternal anti-αIIbantibodies may bind and target fetal HSCs during embryonic development, which contributes significantly to impaired placental and fetal liver development, and fetal death. Destruction of early αIIb+ HSC progenitors may explain the decrease in HSCs (both αIIb+ and αIIb-) and account for the pancytopenia in FNAIT fetuses. Further experiments are in progress to elucidate the mechanism by which destruction of αIIb+ HSCs contributes to fetal pathology.*J.S. and B.E.O contributed equally to this work DisclosuresNo relevant conflicts of interest to declare.

A first WHO reference reagent for the detection of anti-human platelet antigen-15b.

Background and ObjectivesAlloantibodies to human platelet antigen‐15b (anti‐HPA‐15b) have been detected in mothers with foetal–neonatal alloimmune thrombocytopenia and in multiply transfused patients. Assays used to detect this antibody, which aids in disease diagnosis, can be unreliable and vary in sensitivity. The objective was to generate a stable, lyophilized anti‐HPA‐15b preparation and evaluate its suitability as a World Health Organization (WHO) reference reagent for use in the quality control of platelet alloantibody detection assays. Results from an international collaborative study to evaluate the preparation were used to assign a minimum potency at which laboratories can be expected to detect the antibody.Materials and MethodsRecalcified plasma containing anti‐HPA‐15b was aliquotted, lyophilized and coded 18/220. Twenty‐five laboratories in 16 countries tested doubling dilutions of the reconstituted material in glycoprotein‐specific assays such as the monoclonal antibody–specific immobilization of platelet antigen assay and reported the last positive (or endpoint) dilution.ResultsTwenty‐four laboratories (96%) detected antibodies with HPA‐15b specificity in preparation 18/220. Reported endpoint dilutions were normally distributed with a modal dilution of 1 in 16 and ranged from 1 in 2 to 1 in 128. Only two laboratories (8%) failed to detect anti‐HPA‐15b at 1 in 8 dilution.ConclusionsWhen diluted 1 in 8, most laboratories detected anti‐HPA‐15b in preparation 18/220 using HPA‐15bb platelets but not with HPA‐15aa platelets. The participants agreed this to be an appropriate dilution for assignment as the minimum potency. In October 2020, the WHO Expert Committee on Biological Standardization approved 18/220 as an International Reference Reagent.

Placental Complement Activation in Fetal and Neonatal Alloimmune Thrombocytopenia: An Observational Study.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a disease that causes thrombocytopenia and a risk of bleeding in the (unborn) child that result from maternal alloantibodies directed against fetal, paternally inherited, human platelet antigens (HPA). It is hypothesized that these alloantibodies can also bind to the placenta, causing placental damage. This study aims to explore signs of antibody-mediated placental damage in FNAIT. We performed a retrospective study that included pregnant women, their newborns, and placentas. It comprised 23 FNAIT cases, of which nine were newly diagnosed (14 samples) and 14 were antenatally treated with intravenous immune globulins (IVIg) (21 samples), and 20 controls, of which 10 had anti-HLA-class I antibodies. Clinical information was collected from medical records. Placental samples were stained for complement activation markers (C1q, C4d, SC5b-9, and mannose-binding lectin) using immunohistochemistry. Histopathology was examined according to the Amsterdam criteria. A higher degree of C4d deposition was present in the newly diagnosed FNAIT cases (10/14 samples), as compared to the IVIg-treated FNAIT cases (2/21 samples, p = 0.002) and anti-HLA-negative controls (3/20 samples, p = 0.006). A histopathological examination showed delayed maturation in four (44%) placentas in the newly diagnosed FNAIT cases, five (36%) in the IVIg-treated FNAIT cases, and one in the controls (NS). C4d deposition at the syncytiotrophoblast was present in combination with low-grade villitis of unknown etiology in three newly diagnosed FNAIT cases that were born SGA. We conclude that a higher degree of classical pathway-induced complement activation is present in placentas from pregnancies with untreated FNAIT. This may affect placental function and fetal growth.