Blockade of maternal anti-HPA-1a-mediated platelet clearance by an HPA-1a epitope-specific F(ab′)2in an in vivo mouse model of alloimmune thrombocytopenia (original) (raw)
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The Journal of Immunology, 2015
Human platelet Ag (HPA)-1a, located on integrin b3, is the main target for alloantibodies responsible for fetal and neonatal alloimmune thrombocytopenia (FNAIT) in the white population. There are ongoing efforts to develop an Ab prophylaxis and therapy to prevent or treat FNAIT. In this study, an mAb specific for HPA-1a, named 26.4, was derived from an immortalized B cell from an alloimmunized woman who had an infant affected by FNAIT. It is the only HPA-1a-specific human mAb with naturally paired H and L chains. Specific binding of mAb 26.4, both native and recombinant forms, to platelets and to purified integrins aIIbb3 (from platelets) and aVb3 (from trophoblasts) from HPA-1a + donors was demonstrated by flow cytometry and surface plasmon resonance technology, respectively. No binding to HPA-1a 2 platelets or integrins was detected. Moreover, the Ab binds with higher affinity to integrin aVb3 compared with a second HPA-1a-specific human mAb, B2G1. Further in vitro experimentation demonstrated that mAb 26.4 can opsonize HPA-1a + platelets for enhanced phagocytosis by monocytes, inhibit binding of maternal polyclonal anti-HPA-1a Abs, and weakly inhibit aggregation of HPA-1a-heterozygous platelets, the latter with no predicted clinical relevance. Thus, mAb 26.4 is highly specific for HPA-1a and could potentially be explored for use as a prophylactic or therapeutic reagent for FNAIT intervention and as a phenotyping reagent to identify women at risk for immunization.
Rapid detection of HPA-1 alloantibodies by platelet antigens immobilized onto microbeads
Transfusion, 2007
BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is one of the most common bleeding disorders in neonates. It occurs when alloantibodies from an immunized mother react with paternally inherited alloantigens, mostly human platelet antigen 1a (HPA-1a), on the fetal platelets (PLTs). Currently, monoclonal antibody-immobilized PLT antigen (MAIPA) assay represents the standard technique for the serologic diagnosis of NAIT. MAIPA is time-consuming, however, and limited by the availability of monoclonal antibodies (MoAbs). Here, a gel antigen-specific assay (GASA) was developed, which allows rapid detection of HPA-1 alloantibodies without the use of MoAbs. STUDY DESIGN AND METHODS: Glycoprotein (GP) IIb/IIIa was purified by affinity chromatography from outdated PLT concentrates derived from HPA-1aa or HPA-1bb donors. Purified GPs were biotinylated, immobilized onto streptavidin beads, and used for the analysis of HPA-1a alloantibodies by a microtyping system. HPA-1a serum samples derived from mothers with NAIT (n = 36) and from posttransfusion purpura patients (n = 2) as well as HPA-1b (n = 4), HPA-5b (n = 2), HPA-3a (n = 4), and HLA Class I (n = 2) alloantiserum samples from multitransfused patients were investigated in GASA and MAIPA assays. RESULTS: GASA was able to detect all HPA-1a and-1b alloantibodies recognized by MAIPA. Crossreactivity with other PLT-reactive alloantibodies was not observed. Interestingly, 3 of 36 serum samples, which showed only moderate reactivity in MAIPA, reacted strongly in GASA. CONCLUSION: GASA has proved to be a rapid method for the detection of HPA-1a alloantibodies and maybe useful for PLT antibody screening, especially in initial assessment of suspected NAIT cases. ABBREVIATIONS: GASA = gel antigen-specific assay; GP = glycoprotein; MAIPA assay = monoclonal antibody-immobilized platelet antigen assay; NAIT = neonatal alloimmune thrombocytopenia; PTP = posttransfusion purpura.
Journal of Clinical Investigation, 2008
A SNP in the gene encoding integrin β3 causes a clinically important maternal-paternal antigenic difference; Leu33 generates the human platelet antigen 1a (HPA-1a), whereas Pro33 generates HPA-1b. As a potential treatment to prevent fetal intracranial hemorrhage in HPA-1a alloimmunized pregnancies, we generated an antibody that blocks the binding of maternal HPA-1a-specific antibodies to fetal HPA-1a1b platelets by combining a high-affinity human HPA-1a-specific scFv (B2) with an IgG1 constant region modified to minimize Fcγ receptor-dependent platelet destruction (G1Δnab). B2G1Δnab saturated HPA-1a + platelets and substantially inhibited binding of clinical HPA-1a-specific sera to HPA-1a + platelets. The response of monocytes to B2G1Δnab-sensitized platelets was substantially less than their response to unmodified B2G1, as measured by chemiluminescence. In addition, B2G1Δnab inhibited chemiluminescence induced by B2G1 and HPA-1a-specific sera. In a chimeric mouse model, B2G1 and polyclonal Ig preparations from clinical HPA-1a-specific sera reduced circulating HPA-1a + platelets, concomitant with transient thrombocytopenia. As the Δnab constant region is uninformative in mice, F(ab′) 2 B2G1 was used as a proof of principle blocking antibody and prevented the in vivo platelet destruction seen with B2G1 and polyclonal HPA-1a-specific antibodies. These results provide rationale for human clinical studies.
PL2-49, a monoclonal antibody against glycoprotein IIb which is a platelet activator
British Journal of Haematology, 1989
PL2-49 is a murine monoclonal IgGl antibody obtained after immunization of Balb/c mice with EDTA washed platelets. Binding could be detected on Zw"(+) as well as Zwa(-) platelets, but not on type 1 Glanzmann's thrombasthenia platelets using an ELISA screening test. Immunoprecipitation studies showed that PL2-49 bound to glycoprotein IIb when the glycoprotein IIb/IIIa complex dissociation was performed after the monoclonal antibody binding. Experiments with a human alloantibody against Zwa antigen were run in parallel to control the complex dissociation. Ascitic fluid, as well as the purified antibody, induced activation and aggregation of washed platelets and ATP release. PL2-49-induced aggregation did not require exogenous fibrinogen and was inhibited, partially, in the presence of aspirin, apyrase, isosorbide dinitrate. Raising
Transfusion, 2012
BACKGROUND-Recent studies suggest that HPA-1a-specific, low-avidity maternal antibodies not detectable by conventional methods can cause neonatal alloimmune thrombocytopenia (NAIT). We performed studies to further define the incidence and clinical significance of this type of antibody. STUDY DESIGN AND METHODS-Surface plasmon resonance analysis was used to detect low-avidity antibodies in HPA-1a-negative, "antibody-negative" mothers of suspected NAIT cases. The ability of antibodies detected to promote immune destruction of human platelets (PLTs) was examined in a newly developed NOD/SCID mouse model. RESULTS-Among 3478 suspected cases of NAIT, 677 HPA-1a-negative mothers were identified. HPA-1a-specific antibodies were detected by conventional antibody testing in 616 cases (91%). Low-avidity HPA-1a-specific antibodies were identified in 18 of the remaining 61 cases (9%). Clinical follow-up on 13 cases showed that eight were referred because of suspected NAIT and five because the mother's sister had previously had an infant with NAIT. Only six infants born to the 13 sensitized mothers had clinically significant thrombocytopenia at birth. Three of four low-avidity antibodies tested in the mouse caused accelerated clearance of HPA-1a/a but not HPA-1b/b PLTs. Only 3 of 12 mothers with low-avidity HPA-1a antibodies were positive for HLA-DRB3*0101. CONCLUSIONS-The findings confirm previous reports that low-avidity HPA-1a antibodies can cause NAIT but show that the presence of such an antibody does not predict that an infant will be affected. The low incidence of HLA-DRB3*0101 in this cohort (p < 0.0001) suggests that women negative for DRB3*0101 may be predisposed to produce low-avidity HPA-1a antibodies. Neonatal alloimmune thrombocytopenia (NAIT), caused by maternal immunization against platelet (PLT)-specific antigens inherited by a fetus from its father, occurs once in approximately 1000 births. 1-3 Although many cases are mild, approximately half of the affected infants have bleeding symptoms and up to 10% experience intracranial hemorrhage. 4,5 The first human PLT antigen (HPA) shown to be capable of triggering maternal antibodies and causing NAIT was HPA-1a (originally called Pl A1) described by Shulman and colleagues in 1962. 6 Since that time, at least 27 different HPA antigens have been implicated in NAIT. 7-14 However, fetal-maternal incompatibility for HPA-1a continues to be the most common cause of this disorder, even though only approximately
Platelet surface antigens: Analysis by monoclonal antibodies
Blut, 1984
Monoclonal antibodies were produced against human platetets. Four antibodies (PAl, PA2, PA3 and PA4) reacted specifically with platelets and megakaryocytes, but not with peripheral blood lymphocytes, granulocytes, erythrocytes or monocytes. The antibodies belonged to the mouse IgG subclass 2a (PAl, PA2, PA3), or 1 (PA4) respectively. PAl and PA4 did not precipitate, their antigens have not yet fully been characterized. PA3 was directed against the glycoprotein (Gp) complex IIb/IIIa; PA2 precipitated Gp IIb/IIIa, and, in addition, Gp Ia. PA4 revealed specificity against the human platelet alloantigen Zw (a).
Blood Advances, 2018
Antibodies to platelet-specific antigens are responsible for 2 clinically important bleeding disorders: posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The human platelet-specific alloantigen 1a/1b (HPA-1a/1b; also known as PlA1/A2) alloantigen system of human platelet membrane glycoprotein (GP) IIIa is controlled by a Leu33Pro polymorphism and is responsible for ∼80% of the cases of FNAIT. Local residues surrounding polymorphic residue 33 are suspected to have a profound effect on alloantibody binding and subsequent downstream effector events. To define the molecular requirements for HPA-1a alloantibody binding, we generated transgenic mice that expressed murine GPIIIa (muGPIIIa) isoforms harboring select humanized residues within the plexin-semaphorin-integrin (PSI) and epidermal growth factor 1 (EGF1) domains and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a–specific antibodies. Humanizing the PSI dom...
Transfusion, 2009
BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is mostly caused by maternal alloantibodies directed against the human platelet alloantigen (HPA)-1a. Currently, the serologic diagnosis of FNAIT is based on the characterization of the HPA alloantibodies in monoclonal antibody-based antigencapture assays (e.g., MAIPA assay). Accumulated current evidence indicated that such assays may overlook some HPA-1a antibodies. STUDY DESIGN AND METHODS: This study employed surface plasmon resonance (SPR) technology using immunoaffinity-purified glycoprotein IIb/IIIa isoforms immobilized on biosensor chips to study the binding kinetics of HPA-1a alloantibodies from different FNAIT cases in real time. RESULTS: Analysis of HPA-1a alloantibodies from FNAIT cases (n = 9) in SPR showed a moderate relative response (22.2-69.7 resonance units [RU]) and slow antibody dissociation. After the dissociation phase, varying amounts of bound antibodies (41%-79%) remained on the chip. In contrast in HPA-1a alloantibodies from a patient suffering from posttransfusion purpura, a high relative response (~490 RU) was observed at the end of the association phase and no dissociation of antibody binding was detectable. Of particular relevance, by the use of this SPR technique, HPA-1a alloantibodies were detected in two severe FNAIT cases that had determined as false negative by MAIPA assay. In SPR, these HPA-1a alloantibodies showed low-avidity nature characterized by gradual dissociation of antibody during the association phase and complete detachment of antibody binding after the dissociation phase. This high "off-rate" character of lowavidity HPA-1a alloantibodies indicates that such antibody binding is easily detachable by the extensive washing procedure of the MAIPA. CONCLUSIONS: Our results demonstrated that the SPR method can facilitate the diagnosis of clinically relevant low-avidity HPA-1a antibodies. F etal and neonatal alloimmune thrombocytopenia (FNAIT) is induced by maternal immunization against a fetal human platelet alloantigen (HPA). 1 In Caucasians, FNAIT has an incidence of 1:1000 to 1:2000 births. 2,3 Approximately 75 percent of serologically verified cases of alloantibodies against HPA-1a are responsible for the disease. 4 Antibodies against HPA-1a react with the Leu33 but not Pro33 isoform of platelet (PLT) glycoprotein (GP)IIIa or integrin b3 subunit. 5 Recently, the three-dimensional structure of the b3 integrin has been elucidated; 6,7 however, the precise binding site(s) of HPA-1a alloantibodies is not known. A previous study suggested that the amino terminal PSI domain containing the polymorphic residue 33 is sufficient to form HPA-1a epitopes. 8 However, various studies have shown that other residues located in the hybrid and epidermal growth factor domains of the b3 ABBREVIATIONS: D-PBS = Dulbecco's phosphate-buffered saline; FNAIT = fetal and neonatal alloimmune thrombocytopenia; GP(s) = glycoprotein(s); MAIPA assay = monoclonal antibody-based antigen-capture assay; PAIFT = platelet adhesion immunofluorescence test; PTP = posttransfusion purpura; RU = resonance units; SPR = surface plasmon resonance.