Post-transfusion purpura associated with alloimmunization against the platelet-specific antigen, Baka

Post-transfusion purpura (PTP) with severe thrombocytopenia occurred eight days after transfusion in a 28-year-old woman and responded to treatment with prednisone and plasma exchange. In contrast to nearly all previously studied cases of PTP, the patient's platelets were PlA1-positive and anti-PlA1 antibody could not be detected in serum obtained during the thrombocytopenic episode. Her serum was found to contain an antibody specific for a recently described platelet-specific alloantigen, Baka, in addition to multiple HLA-specific antibodies. The patient's platelets, typed following recovery, were Baka-negative. These findings indicate that post-transfusion purpura can occur in association with alloimmunization to platelet-specific antigens other than PlA1. In performing the serologic studies, a close relationship and possible identity between Baka and another recently reported platelet antigen, Leka, was observed. A method for analyzing mixtures of cytotoxic platelet-reactive antibodies without separating the individual antibodies is described.     

Neonatal alloimmune thrombocytopenic purpura associated with sensitization against the platelet-specific antigen Yuk(a)

We report a Japanese newborn who developed alloimmune thrombocytopenia by the antibodies to the newly discovered platelet antigen Yuk(a). The infant recovered uneventfully in 10 days without specific treatment. Antiplatelet alloantibodies in the patient were IgG class detected by mixed passive hemagglutination (MPHA). Family study showed that Yuk(a) antigen was inherited as an autosomal dominant trait. Cases with Yuk(a)-associated alloimmune thrombocytopenia are reviewed.     

Max(a), a new low-frequency platelet-specific antigen localized on glycoprotein IIb, is associated with neonatal alloimmune thrombocytopenia

We have identified a new platelet-specific alloantigen, Max(a), responsible for a typical case of neonatal alloimmune thrombocytopenic purpura. The maternal serum reacted strongly with paternal platelets in the platelet immunofluorescence test, whereas platelet alloantigen typing showed that no known human platelet antigen (HPA)-system was involved. In the monoclonal antibody (MoAb)-specific immobilization of platelet antigens (MAIPA) assay, the new antigen was located on the platelet membrane glycoprotein (GP) IIb-IIIa complex, but immunoprecipitation and immunoblot experiments to further localize the antigen failed. However, in the MAIPA assay, the binding of the anti- Max(a) antibodies from the maternal serum was blocked by two anti-GPIIb MoAbs. Thus, the antigen appeared to be located on GPIIb. Analysis of the family lead to the identification of six additional Max(a+) individuals. Three of these six individuals and the father were tested in the platelet aggregation test and were found to be normal. In the analysis of normal donors, three of 500 were typed positive for the new platelet-specific antigen, indicating a phenotype frequency of 0.6% in the normal population. Platelet RNA was isolated from the newborn's Max(a)+ father and from a healthy donor phenotyped as Max(a-), reverse- transcribed, and the entire GPIIb coding region was amplified by polymerase chain reaction. Subsequent nucleotide sequence analysis showed a single G-->A substitution at position 2,603, predicting a valine-->methionine amino acid substitution at position 837 of the mature glycoprotein. This mutation abolished a BsiYI restriction site at the cDNA level and a BstNI restriction site at genomic DNA level, respectively. The genetic association between the new antigen and this point mutation was confirmed by allele-specific restriction analysis on cDNA and on genomic DNA, as well as by allele-specific primer amplification on genomic DNA. The new mutation is 19 bp upstream of the mutation underlying the HPA-3 system. Therefore, we also evaluated the association between Mas and the HPA-3 polymorphism. So far, all Max(a+) individuals were also found to be HPA-3b, whereas 50 HPA-3a individuals were all Max(a-). This may indicate that Max(a) is a variant of the HPA- 3 allele.     

Human platelet-specific antigen, Siba, is associated with the molecular weight polymorphism of glycoprotein Ib alpha

Platelet-specific antigen Sib(a) has been highly implicated in the pathogenesis of refractoriness to human leukocyte antigen (HLA)-matched platelet transfusions in Japan. We provide evidence that the Sib(a) antigen is located on the glycoprotein (GP) Ib alpha and has a close association with the molecular weight (mol wt) polymorphism of GPIb. In modified antigen-capture ELISA (MACE), anti-Sib(a) antibody reacted only with GPIb/IX held by a murine anti-GPIb/IX monoclonal antibody (MoAb). The reactivity of anti-Sib(a) antibody to Sib(a)-positive (Sib(a+)) platelets was abolished after they were treated with Serratia marcescens protease. Platelets from 50 healthy volunteers were semiquantitatively phenotyped for Sib(a) antigen by MACE and divided into three distinct groups: strongly positive, positive, and negative. They were also analyzed by sodium dodeyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and periodic acid-silver staining for mol wt polymorphism of GPIb, phenotyped as A, B, C, or D. Without exception, Sib(a+) platelets showed larger phenotypes (A or B). Removal of sialic acid from Sib(a+) platelets did not reduce the binding of anti-Sib(a). Finally, anti-Sib(a) antibody specifically immunoprecipitated A and B phenotypes of GPIb from Sib(a+) platelets. Thus, Sib(a) antigen evidently is located in the region of glycocalicin that is present only on the A and B phenotypes of GPIb.     

Studies on the pathophysiology of posttransfusion purpura

Posttransfusion purpura typically occurs in PLA1 negative blood recipients who have been previously immunized to the PLA1 antigen. Following transfusion, severe thrombocytopenia develops with the formation of anti-PLA1. Since the patients' platelets lack the PLA1 antigen, one would not expect this antibody to destroy autologous platelets. In this study we show that PLA1 antigen exists in stored blood and can absorb to PLA1 negative platelets making them PLA1 reactive. Incubating PLA1 (-) platelets with ultracentrifuged plasma from PLA1 (+) blood donors allowed anti-PLA1 to bind to PLA1 (-) platelets. Control plasma from PLA1 (-) blood donors did not lead to anti-PLA1 binding. Using an inhibition assay, we showed that stored blood contains PLA1 material that was not removed by ultracentrifugation. The material absorbing to PLA1 (-) platelets represented the PLA1 antigen, which was confirmed by Western blotting. After incubating plasma containing PLA1 antigen with PLA1 (-) platelets, reactivity at 95,000 D was observed. Native PLA1 (+) platelets showed a similar band. When PLA1 (-) platelets were incubated with plasma from a PLA1 (-) donor, this band was not present. These studies show that a soluble form of PLA1 antigen exists in stored blood that can absorb to PLA1 (-) platelets. Consequently, anti-PLA1 can bind to these platelets leading to thrombocytopenia. These observations may explain the autologous destruction of platelets in posttransfusion purpura.     

Identification of a novel genetic locus associated with immune-mediated thrombotic thrombocytopenic purpura

Immune thrombotic thrombocytopenic purpura (iTTP) is an ultra-rare, life-threatening disorder, mediated through severe ADAMTS13 deficiency causing multi-system micro-thrombi formation, and has specific human leukocyte antigen associations. We undertook a large genome-wide association study to investigate additional genetically distinct associations in iTTP. We compared two iTTP patient cohorts with controls, following standardized genome-wide quality control procedures for single-nucleotide polymorphisms and imputed HLA types. Associations were functionally investigated using expression quantitative trait loci (eQTL), and motif binding prediction software. Independent associations consistent with previous findings in iTTP were detected at the HLA locus and in addition a novel association was detected on chromosome 3 (rs9884090, P=5.22x10^-10, odds ratio 0.40) in the UK discovery cohort. Meta-analysis, including the French replication cohort, strengthened the associations. The haploblock containing rs9884090 is associated with reduced protein O-glycosyltransferase 1 (POGLUT1) expression (eQTL P<0.05), and functional annotation suggested a potential causative variant (rs71767581). This work implicates POGLUT1 in iTTP pathophysiology and suggests altered post-translational modification of its targets may influence disease susceptibility.     

Posttransfusion purpura associated with an autoantibody directed against a previously undefined platelet antigen

Although alloantibody against the PLA1 platelet antigen is usually found in patients with posttransfusion purpura (PTP), the mechanism of destruction of the patient's own PLA1-negative platelets is unexplained. We used a sensitive immunoblot technique to detect antiplatelet antibodies in a patient with classic PTP. The patient's acute-phase serum contained antibodies against three proteins present in control (PLA1-positive) platelets: an antibody that bound to a previously unrecognized platelet protein of mol wt 120,000 [glycoprotein (GP) 120], antibodies that bound to PLA1 (mol wt 90,000), and an epitope of GP IIb (mol wt 140,000). The antibodies against PLA1 and GP IIb did not react with the patient's own PLA1-negative platelets, control PLA1-negative platelets, or thrombasthenic platelets. In contrast, the antibody against GP 120 recognized this protein in all three platelet preparations, but not in Bernard-Soulier or Leka (Baka)-negative platelets. Antibody against GP 120 was not detected in the patient's recovery serum, although the antibodies against PLA1 and GP IIb persisted. F(ab)2 prepared from the patient's acute-phase serum also bound to GP 120. These results suggest that in PTP, transient autoantibody production may be responsible for autologous (PLA1-negative) platelet destruction. In addition, alloantibodies against more than one platelet alloantigen may be found in this disease. The nature of the GP 120 autoantigen and the GP IIb-related alloantigen defined by our patient's serum remains to be determined.     

Neonatal alloimmune thrombocytopenia due to a new platelet-specific alloantibody

An infant with severe neonatal alloimmune thrombocytopenia is described in whom an antibody directed at a new platelet-specific alloantigen, Ca (HPA-6b), is implicated. The new alloantigen is of low frequency in the population and was localized to platelet glycoprotein (GP) IIIa. Immunoprecipitation studies using murine monoclonal antibodies specific for the GP complex IIb-IIIa and GPIIIa alone (AP2 and AP3) suggest that the location of the Ca epitope on GPIIIa may be near the binding site for AP3. Neonatal alloimmune thrombocytopenia associated with Ca is likely to be as severe as that seen in cases due to incompatibilities for the HPA-1 (PIA) and HPA-4 (Pen) platelet alloantigen systems, because each is located on GPIIIa, a densely represented molecule on the platelet surface.     

Identification of the platelet-specific alloantigen, Naka, on platelet membrane glycoprotein IV

We describe the membrane localization of a new platelet-specific alloantigen, designated Naka, that is involved in refractoriness to HLA-matched platelet transfusions. By indirect immunoprecipitation, anti-Naka antibody precipitated a single, radiolabeled platelet membrane protein with a molecular weight (mol wt) of 91 Kd from Naka-positive platelets. When radiolabeled Naka-negative platelets were used as a source of target antigens, no radiolabeled proteins were precipitated. The analyses using nonreduced-reduced two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and using rabbit antiglycoprotein (GP)IV demonstrated that this protein corresponds to GPIV (alternatively GPIIIb). Furthermore, in dot immunobinding, anti-Naka antibody bound to purified GPIV. Our results provide definitive evidence that the Naka alloantigen is carried on GPIV. These results also demonstrate that, on occasion, antibodies against GPIV may play an important role in refractoriness to platelet transfusions.     

Identification of a cell-surface antigen associated with activated T lymphoblasts and activated platelets

We have identified and biochemically characterized an antigen, 8A3, which is expressed on activated T lymphoblasts and activated platelets. Monoclonal antibodies to 8A3 were raised against the primitive lymphoid/myeloid cell line KG1a and additionally bound to the erythroleukemia-derived cell line HEL, whilst exhibiting little or no reactivity with a panel of other hematopoietic cell lines. The 8A3 antigen was expressed on poorly differentiated T-cell leukemias and on phytohemagglutinin-activated T-cells maintained in interleukin-2 (7,000 sites/cell). This antigen, though not detected on resting platelets, was expressed on thrombin-activated platelets (2,000 sites/platelet). Antibodies to 8A3 identified polypeptides of Mr 170,000 and 150,000 in lysates of surface-iodinated KG1a cells, T lymphoblasts, and activated platelets under both reducing and nonreducing conditions. However, peptide mapping and susceptibily to glycosidases indicated that the 8A3 antigen was a monomeric glycoprotein of Mr 170,000 which contained two N-linked endoglycosidase H-sensitive glycans, and that the Mr 150,000 structure was derived from it by proteolytic degradation. The 8A3 antigen was not detectably phosphorylated in KG1a cells in vivo, nor did immune complexes containing it exhibit kinase activity in vitro. Structural and serologic characteristics of the 8A3 antigen indicate that it is different from other previously described leukocyte activation antigens including transferrin receptors, interleukin-2 receptors, members of the integrin family of adhesion molecules, or "restricted" members of the leukocyte-common antigen/CD45 cluster. Furthermore, the 8A3 antigen does not appear to be related to the other previously described activation-specific platelet molecule, GMP140/PADGEM. This antibody may be useful in monitoring T-cell activation status in some clinical situations and in characterizing clinically relevant activation-associated platelet membrane alterations.     

Polymorphism of human platelet membrane glycoprotein IIb associated with the Baka/Bakb alloantigen system

The human Baka/Bakb alloantigen system has been implicated in the pathogenesis of post-transfusion purpura and neonatal alloimmune thrombocytopenic purpura. Human alloantisera specific for either the Baka or Bakb allele have been shown to react exclusively with the heavy chain of membrane glycoprotein (GP) IIb. To investigate the structure of the Bak epitopes, we used the polymerase chain reaction (PCR) to amplify GPIIb cDNA synthesized from platelet RNA samples prepared from individuals of known serologic phenotype. Subsequent DNA sequence analysis of amplified GPIIb cDNAs derived from one Baka homozygous individual and one Bakb homozygous individual revealed a single nucleotide base difference near the 3' end of the mRNA encoding the GPIIb heavy chain. Short 13 base allele-specific oligonucleotides (ASO) containing the putative phenotype-specific base in the middle were then synthesized, end-labeled with digoxigenin-11-dUTP using terminal transferase, and used as probes in subsequent dot-blot hybridization experiments. Platelet RNA was prepared from a panel made up of four Baka/a, three Bakb/b, and two Baka/b individuals, and the mRNA encoding GPIIb was amplified using PCR and spotted onto nylon membranes. ASO hybridization showed that the nucleotide base difference identified above segregated with Bak phenotype in all nine individuals examined (P = .002). The base pair substitution results in an amino acid polymorphism at residue 843 of the mature heavy chain. The Baka form of GPIIb encodes an isoleucine at this position, whereas the Bakb allele contains a serine. Identification of the polymorphism associated with this clinically important alloantigen system should permit new therapeutic and diagnostic approaches for treating and managing patients with alloimmune thrombocytopenic disorders.     

Thrombotic thrombocytopenic purpura associated with a prolactin-producing pituitary adenoma

We report here the case of a 44-year-old woman with thrombocytopenia, anemia, convulsions, hyperprolactinemia, and galactorrhea. The patient died of cardiac failure. Autopsy revealed PAS-positive and von Willebrand factor-positive microthrombi in the arterioles and capillaries of many organs, mainly in the heart and brain, confirming the clinical diagnosis of thrombotic thrombocytopenic purpura. In the pituitary, a prolactin-producing adenoma was identified. To our knowledge, thrombotic thrombocytopenic purpura accompanied by a prolactin-secreting pituitary adenoma has not yet been described. The question of whether the association between the vascular changes and the pituitary adenoma is incidental or causal cannot be answered. Further studies are required to determine whether prolactin released from the pituitary tumor in excess played a role in the formation of microthrombi causing multiple organ failure and the demise of the patient.     

Thrombotic thrombocytopenic purpura associated with ticlopidine

Thrombotic thrombocytopenic purpura is a syndrome characterized by hemolytic anemia, thrombocytopenia, neurological symptoms, fever and renal dysfunction. Although the syndrome is usually associated with various infections, vasculitis and pregnancy, rarely can it be associated with certain neoplasms and drugs such as ticlopidine. A 63-year-old woman, who had undergone coronary angioplasty and had been started on ticlopidine, was admitted to our clinic with a history of vomiting, fatigue, hematuria and deterioration in her cognitive abilities. Thrombotic thrombocytopenic purpura was diagnosed on the basis of neurological changes, an increase in LDH, urea, creatinine, indirect bilirubin levels, anemia and peripheral smear findings. Treatment was initiated with daily plasmapheresis and complete clinical and laboratory recovery developed. The patient was discharged after 14 days.     

Effect of a single injection of humanized anti-CD154 monoclonal antibody on the platelet-specific autoimmune response in patients with immune thrombocytopenic purpura

Blockade of the CD40/CD154 signal is a potential immunomodulatory strategy for T-cell-mediated diseases. As a part of a phase 1, multicenter, dose-escalating trial of humanized monoclonal antibody to CD154 (IDEC-131/E6040) in patients with refractory immune thrombocytopenic purpura (ITP), the autoimmune response to glycoprotein IIb/IIIa (GPIIb/IIIa) was evaluated at successive time points. Five patients each were given a single infusion of 1, 2, 5, or 10 mg/kg IDEC-131/E6040 and followed for 3 months. All adverse events were mild, and there were no severe infections or thromboembolic events. No increase in platelet count was observed in patients treated at 1, 2, or 5 mg/kg, but an increase was observed in 3 patients treated at 10 mg/kg. In only the patients treated at 5 or 10 mg/kg, the frequency of B cells producing anti-GPIIb/IIIa antibodies, GPIIb/IIIa-induced T-cell proliferation, and anti-GPIIb/IIIa antibody production by antigen-dependent T-B-cell collaboration were all suppressed in parallel after the treatment, with a slow return to baseline. In contrast, T-cell response to an irrelevant antigen was not affected. These findings suggest that CD40/CD154 blockade therapy is potentially effective for refractory ITP, through selective suppression of autoreactive T and B cells to platelet antigens.     

Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection.

The cultured T-cell line TIL1200, established from the tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an antigen on most HLA-A2+ melanomas and on all HLA-A2+ cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an antigen recognized by TIL1200 was isolated by screening an HLA-A2+ breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2+ melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pmel17 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types of tumor tested. The cell line transfected with this cDNA also expressed antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible tumor rejection antigen and may be useful for the development of immunotherapies for patients with melanoma.     

Correlation of a quinidine-induced platelet-specific antibody with development of thrombocytopenia

The rapidity by which drug-dependent antiplatelet antibodies can develop is not known, since patients are only studied during or after the episode of thrombocytopenia. This report describes the development of quinidine-induced immune thrombocytopenia in a healthy volunteer during a drug study. The thrombocytopenia developed within two weeks of initiation of quinidine therapy. During the thrombocytopenic episode, but not before receiving the drug, the patient had an IgG antiplatelet antibody that bound to control platelets in the absence of the drug. This antibody was absent when the drug was discontinued and the platelet count rose. The patient's acute serum also induced the release of serotonin from control platelets, and the reaction was enhanced by quinidine. This indicates that drug-dependent antiplatelet antibodies can develop rapidly and supports the hypothesis that quinidine-induced thrombocytopenia is due to a quinidine-dependent platelet-specific IgG.