Phenotype frequency of platelet HPA-1a antigen in a group of Italian blood donors.
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The authors report a case of two siblings who developed a neonatal alloimmune thrombocytopenia (NAITP) due to Bak-System incompatibility. They review the platelet antigen systems involved in NAITP and raise the problem of its present antenatal and postnatal management.
The prediction of neonatal alloimmune thrombocytopenia (NATP) in affected families has, in the past, been based on information about gene frequencies of the antigen systems involved, parental phenotyping, and fetal platelet counts. We explored the feasibility of allele-specific oligonucleotide probe typing for PIA antigens to determine the risk of second or subsequent fetuses in families where one infant had a diagnosis of anti-PIA1-mediated NATP. A total of eight families at risk for delivering an affected fetus were studied with both serologic and oligonucleotide typing. The correlation between serologic and oligonucleotide PIA types was 100%. Similarly, in an additional eight families not at risk for PIA1-mediated NATP, serologic and oligonucleotide typing maintained a perfect correlation. DNA isolated from fetal leukocytes as well as fetal amniocytes was successfully typed using this technology. Oligonucleotide-based typing of fetuses at risk for NATP whose fathers are heterozygous for the PIA antigens allows early recognition of affected fetuses so that prenatal therapy of mothers can be instituted if necessary. When fetuses are found to be unaffected, invasive, and/or expensive, prenatal interventions can be avoided.
Allo-immune neonatal thrombopenia is to platelets what rhesus hemolytic disease is to red blood cells. There is a risk of haemorrhage when fetal platelets decreases below 50,000 per mm3. Such haemorrhage may occur at any time during the thrombopenia period. However they are mostly due to obstetrical traumatisms during delivery. Lethal complications and severe neurologic sequellae is estimated to affect about 30% of newborns: 1/3 these complications occur in utero and 2/3 during delivery. The authors have studied 3 familial cases of foeto-maternal allo-immunisation occurring with the PLA 1 system. They have made a briefing of the physiological, the pathological and the epidemiological aspects of such immunisation before insisting upon case findings of such affected fetus. A capillary blood sample taken from the scalp of the fetus at the beginning of labour allows a safe natural delivery if fetal blood count shows more than 50,000 platelets per mm3. A fetal blood sampling under ultrasonographic guidance will bring about to an early diagnosis and prognosis, this allowing an adequate treatment for fetus with a high risk of thrombopenia.
The prime object in the obstetric management of mothers who have previously delivered an infant with neonatal alloimmune thrombocytopenia is to prevent the occurrence of intracranial haemorrhage, which may occur in subsequent pregnancies in 15-20% of infants with this disease. Elective caesarean section at 38 weeks' gestation may obviate the problem, since it prevents trauma during vaginal delivery but it will not eliminate neurological sequelae in those infants who have already suffered antenatal intracranial bleeding, an entity now well described in these fetuses. A review is presented in which all possible antenatal and post-natal modalities of treatment in the management of these cases are highlighted.
Following a total blood transfusion, a 65 year old woman suffered severe thrombocytopenia characterized by purpura and haemorrhage. An immunological study revealed the existence of IgG and IgM type antibodies in the serum attached to the patient's platelets and to the eluate obtained from these. The specificity of the antibody was Zwa. The patient's platelets are Zwa-negative. Even in the remission phase of the thrombocytopenia it was still possible to obtain antibodies in the platelets of the eluate. Questions raised by this factor are discussed and, despite the difficulties posed, an attempt is made to find a coherent immunological explanation based on the results of the serological study.
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The autosomal recessive transmission of Glanzmann's thrombasthenia (GT) and Bernard-Soulier syndrome (BSS), together with requests of families who already had children with these diseases, prompted us to investigate the feasibility of their antenatal diagnosis. The preliminary step leading to the early detection of GT or BSS was to characterize, in the normal human fetus, the platelet antigens and glycoproteins (GPs) and to define their normal amounts on the membrane surface. Blood samples from 32 fetuses between 18 to 26 weeks of gestation were collected by direct puncture of the umbilical vein using an ultrasound-guided needle. Polyclonal antibodies from human origin directed against PLA1, Leka antigens, and the GPIIb IIIa complex (IgGL), or murine monoclonal antibodies specific for GPIb (AN51, 6D1), GPIIIa (AP-3), or GPIIb IIIa (AP-2) were studied using platelet suspension immunofluorescence tests. The binding of each antibody was quantified using a cytofluorograph (Ortho 50H). PLA1 and Leka antigens were expressed in normal amounts on fetal platelets as early as 16 weeks of intrauterine life. The GPIIb IIIa complex quantified by polyclonal or monoclonal antibodies was in the same range in fetuses (IgGL = 427 +/- 23 AUF, AP-2 = 459.5 +/- 8.5; AP-3 = 536 +/- 14) and in adults (IgGL = 420 +/- 30; AP-2 = 498 +/- 11; AP-3 = 515 +/- 13). The platelet binding of antibodies that recognized GPIb was higher in fetuses (AN51 = 491.5 +/- 14; 6D1 = 479 +/- 15) than in adults (AN51 = 426.5 +/- 9; 6D1 = 449 +/- 8.7). These results suggest that immunological techniques can be applied as early as 18 weeks of gestation for the antenatal diagnosis of GT and BSS.
The cytoadhesin family consists of platelet glycoprotein (GP) IIb-IIIa and the endothelial vitronectin receptor. The beta subunit (GP IIIa) of these complexes expresses the alloantigen Zwa (or PIA1). This alloantigen is not expressed by members of other integrin subfamilies. By using immunoprecipitation and immunoblot techniques, we found that the beta subunit of a heterodimer, expressed by cultured human arterial smooth muscle cells and cultured foreskin fibroblasts, carries the Zwa antigenic determinant. Furthermore, the mobilities of the alpha and beta subunits of these two heterodimers are indistinguishable from those of the alpha and beta subunits of the endothelial vitronectin receptor. Therefore, we propose that the smooth muscle cell and fibroblast heterodimer are members of the cytoadhesin family. In Glanzmann's thrombasthenia, platelet GP IIb-IIIa is absent or severely reduced. Previously, we showed that endothelial cells from a thrombasthenic patient normally synthesize and express a GP IIb-IIIa-related molecule (the vitronectin receptor). Here we show that arterial smooth muscle cells, obtained from the same patient, express a surface molecule indistinguishable from the endothelial vitronectin receptor. We also demonstrate that both the endothelial and the smooth muscle cell GP IIIa-related molecule in this Glanzmann patient express Zwa. Our data indicate that (a) GP IIb-IIIa-related molecules on cell types other than platelets and endothelial cells can express Zwa in vitro, and (b) patients with Glanzmann's disease can express the Zwa antigen. This study substantiates our view that the defect in Glanzmann's disease is restricted to the megakaryocytes/platelets.
The platelet membrane glycoproteins GPIIb and GPIIIa form a calcium-dependent heterodimer that functions as a receptor for adhesive proteins on stimulated platelets. In this study, we have investigated the kinetics of the assembly reaction that result in GPIIb-IIIa dimerization. Pulse-chase experiments analysis performed on human megakaryocytes obtained from liquid cultures of chronic myelogenous leukemic patients with antibodies specific for GPIIIa or GPIIb demonstrated the existence of a pro-GPIIb-GPIIIa complex and of a large pool (60%) of unassociated GPIIIa; nearly all the GPIIb and the pro-GPIIb molecules were found associated with GPIIIa. This free GPIIIa was not exposed on the cell surface. Pulse-chase experiments on a subclone of the human megakaryocytic cell line LAMA-84 revealed that the cells from this subclone produced only the pro-GPIIb, which was neither processed into mature GPIIb nor expressed on the cell surface. The expression of GPIIIa in PMA treated cells resulted in the production of the mature GPIIb form and the expression of the GPIIb-IIIa complex on the cell surface. These results indicate that assembly between the early forms of pro-GPIIb and GPIIIa is an obligatory step for the maturation of the heterodimer and its expression on the cell surface.
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A case of neonatal alloimmune thrombocytopenia due to fetomaternal incompatibility against the platelet specific antigen PlA2 (Zwb) is described. Anti-PlA2 antibodies were present in the maternal serum together with anti-HLA antibodies. Initial identification of the platelet-specific antibodies was achieved by using chloroquine-treated PlA1(+) and PlA1(-) platelets. Antibody specificity was later confirmed using a panel of platelets of known phenotype.
Two newborn infants with severe alloimmunothrombocytopenia due to maternal immunization against fetal PlA1-positive platelets were treated by a combination of i.v.-immunoglobulin (400 mg/kg b.w.) and a transfusion of maternal PlA1-negative platelets. In one infant this regimen was successful; the other one had to be retreated by i.v. immunoglobulin application (400 mg/kg b.w.) over a period of five days.
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beta-thromboglobulin antigen, a platelet-specific secreted protein, occurs in three forms: platelet basic protein, low affinity platelet factor 4, and beta-thromboglobulin. The combined level of beta-thromboglobulin antigen in megakaryocytes, measured by radioimmunoassay, was 13 +/- 7 micrograms/10(6) cells (SD, n = 6). The relative proportions of the three forms of beta-thromboglobulin antigen present within platelets and megakaryocytes were determined in cells lysed with trichloroacetic acid to minimize artifactual proteolysis. Samples were analyzed by isoelectric focusing in polyacrylamide gel with quantitative immunological detection on a nitrocellulose transfer of the gel. In platelets, the major species found was low affinity platelet factor 4 with precursor platelet basic protein as only 25% +/- 11% (SD, n = 16) of total beta-thromboglobulin antigen. In megakaryocytes partially purified both from normal bone marrow aspirates and from whole marrow specimens obtained after surgery, platelet basic protein was a higher proportion of beta-thromboglobulin antigen (49% +/- 13% SD, n = 11) than was the case in platelets. beta-thromboglobulin itself was never detected under the conditions of cell lysis used. Our results suggest that platelet basic protein is synthesized in megakaryocytes and that its cleavage is associated with an earlier stage of cell development than simply maturation to platelets. Further support for the precursor status of platelet basic protein was found in the expression of predominantly this antigenic form in a human erythroleukemia cell line.
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