A 13-mer peptide straddling the leucine33/proline33 polymorphism in glycoprotein IIIa does not define the PLA1 epitope.

We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base-pair primers 105-129 and 452-428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti-PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67-Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK-2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation.     

Serological and biochemical analysis of the PlA1 alloantigen of human platelets

A solid phase platelet antibody assay has been developed which rapidly and sensitively detects Pl^A1 antibodies. The three-step assay is performed by: (1) adhering platelets to the wells of a microtitre plate, (2) incubating the platelets with test serum, and (3) adding radiolabelled Staphylococcal protein A which binds to the Fc domain of IgG antibodies. Immune reactions are detected by overnight autoradiography.
Characterization of the Pl^A1 antigen was performed by using Pl^A1 antisera in immune precipitation assays. A 90 000 dalton molecular weight species was precipitated from Pl^A1 positive human and dog platelets.     

Interdonor variability of platelet response to thrombin receptor activation: influence of PlA2 polymorphism

Considering that platelet response to thrombin receptor activation might be critical for the development of arterial thrombosis, we measured the dense granule release under stimulation by the thrombin receptor activating peptide (TRAP) in a series of 102 healthy volunteers. The threshold TRAP concentration which initiated a secretion ranged from 3 to 20 μ m . A good concordance (79%, k  = 0.677) between two tests performed at a 1 month interval indicated that platelet response to thrombin receptor activation was characteristic of each individual donor. Since the threshold concentration required to initiate secretion corresponded to the threshold concentration which induced a biphasic aggregation, all volunteers were genotyped for the Pl^A2 polymorphism, the Pro³³ variant of GPIIIa. Platelets from subjects with the Pl^A2 polymorphism required higher TRAP concentrations to aggregate than those from subjects with no Pl^A2 allele ( P  = 0.0012). However, they also required a higher ADP concentration to aggregate. In order to exclude any influence of GPIIIa polymorphism on TRAP-induced secretion, we studied the variability of platelet response to TRAP among the 77 individuals with no Pl^A2 allele, and found the same interdonor variability with the same distribution of threshold TRAP concentrations as for the 102 individuals. The results suggest that (i) platelet secretion in response to thrombin receptor activation could be a genetically controlled phenotype independent of the GPIIIa polymorphism; (ii) the Pl^A2 polymorphism is associated with platelet hypoaggregability.     

Inhibition of binding of anti-PLA1 antibodies to platelets with monoclonal antibody LK-4. Evidence for multiple PLA1 receptor sites on platelet GPIIIa

The PLA1 epitope on platelet GPIIIa has a sulfhydryl-dependent conformation and is dependent on a leucine 33/proline33 polymorphism. Monoclonal antibody LK-4 differentiates PLA1/PLA1 from PLA2/PLA2 platelet lysates on solid phase enzyme-linked immunosorbent assay (ELISA), as well as immunoblot. To determine whether LK-4 reacts at or near the binding site(s) for human anti-PLA1, nine such antibodies (Abs) (six neonatal; three posttransfusion) were examined in the presence and absence of LK-4 for binding to platelets, as well as rGPIIIa 1-66, a recombinant glutathione S-transferase fusion peptide. All nine human Abs bound to rGPIIIa 1-66, as well as platelets, in a saturation-dependent manner, employing both solid phase ELISA, as well as flow cytometry. Binding of all nine Abs to rGPIIIa 1-66 or platelets was inhibited by LK-4. IC50's for inhibition of binding of anti-PLA1 to rGPIIIa 1-66 varied from 8 to 160 micrograms/mL (5 x 10(-8)- 1 x 10(-6) mol/L). However, IC50's for LK-4 inhibition of binding to platelets was strikingly different. Six of the nine Abs had IC50's of 1 to 10 micrograms/mL (8-fold to 16-fold greater inhibition than with rGPIIIa 1- 66), whereas three neonatal Abs had IC50's of 380 to 1,013 micrograms/mL (6-fold to 48-fold less inhibition than with rGPIIIa 1- 66). Similar results were noted with intact GPIIIa, rGPIIIa 1-66 blocked the binding of anti-PLA1 Abs to platelets and served to segregate the nine patients into two groups: a sensitive group of anti- PLA1 Abs from six patients in which binding to platelets was progressively inhibited by increasing concentrations of rGPIIIa 1-66 with inhibition at 1 micrograms/mL of 18% and inhibition at 256 micrograms/mL of 78%; a second resistant group of three anti-PLA1 Abs from three patients in which inhibition was first noted at 16 micrograms/mL of 4% with 35% inhibition at 256 micrograms/mL. Thus, LK- 4 binds to GPIIIa at the 1-66 N-terminal region, inhibits binding of anti-PLA1 Ab to platelets, and segregates, anti-PLA1 Abs into two groups. These data are compatible with two or more receptor sites for anti-PLA1 Ab: one that is present on rGPIIIa 1-66 and sensitive to LK-4 inhibition, another that is present on rGPIIIa 1-66, as well as other site(s) on platelet GPIIIa and insensitive to inhibition.     

Brb, a Platelet Alloantigen Involved in Neonatal Alloimmune Thrombocytopenia

Serum from a pregnant woman with the May-Hegglin anomaly contained a platelet-specific antibody. The serum reacted in the platelet indirect immunofluorescence test (PIIFT) with 97.6% of random donor platelets and those of the father but not with the mother's own platelets. This antibody induced a moderate thrombocytopenia in the infant that responded to infusion of intravenous immunoglobulin concentrates. The platelet phenotypes were PLA1+, Baka+, Bra+/Brb- for the mother, PLA1+, Baka+, Bra-/Brb+ for the father, and PLA1+, Bra+/Brb+ for the neonate. Analysis of the maternal serum with an immunoassay based on monoclonal antibody immobilization of platelet antigens (MAIPA) and immunoprecipitation techniques demonstrated the absence of antibodies directed against HLA class I antigens and that the antigen recognized was located on the platelet-GpIa/IIa complex. This antigen was present on 113/115 random donor platelets, in 7 of the 7 unrelated May-Hegglin platelets, and only absent in 3/24 Bra+ individuals, including the mother. No platelet-specific antibody was present in the serum of the 7 unrelated May-Hegglin subjects. The antigen recognized by this platelet-specific antibody thus meets the criteria defining the antithetic allele of Bra, i.e. the Brb alloantigen.     

Platelet Anti-BAKb Antibody Associated with Neonatal Alloimmune Thrombocytopenia

A platelet alloantibody of IgG class but unknown specificity was detected in the serum from the mother of an infant with neonatal alloimmune thrombocytopenia. The antibody reacted by indirect immunofluorescence with 33/65 random donors, 10/11 Baka-negative donors and 23/54 Baka-positive donors. The mother's platelet phenotype was PLA1, Koa, Kob, Baka and the father's was PLA1, Koa, Kob, Baka. Immunoblotting and immunoprecipitation of maternal serum with paternal platelets produced a band at molecular weight 140 kilodaltons identical to the band obtained with Baka antiserum. Family studies confirmed the allelic distribution of Baka and the unknown antigen. The platelet-specific antibody in this patient meets the criteria for an antibody to the new platelet antigen, Bakb.     

Platelet Indirect Radioactive Coombs Test. Its Utilization for Pla1 Grouping

Abstract. A platelet indirect radioactive Coombs test (PIRC) has been described. The technique for purification and labelling the antiglobulin has been precised. This test allowed the typing of platelets in the PLA system by using an absorbed serum from a mother of a thrombocytopenic child. Six other families of neonatal thrombocytopenias were tested. In three of them, the mothers were found PLA₁ negative (PLA₂, PLA₂). Among a panel of 93 platelets, two (0.022) were found PLA₁, negative.
This PIRC test has many advantages compared to other tests such as platelet complement fixation, assay for blocking antibodies or antiglobulin consumption: it gives objective and quantitative results and is highly reproducible; anticomplementary serum may be tested.     

Quantitation of membrane glycoprotein IIIa on intact human platelets using the monoclonal antibody, AP-3

A murine monoclonal antibody specific for glycoprotein (GP)IIIa was prepared by immunization with a GPIIb- and GPIIIa-enriched Triton X-114 extract of platelet membranes. This antibody, designated AP-3, was shown by indirect immunoprecipitation to react solely with GPIIIa derived from either P1A1-positive or -negative individuals. The epitope on GPIIIa recognized by AP-3 is expressed on dissociated GPIIIa as well as on Ca+2-dependent complexes of GPIIb and GPIIIa, as shown by crossed immunoelectrophoresis in the presence or absence of EDTA. A previously described monoclonal antibody specific for the GPIIb/IIIa complex (AP- 2) inhibited platelet aggregation induced by ADP, thrombin, collagen, or arachidonic acid (Pidard et al, J Biol Chem 258:12582-12586, 1983). In contrast, AP-3 had no effect on aggregation induced by any of these reagents, a finding similar to that previously reported for the GPIIb- specific monoclonal antibody, Tab (McEver et al, J Clin Invest 66:1311- 1318, 1980). At saturation, 40,200 AP-3 molecules were bound per platelet, a value similar to that obtained for AP-2 or Tab. Thus, data derived using AP-3 indicate that significant amounts of free GPIIIa are not present, thereby supporting the hypothesis that GPIIb and GPIIIa exist complexed in a 1:1 stoichiometry in the plasma membrane of intact, nonactivated platelets.     

Post-transfusion purpura secondary to passive transfer of anti-P1A1 by blood transfusion

A patient developed severe post-transfusion purpura (PTP) following transfusion of two units of packed red blood cells. The timing of the patient's thrombocytopenia suggested passive immunization rather than the typical anamnestic response associated with classical PTP. Investigation of the blood donors revealed one with evidence of a platelet specific antibody of high titre. This donor was typed as P1A1 negative and the antibody was shown to have anti-P1A1 specificity.     

GPIIIa-related PLA1 antigens with different molecular weights: studies in platelets, endothelial cells, and megakaryocytes

The mol wt of the glycoprotein(s) carrying the PLA1 antigen was examined on platelets, megakaryocytes and endothelial cells by immunoblotting with a human polyclonal anti-PLA1 antibody (BE), as well as on four different monoclonal antibodies (MoAbs; DEK-1, DEK-2C, DEK- 10, and DEK-16) raised against GPIIIa, the 100,000-mol wt platelet glycoprotein known to carry the PLA1 antigen. BE reacted with PLA1 positive but not with PLA1 negative platelets. DEK-1 reacted strongly with PLA1 positive platelets but weakly with PLA1 negative platelets. The remaining three MoAbs reacted equally with PLA1 positive as well as negative platelets. BE, DEK-1, DEK-10, and DEK-16 reacted with a 120,000- as well as 100,000-mol wt band on immunoblot of PLA1 positive platelets. The 120,000-mol wt band copurified with affinity purified 100,000-mol wt GPIIIa. Megakaryocytes had a prominent 120,000- as well as 105,000-mol wt band that reacted with BE on immunoblot (the 100,000- mol wt band was not detectable). Umbilical cord endothelial cells from presumed PLA-positive infants had a prominent 100,000-mol wt band that reacted with BE, DEK-16, and DEK-1 (the 120,000-mol wt band was not visualized). The 120,000- and 100,000-mol wt PLA1-positive bands could be digested with proteolytic enzymes to 55,000- to 65,000-mol wt- resistant fragments that retain PLA1 epitopes. Further digestion with endoglycosidase-H lowered the apparent mol wt by approximately 2,000 to 6,000 daltons without affecting PLA1 reactivity. We conclude that the PLA1 antigen is present on a 120,000- as well as 100,000-mol wt glycoprotein of platelets and megakaryocytes, a 105,000-mol wt band of megakaryocytes, and a 100,000-mol wt glycoprotein of endothelial cells. We postulate that the 120,000-mol wt glycoprotein, which shares three or more epitopes with the 100,000-mol wt GPIIIa, may be a post- translational precursor of this species.     

Autoantibodies against the platelet glycoprotein IIb/IIIa complex in patients with chronic ITP

Chronic idiopathic thrombocytopenic purpura (ITP) is caused by an antibody reactive with platelet-associated antigens. The present studies provide direct evidence that some patients with chronic ITP have autoantibodies against the platelet glycoprotein (GP) IIb/IIIa complex. Microtiter wells, coated with a monoclonal antibody (2G12) specific for GPIIb/GPIIIa were reacted with GPIIb/GPIIIa contained in a platelet extract. Control wells containing the same antibody were reacted with a cell extract containing no GPIIb/GPIIIa. After washing, the wells were reacted with patient or control plasma, and IgG binding was detected using 125I-Fab2-anti-human IgG. Assay values were expressed as binding ratios (cpm GPIIb/GPIIIa wells/cpm control wells). Plasma from 5 of 56 patients with chronic ITP had ratios (1.36-3.14) greater than 3 standard deviations above the mean (+/- SD) of control plasmas--0.93 +/- 0.12. Elevated values were also noted in two patients with anti-P1A1 antibody (ratios greater than 30) and in one patient with Hodgkin's disease and an ITP-like syndrome (ratio 1.53). Normal values were noted in 34 patients with a variety of immune and nonimmune diseases. Plasma from two of the positive ITP patients was reacted with 125I-surface-labeled platelets and, after solubilization, the IgG and bound antigen were precipitated with Staph-A. Autoradiographs from SDS- PAGE electrophoresis of the Staph-A-bound proteins shows two radioactive bands consistent in size with GPIIb and GPIIIa.     

Heterogenous inhibition of platelet aggregation by monoclonal antibodies binding to multiple sites on GPIIIa

Summary. Six monoclonal IgGl-k antibodies (LK2, LK3r, LK4-55, LK5, LK6-55, LK7r) were raised against platelet membrane GPIIIa in order to study the structure-function relationship of this molecule. Antibodies were selected on their ability to react with GPIIIa by ELISA on adherent platelets, by immunoblot on platelet lysates and by fluorescence flow cytometry on intact platelets. Fluorescence reactivity varied from 3- to 202-fold greater than isotype control fluorescence. Two MoAbs reacted on immunoblot under reduced conditions (LK7r and LK3r). Two reacted with a 55 kD chymotrypsin/subtilisin digest of GPIIIa which is likely to exclude amino acids 121-348 (LK4-55 and LK6-55). Four of the MoAbs (LK5, LK3r, LK2 and LK4-55) inhibited tyrosine phosphorylation of one to four distinct bands on immunoblot. LK4-55 reacted with an N-terminal 66 amino acid fusion protein of GPIIIa near the PLA epitope (Leu 33). LK7r reacted with a 212-222 peptide reported to be an RGD fibrinogen binding site. LK2 reacted near a disintegrin-RGD binding site. Except for LK5, all inhibited ADP, collagen and thrombin-induced platelet aggregation in a heterogenous fashion. Percentage inhibition of ¹²⁵I-fibrinogen binding to platelets varied from 18% to 98%. No correlation was noted between inhibition of fibrinogen binding, location of MoAb binding on GPIIIa, reactivity of MoAb binding with GPIIIa, inhibition of thrombin-induced tyrosine phosphorylation or inhibition of platelet aggregation induced by ADP, collagen or thrombin. Thus MoAbs, binding to platelet GPIIIa at different sites, inhibit platelet aggregation in a heterogenous manner.     

Alloimmunization to the PIA1 platelet antigen: results of a prospective study

The natural history of alloimmunization to the PlA1 platelet antigen is uncertain. We followed 50 PlA1-negative pregnant women during pregnancy and for 6 months post-partum in order to determine this natural history. The cohort of PlA1-negative women was obtained by PlA1 typing 5000 women. Three PlA1-negative women formed anti-PlA1 antibodies during this prospective study, two in pregnancy and one in the immediate post-partum period. All three PlA1 antibody producers were HLA-DR3 positive, a histocompatibility phenotype that is strongly associated with alloimmunization to the PlA1 antigen. One of the three infants delivered to these mothers was thrombocytopenic (platelet count 9 x 10(9)/l). The remaining two infants had normal platelet counts at birth (160 and 174 x 10(9)/l). The HLA-A1, -B8, -DR3 and -DRw52 phenotype frequencies in the group of PlA1-negative women who did not form PlA1 antibodies (n = 47) was similar to that found in their husbands, and that expected in a normal Caucasian population. From our data we estimate that alloimmunization to the PlA1 antigen occurs in approximately one out of every 1000 pregnancies in a Caucasian population. It is important to recognize that not all pregnancies in which a mother has formed PlA1 alloantibodies will result in the delivery of a thrombocytopenic infant. These findings are relevant to programs designed to either prevent alloimmunization to the PlA1 antigen (through passive administration of anti-PlA1 immunoglobulin to at-risk PlA1-negative mothers), or to identify women at risk of delivery of thrombocytopenic infants (by antenatal screening to detect women alloimmunized to the PlA1 antigen).     

A human monoclonal antibody specific for the leucine-33 (P1A1, HPA-1a) form of platelet glycoprotein IIIa from a V gene phage display library

IgG alloantibodies to polymorphic platelet glycoproteins (GPs) are known to be responsible for severe thrombocytopenia in the neonate and after transfusion. Platelet GPIIIa can have either a leucine or a proline at residue 33. The most immunogenic platelet alloantigen in thrombocytopenia is the leucine 33 form of GPIIIa. Here, we have generated human monoclonal antibody fragments that are specific for the leucine and not the proline form of GPIIIa and can inhibit the binding of polyclonal human IgG alloantibodies to GPIIIa leucine 33 on platelets. The antibody fragments were selected from a library of single chain Fv fragments displayed on the surface of filamentous phage. The VH gene repertoire was derived from the peripheral blood lymphocytes of an alloimmunized individual and recombined with a VL gene repertoire from a nonimmune source. Antibodies such as these, which are able to distinguish between two variant forms of a native antigen and which have been unobtainable by conventional hybridoma technology, have both diagnostic and potential therapeutic applications.     

Anti-A1 Leb in Serum of a Person of a Blood Group A1h

Abstract. An antibody, anti-A₁ Le^b, has been found in the serum of a person belonging to group A₁h, with complete depression of H on the red cells as well as in the saliva. It differs from earlier examples of the antibody in that it can be neutralized by A₁ nL, secretor saliva as well as O Le^b secretor saliva in addition to A₁ Le^b secretor saliva. Although the proposita secretes A substance, she only secretes Le^a substance and not Le^b substance. No such abnormalities were found in the family investigated. An interpretation of the case is given according to the classical hypothesis of ABO and Lewis system development.     

Post-transfusion purpura (PTP) due to anti-Zwb(-PIA2): the significance of IgG3 antibodies in PTP

The first two patients with post-transfusion purpura (PTP) due to platelet antibodies against the Zwb antigen are reported. The anti-Zwb specificity could be demonstrated only with an enzyme-linked immunosorbent assay (ELISA) but not with the immunofluorescence or the complement fixation test due to coexistent potent multispecific HLA antibodies. One of the patients had never received blood transfusion until 24 d before the development of thrombocytopenic purpura. In both patients, anti-Zwb of IgG1 and IgG3 subclasses defined by monoclonal antibodies were present during the thrombocytopenic period but the antibodies of IgG3 subclass disappeared concomitantly with clinical improvement. The association between the IgG3 subclass of anti-Zw antibodies and the destruction of autologous platelets in Zw-immunized individuals was investigated further. All of four PTP patients had anti-Zw antibodies of the IgG1 and IgG3 subclasses during the thrombocytopenic period while all of 20 mothers of children affected with alloimmune neonatal thrombocytopenia (AINT) had anti-Zwa of only the IgG1 and not IgG3 subclass at the time of delivery of thrombocytopenic children (P less than 10(-4). Thus, the destruction of autologous platelets in PTP is associated with the presence of anti-Zw of the IgG3 subclass which may be of importance in the pathogenesis of PTP.     

Antigen-binding glycosylation inhibiting factor from a human T-cell hybridoma specific for bee venom phospholipase A2.

We obtained human T-cell hybridomas that are specific for bee venom phospholipase A2 (PLA2) and constitutively secrete glycosylation inhibiting factor (GIF). Upon crosslinking of CD3, the hybridoma produced GIF having affinity for PLA2. When affinity-purified PLA2-binding GIF was used as an immunogen, monoclonal antibodies specific for the antigen-binding GIF were obtained. Monoclonal antibody 110BH3 bound the antigen-binding GIF but failed to bind the 13-kDa nonspecific GIF, as determined by both bioassay and ELISA. In contrast, 388F1, a monoclonal antibody against nonspecific GIF, gave ELISA signals with both the nonspecific GIF and the antigen-binding GIF. Gel filtration of affinity-purified antigen-binding GIF revealed the presence of a 72- to 80-kDa protein which gave ELISA signals with both 110BH3 and 388F1 and contained GIF bioactivity. Upon reduction and alkylation, the antigen-binding GIF dissociated into a 62- to 64-kDa protein which gave positive ELISA with antibody 110BH3 but no signal with antibody 388F1, and a 15-kDa protein, which gave ELISA signal with the 388F1 but not with 110BH3. Immunoblotting of a PLA2-binding GIF preparation revealed that under reducing conditions, the antigen-binding GIF dissociated a 13-kDa peptide which reacted with polyclonal antibodies against recombinant GIF. The results indicate that the 13-kDa nonspecific GIF is a subunit of antigen-binding GIF. The PLA2-binding GIF has affinity for an epitope, representing amino acid residues 19-28 in PLA2 which appears to be an external structure in the antigen.     

Measurement of Platelet Surface-Bound IgG by a Monoclonal 125I-Anti-IgG Assay

We have described the use of a monoclonal 125I-labeled anti-IgG (125I-MA) to assay IgG antibody displayed on the surface of platelets from normal and immune thrombocytopenic patients and reported levels of IgG 10-100-fold lower than previous studies. This report describes the immunologic characteristics of the 125I-MA and the assay for surface IgG. The 125I-MA has a high binding affinity for surface-displayed IgG (2.22 X 10(9) M-1), reacts equally well with all four subclasses of IgG and not at all with IgM or IgA. In our assay, the binding of 125I-MA was found to be greater than or equal to 99% specific for IgG (no nonspecific association of 125I-MA with platelets) and the binding ratio of 125I-MA to IgG displayed on the cell surface was 0.91 (close to unity). Finally, platelet lysates were found to contain large amounts of IgG protein (39,597 +/- 27,418 molecules/platelet) as compared to surface-displayed IgG (124 +/- 86 molecules/platelet). This assay has excellent characteristics for quantitation of IgG on platelets and the discrepancy with other techniques may, in part, be due to intentional or inadvertent lysis of platelets during assay conditions.     

Immunochemical characterization of the new platelet alloantigen system Bra/Brb

We report the immunochemical characterization of the new platelet-specific alloantigens Bra and Brb. Bra antibodies were from mothers of children with neonatal alloimmune thrombocytopenia (NAIT), and anti-Brb was found in the serum of a polytransfused patient. By radioimmunoprecipitation, anti-Bra and anti-Brb precipitated two proteins with apparent relative molecular masses in sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 155,000 and 130,000 under non-reduced conditions, and of 165,000 and 148,000 under reduced conditions. In two-dimensional polyacrylamide gel electrophoresis, the two bands moved with isoelectric points ranging from 5.2 to 5.4 and from 4.5 to 4.7, respectively. These features fulfil previously defined criteria for platelet membrane glycoproteins (GP) Ia and IIa. The results were supported by data obtained by an assay employing monoclonal antibody (mab)-specific immobilization of platelet antigens (MAIPA). By this technique, Bra and Brb antigens could be immobilized by mabs specific for the GP Ia/IIa complex (mab Gi 14) or a mab specific for the very late antigen-2 (mab 12 F1), but not by a mab specific for GP IIb/IIIa complex (mab Gi3). Furthermore, platelets from a thrombasthenic patient with complete absence of GP IIb/IIIa expressed the Brb antigen normally as shown in MAIPA; this antigen could be immunoprecipitated with anti-Brb and was identical to that of normal platelets. This confirms that the antigens of the Br system are not associated with the GP IIb/IIIa complex. By direct binding studies using mabs Gi3 and Gi14, we calculated that 51,500 +/- 3900 molecules of anti-GP IIb/IIIa and 6,470 +/- 500 molecules of anti-GP Ia/IIa were bound per platelet at saturation. Our results provide evidence for the first platelet-specific alloantigen system residing on the GP Ia. The difficulty in detecting anti-Bra and anti-Brb by direct binding assays may be related to the small number of GP Ia/IIa complexes on platelets.     

A new platelet alloantigen, Tua, on glycoprotein IIIa associated with neonatal alloimmune thrombocytopenia in two families

We describe immunization of two mothers against a new platelet alloantigen, designated Tu^a, in association with thrombocytopenia in their first born children. The platelet-specific antibodies were identified by a glycoprotein-specific platelet protein assay with husband's platelets. Monoclonal antibodies against glycoprotein complex IIb/IIIa (AP2) and against glycoprotein IIb (SZ22) could be used to immobilize the antigen bearing protein. When monoclonal antibodies against glycoprotein Ib/IX (FMC25) or Ia/IIa (Gi9) were used, no platelet-specific antibodies were detectable. The previously described alloantigens on the glycoprotein IIb/IIIa complex (HPA 1,3,4, Sr^a and Va^a) were not responsible for the reaction. Immunochemical analysis by an immunoblot assay showed that the Tu^a antigen resides on GPIIIa but the antigen was destroyed by reduction of the protein. Altogether 10 individuals belonging to three unrelated families were shown to carry the antigen. The family studies within three generations indicated autosomal codominant inheritance. Thus the Tu^a antigen is apparently different from all previously published platelet alloantigens. One Tu^a positive blood donor was identified in a population study of approximately 150 individuals. This indicates a low frequency in the Finnish population. Extended population studies will be required to determine a more exact frequency of Tu^a antigen.