The human neutrophil-specific alloantigen NB1 was identified as a glycosyl-phosphatidylinositol (GPI)-anchored N-glycosylated protein of M(r) 56-62 kD under reducing conditions. Under non-reducing conditions its M(r) was 49-55 kD. This glycoprotein antigen was found to be expressed by only a subpopulation of normal donor neutrophils, and could not be detected on other blood cells. The allotypic epitope recognized by human anti-NB1 IgG was also recognized by the mouse monoclonal antibody 1B5. The percentage of neutrophils stained by these antibodies varied greatly among healthy donors (range 0-100%). When 16 donors were repeatedly tested, the NB1-positive neutrophil fraction appeared to remain remarkably constant over time in most donors, but significant fluctuations were seen in some. NB1 antigen was found to be expressed not only on the plasma membrane, but also intracellularly on the membranes of small vesicles and specific granules. The neutrophils which expressed NB1 antigen on the plasma membrane were the same as those with intracellular expression of this antigen. Crosslinking of NB1 antigen on the plasma membrane with monoclonal antibody 1B5 and goat-anti-mouse Ig resulted in internalization of the complex, while in-vitro stimulation of neutrophils caused an increase of the intensity of plasma membrane staining with anti-NB1, but only of those cells that were positive already prior to stimulation. The NB1 glycoprotein thus appears to identify a distinct subset of neutrophils, the size of which greatly varies among healthy donors. The function of the NB1 glycoprotein remains unclear, but its behaviour upon crosslinking and chemotactic peptide stimulation suggests a possible role as receptor molecule.

The glycoprotein (GP) Ib/IX complex, a prominent platelet GP complex, is the primary receptor for vWF. Previously, we have established that an antigenic polymorphism of platelets, the HPA-2 or Ko alloantigen system, is located on the 45-kD amino-terminal globular domain of GPIba. With the polymerase chain reaction, we have amplified two segments of the GPIba gene coding for the first 382 amino acids of two HPA-2a and two HPA-2b homozygous individuals. Nucleotide sequence analysis revealed as the only difference a C-T polymorphism at position 434 of the coding region for the mature protein. This base change results in a substitution of threonine (ACG) in HPA-2a (Ko") to methionine (ATG) in HPA-2b (Ko*) at amino acid position 145. The C-T polymorphism is reflected in a difference in restriction enzyme recognition, resulting in an Aha 2-site in the HPA-2b allele and a SfaN1 site in the HPA-2a allele. Restriction fragment length polymorphism analysis of the amplified DNA of 3 HPA-2(a-,b+), 2 HPA-2(a+,b+), and 11 HPA-2(a+,b-) donors showed that these restriction sites were associated with the HPA-2 alleles. DNA-typing for the HPA-2 alloantigen system on genomic DNA obtained from a small number of cells may be applied for determining the genotype of a fetus from an immunized mother or of severely thrombocytopenic patients. (J. Clin. Invest. 1992. 89:381-384.) Key

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Here we describe a new platelet-specific alloantigen that was identified in a case of neonatal alloimmune thrombocytopenia. This antigen has provisionally been called “Mo.” By studying the Mo family, it was shown to be inherited in an autosomal dominant manner. Immunoprecipitation and Western blot analysis showed that the antigen resides on platelet glycoprotein IIIa (GP IIIa). Genomic analysis, performed by applying polymerase chain reaction and sequencing, showed a C-->G substitution of base pair 1267 of the coding region of the DNA for GP IIIa, resulting in a substitution of Proline407 by Alanine407. That this substitution is associated with the antigen could be demonstrated by restriction fragment length polymorphism analysis of cDNA, prepared from platelet RNA, and of genomic DNA. It was confirmed by dot-blot hybridization with allele-specific oligonucleotides. All family members, also those being Mo antigen-positive, were healthy. None of them appeared to suffer from increased tendency of bleeding or thrombosis. Thus, the Mo mutation does not lead to significant platelet dysfunction in vivo with heterozygous carriers. One of 450 random healthy blood donors who were tested was positive for the Mo antigen. Typing was performed by the classical serologic methods as well as by DNA analysis.