HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY A new platelet polymorphism Duva , localized within the RGD binding domain of glycoprotein IIIa, is associated with neonatal thrombocytopenia

We report here the identification and characterization of a new platelet alloantigen, Duva , implicated in a case of neonatal thrombocytopenia. Immunochemical studies demonstrated that the epitope was localized on glycoprotein (GP) IIIa. Sequencing of the exons 2 to 15 of GP IIIa gene polymerase chain reaction products from both parents revealed a single base substitution 517C>T (complementary DNA) present in a heterozygous state in DNA from the father leading to amino acid substitution Thr140Ile (ACC>ATC) within the Arg-Gly-Asp binding domain of GP IIIa. Flow cytometry and immunoprecipitation studies of IIb-C517 or T517 IIIa transfected Cos cells allowed us to demonstrate this mutation was responsible for expression of the Duva epitope. By polymerase chain reaction–single-strand conformational-polymorphism analysis, the mutated allele could not be detected in a population of 100 healthy unrelated donors, indicating a low frequency of occurrence. The Thr140/Ile dimorphism, localized 3 amino acids upstream from the Arg143 involved in the expression of HPA-4a, did not interfere with the binding of an anti–HPA-4a antibody in flow cytometry. Results of functional analysis of wild-type or mutated transfected CHO cells—(1) aggregation in the presence of Ca and soluble fibrinogen after complex activation by dithiothreitol, (2) adhesion on coated fibrinogen, (3) binding of monoclonal antibody PAC-1 or LIBS antibody D3, and (4) outside-in signaling—all suggest that the Thr140Ile polymorphism localized in the Arg-Gly-Asp binding domain of GP IIIa does not affect significantly, if at all, the integrin function. We have shown that the anti-Duva antibody may inhibit platelet GP IIb-IIIa function. (Blood. 2002;99:4449-4456)