790 TRANSFUSION Volume 46, May 2006 Blackwell Publishing IncMalden, USATRFTransfusion0041-11322006 American Association of Blood BanksMay 2006465790799Original Article THE THIRD ALLELE OF HPA-1SANTOSO ET AL. ABBREVIATIONS: CHO = Chinese hamster ovary; EGF = epidermal growth factor; FNAIT = fetal and neonatal alloimmune thrombocytopenia; HPA = human platelet antigen; PTP = posttransfusion purpura. From the Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University, Giessen, Germany; the Department of Haematology, University of Cambridge and National Blood Service, Cambridge, United Kingdom; and the Institute for Clinical Haemostaseology, Duisburg, Germany. Address reprint requests to: Sentot Santoso, PhD, Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University Giessen, Langhansstrasse 7, D-35385 Giessen, Germany; e-mail: sentot.santoso@immunologie.med.uni- giessen.de. This work was supported by grants from the Deutsche Forschungsgemeinschaft to S.S. (SFB 547) and from NBS R&D to A.N.W. and W.H.O. Received for publication June 28, 2005; revision received September 26, 2005, and accepted September 28, 2005. doi: 10.1111/j.1537-2995.2006.00797.x TRANSFUSION 2006;46:790-799. IMMUNOHEMATOLOGY A naturally occurring Leu 33 Val mutation in b3-integrin impairs the HPA-1a epitope: the third allele of HPA-1 Sentot Santoso, Hartmut Kroll, Cornelia L. Andrei-Selmer, Ines Socher, Angela Rankin, Evelyne Kretzschmar, Nicholas A. Watkins, and Willem H. Ouwehand BACKGROUND: Single-amino-acid substitution Leu33Pro in the β3-integrin is responsible for the formation of the human platelet antigen (HPA)-1. Alloimmunization against HPA-1a (β3-Leu33) is the most frequent cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura. STUDY DESIGN AND METHODS: While HPA-1 genotyping a large cohort of patients with thromboembolic disease with a thermal cycler (LightCycler), one patient was identified with a unique HPA-1a melting curve. RESULTS: Sequence analysis revealed a C-to-G transversion at nucleotide 175 in the β3-integrin (ITGB3) gene that alters the Leu33 codon to Val33. Further genotyping of healthy blood donors (n = 2950) identified one nonrelated Pro33Val33-positive individual. To examine whether the presence of Val33 affected the binding pattern of HPA-1 alloantibodies, transfectants were generated expressing recombinant β3-Leu33 or β3- Val33. Interestingly, differences in the reactivity of anti- HPA-1a were observed, with some HPA-1a alloantibodies showing diminished reactivity with β3-Val33 compared to β3-Leu33 and others reacting equally with both types. Similar findings were observed with recombinant human HPA-1a antibodies, with one of the three not binding to β3-Val33. CONCLUSIONS: Our results demonstrate that the naturally occurring Leu33Val mutation in the β3-integrin can disrupt some HPA-1a epitopes. These findings provide evidence for a heterogeneous humoral response against HPA-1a that may have potential clinical implications for alloimmune thrombocytopenia disorders. he platelet (PLT) fibrinogen receptor αIIbβ3 (gly- coprotein IIbIIIa) belongs to the integrin family of heterodimeric transmembrane proteins that mediate cell adhesion and cell migration in numerous fundamental biological processes. 1 On PLT activation, inside-out signaling mechanisms trigger con- formational changes in αIIbβ3 extracellular domains that increase affinity of the integrin heterodimer for fibrinogen and other ligands. The cross-linking of fibrinogen to αIIbβ3 results in PLT aggregation with the subsequent out- side-in signaling leading to clot retraction and the forma- tion of intracellular signaling complexes involving the αIIbβ3 cytoplasmic domains. 2 The β3-subunit is a disulfide bond–rich single polypeptide consisting of 762 amino acid residues. 3 Recently, the crystal structures of both αvβ3 and αIIbβ3 have been resolved. 4-6 These structures show that integrin β3 has an N-terminal PSI (plexins, semaphorins, integrins) domain of 54 amino acids, followed by an immunoglobu- T