A Naturally Occurring Leu33Val Mutation In Beta3 Integrin Impairs the HPA-1a Epitope and Receptor Function: The Third Allele of HPA-1

Abstract number: P2113

Santoso¹ S, Kroll¹ H, Andrei-Selmer¹ LC, Rankin² A, Kretzschmar³ E, Watkins² NA, Ouwehand² WH

¹¹Institute for Clinical Immunology and Transfusion Medicine, Giessen, Germany ¹¹Institute for Clinical Immunology and Transfusion Medicine, Giessen, Germany ²²Department of Haematology, University Cambridge and National Blood Service, Cambridge, UK ³³Institute for Haemostaseology, Dusiburg, Germany

Single amino acid substitution Leu33Pro in the beta3 integrin is responsible for the formation of human platelet antigen (HPA)-1. Alloimmunization against HPA-1a (beta3-Leu33) is the most frequent cause of neonatal alloimmune thrombocytopenia (NAIT) and post-transfusion purpura (PTP). Whilst HPA-1 genotyping of a large cohort of patients with thromboembolic disease using LightCycler, we identified one patient with a unique HPA-1a melting curve. Sequence analysis revealed a C to G transversion at nucleotide 175 in the beta3 integrin that alters the Leu33 codon to Val33. Further genotyping of healthy blood donors (n= 1128) identified two non-related Val33 positive individuals (one each of Leu33/Val33 and Pro33/Val33). To examine whether the presence of Val33 affected the binding pattern of HPA-1 alloantibodies, we generated transfectants expressing recombinant beta3-Leu33 or beta3-Val33. Interestingly, differences in the reactivity of anti-HPA-1a were observed, with some HPA-1a alloantibodies showing diminished reactivity with beta3-Val33 compared to beta3-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 beta3-Val33. Furthermore, functional analysis showed that Val33 mutation impaired integrin function. In comparison to Leu33 and Pro33, Val33 cells showed marked reduction of fibrinogen binding. Our results demonstrate that the naturally occurring Leu33Val mutation in the beta3 integrin can disrupt some HPA-1a epitopes and alters receptor function. These findings may have potential clinical implications for thrombocytopenic disorders.