Polymorphisms in Canine Platelet Glycoproteins Identify Potential Platelet Antigens

Human alloimmune thrombocytopenic conditions caused by exposure to a platelet-specific alloantigen include neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness. More than 30 platelet-specific alloantigens have been defined in the human platelet antigen (HPA) system; however, there is no previous information on canine platelet-specific alloantigens. Using the HPA system as a model, we evaluated the canine ITGB3, ITGA2B, and GP1BB genes encoding GPIIIa (β₃), GPIIb (α_IIb), and GPIbβ, respectively, which account for 21 of 27 HPA, to determine whether amino acid polymorphisms are present in the orthologous canine genes. A secondary objective was to perform a pilot study to assess possible association between specific alleles of these proteins and a diagnosis of idiopathic thrombocytopenic purpura (ITP) in dogs. By using genomic DNA from dogs of various breeds with and without ITP, sequencing of PCR products encompassing all coding regions and exon–intron boundaries for these 3 genes revealed 4 single-nucleotide polymorphisms in ITGA2B resulting in amino acid polymorphisms in the canine genome, 3 previously reported and 1 newly identified (GlyGGG]/ArgAGG] at amino acid position 576 of ITGA2B. Of 16 possible ITGA2B protein alleles resulting from unique combinations of the 4 polymorphic amino acids, 5 different protein isoforms were present in homozygous dogs and explain all of the genotype combinations in heterozygous dogs. There was no amino acid polymorphism or protein isoform that was specific for a particular breed or for the diagnosis of ITP.Abbreviations: HPA, human platelet antigen; ITP, idiopathic thrombocytopenic purpura; PTR, platelet transfusion refractoriness; SNP, single-nucleotide polymorphismHuman alloimmune thrombocytopenic conditions caused by exposure to a platelet-specific alloantigen, through pregnancy or transfusion, include neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness (PTR). More than 30 platelet-specific alloantigens have been defined in the human platelet antigen (HPA) system, with 12 alloantigens grouped into 6 biallelic systems (HPA1 through HPA5 and HPA15) in which alloantibodies against both the common (designated ‘a’) and rare (designated ‘b’) alleles have been identified.^5,12 For the remaining 21 platelet alloantigens (HPA6bw through HPA14bw and HPA16bw through HPA27bw), only antibodies against the rare allele (designated ‘bw’) have been detected. Although HPA traditionally were defined by using immune sera, the molecular basis of these antigens has now been characterized.^5,12 The HPA reside in platelet membrane glycoproteins, the most common being GPIIIa, which accounts for 14 HPA. In all but one (HPA14bw), the platelet alloantigens are defined by a single amino-acid substitution caused by a single-nucleotide polymorphism (SNP) in the gene encoding the relevant membrane glycoprotein.^5,12 The Platelet Nomenclature Committee, which includes members of both the International Society of Blood Transfusion and the International Society of Thrombosis and Haemostasis, has set guidelines for defining new platelet antigens, which include: 1) determining the genetic basis of the alloantigen by genomic DNA sequence analysis, and 2) demonstrating an association between the genetic mutation and the reactivity of alloantibodies with the allelic forms of the protein.¹² For patients suspected of having alloimmune thrombocytopenia, well-defined platelet genotyping methods and serologic assays for the detection of platelet antibodies are available to guide case management.In contrast to the well-characterized HPA system and wealth of information on alloimmune thrombocytopenic conditions in humans, there is no information on canine platelet-specific alloantigens in the literature. However, early serologic studies using antiHPA1a alloantiserum resulted in direct immunoprecipitation of a 90-kDa protein from canine platelets, suggesting that the antigenic determinant for HPA1a has been conserved between humans and dogs.¹⁰ Neonatal alloimmune thrombocytopenia has not yet been documented in dogs, and there is a single case report of suspected posttransfusion purpura in a dog with hemophilia A, in which severe thrombocytopenia was noted 1 to 2 wk after transfusion (cryoprecipitate, fresh whole blood), with an increased amount of platelet surface-associated IgG and rapid resolution (less than 1 wk) of the thrombocytopenia.³⁰ PTR, however, has been well documented in dogs for more than 25 y, because this species has been used as an experimental model to evaluate a variety of methods of preventing platelet alloimmunization.^21-23 The occurrence of platelet alloimmunization in dogs receiving platelets from dog leukocyte antigen-identical littermates indicates that nondog leukocyte antigen immunizing platelet antigens, perhaps platelet-specific antigens, are responsible for the development of PTR.²¹Using the HPA system as a model, our main objective in this study was to evaluate the canine ITGB3, ITGA2B, and GP1BB genes encoding the GPIIIa (β₃), GPIIb (α_IIb), and GPIbβ, respectively, proteins that account for 21 of 27 HPA, to determine whether there are amino acid polymorphisms that could define canine platelet-specific alloantigens. A secondary objective was to perform a pilot study to assess the possible association between canine platelet antigen protein alleles or single amino acid substitutions and primary immune-mediated thrombocytopenia, also referred to as idiopathic thrombocytopenic purpura (ITP), in dogs. The identification of a canine platelet antigen system would improve our understanding of the molecular basis of alloimmune thrombocytopenic conditions in dogs and help guide effective platelet transfusion support of these critical patients.