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IIb and ß3 in Normal Horses and Two Horses with Glanzmann Thrombasthenia
Abstract
Glanzmann thrombasthenia (GT) is an inherited, intrinsic platelet defect characterized by a quantitative or qualitative change in the platelet glycoprotein complex IIb-IIIa (integrin 
IIbß3). The subunits are encoded by separate genes and both subunits must be expressed for a stable complex to form on the platelet surface; therefore, a defect in either gene can result in GT.
Key words: Equine; Glanzmann thrombasthenia; inherited disease; integrin IIbß3; platelet; polymerase chain reaction.
Dr. Eduard Glanzmann, a Swiss pediatrician, first described Glanzmann thrombasthenia (GT) in his patients in 1918; however, the association of a deficiency of the platelet glycoprotein complex IIb–IIIa with GT was not made until 1974.14–16 In the decade that followed, this glycoprotein complex was recognized as being the receptor that mediated platelet aggregation and was termed the fibrinogen receptor. In the 1990s, it was recognized that the fibrinogen receptor of platelets was an integrin and was designated as IIbß3 to comply with the nomenclature for that system. It was recognized at this time that the subunits were encoded by separate genes and that both subunits were required for a stable complex to form on the platelet surface. This disease has been well documented at the clinical and molecular levels in human beings and dogs2,3,7,13 (http://sinaicentral.mssm.edu/intranet/research/glanzmann/menu). The first description of GT at the molecular level in humans was published in 1990, while the molecular basis for GT in dogs was first reported in 1999.4,6
GT was recently described in two horses with histories of chronic, intermittent epistaxis that was unrelated to exercise.12 Horse No. 1 was a 7-year-old thoroughbred cross gelding located in Hatfield, Hertfordshire, England, while horse No. 2 was a 4-year-old quarter horse mare located in Auburn, Alabama. These animals had normal coagulation screening test results, normal platelet numbers, and normal von Willebrand factor antigen levels. Clot retraction and platelet aggregation responses were markedly impaired. Flow cytometric studies indicated a reduction in the IIbß3 integrin on platelet surfaces. Based on these findings, both horses were diagnosed with GT.
The purpose of this study was to determine the cDNA sequences encoding normal equine IIb and ß3 and compare them with established human and canine cDNA sequences and to cDNA sequences determined in both affected horses.
Platelet-rich plasma was obtained by centrifuging 100–200 ml of EDTA-anticoagulated whole blood as previously described.5 Platelets were counted and concentrated into pellets containing 5 x 109 platelets per pellet. Total RNA was harvested from platelet pellets using the Micro to Midi Total RNA Purification System (Invitrogen Corporation, Carlsbad, CA). First strand cDNA synthesis was performed using the SuperscriptTM First-Strand Synthesis System for reverse transcription-polymerase chain reaction (RT-PCR) using oligo(dT) primers (Invitrogen Corporation).
Genomic DNA was harvested from EDTA-anticoagulated whole blood using the QIAamp® DNA Blood Mini Kit (Qiagen, Inc., Valencia, CA). Most primers were designed based on homologous regions of canine (GenBank accession nos. AF153316 and AF116270) and human DNA sequences for IIb and ß3.8,17 Normal equine cDNA sequence was used to design some of the primers.
DNA segments were amplified by PCR in an overlapping fashion using normal equine cDNA or genomic DNA as templates initially (Tables 1 and 2). The experiments were then repeated using cDNA or genomic DNA isolated from horses 1 and 2. In selected experiments, genomic DNA from relatives of horse 2 (dam, sire, and two half-siblings) were also evaluated. Amplification products were separated via electrophoresis on 1.5% agarose gels. DNA was extracted from target bands using the QIAquick Gel Extraction Kit (Qiagen, Inc.). Harvested bands were sequenced directly by a laboratory using an ABI 3100 Genetic Analyzer. Nucleotide and amino acid sequences from different species were compared using the AlignX program of the VectorNTI suite (Informax Invitrogen Life Science Software, Frederick, MD).
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IIb had 82% nucleotide identity with human and 87% with canine sequences. At the amino acid level, percent identities were 82 and 81%, respectively. The cDNA encoding ß3 in horses had 92% nucleotide identity with human and 93% with canine sequences. Amino acid identity for ß3 was 95% when compared with both human and canine sequences. Within the four calcium-binding domains of IIb, equine cDNA exhibited 86–88% identity with human nucleotide sequence. Within the four calcium-binding domains, amino acid identity between equine and human ranged from 83 to 91%. When equine nucleotide sequences were compared with canine sequences within the four calcium-binding domains, nucleotide identities ranged from 83 to 91%, and amino acid identities ranged from 75 to 100%. cDNA sequences for equine IIb and ß3 have been submitted to GenBank (GenBank accession nos. AY322154 and AY999064).
cDNA sequences encoding ß3 in horses 1 and 2 with GT were identical to sequences obtained from a normal horse. cDNA sequences encoding IIb were also identical except for a single guanine to cytosine (CGG to CCG) substitution in codon 41 in exon 2 (Fig. 1A, B). This change would result in the substitution of a proline for an arginine in a highly conserved region of the encoded protein.
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Comparisons of the cDNA sequences that encode IIb and ß3 in humans and dogs have been reported.9,10 Horse cDNA sequence encoding IIb was found to be more similar to dog (87%) than to human (82%), although when translated to the amino acid level, the percent identity with dog and human were very similar (81 and 82%, respectively). Murine monoclonal antibodies generated against human IIb do not cross-react with either the canine or equine platelet integrin subunit (Boudreaux, personal observation). Horse cDNA sequences encoding ß3 were more similar to human and dog sequences at both the nucleotide and amino acid level, with identities ranging from 92 to 95%. In spite of this similarity, monoclonal antibodies to human ß3 that cross-react with the canine subunit do not recognize horse ß3 (Boudreaux, personal observation). The reason for this discrepancy is not known but may be related to the conformation the ß3 subunit achieves when it is complexed with IIb on the platelet surface.
GT has been well characterized at the functional, biochemical, and molecular level in humans and dogs. In dogs, two different mutations, both in the gene encoding IIb, have been reported.1,11 According to the human GT database, maintained by the Mount Sinai School of Medicine, over 60 mutations have been documented in the gene encoding IIb and over 40 mutations have been documented in the gene encoding ß3 (http://sinaicentral.mssm.edu/intranet/research/glanzmann/menu). Over half of the reported cases of GT in humans involving the gene encoding IIb are classified as compound heterozygotes. The findings in this study indicate the likely cause of GT in horse 1 is a single nucleotide change in codon 41 in exon 2 of the gene encoding IIb. This area of the gene is highly conserved among humans, dogs, and horses. While cDNA sequence from horse 2 had the identical base change identified in horse 1, genomic DNA sequence was heterozygous for this base change. This suggests that horse 2 is likely a compound heterozygote. The finding that only the sire and not the dam was heterozygous for this base change further confirms that horse 2 is likely a compound heterozygote. The dam is likely heterozygous for the unidentified mutation. The other unidentified mutation apparently results in either total lack of expression or extreme instability of mRNA coding for IIb, such that message is not well represented in RT-PCR products of platelet RNA. Thus, this mutation may be located within the promoter region or within an intron or other nontranslated portion of the IIb gene. However, a nonsense mutation within the coding portion of the gene could also result in instability of the mRNA, via nonsense-mediated decay.18
The base change documented in exon 2 of horses 1 and 2 would be predicted to result in the change of encoded amino acid 41 from an arginine to a proline. The marked difference in structure and charge of proline compared with arginine, as well as the presence of two adjacent prolines at positions 40 and 41, would be predicted to result in marked instability of the beta-propeller region of the encoded protein. A similar missense mutation in exon 2 of the gene encoding IIb has been described in humans.19 In this case, a single nucleotide change resulted in the substitution of a proline for a leucine at amino acid position 55. Experiments demonstrated that the mutation resulted in severe impairment of expression of the IIbß3 complex on the surface of transfected COS7 cells. The authors hypothesized that the substituted proline caused an aberrant conformation in the encoded protein that prevented association of IIb with ß3, with ultimate lack of expression of the complex on the surface of cells.
This is the first characterization of cDNA sequences encoding platelet IIb and ß3 in normal horses and in two horses with GT. Future studies will be aimed at identifying the second mutation in the apparently compound heterozygote horse 2. Horses that have platelet-type hemorrhage, particularly epistaxis, that do not have thrombocytopenia, vasculitis, or von Willebrand disease, are candidates for the diagnosis of GT. Molecular-based screening assays, based on molecular defects identified in horses with GT, will greatly facilitate the identification of GT in horses with otherwise unexplained platelet-type bleeding.
Acknowledgements
We thank Dr. J. Taintor, Dr. J. Schumacher, Mrs. D. Czerkawski, E. Whatley, S. Spencer, A. Hall, D. Shiver, A. Mitchell, and K. Worley for their help with this project.
References
IIb gene from platelet-derived cDNA. Am J Vet Res 62:1486–1492, 2001[Medline]IIb are associated with type I Glanzmann's thrombasthenia in a Great Pyrenees Dog: a 14-base insertion in exon 13 and a splicing defect of intron 13. Vet Pathol 37:581–588, 2000IIb is responsible for a case of Glanzmann's thrombasthenia. Br J Haematol 118:833–835, 2002[Medline]
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