Platelet gene therapy improves hemostatic function for integrin alphaIIbbeta3-deficient dogs.

Activated blood platelets mediate the primary response to vascular injury. Although molecular abnormalities of platelet proteins occur infrequently, taken collectively, an inherited platelet defect accounts for a bleeding diathesis in ≈1:20,000 individuals. One rare example of a platelet disorder, Glanzmann thrombasthenia (GT), is characterized by life-long morbidity and mortality due to molecular abnormalities in a major platelet adhesion receptor, integrin αIIbß3. Transfusion therapy is frequently inadequate because patients often generate antibodies to αIIbß3, leading to immune-mediated destruction of healthy platelets. In the most severe cases allogeneic bone marrow transplantation has been used, yet because of the risk of the procedure it has been limited to few patients. Thus, hematopoietic stem cell gene transfer was explored as a strategy to improve platelet function within a canine model for GT. Bleeding complications necessitated the use of a mild pretransplant conditioning regimen; therefore, in vivo drug selection was used to improve engraftment of autologously transplanted cells. Approximately 5,000 αIIbß3 receptors formed on 10% of platelets. These modest levels allowed platelets to adhere to αIIbß3's major ligand (fibrinogen), form aggregates, and mediate retraction of a fibrin clot. Remarkably, improved hemostatic function was evident, with ≤135-fold reduced blood loss, and improved buccal bleeding times decreased to 4 min for up to 5 y after transplant. One of four transplanted dogs developed a significant antibody response to αIIbß3 that was attenuated effectively with transient immune suppression. These results indicate that gene therapy could become a practical approach for treating inherited platelet defects.

Characteristics, diagnosis, and treatment of inherited platelet disorders in mammals.

Inherited intrinsic platelet disorders have been identified in dogs, cattle, horses, and cats as well as other animals. The prevalence of mutations in some breeds is high, making these disorders potentially as common as von Willebrand disease in certain breed lineages.

Heterozygosity for P2Y12 receptor gene mutation associated with postoperative hemorrhage in a Greater Swiss Mountain dog.

A 3-year-old, female Greater Swiss Mountain dog developed a hemoperitoneum following an exploratory laparotomy and ovariohysterectomy. Platelet count, PT, APTT, and plasma von Willebrand factor antigen concentration were within RIs. A buccal mucosal bleeding time (BMBT) was prolonged. Given the probability of a hereditary thrombopathia, the dog was administered desmopressin, fresh platelet transfusions, and aminocaproic acid to control hemorrhage. Subsequently, DNA testing for the P2Y12 receptor gene mutation identified the dog as being a heterozygote (carrier). Further platelet function testing was performed following complete recovery. Results of a repeat BMBT and a point-of-care screening test using the Platelet Function Analyzer-100 (collagen/adenosine-diphosphate [ADP] test cartridge) were within RIs. Flow cytometric studies demonstrated a marked reduction in fibrinogen binding to the dog's platelets in response to ADP - adenosine diphosphate activation. Likewise, turbidimetric aggregometry revealed a complete absence of platelet aggregation in response to ADP. However, there were a normal aggregation response to the platelet agonist convulxin and a mild reduction in amplitude in response to γ-thrombin. This is the first report of a dog heterozygous for the P2Y12 receptor gene mutation exhibiting a bleeding tendency and having evidence of impaired platelet function in vitro in response to ADP activation. Given that the mutant allele for the P2Y12 thrombopathia appears to be widespread in the Greater Swiss Mountain dog breed, veterinarians need to be aware that both homozygotes and heterozygotes for this platelet receptor mutation are at risk of developing life-threatening bleeding following trauma or surgery.

Comparison of the activation peptide regions of protease-activated receptors (PAR) in members of the Family Felidae.

BACKGROUND: There is limited information regarding the nucleotides encoding or the predicted amino acid composition of protease-activated receptors (PAR) in cats. OBJECTIVES: The purpose of the study was to determine the nucleotide sequence and predicted amino acid composition of the activation peptide regions of protease-activated receptors PAR1, PAR3, and PAR4 in Felidae family members. METHODS: Genomic DNA isolated from whole blood samples collected from 10 domestic cats and 45 big cats representing 11 species was subjected to PCR using primers flanking the coding regions for the activation peptides of PAR1, PAR3, and PAR4. PCR products were isolated from agarose gels and submitted for sequencing. Nucleotide sequence data was used to predict the amino acid composition of the activation peptide and flanking regions of the 3 receptors. Predicted amino acid sequences were compared between Felidae members and to human beings. RESULTS: Variations in the predicted amino acid composition of the activation peptides and flanking regions of the various PAR were observed when comparing Felidae family members to each other and to human beings. CONCLUSIONS: While the activation peptide regions of the various PAR tend to be conserved, there are differences that may impact the ability of some agonists to mediate biased signaling events documented to occur in human platelets.

Comparison of the gene encoding, and the predicted amino acid composition of, platelet membrane receptor subunit glycoprotein Ibα in members of the family Felidae.

BACKGROUND: There is minimal information regarding platelet receptors in the family Felidae. Comparative studies assist with identifying amino acids critical for protein structure and function. OBJECTIVE: The purpose of the study was to compare the gene encoding, and the predicted amino acid composition of, platelet membrane receptor subunit GPIbα in Felidae family members. METHODS: Genomic DNA samples isolated from whole blood of 13 domestic cats and 50 big cats representing 8 different species were subjected to PCR using primers designed to flank the coding region of GPIbα in overlapping fashion. PCR products were separated via electrophoresis on agarose gels, and extracted products were submitted for sequencing. DNA sequences were used to predict the length and amino acid composition of the protein. RESULTS: Varying protein lengths were predicted in Felidae family members which were primarily due to polymorphisms in the variable number of tandem repeats region encoding the macroglycopeptide region of GPIbα. Other areas of the gene and predicted amino acid compositions were fairly conserved when compared to human sequences and between Felidae family members. CONCLUSION: Various polymorphisms within GPIbα, including length variants encoding the macroglycopeptide region, were identified in members of the family Felidae. More studies are needed to determine if a correlation exists between various polymorphisms and predisposition for hemorrhage or thrombosis as suggested in people.

A novel form of macrothrombocytopenia in Akita dogs.

Blood samples from 3 unrelated Akita dogs with a common history of persistent macrothrombocytopenia in the absence of clinical bleeding were sent to the Auburn University College of Veterinary Medicine (AUCVM) Clinical Pathology Laboratory for evaluation. Due to low platelet counts, one Akita dog had been treated with corticosteroids for presumed immune-mediated platelet destruction, and one Akita dog was treated with doxycycline for one month for presumed infection by a tick-borne agent. In spite of treatment, platelet counts remained low in both dogs. Given the absence of abnormal bleeding in all 3 dogs and lack of response to treatment in 2, congenital macrothrombocytopenia was suspected. Interestingly, platelets from all 3 dogs exhibited a consistent elongated platelet morphology. There were no morphologic abnormalities observed in other cell lines. While there have been anecdotal reports of a possible inherited macrothrombocytopenia in Akita dogs, scientific studies have not been done to verify these reports. This manuscript represents the first case report describing what is likely a congenital macrothrombocytopenia in Akita dogs based on persistently low platelet counts in the absence of clinical signs, and characterized by a unique platelet morphology.

Basset Hound thrombopathia in a 4-month-old female Ba-Shar (Sharp Asset).

A 3-month-old female Basset Hound-Shar Pei mix puppy (Ba-Shar or Sharp Asset) presented with oral bleeding due to a cracked molar. On physical exam, an aural hematoma was also noted that the owner indicated was chronic. The puppy was hospitalized for over 24 h until the bleeding was brought under control. At 4 months of age, the puppy again presented with oral bleeding due to loss of deciduous teeth and was hospitalized until bleeding was controlled. Coagulation screening tests, platelet numbers, and von Willebrand Factor antigen levels were within reference limits. Based on the presence of platelet-type bleeding in the face of normal screening test results, samples were submitted for DNA testing for Basset Hound thrombopathia. The puppy tested as affected for the calcium and diacylglycerol regulated guanine nucleotide exchange factor I (CalDAG-GEFI) mutation causing this disorder. This is the first time thrombopathia has been diagnosed in a "designer" breed.

Abnormal coagulation factor VIII transcript in a Tennessee Walking Horse colt with hemophilia A.

Hemophilia A is an X-chromosome-linked disorder caused by a deficiency in factor VIII (FVIII). Although foals have been diagnosed with hemophilia A based on deficiency in FVIII activity, causative gene mutations have not been identified. The genomic DNA and cDNA encoding FVIII of a Tennesee Walking Horse colt affected with hemophilia A and the genomic DNA of his dam and a normal unrelated horse were analyzed with no splice site or coding sequence abnormalities identified in any of the horses. Polymerase chain reactions (PCR) were then performed on hepatic cDNA from the affected colt and an unrelated normal horse, and no product was obtained for the sequence between and including exon 1 and exon 2 in the affected colt. Based on these results, suspected mutations were identified in the noncoding region of FVIII (intron 1), and genomic sequencing of intron 1 in the dam and the affected colt suggested maternal inheritance.

Characterization of the mutations causing hemophilia B in 2 domestic cats.

The purpose of the present study was to determine the normal sequence for the gene encoding factor IX in cats and to characterize the genetic basis for hemophilia B in 2 unrelated male, domestic, mixed-breed cats. Genomic DNA sequence for the entire coding region of the factor IX gene was determined in the affected cats and compared to the sequence obtained from a healthy cat. The factor IX gene in cats encodes a mature protein consisting of 420 amino acids, unlike genes in humans and dogs that encode 415 and 413 amino acid proteins, respectively. Affected cat 1 had a single nucleotide change in exon 8 at the 1st nucleotide position of the codon encoding an arginine (CGA to TGA) at amino acid position 338. This mutation would be predicted to result in the appearance of a premature stop codon in the portion of the gene encoding much of the catalytic domain of the protein. Affected cat 2 had a single nucleotide change in exon 4 at the 2nd nucleotide position of the codon encoding amino acid 82 (TGT to TAT), which would be predicted to result in the substitution of a tyrosine for a cysteine. This substitution would likely result in disruption of a disulfide bond crucial to normal protein structure and function. This study represents the 1st time hemophilia B has been characterized at the molecular level in cats.

Correction of a large animal model of type I Glanzmann's thrombasthenia by nonmyeloablative bone marrow transplantation.

OBJECTIVE: The purpose of this study was to determine if nonmyeloablative bone marrow transplantation would induce stable hematopoietic chimerism that would correct the bleeding diathesis associated with type I Glanzmann's thrombasthenia (GT). METHODS: Three young dogs (less than 12 weeks of age) with GT were transplanted with DLA-matched bone marrow from littermates. Recipients received a sublethal dose (200 cGy) of total-body irradiation (TBI) prior to infusion with bone marrow (1-4 x 10(8) cells/kg). Recipient dogs were immunosuppressed with cyclosporine (15 mg/kg) and mycophenolate mofetil (10 mg/kg). Chimerism was determined by quantitation of donor microsatellite repeat polymorphisms in peripheral blood DNA and by flow cytometry to detect the presence of glycoproteins IIb and IIIa on platelets. Platelet function was assessed by a clot retraction test. RESULTS: One dog died one week posttransplant due to hemorrhage. Another dog died four weeks posttransplant from an unrecognized congenital heart defect and complications due to canine distemper virus infection. At the time of death, microsatellite analysis indicated 35 to 50% chimerism. Flow cytometry showed 20% of circulating platelets positive for glycoproteins IIb and IIIa. The third dog is alive and doing well approximately two years posttransplant. Hematopoietic chimerism has been sustained at 35 to 60% with approximately 30% of the platelets positive for glycoproteins IIb and IIIa. Platelet function is normal based on clot retraction. The animal does not have clinical signs of bleeding. CONCLUSIONS: These observations suggest that GT and perhaps other severe inherited platelet disorders can be corrected using nonmyeloablative bone marrow transplantation to establish partial chimerism with normal platelets in the platelet compartment.

Evaluation of the genes encoding CD39/NTPDase-1 and CD39L1/NTPDase-2 in horses with and without abnormal hemorrhage and in horses with pathologic evidence of exercise-induced pulmonary hemorrhage.

BACKGROUND: Exercise-induced pulmonary hemorrhage (EIPH) is a common disorder of equine athletes. The role of polymorphisms in genes encoding hemostasis-regulatory proteins in horses with abnormal hemorrhage is unknown. OBJECTIVES: The goal of this study was to evaluate the genes encoding 2 ectonucleotidases, CD39/NTPDase-1 and CD39L1/NTPDase-2, and one ecto-5' nucleotidase, CD73, in horses with abnormal hemorrhage or pathologic changes consistent with EIPH. METHODS: Twenty-three horses with histories of abnormal hemorrhage, 8 horses with gastrointestinal signs, and 45 healthy horses were evaluated using polymerase chain reaction-based techniques. Formalin-fixed tissues from 21 horses with pathologic changes consistent with EIPH were also evaluated. RESULTS: Three single nucleotide polymorphisms (SNPs) were identified in the gene encoding CD39 and one SNP was identified in the gene encoding CD39L1. No SNPs were identified in the gene encoding CD73. CD39 SNPs were identified in 19 of 20 (95%) horses with unexplained hemorrhage and 20 of 21 (95%) horses with pathologic features consistent with EIPH. CD39L1 SNPs were identified in 6 of 20 (30%) horses with unexplained hemorrhage and 8 of 21 (38%) horses with pathologic features consistent with EIPH. CD39 and CD39L1 SNPs were identified in 5 of 8 (62.5%) and one of 8 (12.5%) horses, respectively, presenting with colic or weight loss. CD39 and CD39L1 SNPs were identified in 28 of 45 (62%) and 13 of 45 (28.8%) healthy horses, respectively. CONCLUSIONS: CD39 and CD39L1 are critically important in maintaining normal hemostasis and limiting inflammation. Further studies are needed to evaluate their role in the pathogenesis of equine EIPH.

Successful ovariectomy in a dog with Glanzmann thrombasthenia.

Ovariectomy was performed in a 7-month-old Great Pyrenees with a severe congenital bleeding disorder. A diagnosis of Glanzmann thrombasthenia, a rare, congenital bleeding disorder characterized by a functional platelet defect, was later confirmed by isolation of genomic DNA from blood and amplification of exon 13 and intron 13 of the gene encoding for platelet glycoprotein subunit alphaIIb. Perioperative management consisted of administration of platelet-rich plasma prior to surgery and the use of high-frequency electrocoagulation to minimize tissue trauma. In addition, ovariectomy, rather than ovariohysterectomy, was performed to minimize surgical exposure required and manipulation of the urogenital tract. Results in this dog suggest that a combination of preoperative transfusion with functional platelets and use of techniques to minimize tissue trauma may allow abdominal surgery to be performed successfully in dogs with functional platelet disorders.

Babesia gibsoni infection among dogs in the southeastern United States.

OBJECTIVE: To identify subclinical Babesia gibsoni infection in American Pit Bull Terriers from the southeastern United States and to determine the genetic sequence of parasite DNA isolated from these dogs. DESIGN: Case series. ANIMALS: 33 American Pit Bull Terriers and 87 dogs of various other breeds. PROCEDURE: Blood smears were examined for microscopic evidence of the parasite, and DNA was extracted from blood samples and used in a polymerase chain reaction (PCR) assay designed to amplify the small subunit ribosomal RNA gene sequence of B. gibsoni. Amplification products of the expected size were sequenced, and sequences were compared with published sequences for B. gibsoni isolates. Hematocrit, platelet count, mean platelet volume, WBC count, and eosinophil count were compared between dogs with positive PCR assay results and dogs with negative results. RESULTS: Results of the PCR assay were positive for 18 of the 33 (55%) American Pit Bull Terriers, including all 10 dogs with microscopic evidence of parasitemia. Only 1 of these dogs was clinically ill at the time blood samples were collected. Results of microscopic evaluation of blood smears and of the PCR assay were negative for the 87 other dogs. Hematocrit and platelet count were significantly lower in dogs with positive PCR assay results than in dogs with negative results. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that American Pit Bull Terriers in the southeastern United States may be subclinically infected with B. gibsoni. However, subclinical infection was not identified in dogs of other breeds from the same geographic area.

Evaluation of Effects of Dietary n-6 to n-3 Fatty Acid Ratios on Leukotriene B Synthesis in Dog Skin and Neutrophils.

Résumé- Les effets d'alimentations supplémentées avec des ratios croissants decides gras polyinsaturés n - 6/n - 3 sur la synthèse de leucotriènes B dans la peau du chien et les neutrophiles sont présentés. Trente chien Beagles ont reçu pendant 2 mois avec une alimentation ayant un ratio n -6/n -3 de 28:1. Des aliments expérimentaux contenant des ratios de 5:1, 10:1, 25:1, 50:1 et 100:1 (six chiens par groupe) ont été administrés ensuite pendant 12 semaines. A la fin des deux mois d'alimentation témoin et au bout de 6 et 12 semaines d'alimentation expérimentale, les concentrations de LTB4 et LTB5 dans la peau et les neutrophiles ont été déterminées. Les neutrophiles de chiens ayant mangé des aliments de ratio 5:1 et 10:1 ont synthétisé 30-33 pourcent moins de LTB4 (P <0,05) et 370-500 pourcent plus de LTB5 (P <0,05) à 6 et 12 semaines, mais le relargage d'anions superoxide était inchangé. La peau des chiens stimulée par un lipopolysaccharide a synthétisé 48 à 62 pourcent moins de LTB4 (P < 0,05) et 48 à 79 pourcent moins de LTB5 (P <0,05) à 12 semaines. [Vaughan, D. M., Reinhart, G. A., Swaim, S. F., Lauten, S. D., Garner, C. A., Boudreaux, M. K., Spano, J. S., Hoffman, C. E., Conner, B. Evaluation of effects of dietary n -6 to n - 3 fatty acid ratios on leukotriéne B synthesis in dog skin and neutrophils. (Evaluation de l'effet du ratio d'acides gras n - 6/n - 3 dans l'alimentation sur la synthèse de leucotriènes B dans la peau du chien et les neutrophiles). Resumen- Evaluamos los efectos producidos por el aumento en la proportion de ácidos grasos poli-insatu-rados n-6 a n-3 en la dieta sobre la sintesis de leucotrienos B en la piel del perro y en los neutrófilos. Se administró durante dos meses una dieta con una proporión 28:1 de ácidos grasos n-6 a n-3 a un grupo de treinta Beagles. Se administraron dietas experimentales con proporciones de 5:1, 10:1, 25:1, 50:1 y 100:1 durante 12 semanas (seis perros por grupo). Se cuantificaron los niveles de leucotrienos B4 y B5 en la piel y en los neutrófilos al final de los dos meses de dieta control y a las 6 y 12 semanas de la dieta-tratamiento. Los neutrófilos de perros con dietas 5:1 y 10:1 sintetizaron 30 a 33% menos leucotrieno B4 (P < 0.05) y 370 a 500% más leucotrieno B5 (P <0.05) a las 6 y 12 semanas pero no alteraron la liberación de aniones superóxido durante la espiracion. La piel de perro estimulada con lipopolisacáridos sintetizó de 48 a 62% menos leucotrieno B4 (P < 0.05) y 48 a 79% más leucotrieno B5 (P < 0.05) a las 12 semanas. [Evaluation of effects of dietary n-6 to n-3 fatty acid ratios on leukotriene B synthesis in dog skin and neutrophils (Effecto de la proporción de ácidos grasos n-6 a n-3 en la dieta sobre la sintesis de leucotrienos B en la piel del perro y en los neutrófilos). Abstract- The effects of diets supplemented with increasing ratios of n-6 to n-3 polyunsaturated fatty acids on leukotriene B synthesis in dog skin and neutrophils were evaluated. Thirty Beagles were conditioned for 2 months on a diet with an n-6 to n-3 fatty acid ratio of 28:1. Experimental diets, containing n-6 to n-3 ratios of 5:1, 10:1, 25:1, 50:1 and 100:1 (six dogs/group), were fed for 12 weeks. At the end of the 2 month control diet period, and again at 6 and 12 weeks of treatment feeding, leukotriene B4 and leukotriene B5 were quantitated in skin and neutrophils. Neutrophils from dogs fed the 5:1 and 10:1 diets synthesized 30-33 per cent less leukotriene B4 (P < 0.05) and 370-500 per cent greater leukotriene B5 (P < 0.05) at 6 and 12 weeks, but had no change in the release of superoxide anions during respiratory burst. Lipopolysaccharide-stimu-lated dog skin synthesized 48-62 per cent less leukotriene B4 (P < 0.05) and 48-79 per cent more leukotriene B5 (P <0.05) at 12 weeks.

Two novel missense mutations associated with hemophilia A in a family of Boxers, and a German Shepherd dog.

BACKGROUND: Hemophilia A is an X-linked disorder caused by a deficiency in coagulation factor VIII. Over 2300 unique mutations in the gene-encoding factor VIII have been documented in people, but limited information is known in dogs. An 11-week-old male Boxer and a 5-year-old male German Shepherd were diagnosed with hemophilia A based on diminished factor VIII activity. OBJECTIVE: The purpose of the study was to identify genetic mutations associated with hemophilia A in both dogs. METHODS: Genomic DNA was isolated from EDTA blood samples from the affected German Shepherd and Boxer, the Boxer's dam, 3 female siblings, and one asymptomatic male sibling. Primers were designed in noncoding regions to amplify the 26 exons of the factor VIII gene via PCR. RESULTS: The affected Boxer sequence revealed a single nucleotide change, cytosine to guanine, at nucleotide position 1412 (1412C>G) in exon 10. The change is predicted to result in the substitution of arginine for proline at amino acid 471 (P471R) in the A2 domain of factor VIII. The dam and female siblings were carriers, the male sibling did not have the mutation. The German Shepherd dog had a single nucleotide change of a guanine to adenine at position 1643 (1643G>A) in exon 11, predicting the substitution of tyrosine for cysteine at amino acid 548 (C548Y) in the A2 domain. CONCLUSIONS: Here we document 2 mutations associated with canine hemophilia A associated with < 1% factor VIII activity, similar to that in people. Another related Boxer with the P471R mutation was later identified.

A novel point mutation in the ß1-tubulin gene in asymptomatic macrothrombocytopenic Norfolk and Cairn Terriers.

BACKGROUND: An asymptomatic macrothrombocytopenia, phenotypically similar to asymptomatic inherited macrothrombocytopenia in Cavalier King Charles Spaniels, was described in a group of Norfolk Terriers (NT) from Northern Italy, and isolated cases were also reported in Cairn Terriers (CT). OBJECTIVES: The purpose of this work was to evaluate for the presence of a genetic defect in the ß1-tubulin gene in macrothrombocytopenic NT and CT. METHODS: Samples from 20 healthy dogs (13 NT and 7 CT) were collected at different institutions in Italy (n = 8), United Kingdom (n = 3), and United States (n = 9). Genomic DNA was harvested from EDTA-anticoagulated blood and all coding areas and exon-intron splice sites in the gene encoding ß1-tubulin were amplified and sequenced. RESULTS: Twelve dogs (9 NT and 3 CT) showed a single nucleotide polymorphism (SNP) in exon 1 at nucleotide position 5 (G5A) that would result in the change of an arginine to a histidine at amino acid position 2 (R2H). Four dogs (3 NT and one Cairn Terrier) were heterozygous for the SNP, and 4 dogs (one Norfolk Terrier and 3 CT) matched the normal canine genome. Homozygous dogs for the SNP were macrothrombocytopenic with platelet counts ranging from 19,000 to 110,000/µL. Heterozygous and normal dogs had normal platelet counts and morphology. None had the CKCS point mutation. CONCLUSIONS: The ß1-tubulin N-terminal amino acids form the nucleotide-binding domain and thus this mutation could affect GTP binding enough to influence platelet formation in homozygous but not in heterozygous dogs. The presence of macrothrombocytopenia only in homozygous affected dogs reveals an association between the SNP and the phenotype.

Identification of potential platelet alloantigens in the Equidae family by comparison of gene sequences encoding major platelet membrane glycoproteins.

BACKGROUND: Platelet alloantigens in horses may play an important role in the development of neonatal alloimmune thrombocytopenia (NAIT). OBJECTIVE: The objective of this study was to evaluate genes encoding major platelet glycoproteins within the Equidae family in an effort to identify potential alloantigens. METHODS: DNA was isolated from blood samples obtained from Equidae family members, including a Holsteiner-Oldenburg cross, a Quarter horse, a donkey, and a Plains zebra (Equus burchelli). Gene sequences encoding equine platelet membrane glycoproteins IIb, IIIa (integrin subunits αIIb and ß3), Ia (integrin subunit α2), and Ibα were determined using PCR. Gene sequences were compared to the equine genome available on GenBank. Polymorphisms that would be predicted to result in amino acid changes on platelet surfaces were documented and compared with known alloantigenic sites documented on human platelets. RESULTS: Amino acid differences were predicted based on nucleotide sequences for all 4 genes. Nine differences were documented for αIIb, 5 differences were documented for ß3, 7 differences were documented for α2, and 16 differences were documented for Ibα outside the macroglycopeptide region. CONCLUSIONS: This study represents the first effort at identifying potential platelet alloantigens in members of the Equidae Family based on evaluation of gene sequences. The data obtained form the groundwork for identifying potential platelet alloantigens involved in transfusion reactions and neonatal alloimmune thrombocytopenia (NAIT). More work is required to determine whether the predicted amino acid differences documented in this study play a role in alloimmunity, and whether other polymorphisms not detected in this study are present that may result in alloimmunity.

Leukocyte adhesion deficiency type I in a mixed-breed dog.

A 6-month-old, neutered male, mixed-breed dog was examined for a 2-month persistent fever, nonhealing dermal metacarpal area wound, and leukocytosis (47.0-198.0 × 10(3)/µl). Serum chemistry findings included hypoalbuminemia, hyperglobulinemia, hyperphosphatemia, and hyperphosphatasemia. Complete blood cell count results revealed a moderate microcytic, hypochromic nonregenerative anemia with a profound leukocytosis (198.5 × 10(3)/µl), characterized by neutrophilia with toxicity and hypersegmentation, and significant band cells. Tick-borne disease titers (genera Anaplasma, Ehrlichia, and Borrelia) were negative, as were polymerase chain reaction for other infectious agents (genera Hepatozoon, Mycobacterium, Mycoplasma; and Canine distemper virus). No agents were identified in a deep dermal biopsy (conventional and special histochemical stains) of the chronic draining, metacarpal region lesion. Cytology of the draining tract revealed numerous mixed bacteria and a surprising lack of neutrophils. Chronic occult blood loss with iron deficiency was considered a possible cause of the anemia. Differentials for the leukon were chronic established inflammation (occult infectious agent), chronic neutrophilic leukemia, paraneoplastic leukocytosis (neoplastic source of granulocyte colony-stimulating factor [CSF] or granulocyte-macrophage CSF), and leukocyte adhesion deficiency (LAD). The possibility of a LAD disorder was further investigated because of the noted hypersegmented neutrophils, absence of neutrophils in the cytology sample, the animal's young age, and persistence of clinical and laboratory signs. Flow cytometry of blood neutrophils showed a 60% reduction in surface expression of the ß2-integrin (CD18) subunit, whereas neutrophil function tests (oxidative burst and phagocytosis) were normal. Genetic testing revealed a homozygous missense mutation in the ß2-integrin subunit gene, previously recognized only in purebred Irish Setters, leading to a diagnosis of LAD type 1 disorder in this mixed-breed dog.