Characterization of the cDNA and genomic DNA sequence encoding for the platelet integrin alpha IIB and beta III in a horse with Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is characterized by a defect of platelet aggregation. This autosomal recessive genetic disorder is caused by an abnormality of the platelet glycoprotein receptors alpha IIb or beta III. Recently, we identified a horse with clinical and pathological features of GT. The aim of this study was to describe this case of GT at the molecular level. A point mutation from G to C in exon 2 of ITGA2B causing a substitution of the expected amino acid arginine 72 (Arg(72)) by a proline (Pro(72)) was encountered. This amino acid change may result in abnormal structural conformations that yield an inactive alpha IIb subunit. The genomic DNA analysis showed that this horse was homozygous for the missense mutation.     

Characterization of the cDNA Encoding alphaIIb and beta3 in normal horses and two horses with Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is an inherited, intrinsic platelet defect characterized by a quantitative or qualitative change in the platelet glycoprotein complex IIb-IIIa (integrin alpha(IIb)beta3). 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.     

Clinical, biochemical, and molecular aspects of Glanzmann's thrombasthenia in humans and dogs

Glanzmann's thrombasthenia (GT) is an inherited, intrinsic platelet function defect that involves the platelet glycoprotein complex IIb-IIIa, also known as the fibrinogen receptor and the integrin alphaIIbbeta3. The defect was originally described by Dr. Glanzmann in humans in 1918 as a bleeding disorder that differed clinically from other known coagulopathies. Over the decades that followed, researchers determined the biochemical and molecular basis for the disease in humans. Otterhounds with thrombasthenic thrombopathia, described in the 1960s, were the only animal model that closely resembled the disease described in humans until 1996. At that time, a Great Pyrenees dog was identified with unequivocal clinical and biochemical features of Type I GT The cDNA encoding for glycoproteins IIb and IIIa were sequenced in normal dogs in 1999, allowing for identification of specific mutations causing Type I GT in both Otterhounds and Great Pyrenees dogs. Knowing the molecular basis for Type I GT in dogs as well as the cDNA sequences in normal dogs should enhance the understanding of structure/function relationships of the alphaIIbbeta3 integrin and provide an excellent animal model for studies aimed at correction of GT in humans. The following review focuses on the structure and function of this platelet receptor and reviews the molecular, biochemical, and clinical aspects of Glanzmann's thrombasthenia in humans and dogs.     

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.     

Two genetic defects in alphaIIb 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

Glannzmann's thrombasthenia (GT) is an autosomal recessive bleeding disorder caused by qualitative or quantitative deficiencies of the platelet membrane glycoprotein alphaIIbbeta3. This is the first report of a molecular genetic basis for type I GT in dogs. As previously reported, a thrombasthenic Great Pyrenees dog (dog No. 1) experienced uncontrolled epistaxis despite results of coagulation screening tests, platelet quantitation, and von Willebrand factor quantitation that were within reference ranges. Platelet aggregation was minimal in response to agonists. Flow cytometry, autoradiography, and immunoblot experiments demonstrated either marked reduction or absence of glycoproteins alphaIIb and beta3. In this study, we report the presence of a 14-base insertion in exon 13 and defective splicing of intron 13 in the alphaIIb gene of two thrombasthenic dogs (Nos. 1 and 8). The insertion disrupted the fourth alphaIIb calcium-binding domain, caused a shift in the reading frame and resulted in a premature termination codon. Possible consequences of this mutation include decreased alphaIIb mRNA stability and production of truncated alphaIIb protein that lacks the transmembrane and cytoplasmic domains and a large portion of the extracellular domain. We identified the dam, sire, and three littermates of dog No. 8 as carriers of the alphaIIb mutation. Canine alphaIIb and beta3 genes share significant homology with the genes in human beings, making canine GT an excellent translational model for human GT. A defined molecular basis for canine GT will enhance ongoing gene therapy research and increase the understanding of structure-function relationships of this integrin.     

Glanzmann thrombasthenia in an Oldenbourg filly

An 18-month-old Oldenbourg filly was presented with a bleeding diathesis. Laboratory testing included platelet count, gingival bleeding time, prothrombin time (PT), activated partial thromboplastin time (aPTT), von Willebrand factor (vWf) antigen, clottable fibrinogen, clot retraction time, PFA-100 closure time, platelet aggregometry (on platelet-rich plasma), and thrombelastography (TEG). TEG was performed by using kaolin and tissue factor as coagulation activators. Expression of the platelet receptor for fibrinogen was assessed by flow cytometry by using anti CD41 (alpha(IIb) or glycoprotein IIb)/CD61 (beta(III) or glycoprotein IIIa) and anti-CD41 antibodies. Abnormal laboratory findings included prolonged oral mucosal bleeding time (>12 hours), prolonged closure time with collagen/ADP (>300 seconds), and absence of clot retraction after 60 minutes. TEG reaction times were similar with kaolin and tissue factor in the patient and a control horse. However, maximum amplitudes in the patient were decreased with both kaolin (43.7 mm; control, 63.9 mm) and tissue factor (37.7 mm; control, 57.8 mm). Platelet aggregation responses to ADP and collagen were profoundly reduced in the affected horse compared with a control. Flow cytometry showed an absence of CD41 and decreased expression of CD41/CD61-reacting antigen on the patient's platelets compared with those from a control horse. The laboratory findings supported a diagnosis of Glanzmann thrombasthenia, likely caused by a mutation in the gene encoding the GPIIb subunit.     

Investigation of a novel, heritable bleeding diathesis of Thoroughbred horses and development of a screening assay

BACKGROUND: Bleeding in racing horses associated with exercise appears to be multifactorial, and clinical investigation into severe cases rarely occurs. Previously, we reported a severe bleeding diathesis in a Thoroughbred mare. Herein, we describe the cellular physiology of this defect, provide a diagnostic tool for identifying it, and demonstrate that the dysfunction is heritable. HYPOTHESIS: The subject has a heritable defect in platelet secretion that reduces thrombin generation in the absence of additional plasma factors and delays the onset of thrombin production even in the presence of these factors. ANIMALS: The study included 3 clinically normal Thoroughbred horses: the subject and her offspring. METHODS: Washed platelets were examined for their ability to (1) translocate phosphatidylserine to the outer leaflet of the platelet membrane as determined by annexin-V binding, (2) generate thrombin as assessed by the activity of the prothrombinase enzyme complex, and (3) bind fibrinogen and form aggregates as determined by flow cytometry. RESULTS: Subject and offspring platelets created procoagulant surfaces by translocating phosphatidylserine. The subject's platelets demonstrated reduced prothrombinase activity, resulting in decreased production of thrombin relative to control platelets. Subject and offspring platelets bound less fibrinogen than control platelets when stimulated with thrombin. CONCLUSIONS AND CLINICAL IMPORTANCE: The subject mare has a transmissible defect that involves reduced generation of thrombin by activated platelets, resulting in decreased aggregation and ineffective clotting. A flow cytometric assay of fibrinogen binding to washed platelets discriminates individuals with this platelet dysfunction and may be useful for discerning subclinical congenital or acquired platelet dysfunctions.     

Prophylactic use of a lyophilized platelet product for rhinoscopic diagnosis and treatment of sinonasal aspergillosis in a dog with a P2Y12 platelet receptor mutation

Objective

To describe the periprocedural use of a lyophilized platelet product during rhinoscopic diagnosis and treatment of sinonasal aspergillosis in a Greater Swiss Mountain Dog with a P2Y12 platelet receptor disorder.

Case Summary

After the development of severe epistaxis, a Greater Swiss Mountain Dog was diagnosed with thrombopathia secondary to a P2Y12 receptor gene mutation. Concurrent primary nasal disease was also suspected due to persistent mucopurulent nasal discharge. One month after the initial presentation for epistaxis, the dog was readmitted for workup of nasal disease. Computed tomography of the head showed turbinate lysis and regional lymphadenopathy. Because of concern for a high risk of bleeding in a thrombopathic patient subjected to rhinoscopy and nasal biopsies, a lyophilized platelet product was administered prior to the procedure. Rhinoscopic exam revealed fungal plaques consistent with Aspergillus spp. that were later confirmed on fungal culture to be Aspergillus fumigatus. Rhinoscopic biopsies were performed as well as debridement of the fungal plaques, followed by topical administration of clotrimazole solution. Bleeding was minimal during and after the procedure, and the dog recovered uneventfully.

New or Unique Information Provided

This is the first report of the prophylactic use of lyophilized platelets in a thrombopathic patient undergoing an invasive procedure with potential for significant hemorrhage. Minimal bleeding occurred during the procedure, suggesting that lyophilized platelets could be used for the prevention of bleeding in thrombopathic patients undergoing invasive procedures.     

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

A novel hereditary disorder of platelets was identified across 5 generations of a family of Greater Swiss Mountain dogs. The first dog identified with the mutation bled excessively following routine ovariohysterectomy and required multiple transfusions. Coagulation screening assays, platelet counts, and von Willebrand factor antigen activity were within reference intervals. Flow cytometric studies indicated that platelets from the affected dog expressed normal levels of glycoproteins IIb and IIIa and responded to 2 platelet-activating agents, convulxin and platelet-activating factor, but not to ADP. Based on DNA studies, a 3 base-pair deletion predicted to result in elimination of a serine from the extracellular domain was identified in the gene encoding P2Y12, an ADP receptor protein located on platelet membranes. Flow cytometric analysis of platelets and studies of DNA performed concurrently on 2 unrelated Greater Swiss Mountain dogs were unremarkable. The mutation was subsequently identified in the sire, the maternal grand-dam, a maternal great grandparent, a paternal great grandparent, and a great-great grandparent. The sire was homozygous, but had not yet been identified as having a hemostatic disorder; the other 4 dogs were carriers. This is the first report of a mutation in the gene encoding the ADP receptor P2Y12 in a domestic animal. P2Y12 is the same receptor targeted by ticlopidine and clopidogrel, platelet inhibitors used in lieu of aspirin in people at risk for cardiovascular disease; thus, spontaneous bleeding is not expected unless there are other contributing factors. This disorder is particularly troublesome because spontaneous hemorrhage is absent to mild in affected dogs; however, following routine surgical procedures or trauma, excessive bleeding could occur and have possible fatal consequences.     

Thrombopathies causing bleeding in a boxer and mixed-breed dog

Hereditary platelet function disorders are clinically characterized by recurrent surface bleeding and prolonged bleeding time, despite normal platelet count and coagulation tests. The authors describe persistent thrombopathies in two young dogs with increased bleeding tendencies but with normal plasma coagulation times and von Willebrand factor (vWf) concentrations. Buccal mucosal bleeding times were prolonged in both dogs. In aggregation studies, platelets underwent only a shape change or minimal aggregation in response to adenosine diphosphate and collagen. Whole-platelet adenine nucleotide concentrations were normal. Electron microscopic evaluation of fibrinogen and vWf binding to the platelets of case no. 1 demonstrated the presence of glycoprotein IIb/IIIa and Ib receptors. Thus, the intrinsic platelet function defects may be different in these two dogs and may likely represent secretion/signal transduction disorders.     

Preliminary studies of a platelet function disorder in Simmental cattle.

A bleeding disorder due to abnormal platelet function occurs in Simmental cattle. Whole blood from these animals underwent good clot retraction. Platelet aggregation in response to adenosine diphosphate (ADP) and collagen in a whole blood aggregation system was markedly impaired. Normal bovine platelets in a whole blood aggregation system showed very little aggregation in response to epinephrine and arachidonic acid. Aggregation in platelet-rich plasma was negligible in response to ADP, collagen and thrombin. Dense granule release of radiolabelled serotonin from the platelets of one affected cow was similar to that of normal bovine platelets. Platelet membrane glycoprotein electrophoresis with the platelets of one affected cow revealed no quantitative abnormalities. These findings reveal similarities and differences in thrombopathic Simmental platelet function when compared to human Glanzmann's thrombasthenia and Basset Hound thrombopathia.     

Identification of an Intrinsic Platelet Function Defect in Spitz Dogs

A recently identified intrinsic platelet function defect in 2 Spitz dogs is described. Both affected dogs had a history of chronic intermittent bleeding primarily from the nasal, oral, and gastrointestinal mucosa. Platelet aggregation in response to adenosine diphosphate (ADP), collagen, and platelet activating factor (PAF) was absent; however, platelet shape change did occur. Platelets aggregated in response to gamma thrombin, although a delayed onset and a reduced velocity of aggregation were present. Platelet ¹⁴C-serotonin release was diminished in response to collagen and PAF. Glycoprotein Illa was detected on the surface of platelets by flow cytometry. Platelets were morphologically normal under light and electron microscopy. Two male Spitz dogs, related to one of the affected dogs, did not have a bleeding diathesis. Collagen-induced platelet aggregation, however, was diminished in these 2 dogs. This platelet defect most closely resembles the defect described in Basset hounds.     

Immune thrombocytopenia (ITP): Pathophysiology update and diagnostic dilemmas

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder. The understanding of ITP pathogenesis is rapidly evolving. We now recognize ITP as a complex and heterogeneous syndrome that results from a combination of humoral and cell‐mediated attacks on platelets peripherally and megakaryocytes in the bone marrow. Autoantibody‐mediated ITP also varies in the pathway used to clear platelets, which depends on the platelet glycoprotein being targeted. Moreover, ITP patients present with variable bleeding severities and treatment responses that do not closely correlate with platelet count. A gold standard diagnostic test for ITP is lacking, and biomarkers to assess disease severity are in their infancy. This review provides an update on the immunopathogenesis of ITP and summarizes currently available tests for ITP diagnosis, prediction of disease severity, and treatment responses. Given the heterogeneous pathogenesis and clinical presentation of ITP, we highlight the need for the development of diagnostic and prognostic tests that would allow for the individualized management of a complex disease.     

Evaluation of glycoprotein Ib expression on feline platelets

OBJECTIVE: To determine whether platelets obtained from cats expressed glycoprotein Ib (GPIb). SAMPLE POPULATION: Platelets obtained from 11 specific-pathogen-free cats. PROCEDURE: Platelets were analyzed by use of immunofluorescence microscopy, flow cytometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western immunoblot analysis, and immunoprecipitation. RESULTS: Immunofluorescence microscopy and flow cytometry revealed the protein on the surface of feline platelets. Biochemical studies (western immunoblot analysis and immunoprecipitation) revealed a 140-kd membrane glycoprotein. Additional biochemical studies revealed that feline GPIb was sensitive to proteolysis, because platelet cytoskeletons prepared with low concentrations of a calpain inhibitor (ie, leupeptin; 100 microg/ml) had substantial proteolysis, and there was an association of protein fragments with the actin cytoskeleton. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of these results indicate that feline platelets express a 140-kd membrane protein that is recognized by monoclonal antibodies developed against GPIb. Application of standardized ELISA to quantitate glycocalicin, the water-soluble fragment of GPIb, may provide important information on the production of microvesicles, increased platelet turnover, and abnormal proteolysis.     

Repair of an incompetent urethral sphincter in a mare

Objective: To describe successful surgical treatment of urinary incontinence caused by a ruptured and/or transected urethral sphincter in a mare. Study Design: Clinical report. Animal: A 7‐year‐old, Swedish Warmblood mare with urinary incontinence. Methods: The urethral sphincter, which had been damaged during removal of a cystic urolith, was repaired by apposing the ends of the disrupted urethralis muscle and tunica muscularis. Results: The mare was no longer incontinent after repair of the defect by apposition of the ends of the urethralis muscle and tunica muscularis. Conclusions: Transection and/or rupture of the urethral sphincter of a mare may result in urinary incontinence. Clinical Relevance: Apposition of the ends of the ruptured or transected urethralis muscle and tunica muscularis can correct urinary incontinence caused by this defect.     

Platelet size,platelet surface-associated IgG,and reticulated platelets in dogs with immune-mediated thrombocytopenia

Abstract: Immune-mediated thrombocytopenia (IMT) is a disorder in which bound IgG on the surface of platelets results in platelet removal and alterations in mean platelet volume. Using flow cytometry, alterations in platelet size, platelet surface-associated IgG (PSAIgG), and numbers of reticulated platelets were determined in 13 dogs with primary IMT and 4 dogs with secondary IMT induced by experimental infection with Babesia gibsoni . Effects of sample age on platelet parameters also were determined, using samples from 20 dogs with normal platelet counts analyzed within 4 hours and after 24, 48, and 72 hours of storage in EDTA. No significant changes in platelet count, platelet size, or reticulated platelet percentage were observed in samples assayed within 4 and 24 hours of blood collection; whereas PSAIgG values increased 3 to 7 fold in samples stored for 24–72 hours. Using reference values for freshly collected or 24-hour-old samples, 10 of 13 (77%) dogs with primary IMT and all B gibsoni-inf ected dogs had increased PSAIgG levels. In 12 (75%) of the 16 dogs with thrombocytopenia the percentage of reticulated platelets was increased; however, absolute numbers of reticulated platelets were within reference values. Moreover, PSAIgG level and the percentage of reticulated platelets were not always increased concurrently in dogs with primary and secondary IMT. Platelet microparticles were detected in all B gibsoni-infected dogs, 8 of 13 (62%) dogs with primary IMT, and transiently in a dog that responded to immunosuppressive treatment. The results of this study indicate that sample age and time of sampling during disease affect interpretation of platelet parameters in dogs with IMT.     

An Inherited Platelet Function Defect in Basset Hounds

An inherited platelet function defect occurring in a family of basset hounds has been described. The trait is transmitted as an autosomal characteristic and appears to be expressed clinically only in the homozygous state. The characteristics of this platelet defect include:1) marked bleeding tendencies and prolonged skin bleeding times in either male or female dogs.2) normal blood coagulation mechanism.3) adequate numbers of circulating platelets which appear morphologically normal by light microscopy.4) normal whole blood clot retraction.5) deficient in vivo platelet consumption and in vitro platelet retention in glass bead columns.6) defective ADP-induced platelet aggregation in homozygotes, apparently normal ADP response in heterozygotes, and defective collagen-induced platelet aggregation in both.     

IMAGING DIAGNOSIS—EQUINE NASAL SEPTAL THICKENING DUE TO CHRONIC CHONDRITIS

We describe the use of computed tomography (CT) in a 6-year-old mare with upper airway obstruction due to an abnormally thick nasal septum. Upon CT imaging, multifocal, expansile cyst-like lesions were detected in the nasal septum. The histopathologic diagnosis was chronic dissecting chondritis. Resection of the abnormal nasal septum resulted in resolution of the clinical signs.     

Aggregation of bovine platelets by acetyl glyceryl ether phosphorylcholine (platelet activating factor)

Platelet activating factor is a multifaceted mediator of inflammation capable of stimulating platelet aggregation as well as anaphylaxis, neutropenia and numerous other in vitro and in vivo cellular changes. This lipid mediator, or autocoid, is released by a wide variety of inflammatory cells following an equally diverse group of cellular stimuli including phagocytosis or antigenic stimulation. The synthesized form of PAF is acetyl glyceryl ether phosphorylcholine (AGEPC). In this study AGEPC aggregated bovine platelets in a dose dependent manner. Maximal, irreversible aggregation occurred at 3.6 × 10^?11 M AGEPC with unwashed platelets and at 8.8 × 10^?12 M AGEPC with washed platelets. Aggregation failed to occur when platelets were tested with the biologically inactive structural analog of AGEPC. The possible contribution by platelet cyclooxygenase products was eliminated by showing lack of platelet aggregation to arachidonic acid and also by pretreating platelets with aspirin.     

Surgical repair and reconstruction of a nasal fistula following intra-lesional formalin injection

A 6-year-old Cleveland Bay mare was referred to the University of Liverpool Equine Hospital for repair of an iatrogenic nasocutaneous fistula. The fistula had developed following attempted ablation of an epidermal inclusion cyst located in the right nasal diverticulum with intra-lesional formalin 6 weeks previously. Subsequent tissue necrosis resulted in the formation of a full-thickness defect in the overlying tissues and the creation of a 5 × 5 cm nasocutaneous fistula. Surgical management consisted of initial debridement of the fistula and use of a commercial dermal regeneration mesh to bridge the defect, acting as a tissue scaffold. The levator labii superioris muscle was utilised to cover the scaffold by transecting it close to its proximal origin and reflecting it rostrally. Finally, a transposition skin flap was created adjacent to the site and was rotated to cover the defect. The mare made an excellent recovery, and follow-up confirmed complete healing of the surgical site with an excellent cosmetic outcome.