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.     

Glanzmann thrombasthenia in a 17-year-old Peruvian Paso mare

A 17-year-old Peruvian Paso mare was evaluated for bilateral epistaxis that had been present for at least 3 years. The mare had mild anemia, platelet count within the reference interval, unremarkable coagulation times, and a negative Coggins test. On endoscopic examination, structural abnormalities were not observed in the nasal cavities, pharynx, larynx, trachea, or either guttural pouch, but petechiation was noted in the nasal mucosa. Additional tests revealed prolonged cutaneous bleeding time, normal concentration of von Willebrand factor antigen, an abnormal clot retraction test, and failure of plalelet aggregation in response to agonists, suggesting a functional disorder of platelets. Genetic analysis indicated the horse was homozygous for a 10-base-pair deletion that included the last 3 base pairs of exon 11 and the first 7 base pairs of intron 11 of the gene encoding glycoprotein IIb. The diagnosis was Glanzmann thrombasthenia (GT) caused by a structural defect in glycoprotein IIb. GT is an autosomal recessive disorder caused by a defect in the glycoprotein IIb-IIIa complex on platelet surfaces. Separate genes encode each glycoprotein, and mutations in either gene can result in GT. This case of GT is unique given the age of the mare at the time of diagnosis. We conclude that GT, although an inherited disorder, should be considered in horses with suspected dysfunctional platelets, regardless of age.     

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.     

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.     

An investigation of the ability of the glutaraldehyde test to distinguish between acute and chronic inflammatory disease in horses

The glutaraldehyde test (GT), a rapid and inexpensive test, has been utilized empirically for many years in bovine practice for diagnosing inflammatory diseases. GT is used primarily to demonstrate increased serum concentrations of fibrinogen and globulin. Glutaraldehyde binds with free amino groups in fibrinogen and immunoglobulin to create a clot in a first degree chemical reaction. The clotting time of the GT estimates the content of proteins produced in response to inflammation. The applicability of GT for diagnosing inflammation in the horse has never been investigated. The objective of this study was to determine the ability of GT to distinguish between acute and chronic inflammatory disease in horses. Thirty-seven horses with suspected inflammatory diseases were evaluated using the GT, history, complete clinical examination and routine blood analysis. GT-times, laboratory results and clinical outcome were compared statistically. Horses that were determined to be acutely affected (based on history, clinical examination and routine blood analysis) tended to have a negative GT (75%). Results of the GT did not correlate with blood fibrinogen concentration. Positive GT also predicted a fatal outcome in 69% of the clinical cases. The results of this trial indicate that GT can be a useful screening test to distinguish between acute and chronic inflammatory disease in horses.     

Hemostatic studies in racing standardbred horses with exercise-induced pulmonary hemorrhage. Hemostatic parameters at rest and after moderate exercise.

The purpose of this study was to determine whether a defect in hemostasis might be a factor in the etiology of exercise-induced pulmonary hemorrhage (EIPH). Hemostatic parameters were evaluated in 22 EIPH-positive and ten EIPH-negative racing horses while in a rested state. Nineteen EIPH-positive and ten EIPH-negative horses were further evaluated just before and immediately after a 15 min exercise period on a 260 m oval track. When EIPH-positive and EIPH-negative horses were compared at rest, there was no significant difference in any of the coagulation and fibrinolytic parameters studied. There was however, a significant difference in platelet function as assessed by aggregometry. The platelets from affected horses were significantly less responsive than those from nonaffected horses when exposed in vitro to the platelet agonists adenosine diphosphate, collagen and platelet activating factor. Exercise tended to increase the packed cell volume and factor VIII/von Willebrand factor and to decrease platelet aggregation responses to low concentrations of adenosine diphosphate. These effects of exercise however were quantitatively similar in both EIPH-positive and EIPH-negative horses. Reduced platelet function may therefore be a contributing factor in the bleeding characteristic of horses with EIPH.     

Osteoinductivity of gelatin/β-tricalcium phosphate sponges loaded with different concentrations of mesenchymal stem cells and bone morphogenetic protein-2 in an equine bone defect model

Fracture is one of the most life-threatening injuries in horses. Fracture repair is often associated with unsatisfactory outcomes and is associated with a high incidence of complications. This study aimed to evaluate the osteogenic effects of gelatin/β-tricalcium phosphate (GT) sponges loaded with different concentrations/ratios of mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) in an equine bone defect model. Seven thoroughbred horses were used in this study. Eight bone defects were created in the third metatarsal bones of each horse. Then, eight treatments, namely control, GT, GT/M-5, GT/M-6, GT/M-5/B-1, GT/M-5/B-3, GT/M-6/B-1, and GT/M-6/B-3 were applied to the eight different sites in a randomized manner (M-5: 2?×?10⁵ MSCs; M-6: 2?×?10⁶ MSCs; B-1: 1 μg of BMP-2; B-3: 3 μg of BMP-2). Repair of bone defects was assessed by radiography, quantitative computed tomography (QCT), and histopathological evaluation. Radiographic scores and CT values were significantly lower in the control group than in the other groups, while they were significantly higher in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The amount of mature compact bone filling the defects was greater in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The present study demonstrated that the GT sponge loaded with MSCs and BMP-2 promoted bone regeneration in an equine bone defect model. The GT/MSC/BMP-2 described here may be useful for treating horses with bone injuries.     

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.     

Oligonucleotide probes for DNA fingerprinting in horses

SUMMARY: 10 different oligonucleotide probes were evaluated for DNA fingerprinting in horses. Five probes were able to detect polymorphic bands. The probes (GT)(8) , (GTG)(5) and (GGAT)(4) are most informative for individual identification and were used to analyze a population of Hannoveranian horses. The probability that two individuals have the same DNA fingerprint pattern is 1.2 × 10(-8) , 5.2 × 10(-10) and 1.5 × 10(-7) respectively. Using a combination of the three probes, paternity tests were performed with exclusion probabilities between 0.08% and 4%. ZUSAMMENFASSUNG: Oligonukleotide-Sonden für DNS-Fingerprints von Pferden Zur Darstellung von DNA-Fingerprints beim Pferd wurden zehn verschiedene Oligonukleotid-Sonden verglichen. Mit fünf Sonden konnten polymorphe Banden nachgewiesen werden. Die Sonden (GT)(8) , (GTG)(5) und (GGAT)(4) besa?en die gr??te Informativit?t für den Identit?tsnachweis und wurden für die Analyse einer Population von Hannoverschen Pferden benutzt. Die Wahrscheinlichkeit, da? zwei Individuen dieselben Fingerprint-Muster aufweisen, liegt bei 1,2 × 10(-8) , 5,2 × 10(-10) bzw. 1,5 × 10(-7) . Bei Verwendung einer Kombination der drei Sonden wurden Vaterschaftskontrollen mit Ausschlu?wahrscheinlichkeiten zwischen 0,08% und 4% erreicht.     

Mean platelet component as an indicator of platelet activation in foals and adult horses

BACKGROUND: Mean platelet component (MPC) is a new platelet variable, measured by modern commercial complete blood count analyzers, that is reduced during platelet activation in humans and small animals. HYPOTHESIS: MPC decreases in horses with clinical conditions that cause platelet activation and disseminated intravascular coagulation (DIC). ANIMALS: We obtained 418 CBCs from 100 sick and 20 healthy neonates and 178 sick and 45 sound adult horses. Sick neonates were classified into septic and nonseptic, and DIC and non-DIC groups. Adults were grouped by diagnoses (systemic inflammatory disorders, gastrointestinal problems, and thrombocytopenia). METHODS: MPC together with platelet count, mean platelet volume, platelet distribution width, and platelet component distribution width were measured with a commercial analyzer and compared between the different disease and control groups in neonates and in adults. RESULTS: MPC values were significantly lower in the septic and nonseptic neonates (24.0 +/- 3.5 g/dL and 26.6 +/- 2.6 g/dL, respectively) than in the control group (28.1 +/- 1.7 g/dL). Neonates with DIC had the lowest MPC values (23.8 +/- 6.3 g/dL). MPC values in adult horses were significantly lower in the inflammatory (23.5 +/- 4.7 g/dL), gastrointestinal obstruction (23.0 +/- 5.0 g/dL), enteritis (23.6 +/- 4.6 g/dL), ischemic (23.9 +/- 5.1 g/dL), and thrombocytopenia (20.2 +/- 5.7 g/dL) groups when compared with control horses (26.2 +/- 3.5 g/dL). Other platelet variables were not different between the control and the disease groups. CONCLUSION AND CLINICAL IMPORTANCE: MPC might be a useful variable for quickly and easily detecting platelet activation in sick neonates and adult horses.     

Platelet transfusion in thrombocytopenic horses

Platelet transfusions might be indicated in horses with thrombocytopenia. The need for a transfusion cannot be determined by platelet numbers alone, as primary or co‐existing disease processes, platelet function and age of the recipient also need to be considered. In patients with no co‐morbidities, relevant bleeding is uncommonly observed with platelet counts >10 x 10⁹/l and a therapeutic approach with initiation of treatment when signs of bleeding are observed might be justified.     

Effects of clopidogrel and aspirin on platelet aggregation, thromboxane production, and serotonin secretion in horses

Background: Critically ill horses are susceptible to thrombotic disease, which might be related to increased platelet reactivity and activation. Objectives: To compare the effect of oral clopidogrel and aspirin (ASA) on equine platelet function. Animals: Six healthy adult horses. Methods: Horses received clopidogrel (2 mg/kg PO q24h) or ASA (5 mg/kg PO q24h) for 5 days in a prospective randomized cross‐over design. Platelet aggregation responses to adenosine diphosphate (ADP) and collagen via optical aggregometry, and platelet secretion of serotonin (5HT) and production of thromboxane B₂ (TXB₂) by ELISA were evaluated. In horses receiving clopidogrel, high‐performance liquid chromatography analysis for clopidogrel and its carboxylic‐acid metabolite SR 26334 was performed. Results: SR 26334 was identified in all clopidogrel‐treated horses, although the parent compound was not detected. Clopidogrel resulted in decreases in ADP‐induced platelet aggregation persisting for 120 hours after the final dose. ADP‐induced platelet aggregation decreased from a baseline of 70.2 ± 14.7% to a minimum of 15.9 ± 7.7% 24 hours after the final dose (P < .001). Collagen‐induced aggregation decreased from a baseline of 93 ± 9.5% to a minimum of 70.8 ± 16.9% 48 hours after the final dose (P < .001). ASA did not decrease platelet aggregation with either agonist. ASA decreased serum TXB₂ from a baseline value of 1310 ± 1045 to 128 ± 64 pg/mL within 24 hours (P < .01). Conclusions and Clinical Importance: Clopidogrel effectively decreases ADP‐induced platelet aggregation in horses, and could have therapeutic applications for equine diseases associated with platelet activation.     

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.     

Modulation of peripheral markers of the serotoninergic system in healthy horses

To evaluate the changes in plasma and platelet serotonin (5-HT) as markers of the serotoninergic system in equines, 5-HT content was measured by high performance liquid chromatography (HPLC) in deproteinized plasma obtained from peripheral blood samples of 12 clinically healthy horses, before and after feeding. 5-HT was measured in platelet rich plasma (PRP) and in platelet poor plasma (PPP). 5-HT in platelets (p5-HT) was obtained by subtracting free 5-HT in PPP (f5-HT) from 5-HT in PRP. After food ingestion, significant increases in p5-HT and f5-HT (p < 0.001), and no changes in the f5-HT/p5-HT ratio were recorded. Increase in the total circulating 5-HT might account both for initiating peristaltic activity and for increasing the f5-HT levels. Augmented 5-HT uptake by platelets could reflect the hypothetical increased activity of the serotoninergic neurons. Besides showing the feasibility to obtain f5-HT and p5-HT through HPLC determination of 5-HT in PRP and PPP equine plasma, these findings are consistent with the postulation that 5-HT is released from enterochromaffin cells following a mechanical and chemical stimulation.     

Suspected aspirin resistance in individual healthy adult warmblood horses

The reasons for this prospective experimental study were to determine a dosing scheme with loading and maintenance dose of aspirin inducing inhibition of platelet function measured by whole blood impedance aggregometry. Ten horses received aspirin orally in the morning with one loading dose of 4.7–5 mg/kg and maintenance doses of 1–1.3 mg/kg daily the following 4 days. Aggregometries (COLtest, ASPItest, ADPtest) and serum salicylic acid were measured. ASPItest showed significant difference in inhibition at 24 and 48 hr (p < .05) and 96 hr (p < .01). Significant change for ADPtest and COLtest couldn't be detected. Serum salicylic acid concentrations were significantly (p < .01) increased at 6 and 12 hr. Despite this, three horses failed any inhibitory effect of platelet function, suspecting an aspirin resistance. Regarding the other seven horses platelet aggregation induced by ASPItest was reduced between 37% and 100% from baseline at 6 and 12 hr and between 0 and 98% during the next 4 days. Correlations of serum concentration of salicylic acid and aggregometries couldn't be detected. It can be presumed that equine platelets are less susceptible to aspirin what may compromise eventually the anticoagulatory effects and efficacy in preventing and treating diseases with increased platelet activation as endotoxaemia or laminitis.     

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.     

Studies on vitamin E and selenium deficiency in young pigs. IV. Effect on coagulation system.

An evaluation of the coagulation system has been conducted in vitamin E and/or selenium deficient swine. The partial thromboplastin time, plasma fibrinogen concentration, platelet lipid peroxides, as well as the fibrinogen/fibrin degradation products were not found to be significantly affected by either vitamin E deficiency, selenium deficiency, or deficiency of both. With selenium deficiency, the prothrombin time was shortened (p less than 0.05). The platelet count and platelet turnover were greatly decreased by both vitamin E (p less than 0.001) and selenium deficiency (p less than 0.005). Further-more, the survival of platelets labelled with 75Se-selenomethionine and the per cent isotope incorporated into platelets were reduced (p less than 0.05 and p less than 0.005) in association with vitamin E deficiency, but not with selenium deficiency. These results were interpreted as evidence of a platelet production defect and possibly a platelet function defect in vitamin E deficient animals. Selenium deficiency were also associated with decreased (p less than 0.05) survival of fibrinogen labelled with 75Se-selenomethionine and increased (p less than 0.05) turnover of fibrinogen. From these fibrinogen kinetic findings, it was considered that chronic low grade disseminated intravascular coagulation possibly occurs in selenium deficient animals, probably in relation to the development of hepatosis dietetica or widespread microvascular damage. However, other possibilities such as increased fibrinogenolysis in relation with hepatosis dietetica or an intrinsic fibrinogen defect due to selenium deficiency also need to be taken into consideration and have not been ruled out in the present study.     

Poor reproducibility of template bleeding time in horses

BACKGROUND: Template bleeding time (TBT) is considered to be a useful test for detecting platelet function disorders and the effect of platelet-activating drugs, but studies in human medicine have concluded that the test has poor reproducibility and sensitivity. HYPOTHESIS: TBT has poor reproducibility in horses and has insufficient sensitivity to detect the effect of etamsylate on platelet function. ANIMALS: Twenty healthy horses. METHODS: TBT was determined and repeated 2 hours and 30 days later. TBT was also performed 2 hours after IV administration of etamsylate. RESULTS: Although no statistical differences were seen between the TBT values obtained at different times, the coefficients of variation for TBT replicates ranged from 26.8% to 45.5%. The reference range for TBT was 138.4-860.4 seconds. No statistically significant shortening of the mean TBT value was observed after etamsylate administration. CONCLUSION AND CLINICAL IMPORTANCE: TBT has poor reproducibility, and the reference range is too wide to make TBT useful in a clinical setting. Other tests with higher reproducibility should be considered when assessing platelet function disorders in horses.     

Assessment of platelet function in horses: ultrastructure, flow cytometry, and perfusion techniques

We studied equine platelet function and activation using ultrastructural examination, flow cytometry, and perfusion. The main aim of the study was to evaluate hemostatic mechanisms in horses using these techniques. Ultrastructural observations were done on resting and activated platelets. Flow cytometry was used to evaluate binding of antibodies to major platelet glycoproteins (GPIIb-IIIa, GPIV, and GPIb) and activation-dependent antigens (P-selectin and lysosomal integral membrane protein [LIMP]). Perfusion techniques were used to evaluate the interaction between platelets and damaged subendothelium. Aggregation experiments were done to identify the best agonists for flow cytometry. Ultrastructural observations confirmed that equine platelets lack a developed open canalicular system and that release of granule contents occurs by fusion of adjacent granule membranes that ultimately connect with external membranes. Flow cytometry identified a 2-fold increase in binding of antibodies against GPIIb-IIIa and GPIV after activation. Binding of antibodies against P-selectin and LIMP increased from 2.12 and 1.74% to 15.5 and 11.6%, respectively, in response to thrombin and to 21.86 and 10.50%, respectively, in response to collagen. Annexin V binding increased moderately after activation. Perfusion experiments with citrated blood indicated that equine platelets react more strongly to subendothelium than do human platelets. When blood was anticoagulated with low molecular weight heparin, a marked impairment of platelet interactions was observed. In conclusion, although some differences were observed between human and equine platelet function, some techniques currently used to assess human platelet function may be useful to assess equine platelets.     

Platelet Function Defect in a 5-Day-Old Simmental Calf

A 5-day-old Simmental Heifer was evaluated for excessive bleeding from the skin following horse fly bites. A coagulation profile and platelet numbers were normal. In vitro platelet function, measured by whole blood aggregometry, was found to be abnormal when compared with age-matched controls. Therefore, this defect in hemostasis was attributed to a qualitative defect in platelet function.