Much progress has been made in the last 10 years related to the diagnosis of inherited intrinsic platelet disorders in animals with respect to clinical, functional, biochemical, and molecular characteristics. Owing to lack of awareness among veterinary practitioners, these disorders may be misdiagnosed and inappropriately managed. The purpose of the information reported here is to provide a brief description of platelet anatomy and function, followed by descriptions of methods of evaluation of platelet function, specific platelet disorders, and methods of diagnosis. Information related to case management and treatment is also provided.
Platelet Anatomy and Function
Intrinsic versus extrinsic platelet disorders—Intrinsic platelet disorders are characterized by defects in some part of the platelet anatomy that result in platelet dysfunction (thrombopathia) or thrombocytopenia. Defects involving membrane glycoproteins, granules, signal transduction proteins, and structural proteins can exist at functional, biochemical, and molecular levels.1–27 The inheritance pattern for all reported congenital intrinsic platelet disorders in animals is autosomal recessive. Extrinsic platelet disorders, in contrast to intrinsic platelet disorders, are characterized by the lack, reduction, or dysfunction of a protein vital for mediating platelet adhesion or aggregation. The platelets in these disorders are functionally normal. Extrinsic platelet disorders include hypofibrinogenemia or dysfibrinogenemia and vWD. Although fibrinogen disorders are rare in veterinary medicine, vWD is relatively common, particularly in dogs.28
Clinically, intrinsic and extrinsic platelet disorders are indistinguishable; BMBTs are usually prolonged in both types of disorders, whereas results of coagulation screening tests such as PT and APTT are normal. Bleeding patterns are usually typical of platelet-related bleeding problems and include petechial and ecchymotic hemorrhages and mucosal bleeding such as gingival bleeding, epistaxis, and urinary and gastrointestinal hemorrhage. Internal bleeding within organs such as the kidney, spinal cord, and brain can also occur. Excessive bleeding during the eruption of permanent teeth can occur in dogs between the ages of 4 and 6 months and is usually the first indication that a bleeding diathesis exists. Clinical signs in affected animals older than 6 months can vary considerably. Signs may be subtle or nonspecific, making detection or diagnosis difficult. Periodic bruises on the abdomen or petechial hemorrhages on mucous membranes may be the only clinical signs. Internal hemorrhage within the brain or spinal cord may result in seizures or paralysis.
Affected animals may also have chronic epistaxis. Epistaxis can happen at any age and is more likely to occur in affected animals treated with platelet-inhibitory medications or in affected animals with other underlying problems (inherited or acquired). Trauma or surgery can result in severe hemorrhage with intrinsic or extrinsic platelet disorders. Transfusion products for the 2 types of disorders are markedly different; therefore, distinguishing between intrinsic and extrinsic platelet disorders is imperative prior to conducting surgical procedures on affected animals.
Normal platelet anatomy and function—Platelets are derived from the cytoplasm of megakaryocytes, and their structure is quite complex (Figure 1). Platelet membranes are similar to those of other cells and are characterized by phospholipids arranged in a bilayer forming a hydrophobic core. Interspersed within the fluid lipid matrix are proteins and glycoproteins; some of these proteins serve as specialized receptors important in platelet responses. One glycoprotein complex that has been well characterized is glycoprotein IIb-IIIa, also known as integrin αIIbβ3.29 Glycoprotein IIb-IIIa complexes primarily function as fibrinogen receptors important for mediating platelet aggregation, although these complexes can also bind vWF and assist with early stages of platelet adhesion to the subendothelium (Figure 2). Glycoprotein IIb-IIIa complexes on platelets must be induced to undergo a conformational change before they can bind fibrinogen. This conformational change is induced by signaling molecules generated during platelet activation (Figure 3).
Illustration of the microarchitecture of a platelet. Platelet shape is maintained by a circumferential band of microtubules, which are also important in mediating platelet release from megakaryocytes. Platelet granules include α, dense, and lysosomal granules; lysosomal granules are not readily detectable even via electron microscopy and are not depicted. The α granules, the only granules visible by light microscopy, primarily contain proteins stored within 2 main compartments. The outer more lucent area of the α granule is the storage site for vWF and fibrinogen. Dense granules, which appear dense via electron microscopy, primarily store adenine nucleotides, serotonin, calcium, and inorganic phosphates. Platelets contain glycogen stores, mitochondria, and microfilaments important for mediating granule movement and clot retraction during platelet activation. The open canalicular system (OCS), a system of open interconnecting channels within the platelet cytoplasm, does not exist or is poorly formed in ruminant and equine platelets.
Citation: Journal of the American Veterinary Medical Association 233, 8; 10.2460/javma.233.8.1251
Illustration of the microarchitecture of a platelet. Platelet shape is maintained by a circumferential band of microtubules, which are also important in mediating platelet release from megakaryocytes. Platelet granules include α, dense, and lysosomal granules; lysosomal granules are not readily detectable even via electron microscopy and are not depicted. The α granules, the only granules visible by light microscopy, primarily contain proteins stored within 2 main compartments. The outer more lucent area of the α granule is the storage site for vWF and fibrinogen. Dense granules, which appear dense via electron microscopy, primarily store adenine nucleotides, serotonin, calcium, and inorganic phosphates. Platelets contain glycogen stores, mitochondria, and microfilaments important for mediating granule movement and clot retraction during platelet activation. The open canalicular system (OCS), a system of open interconnecting channels within the platelet cytoplasm, does not exist or is poorly formed in ruminant and equine platelets.
Citation: Journal of the American Veterinary Medical Association 233, 8; 10.2460/javma.233.8.1251
Illustration of the microarchitecture of a platelet. Platelet shape is maintained by a circumferential band of microtubules, which are also important in mediating platelet release from megakaryocytes. Platelet granules include α, dense, and lysosomal granules; lysosomal granules are not readily detectable even via electron microscopy and are not depicted. The α granules, the only granules visible by light microscopy, primarily contain proteins stored within 2 main compartments. The outer more lucent area of the α granule is the storage site for vWF and fibrinogen. Dense granules, which appear dense via electron microscopy, primarily store adenine nucleotides, serotonin, calcium, and inorganic phosphates. Platelets contain glycogen stores, mitochondria, and microfilaments important for mediating granule movement and clot retraction during platelet activation. The open canalicular system (OCS), a system of open interconnecting channels within the platelet cytoplasm, does not exist or is poorly formed in ruminant and equine platelets.
Citation: Journal of the American Veterinary Medical Association 233, 8; 10.2460/javma.233.8.1251
Illustration of platelet adhesion to the subendothelium of blood vessels. Adhesion is mediated by binding of platelet glycoprotein (GP) Ib-IX-V to vWF bound to subendothelial collagen. Shearing, as a result of blood flowing over the bound vWF protein, results in exposure of epitopes on vWF for GP Ib-IX-V. As the platelet is tethered and slowed by GP Ib-IX-V binding, GP IIb-IIIa becomes activated and also binds to vWF, resulting in more stable platelet adhesion. Activation of GP IIb-IIIa results in a change in conformation of the IIb-IIIa complex that allows fibrinogen binding and linking of platelets together (platelet aggregation). Each platelet possesses 40,000 to 80,000 GP IIb-IIIa receptors.
Citation: Journal of the American Veterinary Medical Association 233, 8; 10.2460/javma.233.8.1251
Illustration of platelet adhesion to the subendothelium of blood vessels. Adhesion is mediated by binding of platelet glycoprotein (GP) Ib-IX-V to vWF bound to subendothelial collagen. Shearing, as a result of blood flowing over the bound vWF protein, results in exposure of epitopes on vWF for GP Ib-IX-V. As the platelet is tethered and slowed by GP Ib-IX-V binding, GP IIb-IIIa becomes activated and also binds to vWF, resulting in more stable platelet adhesion. Activation of GP IIb-IIIa results in a change in conformation of the IIb-IIIa complex that allows fibrinogen binding and linking of platelets together (platelet aggregation). Each platelet possesses 40,000 to 80,000 GP IIb-IIIa receptors.
Citation: Journal of the American Veterinary Medical Association 233, 8; 10.2460/javma.233.8.1251
Illustration of platelet adhesion to the subendothelium of blood vessels. Adhesion is mediated by binding of platelet glycoprotein (GP) Ib-IX-V to vWF bound to subendothelial collagen. Shearing, as a result of blood flowing over the bound vWF protein, results in exposure of epitopes on vWF for GP Ib-IX-V. As the platelet is tethered and slowed by GP Ib-IX-V binding, GP IIb-IIIa becomes activated and also binds to vWF, resulting in more stable platelet adhesion. Activation of GP IIb-IIIa results in a change in conformation of the IIb-IIIa complex that allows fibrinogen binding and linking of platelets together (platelet aggregation). Each platelet possesses 40,000 to 80,000 GP IIb-IIIa receptors.
Citation: Journal of the American Veterinary Medical Association 233, 8; 10.2460/javma.233.8.1251
Illustration of platelet signaling leading to change in conformation of the platelet GP complex IIb-IIIa. Most platelet-activating agents, including ADP, collagen, and thrombin, mediate platelet activation by inducing phosphatidylinositol metabolism, mobilizing calcium, generating DAG, and reducing cAMP. Activation of phospholipases, protein kinases, GEFs, and guanosine triphosphatases are all critical to mediating platelet activation events. Rap1b is a guanosine triphosphatase that plays a critical role in mediating the change in conformation of GP IIb-IIIa necessary for binding of fibrinogen. Binding of calcium and DAG to CalDAG-GEFI results in activation of GEF, which in turn activates Rap1b. Thrombin, by binding to PAR4, can bypass the need for CalDAG-GEFI in the activation of GP IIb-IIIa. As a result, animals with CalDAG-GEFI thrombopathia have platelets that respond to thrombin and are able to mediate normal clot retraction.
Citation: Journal of the American Veterinary Medical Association 233, 8; 10.2460/javma.233.8.1251
Illustration of platelet signaling leading to change in conformation of the platelet GP complex IIb-IIIa. Most platelet-activating agents, including ADP, collagen, and thrombin, mediate platelet activation by inducing phosphatidylinositol metabolism, mobilizing calcium, generating DAG, and reducing cAMP. Activation of phospholipases, protein kinases, GEFs, and guanosine triphosphatases are all critical to mediating platelet activation events. Rap1b is a guanosine triphosphatase that plays a critical role in mediating the change in conformation of GP IIb-IIIa necessary for binding of fibrinogen. Binding of calcium and DAG to CalDAG-GEFI results in activation of GEF, which in turn activates Rap1b. Thrombin, by binding to PAR4, can bypass the need for CalDAG-GEFI in the activation of GP IIb-IIIa. As a result, animals with CalDAG-GEFI thrombopathia have platelets that respond to thrombin and are able to mediate normal clot retraction.
Citation: Journal of the American Veterinary Medical Association 233, 8; 10.2460/javma.233.8.1251
Illustration of platelet signaling leading to change in conformation of the platelet GP complex IIb-IIIa. Most platelet-activating agents, including ADP, collagen, and thrombin, mediate platelet activation by inducing phosphatidylinositol metabolism, mobilizing calcium, generating DAG, and reducing cAMP. Activation of phospholipases, protein kinases, GEFs, and guanosine triphosphatases are all critical to mediating platelet activation events. Rap1b is a guanosine triphosphatase that plays a critical role in mediating the change in conformation of GP IIb-IIIa necessary for binding of fibrinogen. Binding of calcium and DAG to CalDAG-GEFI results in activation of GEF, which in turn activates Rap1b. Thrombin, by binding to PAR4, can bypass the need for CalDAG-GEFI in the activation of GP IIb-IIIa. As a result, animals with CalDAG-GEFI thrombopathia have platelets that respond to thrombin and are able to mediate normal clot retraction.
Citation: Journal of the American Veterinary Medical Association 233, 8; 10.2460/javma.233.8.1251
Just beneath the platelet membrane is a circumferential band of microtubules.30 Microtubules are hollow, cylindric structures composed of protofilaments formed by α-β tubulin dimers arranged in a helical head-to-tail fashion. Microtubules are responsible for the maintenance of the disk-shaped form of the circulating platelet.31 In normal physiologic conditions, β-1 tubulin is solely expressed as a component of microtubules within megakaryocytes and plays a role in the orderly fragmentation of platelets from the cytoplasm of megakaryocytes.32
Connected to the surface of the platelet and extending deep within its cytoplasm is a tortuous maze of open interconnecting channels called the open canalicular system.33 This system can act as a conduit for the release of granule contents by activated platelets and can also serve as a method for platelets to take up particles such as viruses34 from the surrounding area. The open canalicular system is everted to some extent during platelet activation. It can thus serve to increase the number of surface receptors available for ligand binding because many of the types of receptors on the platelet surface also line the open canalicular system.35 Interestingly, ruminant and equine platelets do not possess an open canalicular system.36 Granule contents are released in these species by fusion of the granules directly to the outer membrane.
Another specialized cytoplasmic organelle is the dense tubular system. This system is derived from elements of the smooth endoplasmic reticulum in the megakaryocyte and is the site for prostaglandin synthesis and calcium sequestration by the platelet. Numerous microfilaments are also located throughout the cytoplasm. The microfilaments have contractile properties and play a role in concentrating the granules in the center of the platelet, in degranulation of the platelet, and in clot retraction. The microfilaments have been identified as consisting of actin, myosin, and tropomyosin-like proteins. Located more centrally within the cytoplasm of the platelet are scattered mitochondria, small accumulations of glycogen, and numerous storage granules.
Three main types of storage granules exist—dense, α, and lysosomal—and they are heterogeneous in content and morphology.37 The dense granules or dense bodies, as the names imply, are electron dense when viewed with an electron microscope and serve as storage sites for adenine nucleotides, serotonin, calcium ions, and inorganic phosphates. The α granules, the largest and most numerous of the platelet granules, correspond to the azurophilic granules viewed by light microscopy. They primarily contain proteins such as β-thromboglobulin, platelet factor 4, fibrinogen, platelet-derived growth factor, factor V, vWF, fibronectin, and thrombospondin. Lysosomal granules contain hydrolases similar to those in neutrophil granules. Lysosomal and dense granules cannot be seen via light microscopy.
Platelet membranes contain numerous receptors important for mediating platelet responses to agonists such as ADP, collagen, thrombin, and thromboxane.38–41 Protease-activated receptors are thrombin receptors located on platelets. There are 3 main types of PARs designated as PAR1, PAR3, and PAR4. Molecular evidence suggests that canine platelets possess PAR1, PAR3, and PAR424; however, PAR1 and PAR4 are considered the primary receptors involved in thrombin-induced platelet activation in dogs and in humans. This contrasts with rat platelets, in which PAR3 and PAR4 are considered the primary thrombin receptors.42
Many platelet receptors mediate platelet activation by inducing phosphatidylinositol metabolism, mobilizing calcium, generating DAG, and reducing cAMP. Activation of phospholipases, protein kinases, GEFs, and GTPases are all critical to mediating platelet activation events.43 Platelets not only have the capacity to mobilize and release preformed products but also have the capacity to synthesize new proteins important for sustaining hemostatic and other biological events.44 The series of events that include binding of agonists to receptors and generation of second messengers, which ultimately leads to changes in platelet receptor conformation, binding of fibrinogen, and release of granule contents, are included in the general category of signal transduction. Disorders of platelet signal transduction are the most common cause of inherited platelet disorders in humans and are likely the most common cause in other mammals as well.45
Evaluation of Platelet Function
Platelet function testing—Methods for evaluating platelet function include global assays, which serve to confirm that a platelet disorder is likely present, and directed assays, which are useful for identifying the specific type of disorder present. Global assays are becoming more available as part of routine diagnostic methods in clinical pathology laboratories, whereas directed assays tend to be specialized in nature and are not readily available outside of research laboratories.
Thromboelastography is an example of a global assay that evaluates clot initiation and clot formation and stability.46,47 Readouts from the instrument provide a graphic depiction of clot formation and clot lysis over time, thus allowing the operator to identify hypo- or hypercoagulable states. Abnormalities in coagulation proteins, fibrinolytic proteins, and platelets can all result in altered thromboelastographic findings; however, the technology is not useful for specifically identifying a particular aspect of platelet dysfunction.
Aperture closure instruments are becoming increasingly available and are used to detect high shear-dependent platelet adhesion and aggregation.48,49 The instruments aspirate citrated whole blood under high shear forces through a capillary tube and a compartment containing test cartridge membranes. The test cartridge membranes have a small, centrally located aperture and are coated with ADP and collagen or collagen and epinephrine. As blood flows under high shear forces across the membrane and through the aperture, platelets become activated and begin to adhere and aggregate, resulting in closure of the aperture within 1 to 3 minutes. The instrument measures the decrease in flow rate with time until flow completely stops. The final closure time and volume of blood flow are recorded by the instrument. The closure time is affected by many variables, including platelet count; Hct; medications; vWD; intrinsic platelet disorders; and improper sample handling, including errors in anticoagulant-to-blood ratios. These instruments are not useful for specifically identifying a particular aspect of platelet dysfunction and cannot be used to distinguish between vWD and an intrinsic platelet disorder.
The BMBT test is another global assay of platelet function.50 Bleeding time is evaluated on the buccal mucosa by use of a spring-loaded cassette that incises the mucosa to a precise depth and length. The buccal mucosa is everted and held gently in place with a gauze strip during the performance of the test. The incisions are allowed to close naturally, primarily through platelet adhesion and aggregation, and care is taken not to disturb the incisions during the procedure. The technique may require that an animal be anesthetized, particularly if the animal is fractious, shakes its head, or licks at the incision. A normal BMBT is 2 to 3 minutes. The BMBT can be prolonged with thrombocytopenia, acquired platelet function defects, vWD, or intrinsic platelet function disorders.
Examples of directed assays of platelet function or structure include platelet aggregation, clot retraction, flow cytometry, and electron microscopy. Platelet aggregation testing is the gold standard for evaluation of platelet function.51 Platelet aggregometers assess the ability of platelets to form aggregates and make use of light transmission (platelet-rich-plasma) or impedence (whole blood) as monitoring methods. Light transmission methods are more sensitive to subtle changes in platelet function than impedence methods. Platelet aggregation methods can be accompanied by methods that evaluate platelet release. Examples include 14C-serotonin release and luciferin-luciferase assays used to detect ATP release. Varying concentrations of plateletactivating agents such as ADP, collagen, and thrombin can often be used to dissect specific pathways of platelet signaling via directed assays of platelet function. This can greatly facilitate the biochemical and molecular characterization of inherited platelet disorders. Platelet aggregation methods require specialized equipment and expertise in the techniques of platelet isolation in various species (for light transmission methods) and evaluation of results.
The clot retraction assay is a test of platelet function that relies on the normal interaction between thrombin, platelet receptors, and fibrinogen.12 Because it is a fairly specific test of platelet function, not all animals with platelet function defects will have abnormal clot retraction assay results. Animals with CalDAG-GEFI thrombopathia have platelets that respond to thrombin, leading to conformational change of the glycoprotein IIb-IIIa receptors necessary for fibrinogen binding; therefore, results for the clot retraction assay in these animals are normal. Animals with Glanzmann thrombasthenia have platelets that lack the glycoprotein IIbIIIa receptor and are unable to bind fibrinogen. These animals have abnormal clot retraction.
Flow cytometric assays are useful for identifying specific receptor deficiencies or functional abnormalities.52–54 Monoclonal antibodies that recognize glycoproteins IIb and IIIa or polyclonal antibodies that recognize the entire IIb-IIIa complex are available commercially and can be used to diagnose or rule out Glanzmann thrombasthenia. Antibodies that bind specifically to activated platelets and minimally to nonactivated platelets are considered activation-specific antibodies. Antibodies that bind preferentially to surface-bound rather than soluble ligand because of antigens on the ligand that become expressed as a result of binding a receptor are termed receptor-induced binding-site antibodies. The monoclonal antibody CAP-1 recognizes a receptorinduced binding-site epitope on canine fibrinogen.55 This antibody binds minimally to soluble fibrinogen but binds strongly to fibrinogen that has become bound to the glycoprotein IIb-IIIa complex. This antibody can be used to evaluate the functionality of the IIb-IIIa receptor in response to various platelet-activating agents, including ADP and collagen, in dogs. Annexin V binding can be used as a marker for the expression of phosphatidylserine on the surface of activated platelets.56 Activated platelets provide a negatively charged surface for the assembly of coagulation protein complexes. This procoagulant surface is expressed primarily because of exteriorization of phosphatidylserine. Electron microscopy can be used to evaluate platelet structure and can be useful in diagnosing gross abnormalities in α or dense granule structure. Electron microscopy can also be useful for identification of microtubules.
Platelet Disorders
Glanzmann thrombasthenia—Glanzmann thrombasthenia is a platelet disorder resulting from lack of or reduction in glycoprotein IIb-IIIa (αIIbβ3 integrin) receptors on the surface of platelets.57 Glycoproteins IIb and IIIa must both be synthesized and transported to the platelet surface to form a stable complex. Because the 2 glycoproteins are encoded by separate genes, mutations in either gene can result in Glanzmann thrombasthenia. More than 30 mutations have been identified in each gene in humans with Glanzmann thrombasthenia. A database of causative mutations in humans is available online.58 In veterinary medicine, all of the gene mutations identified thus far have been in the gene that encodes glycoprotein IIb. To date, none of the mutations identified in the gene that encodes IIIa have resulted in Glanzmann thrombasthenia. The disorder has been diagnosed in Great Pyrenees,12 Otterhounds17 (formerly known as thrombasthenic thrombopathia in Otterhounds59), a Thoroughbred-cross,21,22 a Quarter Horse,21,22,25 and an Oldenbourg filly.60 The mutations in Great Pyrenees16 and Otterhounds17 are distinct, and both are located within gene segments that encode highly conserved calcium-binding domains within the IIb subunit. Two distinct mutations have been identified in horses.22,25 The aforementioned Quarter Horse was identified as a compound heterozygote and carried both mutations. Information on molecular genetics pertaining to the Oldenbourg horse has not been published, and it is not known whether the mutation is identical to the ones described for the Quarter Horse or represents a distinct (third) mutation (Table 1).
Summary of intrinsic platelet disorders and associated genetic mutations in specific breeds of dogs, horses, and cattle.
| Disorder, by breed | Affected gene | Site of mutation | Nature of mutation |
|---|---|---|---|
| Glanzmann thrombasthenia | |||
| Great Pyrenees | lib | Exon 13 | 14-base pair repeat16 |
| Otterhound | lib | Exon 12 | GAC to CAC (D to H)17 |
| Thoroughbred | lib | Exon 2 | CGG to CCG (R to P)22 |
| Quarter Horse | lib | Exon 11-lntron 11 splice site region | 10 base deletion25 |
| Oldenbourg | Not known | Not known | Not known60 |
| Thrombopathia | |||
| Basset Hound | CalDAG-GEFI | Exon 5 | 3 base deletion (F deletion)24 |
| Eskimo Spitz | CalDAG-GEFI | Exon 5 | 1 base duplication (frameshift, premature stop codon)24 |
| Landseer-ECT | CalDAG-GEFI | Exon 8 | CGA to TGA (R to stop codon)24 |
| Simmental cattle | CalDAG-GEFI | Exon 7 | CTC to CCC (L to P)23 |
| Macrothrombocytopenia | |||
| Cavalier King Charles Spaniel | β1-Tubulin | Exon 4 | GAC to AAC (D to N)27 |
| Chediak-Higashi syndrome | |||
| Japanese Black cattle | LYST | Nucleotide 6065 | CAT to CGT (H to R)61 |
| Cyclic hematopoeisis | |||
| Gray Collies | AP3B1 | Exon 20 | 1 base duplication (frame shift, premature stop codon)62 |
| Procoagulant expression | |||
| German Shepherd Dog | Not known | Not known | Not known18,63 |
| Selective ADP deficiency | |||
| Cocker Spaniel | Not known | Not known | Not known11 |
IIb = Platelet glycoprotein IIb. D = Aspartic acid. H = Histidine. R = Arginine. P = Proline. F = Phenylalanine. L = Leucine. N = Asparagine. AP3B1 = Adaptor protein complex 3, β-subunit. LYST = Lysosomal trafficking regulator.
Glanzmann thrombasthenia is characterized functionally by lack or severe impairment of platelet aggregation responses to all platelet-activating agents, including thrombin. Clot retraction is also markedly impaired to nonexistent. Diagnosis of novel cases of Glanzmann thrombasthenia can be accomplished via evaluation of platelet function (aggregation response values will be markedly less than reference range values in response to ADP, collagen, and thrombin) and flow cytometry (glycoproteins IIb and IIIa will be absent or markedly reduced on platelet surfaces). Unfortunately, these techniques are not readily available and usually require that the affected animal be evaluated on the premises of the testing facility. Molecular testing is available at Auburn University for detection of affected or carrier Great Pyrenees and Otterhounds with Glanzmann thrombasthenia. Tests are also available at Auburn University for detection of the mutations reported for horses.
Otterhound breeders, recognizing the limited gene pool of this breed, have been fairly aggressive with regard to molecular testing and have made an effort to test all dogs used for breeding, regardless of whether there is a history of prior bleeding problems in their lineage. As of February 2008, 104 Otterhounds had been evaluated for Glanzmann thrombasthenia on a molecular basis. The prevalence of the mutation in the carrier population is estimated at 30%, on the basis of dogs tested at Auburn University and the inclusion of untested obligate carriers. The first Great Pyrenees with Glanzmann thrombasthenia was reported in 1996.12 Since then, although Great Pyrenees affected with Glanzmann thrombasthenia have been identified in several states, testing for carrier status has been sporadic and is usually driven by diagnosis of the disorder in an affected puppy within a breeding line. Lack of awareness and misdiagnosis have likely resulted in limited reporting of this disorder in affected Great Pyrenees. For every puppy affected with Glanzmann thrombasthenia, the parents and half of the siblings within the litter are obligate carriers without clinical signs; considering this fact, the likelihood of the mutation becoming more prevalent is high. Carriers have been detected in several states, including Mississippi, Alabama, Florida, Missouri, Minnesota, Indiana, Illinois, Oklahoma, and Washington. The prevalence of mutations causing Glanzmann thrombasthenia in horses is also unknown. Testing of horses will likely be prompted by detection of affected animals, similar to the situation in Great Pyrenees.
Signal transduction disorders—Signal transduction platelet disorders have been documented in dogs (Basset Hounds,1,4 Eskimo Spitz,8 and Landseers-ECT) and Simmental cattle.5,7,9,13,14 These platelet disorders are characterized functionally by a decreased to absent response to most platelet-activating agents, including ADP and collagen. Platelets of these animals do respond to thrombin; however, the kinetics of the response is impaired. Results of clot retraction assays are within reference ranges in affected animals and are related to the ability of the platelets to respond to thrombin. Results of flow cytometric assays confirm the existence of glycoprotein IIb-IIIa complexes on the platelet surface, indicating that the dysfunction is related to the inability of platelets to relay essential signaling information in response to most agonists. The molecular basis for these disorders involves distinct mutations in the gene encoding CalDAG-GEFI, which is a GEF vital for normal platelet signal transduction.24 This factor activates the protein Rap1b, a guanosine triphosphatase that in turn plays a role in inducing the conformational change in glycoprotein IIb-IIIa, which is necessary for binding of fibrinogen64 (Figure 3). It is not known whether Rap1b is directly activated by the PAR4 pathway or whether other signaling molecules are involved leading to activation of GPIIb-IIIa. The ability of thrombin to activate mutant platelets is believed to be attributable to the ability of PAR4 signaling to bypass CalDAG-GEFI and activate Rap1b directly or act via a pathway independent of Rap1b.24,65
Mutations have been identified in areas of the gene encoding structurally conserved regions within the catalytic domain of CalDAG-GEFI in dogs and Simmental cattle (Table 1).23,24 Molecular-based assays are available for diagnosis of these disorders at Auburn University. Breeders of Basset Hounds in the United States and Landseers-ECT in several European countries are currently testing their breeding stock. Landseer-ECT has been recognized as a breed in Europe since 1960 and is distinct from the Landseer-Newfoundland breed recognized in the United States. The testing strategy used by these breeders is similar to that of breeders of Otterhounds in that testing is performed regardless of whether there is a history of bleeding in the lineage. As of February 2008, the estimated prevalences of carrier Basset Hounds and Landseers-ECTa among their breeds were 27% and 28%, respectively. Other than the original 2 Eskimo Spitz reported in 1994, no other Eskimo Spitz has been identified as affected by or being carriers of the mutation identified in the CalDAG-GEFI gene. Whether this small number is owing to a very low prevalence or lack of recognition of the disorder is not known. The prevalence of the CalDAG-GEFI gene mutation in Simmental cattle is also unknown; testing for the disorder has not been mandated by breed organizations.
These disorders, previously referred to as canine, Basset Hound, Spitz, Landseer, or Simmental thrombopathia, should probably be referred to as CalDAG-GEFI thrombopathias or CalDAG-GEFI platelet disorders because the specific cause of the disorders is now known. Two children are reported as having CalDAG-GEFI platelet disorders.66 Interestingly, the 2 affected children reportedly had a bleeding diathesis, problems related to defective neutrophil function, and overwhelming bacterial infections. The protein CalDAG-GEFI may play an important role in signal transduction events necessary for activation of integrin-type adhesion molecules in neutrophils and platelets in humans; more studies are needed to explore this possibility. All CalDAG-GEFI mutations identified thus far in dogs and cattle have solely been associated with a bleeding diathesis; problems with infections have not been observed. Although CalDAG-GEFI is also expressed in the brain, neurologic signs in humans or other animals with CalDAG-GEFI disorders have not been reported.
Granule disorders—Granule disorders of several species have been described and include Chediak-Higashi syndrome,2,3,15,19,26 cyclic hematopoiesis of gray Collies,6 and selective ADP deficiency in Cocker Spaniels.11 Leukocyte and melanocyte abnormalities also exist in Chediak-Higashi syndrome, which has been diagnosed in Persian cats, mice, foxes, killer whales, and cattle. Platelets of affected animals lack discernible dense granules and are deficient in storage pools of adenine nucleotides, serotonin, and divalent cations. Results of studies of platelet ultrastructure indicate that platelets of animals with Chediak-Higashi syndrome do not form tight aggregates in response to ADP in vitro.3
In 1 lineage of Persian cats, all cats with Chediak-Higashi syndrome had a blue-smoke hair color and pale irises, and several also had bilateral nuclear cataracts.2,67,68 These affected cats experienced prolonged bleeding at incision sites and the development of hematomas following venipuncture. In humans and Japanese black cattle, Chediak-Higashi syndrome results from mutations in the lysosomal trafficking regulator gene, which encodes a 425-kd cytoplasmic protein that may be involved with incorporation of proteins into lysosomal membranes.61 The molecular cause of Chediak-Higashi syndrome in Brangus and Hereford cattle is not known, but distinct mutations within the gene that encodes lysosomal trafficking regulator protein are likely.
Cyclic hematopoiesis, which has been described in gray Collies, also affects leukocytes and melanocytes. The disorder is characterized by cyclic fluctuations in numbers of circulating neutrophils, reticulocytes, and platelets.69,70 The mortality rate is high among affected dogs; most puppies die prior to 6 months of age because of fulminating infection. Platelet numbers are typically no lower than the lower reference limit and usually fluctuate between 300,000 and 700,000/ML. Platelet reactivity to collagen and, possibly, thrombin is defective. Platelet content of serotonin, ATP, and ADP is reduced. Clot retraction and platelet adhesiveness are impaired.6 A mutation in the gene encoding the B-subunit of adaptor protein complex 3 has been identified. The protein complex directs trans-Golgi export of transmembrane cargo proteins to granules.62 Selective platelet ADP deficiency in a family of Cocker Spaniels has been reported.11 In those dogs, uptake and release of serotonin were not impaired, suggesting that dense granules were present. Affected Cocker Spaniels from outside this family have not been identified, and molecular evaluation of potential candidate genes has not been conducted.
Procoagulant expression disorder—Platelet activation results in the flip-flop of phospholipids from the inside to the outside and from the outside to the inside of the outer platelet membrane. Expression of phosphatidylserine on the platelet surface results in an overall negative charge, which is necessary, along with calcium, for assembly and support of the coagulation proteins leading to the production of fibrin.71 Scott syndrome is a rare disorder of humans in which platelets do not express procoagulant activity.72 A similar disorder in a family of German Shepherd Dogs has been described.18,63 Results of platelet function tests, including aggregation and release, clot retraction, and bleeding times, are within reference ranges, as are platelet numbers. Affected platelets are impaired in their ability to express phosphatidylserine or prothrombinase activity on their surface. Binding of annexin V to affected platelets in response to calcium ionophore is markedly reduced. Although this disorder is a platelet disorder, clinical signs are more typical of a coagulopathy because of the inability of platelets to generate and support a surface for coagulation proteins. Results of coagulation screening tests, however, are within reference ranges because these assays do not use platelets from affected dogs to support the reactions evaluated in the assays. The molecular basis for procoagulant expression disorder in humans and German Shepherd Dogs is not known.
Inherited macrothrombocytopenia—Many types of inherited macrothrombocytopenias have been identified and evaluated in humans.73,74 In most instances, these inherited macrothrombocytopenias are associated with a bleeding diathesis. In veterinary medicine, Cavalier King Charles Spaniels can inherit macrothrombocytopenia as an autosomal recessive trait.10,20,75,76
Affected dogs typically have large platelets, with platelet numbers ranging between 50,000 and 100,000/ML. These dogs do not have bleeding tendencies; however, because the cause of their thrombocytopenia may be misdiagnosed, they are at risk for receiving inappropriate medical (corticosteroids or antimicrobials) or surgical (splenectomy) treatments.77 The molecular basis for the inherited macrothrombocytopenia of Cavalier King Charles Spaniels is a mutation in the gene encoding β1-tubulin.27 It is speculated that the mutation results in microtubule instability, which alters megakaryocyte proplatelet release. Molecular assays are available at Auburn University and should assist veterinarians in distinguishing the cause of the thrombocytopenia in a particular animal. Recently, 60 Cavalier King Charles Spaniels from the United States and 40 others from Dublin were evaluated for the mutation.27 Investigators in that study identified that the mutation existed in approximately 90% of the US dogs evaluated; approximately 50% of those dogs were carriers, and 50% were affected. In contrast, 50% of Dublin dogs were carriers, whereas only 12.5% were affected with macrothrombocytopenia.
Diagnosis
Diagnosis of novel intrinsic platelet disorders is often made on the basis of exclusion because of the limited availability of specialized techniques required for specific diagnosis. Animals with platelettype bleeding, values for coagulation screening tests (APTT, PT, and thrombin time) and platelet numbers that are within reference ranges, and vWF concentrations that are not sufficiently low to explain the amount of hemorrhage (typically > 15% vWF antigen plasma concentrations) are likely candidates for the diagnosis of an intrinsic platelet disorder. Molecular assays have become available for diagnosis of identified intrinsic platelet disorders in dogs, horses, and cattle. These assays can greatly assist veterinarians in diagnosis of disease and detection of carriers, thus providing a critical service for clients involved in breeding animals. Uncharacterized intrinsic platelet disorders are likely to exist, necessitating that veterinarians be vigilant and knowledgeable in their approach to unexplained bleeding disorders in animals to avoid misdiagnosis. This becomes critically important in case management because control of a bleeding episode in an animal with an intrinsic platelet disorder requires transfusion of platelet-rich plasma or platelet concentrates (Table 2). Whole blood transfusions may be required in animals that have Hct values < 15%; however, whole blood transfusions often will not provide enough functional platelets to curtail the hemorrhagic episode. Details concerning transfusion requirements are beyond the scope of this report, and readers are encouraged to obtain more detailed information elsewhere.78
Comparison of platelet disorders, associated diagnostic assays, and appropriate transfusion products.
| Platelet disorder | Diagnostic assays | Transfusion product* |
|---|---|---|
| vWD | vWF antigen (ELISA); molecular assays available for some breeds | Cryoprecipitate or plasma |
| Intrinsic platelet function disorders | Platelet function testing†; molecular assays available for some breeds | Platelet-rich plasma or platelet concentrates |
| Inherited macrothrombocytopenia | Platelet count and smear evaluation; molecular assay available | Treatment not necessary |
Whole blood should be administered to animals with PCVs < 15%; however, whole blood may not contain a sufficient number of platelets to curtail bleeding associated with severe acquired thrombocytopenia or a platelet function disorder.
Diagnosis of novel congenital intrinsic platelet function disorders requires specialized testing (eg, platelet aggregometry, flow cytometry, platelet release assays, or electron microscopy), and a definitive diagnosis usually requires that the affected animal be on or near the premises of the testing facility. Veterinary colleges in the United States that routinely perform platelet function testing on animals that the author is aware of include those at Auburn University; the University of California, Davis; Cornell University; Michigan State University; and the University of Pennsylvania.
Case Management
The author has extensive experience with the care of dogs with inherited platelet disorders, particularly Great Pyrenees with Glanzmann thrombasthenia. Elective surgical procedures in thrombopathic animals should be avoided or accompanied by appropriate transfusion products. Simple procedures in clinically normal animals may not be simple procedures in thrombopathic animals. Ear and teeth cleaning can result in severe hemorrhage that may be difficult to control without transfusion. Hemorrhage into the ear canal can occur with ease in a thrombopathic animal. Even minor trauma should be avoided because this can lead to hemorrhage, followed by scratching, resulting in more trauma and more hemorrhage—a vicious cycle that will be difficult to curtail. Medications that inhibit platelet reactivity should be avoided, including the use of topical medicated powders on open wounds. Aerosolized irritants, such as those associated with cedar shavings, detergents, or other harsh odor–producing substances, can elicit hemorrhage on mucosal surfaces of the respiratory tract. Insidious blood loss through the gastrointestinal or urinary tract can result in severe iron deficiency anemia and is of particular concern in young growing animals in which demands for iron are increased.
Owners should be taught to evaluate the color of mucous membranes on a regular basis. Veterinarians may want to evaluate Hct values or serum iron concentrations in thrombopathic dogs every 4 to 6 months (or more frequently in young, growing animals). Properly managed thrombopathic animals can live long lives; the diagnosis of thrombopathia is not a death sentence. The first Great Pyrenees with Glanzmann thrombasthenia lived to be 12 years old in a home setting. Thrombopathic Basset Hounds have also lived to be > 10 years of age in laboratory and home settings.
Conclusions
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 vWD in certain breed lineages. Hemorrhage may be difficult to identify or detect in certain situations, making diagnosis difficult. For example, the author is aware of 2 Basset Hounds with CalDAGGEFI thrombopathias that were evaluated for paralysis secondary to spinal cord hemorrhage. Clinical signs mimicked, to some extent, the clinical signs associated with a herniated disc. An intrinsic platelet disorder should be suspected whenever hemorrhage exists despite results for coagulation screening assays and platelet counts that are within reference ranges and whenever vWF antigen concentrations in plasma are > 15%. Although coinheritance of vWD and an intrinsic platelet disorder has not yet been recognized, this may occur, particularly in some breeds, and may account for some of the variability in clinical signs reported for the various inherited platelet disorders.
ABBREVIATIONS
| APTT | Activated partial thromboplastin time |
| BMBT | Buccal mucosa bleeding time |
| CalDAG-GEFI | Calcium diacylglycerol guanine nucleotide exchange factor I |
| DAG | Diacylglycerol |
| ECT | European continental type |
| GEF | Guanine nucleotide exchange factor |
| PAR | Protease-activated receptor |
| PT | Prothrombin time |
| vWD | von Willebrand disease |
| vWF | von Willebrand factor |
Landseer-ECT data include results for 41 DNA samples provided by Dr. Peter Leegwater, Utrecht University, Utrecht, The Netherlands.
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