Platelet disorders (Proceedings)

Platelets are essential for normal hemostasis and serve three main functions. They maintain vascular integrity by forming platelet plugs and promoting endothelial vasoconstriction in areas of disrupted endothelium. Second, they assist with procoagulant activity to help in secondary hemostasis and fibrin formation. Third, they promote vascular healing through platelet-derived growth factor, which works to stimulate endothelial cell migration and smooth muscle production.

Platelets form a hemostatic plug by adhering to exposed subendothelial collagen. This adherence is mediated by vonWillebrand's factor (vWF), which is formed by endothelial cells and megakaryocytes and is found free in the plasma as well as within platelet alpha granules. Substances such as thromboxane A2, thrombin, and endotoxin activate platelets. After platelets adhere to the subendothelial collagen, they go through a shape change, exposing surface receptors to soluble fibrinogen. Platelets release ADP, which activates synthesis of thromboxane A2 and causes irreversible platelet aggregation and intense local vasoconstriction. Decreased number or decreased function of platelets is relatively common causes of bleeding disorders in our patient population.

Diagnosis of Bleeding Disorders

Correct and rapid diagnosis of bleeding disorders is extremely important as delay can lead to possible death of the patient. A thorough history, detailed physical examination, and careful selection of laboratory tests will greatly facilitate a diagnosis. Patients may present with overt bleeding, signs of anemia or hypovolemia, or no obvious symptoms. If there is no explanation for the bleeding, if bleeding is occurring at multiple sites, or if the bleeding seems disproportionate to the injury, a hemostatic defect should be considered.

A thorough, detailed history is vital to the diagnosis. Ascertain the duration of the problem, history of previous bleeding disorders, toxin or medication exposure, and familial history. The age and breed of the patient and the age at which symptoms first appeared may lead the clinician to form a more accurate differential list.

A careful physical examination should be performed. The hallmark clinical sign of thrombocytopenia is the presence of petechiae or ecchymoses. These are most often noted on the abdomen, inguinal area, ear pinnae, sclera, and mucous membranes. Petechiae and ecchymoses are less common in thrombopathias, but they may occur. Signs of clinical hemorrhage in these patients may include melena, hematuria, epistaxis, hyphema or retinal hemorrhage, and prolonged or excessive bleeding at venipuncture, trauma, or surgical sites.

Laboratory tests

The normal platelet count is 200,000-500,000/μl. Simple in-house screens for platelet disorders include the blood smear and the buccal mucosal bleeding time (BMBT). In order to perform a good blood smear, blood should not remain in anticoagulant for more than a few hours or some platelet clumping may occur. The entire smear should be scanned for the presence of platelet clumps, which might result in pseudothrombocytopenia. Clumps are very common in blood smears from cats. Platelets should be counted in the monolayer area of the smear, and the number of platelets counted in the high-powered field (oil) should be averaged. Normal is 8-15 platelets/hpf in dogs and 10-12 platelets/hpf in cats. Each platelet represents approximately 15,000 platelets/μl, so a platelet estimate can be obtained by multiplying the average platelets per hpf by 15,000. Animals rarely bleed spontaneously until platelet count is less than 40,000/μl.

The smear should be evaluated for reticulated or immature platelets and macroplatelets. Small platelets may be seen with early immune-mediated platelet destruction, and large platelets often indicate a regenerative thrombopoiesis. Mean platelet volume (MPV) can be determined from a blood sample collected in citrate anticoagulant and stored at 37°C until evaluation. Platelet distribution width (PDW) may also be performed to assess platelet size.

The BMBT is used to assess platelet and vascular function if the platelet count is within normal range. The Simplate II (Organon Teknika, Durham, NC) contains a triggered blade that makes an incision in the buccal mucosa. Ideally, the upper lip of the patient should be tied in place, as fingers may accidentally move. As bleeding occurs from the site, it is wicked away using filter paper and taking care not to touch the incision site. The time from incision to a clot formation is timed. Normal is less than 4 minutes in the dog. If the BMBT is prolonged, and the platelet count is normal, a platelet or vascular dysfunction syndrome should be suspected.

Further platelet function tests include clot retraction, antiplatelet antibody, platelet adhesion, platelet aggregation, and thromboelastography.

Treatment of Bleeding Disorders

Treatment for hypovolemia or anemia secondary to blood loss must be instituted if these patients present symptomatic. Patients should be handled carefully and kept in a well-padded cage when in hospital. When treating at home, exercise should be restricted, and soft food should be fed to prevent gingival trauma. IM injections and jugular venipuncture should be avoided. Any venipuncture site should have mild pressure applied for at least 5 minutes. Avoid drugs that decrease platelet function. For pain control, NSAIDs should be avoided and narcotics used instead. If antibiotics are needed, avoid penicillins, tetracyclines, gentamicin, and sulfa drugs. If anesthesia or sedation is needed, avoid acepromazine, diazepam, ketamine, propofol and halothane. H1 and H2 blockers impair platelet aggregation.

Blood transfusions are generally administered for symptomatic support for the blood loss rather than for treatment, as most transfusions supply little, if any, viable platelets to the patient. A whole blood or packed red blood cell transfusion should be considered if the patient is symptomatic for anemia or has ongoing losses of a substantial amount of blood.

Platelet transfusions are challenging, however there are a few alternatives available. The clinician needs to understand that a platelet transfusion will only be a short fix. Once the platelets are consumed, the benefit is over. In animals with immune-mediated platelet destruction, the half-life of the platelets will be even shorter. There is also some question as to the viability of the platelets in a transfusion. Therefore, the cost to benefit ratio needs to be considered carefully before choosing to go forward with a platelet transfusion. Consider platelet transfusions for those patients who are undergoing a life-threatening hemorrhagic secondary to thrombocytopenia. A platelet transfusion may help arrest this hemorrhage long enough to get definitive treatments started. Another possible time to consider a platelet transfusion is in a thrombocytopenic patient who is about to undergo an invasive procedure (i.e. a splenectomy for a IMT dog). In this case, the platelets should be administered immediately prior to beginning the procedure.

Options for platelet transfusions include fresh whole blood, platelet-rich plasma, platelet concentrate, and lyophilized platelets. Fresh whole blood is a minor platelet source, and is best chosen in patients that have significant, ongoing hemorrhage, as a large volume must be transfused in order to get a measurably significant amount of platelets.

Platelet rich plasma is harvested from a unit of fresh whole blood that is less than 8 hours old and has not been refrigerated. Refrigerated platelets do not maintain function or viability as well as those stored at room temperature. Also, platelets start to lose viability after 24-hours, so it is best to make a unit just prior to administration. Under ideal situations, platelets from a single unit of PRP administered to a 30kg dog would raise the platelet count by 10,000/μl.

Platelet concentrates are preserved in 6% DMSO and may conserve for up to 3 years. However, this method of storage has been shown to decrease platelet viability, decrease aggregation, and reduce clinical efficacy compared to fresh platelets. A frozen platelet concentrate has been produced and is commercially available for dogs (Midwest Animal Blood Services, Stockbridge, MI).

Lyophilized platelets are cryopreserved platelets and have recently become available from Midwest. These are undergoing clinical trials for efficacy at this time and have not been released for general use.

Cryoprecipitate does not contain platelets, but is used to treat the inherited thrombopathia von Willebrand Disease (vWD) as well as the inherited coagulopathy hemophilia A (factor VIII deficiency) and fibrinogen deficiencies. Cryoprecipitate is the precipitate from plasma as it is slowly thawed, and it contains concentrated vWF, factor VIII, fibrinogen, and fibronectin.

Specific Disorders of Primary Hemostasis

Primary hemostatic disorders include thrombocytopenias, thrombopathias, and vascular disorders. Thrombocytopenias can arise through three main mechanisms; decreased production, increased destruction, or sequestration/consumption. Decreased platelet production can occur with bone marrow disorders such as infiltration by infectious or neoplastic cells, aplastic marrow, exposure to marrow toxins (chemotherapeutics, sulfadiazine, estrogen), retroviruses (FeLV, FIV), vaccinations, and Ehrlichia platys. Decreased production is a relatively rare cause of thrombocytopenia, and these patients often present with a bi- or pancytopenia.

Platelet destruction can be immune-mediated, either autoimmune or secondary to a haptogen, such as a drug or infectious organism. Causes of platelet destruction include Ehrlichia, bacterial, or viral infections; and neoplasia. Platelet consumption/sequestration can occur with disseminated intravascular coagulation, hypersplenism, endotoxemia, or severe hemorrhage.

Thrombopathias may be acquired or inherited. Etiologies for acquired thrombopathias include uremia, infectious agents such as Ehrlichia and FeLV, snake venom, hepatic and pancreatic disease, neoplasia, and drugs such as aspirin, antibiotics, anesthetics antihistamines, and calcium channel blockers. Inherited thrombopathias are discussed below.

Thrombocytopenias, Acquired Disorders

Immune-Mediated Thrombocytopenia (IMT)

Antibodies are directed towards platelet surface antigen, and as a result of antibody binding, there may be reduced circulating platelet lifespan and/or impairment of platelet function. Primary (idiopathic) IMT disorder is seen in dogs and is more prevalent in certain breeds such as the Cocker Spaniel, English Sheepdog, German Shepherd, and Poodle. Antiplatelet or antimegakaryocytic antibodies may be detected. Secondary IMT is more common and may be associated with systemic autoimmune disease (SLE), neoplasia (hemangiosarcoma or lymphoproliferative tumors), infectious disease (ehrlichiosis, RMSF, FeLV, FIV, babesiosis, dirofliariasis), drugs (heparin), modified live vaccinations, or immune-complex vasculitis.

Clinical signs usually develop when the rate of platelet destruction exceeds the rate of production, and the platelet count falls below 40,000/μl. Normal platelet lifespan is approximately 7 days, but with IMT it can fall to as short as 1 hour.

Diagnosis: Clinical signs include those listed above for thrombocytopenia. Weakness, pale mucous membranes, and lethargy are common symptoms as well. There is not a specific diagnostic test for IMT. Thrombocytopenia should be confirmed via a CBC, blood smear, and platelet estimate. Thoracic radiographs and abdominal ultrasound should be performed to rule out neoplasia as an underlying cause. Coagulation tests should be performed to rule out coagulopathies and DIC. A heartworm antigen test is recommended. ANA should be performed in those patients suspected of having SLE. Tests to detect anti-platelet antibodies may be useful, although these tests are not readily available and sensitivity varies. A bone marrow aspirate and biopsy may be indicated to evaluate for megakaryocytes.

Treatment: Secondary IMT generally resolves itself once the underlying pathology has been treated or removed. However, immune-suppression may be needed in severe cases until the condition reverses itself. Primary IMT requires immune-suppressive drugs to stop the destruction of self-antigens. Glucocorticoids are the first line drugs for IMT. Prednisone or prednisolone at 2-4 mg/kg PO, SQ, IM every 12-24 hours until normalization of platelet count, followed by a slow taper every 2-3 weeks until lowest effective dose. Dexamethasone (0.25-0.3mg/kg IV, SQ) may also be used in hospital in a vomiting animal if needed.

Other immunosuppressive drugs that may be considered include azathioprine (Imuran®) at 2mg/kg PO q 24 hr. Bone marrow suppression and hepatotoxicity are possible side effects. Azathioprine can be combined with prednisone and may result in longer survival times. Cyclophosphamide (Cytoxan®) at 1-2 mg/kg PO four days/week can also be added. Side effects may include myelosuppression, GI disturbances, and hemorrhagic cystitis. This drug has not shown any benefit when combined with prednisone, so at this time it is not recommended. Cyclosporine (Neoral®) at 5-10 mg/kg/day PO divided BID is a strong immunosuppressant that may be added in stubborn cases. This drug is not myelosuppressive, so it can also be used in cases with bone marrow dysfunction. It is costly and does require therapeutic blood monitoring.

Vincristine has been shown to help increase the platelet count and provide a faster recovery time when combined with prednisone. The dose is 0.02 mg/kg IV once. Increased platelet numbers may be seen in 5-7 days. Vincristine both increases production and decreases destruction of platelets. Vincristine can be repeated once in two weeks if needed.

Human immunoglobulin at 0.5-1.0 g/kg may be used in IMT refractory to therapy. It works by blocking the Fc receptors on mononuclear phagocytic cells. It can be safely administered 1-2 times with minimal side effects. It has been demonstrated to increase short-term survival with little effect on long-term survival.

Other treatments that may be considered include plasmapheresis and splenectomy. Plasmapheresis requires specialized equipment and training, so has very limited availability. Splenectomy has shown some success, however surgery on a thrombocytopenic patient carries its own risks.

The prognosis is fair to good for primary IMT that is treated properly. Long-term recurrence rate is 25% with 50% of these patients becoming refractory to immunosuppressive therapy.

Thrombopathias, Inherited Disorders

Von Willebrand Disease

Von Willebrand Disease (vWD) is the most common inherited bleeding disorders in the dog. The disease occurs when there is a deficiency of von Willebrand factor (vWF), which is a glycoprotein that mediates platelet adhesion to exposed endothelial tissue following vessel damage and promotes platelet aggregation. vWF also serves as a carrier protein for factor VIII.

Three types of vWD disease are recognized in dogs. Type I vWD is a partial deficiency of vWF, and is common in the Doberman Pinscher breed. Type II vWD involves a deficiency or low number of the high molecular weight multimers of vWF and has been identified in the German Shorthair and Wirehaired Pointers. Type III vWD is a severe deficiency in all multimers and has been seen in Scottish Terriers, Shetland Sheepdogs, and Chesapeake Bay Retrievers.

The diagnosis of vWD is based on clinical signs, in-house screening tests, vWF analysis, and genotypic analysis. The clinical symptoms include bleeding excessively following surgery or injury and bleeding from mucosal surfaces. Petechiae are not common with vWD. Type II and III are associated often with the most severe bleeding.

Screening tests include the BMBT and platelet function analyzer (PFA-100; Dade-Behring, Deerfield, IL). The BMBT is more commonly available and cheaper, although it may be less sensitive. vWF analyses are done by ELISA. Genetic tests are also available now in several breeds. To identify which multimers are involved, a multimeric analysis is also available, and a collagen binding assay is now available for functional analysis.

Treatment ranges from symptomatic support (i.e. local pressure or cautery at a bleeding wound) to transfusion and DDVP therapy. Desmopressin (DDAVP) can increase factor concentration in type I dogs and may be used to boost the vWF concentration in donor dogs. The beneficial hemostatic effects of DDAVP are evident within 30 minutes following administration of 1 μg/kg subcutaneously (nasal spray preparation) and may last for up to 4 hours. If needed, DDAVP can be repeated in 24 hours. Cryoprecipitate is used to control active bleeding in affected dogs or pre-surgically as a preventative. The effects are short lived, and there is no long term or definitive treatment for vWD.

Canine Thrombasthenic Thrombopathia of Otter Hounds

This is a variant of human Glanzmann's thombasthenia. There is an absence or marked reduction in the platelet GpIIb/IIIa receptor. This receptor is required for shear rate dependent adhesion and aggregation. A similar defect is reported in the Great Pyrenese.

Chediak-Higashi Syndrome

This is an autosomal recessive disorder of Persian cats and other species. The bleeding tendency is secondary to a platelet storage pool deficiency. These platelets lack dense granules eliminating important platelet agonists that perpetuate clot formation. Humans and animals with this syndrome also have hypopigmentation of the skin, hair and eyes.

Canine Thrombasthenia of Basset Hounds

Functional abnormalities are associated with impaired cyclic AMP (cAMP) metabolism and abnormal platelet phosphodieterase activity. cAMP is elevated which is an inhibitory signal to platelet activation. Platelet aggregation in response to most agonists is abnormal.

Vasculitis

Endothelial injury may be induced by infectious agents, hypoxia, acidosis, inflammation, and immune mediated disease. Vasculitis consumes platelets and leads to generalized microangiopathic disease. Systemic vasculitis can occur secondary to diseases such as tick borne disease, systemic inflammatory response syndrome (SIRS), or sepsis.

References available upon request