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Chasing after the causes of platelet disorders June 2002 The following article was adapted from the Archives of Pathology & Laboratory Medicine (2002;126:133-146). Multiple etiologies exist for platelet-derived bleeding disorders. The laboratory evaluation of these disorders can range from simple to complex and should initially include a thorough evaluation of the patient's medical history, concentrating on personal and familial bleeding disorders and current medications. With this as a starting point, the algorithms presented here may be helpful in elucidating the underlying etiology for platelet-derived bleeding. Role of platelets in hemostasis Platelets are small (2 µm-diameter), non-nucleated blood cells produced in the bone marrow from megakaryocytes. They are rapidly activated by blood vessel injury and are a crucial component of the primary hemostatic response. In their unactivated state, platelets are roughly discoid in shape and contain cytoplasmic organelles, cytoskeletal elements, invaginating open-canalicular membrane systems, and platelet-specific granules called alpha and dense granules. Platelets have numerous intrinsic glycoproteins attached to the outer surface of their plasma membrane that are receptors for such ligands as fibrinogen, collagen, thrombin, and thrombospondin to von Willebrand factor and fibronectin.1 Platelets promote hemostasis by the following interconnected mechanisms:
Platelet adhesion to subendothelium is the initial step in platelet activation. The subendothelium is composed of extracellular matrix proteins, many of which are ligands for receptors on the platelet surface. These adhesive proteins are exposed when the endothelial layer is disrupted. Due to the large number of extracellular matrix proteins and a high density of platelet surface receptors, platelets adhere rapidly to areas of vascular injury. Von Willebrand factor, a large, multimeric protein secreted into the extracellular matrix from endothelial cells, facilitates platelet adhesion by binding to platelet surface glycoprotein Ib/IX/V, especially at high shear rates.2 Platelets can also adhere to vascular wall-associated fibrin or fibrinogen by interacting with platelet surface glycoprotein IIb/IIIa.3 After adhering to the subendothelium, platelets undergo cytoskeletal activation, which leads to a change in their shape and the development of pseudopods. Intracellular signaling processes leading to increased cytoplasmic calcium then initiate a secretory release reaction that releases products from the alpha granules (platelet factor 4, β-thromboglobulin, thrombospondin, platelet-derived growth factor, fibrinogen, VWF) and dense granules (adenosine diphosphate [ADP], serotonin).4 The release of ADP combined with calcium mobilization leads to a conformational change of the fibrinogen receptor, the GP IIb/IIIa receptor complex (integrin αIIbβ3). This initiates the process of aggregation, in which a GP IIb/IIIa receptor on one platelet is bound in a homotypic fashion to the same receptor on adjacent platelets via a central fibrinogen molecular bridge. Beside ADP, other agonists, such as epinephrine, thrombin, collagen, and platelet-activating factor, can initiate platelet aggregation by interacting with membrane receptors. This platelet-release reaction and aggregation recruits many other platelets to the vessel wall and forms a hemostatic platelet plug. Activated platelets also play a vital procoagulant role that serves as a link between platelet function and coagulation activation. Platelet membrane phospholipids are rearranged during activation, and phosphatidyl serine is transferred from the inner table to the outer table of the platelet membrane, providing a binding site for phospholipid-dependent coagulation complexes that activate factor X and prothrombin. Laboratory tests for evaluating platelet function Clinical history Platelet count and peripheral blood smear The mean platelet volume is an indication of platelet size. Normal MPV ranges are approximately 7 to 11 fL. The MPV can be an indication of platelet turnover because younger platelets tend to be larger. A spectrum of platelet sizes is seen in patients with rapid turnover, while true congenital macrothrombocytopenias usually have uniformly large platelets. Initial platelet function tests or bleeding time Bone marrow examination Platelet aggregation Coagulation testing and von Willebrand assay Electron microscopy Newer methods of platelet evaluation The PFA-100 measures platelet-related primary hemostasis in citrated whole blood specimens.9 It uses two disposable cartridges that contain a membrane with a central aperture (147 µm) coated with aggregation agonists (collagen and epinephrine, and collagen and ADP), through which platelets are passed at high shear rates (5,000 to 6,000 s-1). The instrument measures the "closure time" required for platelets to adhere to the membrane, aggregate, and occlude the aperture. The collagen-epinephrine cartridge detects platelet dysfunction induced by intrinsic platelet defects, von Willebrand disease, or platelet-inhibiting agents. The collagen-ADP cartridge usually produces abnormal results with platelet disorders and von Willebrand disease but produces a normal closure time with aspirin-like drugs because of their high ADP concentrations. Von Willebrand disease, intrinsic platelet dysfunction, and nonaspirin drugs may produce an abnormal closure time with both cartridges. The rapid platelet-function assay Ultegra is an automated turbidimetric whole blood assay designed to assess platelet aggregation based on the ability of activated platelets to bind fibrinogen.10 Fibrinogen-coated polystyrene microparticles agglutinate in whole blood in proportion to the number of available platelet glycoprotein IIb/IIIa receptors.10 The Ultegra specifically is designed to measure the effect of glycoprotein IIb/IIIa antagonist drugs, such as abciximab, tirofiban, or eptifibatide. It is not sensitive to such drugs as aspirin, clopidogrel, or ticlopidine, and it is not designed to detect platelet function disorders or von Willebrand disease. Plateletwork's rapid platelet aggregometer is designed to determine the percentage of platelet aggregation in fresh whole blood samples taken during interventional cardiac procedures. It measures the change in the platelet count due to aggregation of functional platelets in the blood sample. It is the first bedside test to simultaneously measure platelet count and platelet aggregation. Flow cytometry has been used to study platelet structure and function, but only in specialized centers. It detects platelet activation using antibodies to proteins newly expressed on the platelet surface during activation. Platelet flow cytometry can be used to diagnose deficiencies of platelet surface glycoproteins. It also can be used to measure platelets with increased RNA content using the dye thiazole orange, which binds to RNA and DNA. This technique is used to evaluate whether thrombocytopenia is due to increased platelet destruction or decreased platelet production since newly released platelets have increased RNA content. Categories of platelet-derived bleeding diathesis Platelet dysfunction can lead to a clinical bleeding disorder that is congenital or acquired in nature. Laboratory evaluation of platelet dysfunction is often complex, but it can be simplified by using a diagnostic algorithm and classifying such disorders as platelet dysfunction associated with normal, decreased, or increased platelet counts (Figs. 2,3,4). A thorough medical, family, and drug history is essential in establishing the etiology of platelet dysfunction, as is exclusion of coagulation and fibrinolytic disorders. In all of the disorders discussed below, the results of the coagulation screening tests PT and APTT should be considered normal. Platelet dysfunction with normal platelet count Drug-induced platelet dysfunction is probably the most common cause of platelet-mediated bleeding and will also demonstrate platelet dysfunction with a normal platelet count, so it is extremely important to take a careful drug history.5 A list of drugs that cause platelet dysfunction can be found in Table 1. Platelet aggregation abnormalities typically found with drugs such as aspirin, GP IIb/IIIa antagonists, or the thienopyridines can be found in Table 2. Glanzmann thrombasthenia is a congenital deficiency or dysfunction of GP IIb/IIIa, the receptor for fibrinogen, and is responsible for mediating platelet aggregation.11 It is an autosomal recessive disorder that manifests in lifelong mucocutaneous bleeding. Mutations of GP IIb and GP IIIa have been implicated.12 The initial platelet function test will be abnormal in patients with Glanzmann thrombasthenia. No aggregation response will result from adding ADP, collagen, epinephrine, and arachidonic acid-aggregating agents, whereas the ristocetin-induced aggregation is normal11 (Table 2). This finding is virtually diagnostic of Glanzmann thrombasthenia, but the disorder can be confirmed by platelet flow cytometry or crossed immunoelectrophoresis of platelet membrane proteins. Abnormalities of platelet secretion can be due to deficiency of platelet granules or defects in the signal transduction events that regulate secretion or aggregation.13 Platelet storage pool disorders can be congenital or acquired and result from a deficiency of granules (alpha or dense granules, or both) or a defective release of granules at platelet activation.14 Dense granule storage pool disorders (δ-SPDs) can appear as a singular clinical entity or as part of other hereditary disorders, such as Chediak-Higashi, Hermansky-Pudlak syndrome, thrombocytopenia with absent radii (TAR syndrome), or Wiskott-Aldrich syndrome.14 δ-SPD often shows decreased aggregation response to ADP, epinephrine, and collagen and normal aggregation to arachidonic acid and ristocetin (Table 2). Decreased ATP release by lumiaggregometry and decreased mepacrine uptake/release by flow cytometry are observed. Ultrastructural abnormalities in these disorders usually show decreased dense granules. Furthermore, α-SPD (gray platelet syndrome) has decreased alpha granules and is usually considered a macrothrombocytopenia (see the subheading Platelet disorders with thrombocytopenia).15 A rare α/δ-SPD that has features of both disorders has been described in the literature. Acquired platelet storage pool disorders can be seen with underlying myeloproliferative disorders or in clinical scenarios where there is ongoing in vivo platelet activation, such as cardiopulmonary bypass, disseminated intravascular coagulation, and thrombotic thrombocytopenic purpura/hemolytic uremic syndrome. In addition to being seen in storage pool disorders, platelet release defects can be found with defects of platelet signal transduction. These generally are a poorly defined group of disorders, but they may constitute a significant percentage of patients with abnormal secondary wave of aggregation and decreased granule release in whom alpha and dense granules are not deficient.13 Other significant disorders of platelet function that have platelet counts in the normal range usually are acquired with the presence of another disease or drug therapy.5 These are far more common than the aforementioned disorders. Platelet dysfunction is often observed with chronic renal failure or liver disease in patients suffering from a variety of myeloproliferative and lymphoproliferative disorders-for example, polycythemia vera, myelofibrosis, paroxysmal nocturnal hemoglobinuria, acute myelogenous leukemia, and hairy cell leukemia. Platelet dysfunction also may be associated with a variety of clinical scenarios, such as previous cardiopulmonary bypass, implantation of prosthetic materials, including vascular grafts and prosthetic heart valves, and use of ventricular assistance devices. Platelet dysfunction in these disorders usually is difficult to characterize because nonspecific defects of platelet aggregation typically are observed. Platelet disorders with thrombocytosis In patients with thrombocytosis, the differential diagnosis is primarily between a reactive thrombocytosis and a myeloproliferative process (essential thrombocytosis, chronic myelogenous leukemia, polycythemia vera, and myelofibrosis). The algorithmic approach to the diagnosis of thrombocytosis is shown in Fig. 2. Patients with a myeloproliferative disorder typically have platelet counts greater than 1 x 106/µL, and patients with reactive thrombocytoses have lower counts, but there is a great deal of overlap. For myeloproliferative disorders, characteristic features of a specific disease can be discerned by examining the peripheral blood smear and bone marrow and with cytogenetic studies. Platelet aggregation studies alone can suggest an underlying myeloproliferative disorder, particularly when epinephrine-induced aggregation alone is reduced or absent.16 The decreased epinephrine-induced aggregation is thought to be due to down-regulation of α2-adrenergic receptors. Other patterns of platelet dysfunction with myeloproliferative disorders include decreased platelet aggregation to ADP or collagen, dense-granule storage pool pattern, abnormal platelet morphology, abnormalities of the arachidonic acid pathway, decreased receptors for prostaglandin D2, or increased aggregation with various agonists. In the clinical evaluation of patients with myeloproliferative disorders, it is important to remember that bleeding and thrombosis can be observed in these patients and that the results of the platelet function tests will not necessarily distinguish whether a patient is at risk for bleeding or thrombosis. In contrast to patients with myeloproliferative disorders, patients with reactive thrombocytosis usually have normal platelet function. A reactive, or secondary, thrombocytosis can be associated with many clinical entities, including iron deficiency, inflammatory and infectious disorders post-splenectomy in such malignancies as carcinomas or lymphomas, as well as myelodysplastic disorders, smoking, and exercise. It can also be observed as a rebound thrombocytosis following splenectomy, treatment for idiopathic thrombocytopenic purpura, pernicious anemia, or after cessation of myelosuppressive drugs. Platelet disorders with thrombocytopenia
Thrombocytopenia with small platelets can be seen in patients with Wiskott-Aldrich syndrome.17 This is an X-linked recessive disorder characterized by immunologic abnormalities, recurrent infections, eczema, and thrombocytopenia. The mean platelet volume, a measure of platelet size included in most CBCs, is often low (about half normal size). Platelet dysfunction is severe; the platelets are unable to aggregate and a storage pool-like pattern is often seen. Patients with thrombocytopenia due to marrow aplasia may also have small platelets, but the MPV is usually low-normal, not decreased. The rare macrothrombocytopenia disorders are congenital in nature and most are inherited in an autosomal dominant fashion. They usually are due to congenital defects in platelet production by megakaryocyte or demarcation membrane systems, although the structural or genetic abnormalities are known in only a few disorders18 (Fig. 3). Some patients with acquired platelet destruction and turnover, such as idiopathic thrombocytopenic purpura, may have high MPVs due to the rapid release of new platelets, but the macrothrombocytopenia syndrome platelets generally are much larger and more uniform in size. Bernard-Soulier disease, the most well-characterized of the macrothrombocytopenia disorders, is a congenital deficiency of the platelet glycoprotein Ibα/Ibβ/IX/V receptor, the surface receptor for VWF-mediated platelet aggregation.19 Many patients with Bernard-Soulier disease have severe bleeding with moderately severe thrombocytopenia and large platelets. Most of the Bernard-Soulier genetic defects are due to mutations of the GPIbα gene, but they may also be due to defects of the GP Ibβ or GP IX genes. Normal platelet aggregation is noted with exposure to ADP, collagen, epinephrine, and arachidonic acid, but aggregation is absent when ristocetin is added ( ). The glycoprotein abnormality can be confirmed with flow cytometry or crossed immunoelectrophoresis. Additional laboratory studies show normal VWF antigen and ristocetin cofactor activity distinguish Bernard-Soulier syndrome from von Willebrand disease. Patients who are heterozygous for the disease will show only giant platelets on a blood smear without hypoplatelet function, thrombocytopenia, or bleeding. These heterozygous patients may have associated velopharyngeal insufficiency, conotruncal heart disease, and learning disabilities and are classified as having the velocardiofacial syndrome. Gray platelet syndrome is an autosomal dominant α-SPD characterized by mild bleeding symptoms, reticulin fibrosis of the bone marrow, variable thrombocytopenia, and large (mean, 13 fL) platelets that appear gray on the peripheral blood smear due to decreased alpha granules.15 Pale platelets can also be seen with ongoing platelet activation and circulating "exhausted" platelets, but these patients will have a mixture of normal and pale platelets. Other rare macrothrombocytopenias are listed in Fig. 3. Several macrothrombocytopenia disorders are characterized by neutrophilic inclusions. May-Hegglin anomaly is the most common macrothrombocytopenia. It is an autosomal dominant disorder characterized by Dohle body inclusions in neutrophils with a mild bleeding disorder.18 A peripheral smear shows a modest thrombocytopenia with uniformly large platelets. Laboratory studies will usually show normal platelet aggregation and a normal bleeding time, attesting to the increased functionality of the larger platelets. Electron microscopic analysis of platelets in May-Hegglin anomaly will often show disorganized microtubles. Electron microscopic analysis of the neutrophilic inclusions shows them to lack a limiting membrane, to be free of specific granules, and to contain parallel bundles of ribosomes, microfilaments, and segments of endoplasmic reticulum. The two other macrothrombocytopenia disorders with neutrophilic inclusions are Fechtner syndrome and Sebastian syndrome. Fechtner syndrome can be distinguished by hereditary nephritis, deafness, cataracts (Alport syndrome), and macrothrombocytopenia. Sebastian syndrome, on the other hand, has no specific clinical associations. In thrombocytopenic platelet disorders with normal platelet morphology and size, marrow examination may be helpful in differentiating the underlying causes. This group of disorders includes congenital and acquired thrombocytopenias that usually are due to decreased platelet production or increased platelet destruction (Fig. 4). The number of megakaryocytes on the bone marrow can help distinguish between these etiologies, but analysis of platelet turnover by mRNA analysis (analogous to a platelet reticulocyte count) may also be helpful. The finding of adequate or increased megakaryocytes on the bone marrow or increased reticulated platelets suggests peripheral platelet destruction. Platelet function tests typically do not help in differentiating between the entities in this class of disorders because most function studies will give abnormal results simply due to the low platelet number. The overall MPV is usually normal with destructive thrombocytopenia, but platelet sizes typically vary, and many large platelets are seen, indicating rapid platelet turnover. These disorders are invariably acquired, and an underlying abnormality should be sought. The clinical scenario generally is the most helpful in classifying these disorders. Idiopathic thrombocytopenic purpura is known to be due to platelet sensitization, with autoantibodies leading to platelet destruction in the reticuloendothelial system. Peripheral smears may show variable macrothrombocytopenia, and autoantibodies to specific surface glycoproteins can be detected by flow cytometry or immunoassay,20 although diagnosis is largely from clinical findings. Other immune thrombocytopenias include post-transfusion purpura and neonatal alloimmune thrombocytopenia, which often occur because of polymorphisms of platelet antigens, such as PLA1 (HPA-1).21 The thrombocytopenia of thrombotic thrombocytopenic purpura is thought to be secondary to deficiency of a VWF-cleaving metalloproteinase in many patients, leading to diffuse thrombus formation in small vessels and a decline in circulating platelets.22 These patients will show characteristic clinical symptoms, such as renal failure, mental status changes, fever, and hemolysis with prominent schistocytes on the peripheral blood smear but normal screening coagulation studies. An assay for the VWF-cleaving metalloproteinase has been developed, but because it is based on the VWF multimer assay, it is not readily available in most laboratories. Drug-induced thrombocytopenias attributed to immunologic platelet destruction can be seen with many drugs, but the most common offenders are quinidine, quinine, heparin, sulfonamide drugs, and gold salts. Drug-induced thrombocytopenias can be diagnosed by detecting platelet-associated antibody by flow cytometry, although this is a nonspecific finding that can also be seen with infections and autoimmune disorders. The drug dependence of the antibody binding can be demonstrated by incubating platelets with patient plasma in the presence of the drug. Heparin-induced thrombocytopenia is a distinctive drug-induced thrombocytopenia associated with heparin therapy in which antibodies are formed to heparin-platelet factor 4 complexes, leading to platelet aggregation, platelet microparticle formation, endothelial injury, and paradoxical thrombosis.23 The thrombocytopenia is often delayed five to 12 days after starting heparin therapy and usually resolves after heparin therapy is stopped. Specific laboratory tests, such as heparin-induced platelet aggregation, serotonin release, and heparin-platelet factor 4 enzyme-linked immunosorbent assay, are available to diagnose this disorder. Conclusion Platelet-derived bleeding diatheses have multiple causes. Laboratory evaluation of these disorders can range from simple to complex but should initially include an extensive evaluation of the patient's medical history, concentrating on personal and familial bleeding disorders and medications being taken. Using this information and the aforementioned algorithms may help clinicians or pathologists determine the underlying origin for platelet-derived bleeding.n References: Dr. Corcoran is a pathology of laboratory medicine resident, Cleveland (Ohio) Clinic Foundation. Dr. Kottke-Marchant is section head of hemostasis and thrombosis in the Department of Clinical Pathology, Cleveland Clinic Foundation. She is also a member of the CAP Coagulation Resource Committee. |
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