Thrombopoiesis, the generation of platelets from megakaryocytes in the bone marrow, is complex and incompletely understood. Megakaryocytes are large end-stage cells from which platelets bud. The earliest recognized committed progenitor is the burst-forming unit (BFU)-Meg.¹ Fig. 33.1 shows megakaryocyte development from stem cell stage through to platelet production. BFU-Megs develop into colony-forming unit (CFU)-Megs in the presence of growth factors thrombopoietin (TPO), interleukin-3 (IL-3) and IL-11. Megakaryocyte nuclei are large polyploid structures with chromosome contents between diploid (2N) to 64N. Such polyploid status is achieved through a process termed nuclear endoduplication: that is, successive doubling of chromosome content in the absence of cell division. Platelets are produced from megakaryocytes that are 8N or greater.² A single megakaryocyte can generate around 3000 platelets of which 20–30% are pooled in the spleen. In health the peripheral blood platelet count is 150–400 × 10⁹/l but this fluctuates, for example, following heavy exercise, ‘stress’, and around the menstrual cycle. This transient rise in platelet count may be caused by mobilization of platelets pooled in the spleen. There are also racial differences in the ‘normal’ platelet count and some Mediterranean populations have platelet counts as low as 80 × 10⁹/l in health. Platelets are produced at a rate of 35 000–44 000 per microliter per day³ and have a lifespan of 9–10 days.

Fig. 33.1 Hematopoietic lineages, showing megakaryocyte development and platelet production.

(From Provan D, Gribben JG 2000 (eds), Molecular Haematology. Blackwell Science, Oxford, with permission.)

Platelet structure and function

Normal platelet function requires the presence of key membrane proteins and two major types of cytoplasmic granules. Because of their limited metabolic activity, and the presence of polymorphic glycoproteins on their exterior surface, platelets are vulnerable to attack by many agents including drugs, toxins, viruses and the immune system. In addition, drugs or diseases that interfere with platelet function do so for the lifetime of the platelet and it may take several days for the effects of any interfering drugs to diminish once the offending agent is stopped.

Platelet membrane constituents

The platelet plasma membrane contains a variety of polymorphic glycoprotein molecules which interact with ligands such as coagulation factors, vessel wall components and other molecules in order to generate the primary hemostatic plug.

The integrin family of proteins

Integrins are key platelet membrane proteins and have been characterized on a large number of leukocytes and many other cells. For example, the fibronectin receptor on mammalian fibroblasts is one of the best characterized matrix receptor proteins ⁴ (Fig. 33.2). This receptor, in common with all other integrins, is a heterodimer consisting of a non-covalently associated complex of two distinct high-molecular-weight polypeptides, α and β. The receptor functions as a transmembrane linker which mediates the interaction between the intracellular actin cytoskeleton and fibronectin in the extracellular matrix. Like all integrins, the fibronectin receptor recognizes so-called RGD (Arg-Gly-Asp) sequences in matrix components. In platelets, integrins recognize and bind a variety of proteins in order to form a hemostatic plug through a complex mechanism of platelet adhesion, shape change and activation of the clotting pathway.

Fig. 33.2 Fibronectin receptor on mammalian fibroblasts: the platelet integrins share homology with the fibronectin receptor.

Platelet integrins and related proteins

Platelets contain five integrin α subunits and two β subunits producing: α_IIbβ₃, α_νβ₃, α₂β₁, α₅β₁ and α₆β₁.^5,^6,⁷ These are shown in Fig. 33.3. Further detail is provided by Table 33.1. These proteins are essential for normal platelet function, and are often the target of immunological attack in disorders such as idiopathic thrombocytopenic purpura (ITP).

Fig. 33.3 Cartoon of platelet membrane, showing platelet glycoproteins.

(Courtesy of Peg Berrity, Science Photo Library.)

Table 33.1 Platelet integrins and related proteins

Platelet alloantigens

These can be platelet-specific or shared with other cells. Important shared antigens include HLA class I and ABH (blood group A and B) antigens. Platelet-specific antigens fall into five well-defined human platelet antigen (HPA) groups (Table 33.2; see also Chapter 37): HPA-1, HPA-2, HPA-3, HPA-4 and HPA-5, each of which has an α and β allele. Each platelet allotype represents a single amino acid substitution in the platelet glycoprotein molecule. Because some platelet glycoproteins carry epitopes that play a major role in platelet function, platelet alloantibodies may not only cause thrombocytopenia but also affect primary hemostasis.

Table 33.2 Human platelet alloantigen system.

Cytoplasmic platelet constituents

Platelets contain two principal types of granule: dense bodies and α granules (Fig. 33.4). Dense bodies contain ADP, ATP, 5-HT, calcium and pyrophosphate. The α granules contain more than 300 releasable proteins including adhesion molecules, chemokines, cytokines, coagulation factors, fibrinolytic regulators, growth factors and pro- and anti-angiogenic factors such as PF4, β-thrombospondin, PDGF, vWF, fibrinogen, factor V and fibronectin. The contents of these granules are integral components of the platelet’s biological activities.

Fig. 33.4 Schematic representation of the platelet showing dense granules and alpha granules, with details of the contents of each.

Biological function of platelets

The primary role of the platelet is the prevention of blood loss from damaged tissues and vessels, i.e. primary hemostasis. This is achieved through platelet activation, adhesion, shape change and aggregation. Platelets may also play a role in the maintenance of vascular integrity by the constitutive release of cytokines and growth factors from their granules that bind to endothelial cell surface receptors resulting in intracellular signaling that stabilizes the molecular complexes that form the junctions between adjacent endothelial cells.⁸

Platelet adhesion (Fig. 33.5A)

This process involves interaction between the platelet integrins and the subendothelium. The main integrin involved in platelet adhesion is GPIIb/IIIa which can be activated via a number of signals including exposure to extracellular matrix components such as collagen, in addition to other key activators such as adenosine diphosphate (ADP). Known ligands for platelet integrins include molecules such as fibrinogen, fibronectin and von Willebrand factor (vWF).

Fig. 33.5 (A, B) (A) Platelet adhesion to subendothelium: the platelet membrane contains a variety of glycoprotein receptors to adhesive proteins. VN, vitronectin; Lm laminin receptor; ABP, actin-binding protein; a-A, a-actin; vWF, von Willebrand factor; TSP, thrombospondin; FN, fibronectin. (B) Platelet aggregation: the terminal event in platelet aggregation is thought to be the binding of fibrinogen to GPIIb/IIIa receptors on adjacent platelets. The fibrinogen bridge may be stabilized by thrombospondin bound to its receptor (GPIV).

(From Mackie IJ. The biology of hemostasis and thrombosis. In: Ledingham JGG, Warrell DA, editors. Concise Oxford Textbook of Medicine. Oxford: Oxford University Press; 2000, with permission.¹⁴²)

Platelet activation

If there is damage to the endothelial lining of blood vessels, platelets become exposed to subendothelial structures. Platelets release a variety of attractants and other chemicals and recruit other platelets which are attracted to the site of injury. Important platelet activators include ADP, thromboxane A₂ and thrombin. The activated platelets provide a strong procoagulant surface on which the main clotting cascade is amplified, with the final product being the generation of thrombin and the production of a fibrin clot, composed of platelets enmeshed within a fibrin network.

Platelet shape change

After adhesion to the subendothelium, platelets undergo a major shape change, from a discoid shape to one which is irregular, with projections (Fig. 33.6). This process is initially reversible but ultimately becomes irreversible.

Fig. 33.6 Platelet shape change. Scanning electron micrograph of platelet shape transformation during adhesion to the arterial wall. Assemblies of actin filaments create spike-like attachment points and arrange themselves in an expansive webwork that mantles the neighboring space.

(From Roth GJ. Developing relationships: arterial platelet adhesion, glycoprotein Ib, and leucine-rich glycoproteins. Blood. 1991;77:5–19, with permission.¹⁴³)

Platelet aggregation (Fig. 33.5B)

There are a number of compounds that can induce platelet aggregation, including ADP, collagen, thrombin, adrenaline, vasopressin and others. Aggregation occurs following the shift of GPIIb/IIIa, the dominant integrin on the platelet surface, from a low affinity to a high affinity state and is mediated through the binding of fibrinogen or von Willebrand factor to adjacent platelets.

Quantitative platelet abnormalities: thrombocytopenia

Thrombocytopenia, a reduction in platelet count, may be caused by:

• impaired production

• increased destruction

• altered distribution

• a combination of these.

Pseudothrombocytopenia

This describes patients in whom the peripheral blood platelet count is found to be spuriously low, and is caused by a variety of mechanisms. Platelet clumping caused by the anticoagulant ethylenediaminetetraacetic acid (EDTA) is the commonest cause, with an incidence of around 0.1%. EDTA-induced pseudothrombocytopenia is easily excluded by examination of a blood film which will confirm the presence of numerous large platelet clumps.^9,¹⁰ Other causes of pseudothrombocytopenia include the presence of giant platelets, which are not counted as platelets by modern automated counters, and platelet satellitism (platelets attach themselves to monocytes or granulocytes) (Fig. 33.7).

Fig. 33.7 Platelet satellitism. The figure shows a neutrophil surrounded by adherent platelets.

(From Lewis SM, Bain B, Bates I, Dacie J 2000 (eds), Practical Haematology, 9th edn. Churchill Livingstone, Edinburgh, with permission.)

Pooling of platelets in the spleen

This accounts for the thrombocytopenias seen in patients with hepatic cirrhosis and portal hypertension. The term hypersplenism is used for patients in whom there is thrombocytopenia through excessive splenic pooling of platelets.

Mechanism involved

In health, the spleen may pool up to one third of the total platelet mass, and in disease states this may rise to 90%.¹¹ Although the peripheral blood platelet count may only be a fraction of the normal range, the patient generally has an overall normal platelet mass since production is entirely normal but the low peripheral counts simply reflect a larger than normal mass of platelets pooled in the spleen.

Thrombocytopenia due to failure of platelet production

Acquired amegakaryocytic thrombocytopenia

This describes severe thrombocytopenia caused by a selective reduction in megakaryocytes in an otherwise normal bone marrow, and is the result of damage to the megakaryocyte stem cell. The disorder is analogous to acquired pure red-cell aplasia (PRCA). Acquired amegakaryocytic thrombocytopenia may be caused by drugs, toxins and connective tissue disorders.¹²^–¹⁴

Drug-induced megakaryocyte hypoplasia

All drugs that are myelosuppressive will inhibit megakaryocyte stem cells. Agents that are cell cycle phase-specific have the most profound effects on megakaryocytes (e.g. methotrexate and cytosine arabinoside). Other drugs may selectively ‘poison’ megakaryocytes, and induce profound hypoplasia, for example thiazide diuretics, ethanol and estrogens.

Bone marrow failure syndromes

The bone marrow failure (BMF) disorders are those in which there is a failure of bone marrow precursor (stem) cells in contrast to those disorders such as myelodysplastic syndromes in which normal or increased numbers of abnormal cells are produced, or those in which the survival of the cells is reduced. The BMF syndromes are a diverse groups of disorders with a common endpoint in which there is loss of hematopoietic stem cells. BMF can be acquired or inherited (see Table 33.3).

Table 33.3 Acquired bone marrow failure syndromes

Aplastic anemia

This is discussed in Chapter 13.

Thrombocytopenia due to increased platelet destruction

Causes may be immunologic or non-immunologic.

Non-immunologic causes of thrombocytopenia

Disseminated intravascular coagulation (DIC)

DIC is characterized by excessive activation of the coagulation cascade (see also Chapter 35). In most cases DIC is an acute event, but chronic DIC is well described, although clinically less important. The main problem faced in patients with DIC is bleeding, which may be mild but is often severe with generalized oozing from venepuncture sites, central lines and other indwelling cannulae, gastrointestinal and genitourinary tracts. Microthrombi are found in 5–10% of cases, often affecting digits, with resulting peripheral gangrene.

Pathogenesis

DIC is triggered by the release or exposure of tissue thromboplastins which contain a high concentration of phospholipids following trauma, surgery, mismatched blood transfusion and a variety of other triggers (Table 33.4). In addition to systemic activation of coagulation (leading to fibrin clot formation, organ failure and consumption of platelets and coagulation factors that may cause bleeding), there is dysregulation of natural anticoagulant systems and fibrinolysis.

Table 33.4 Triggers for disseminated intravascular coagulation (DIC)

Trauma	Including surgical
Dissemination of cancer cells	Malignancy, following administration of chemotherapy
Massive hemolysis	Post mismatched blood transfusion
Venoms	e.g. snake venoms
Endothelial injury	Gram-negative sepsis
Infections
Burns
Septicemia

TTP is a disseminated microangiopathy, first described by Moschowitz in 1924.¹⁷ It is uncommon, occurring primarily in adults and with an annual incidence in the US of 4–11 cases per million people. HUS has clinical and laboratory features that often overlap with those of TTP. However, HUS is more common in children, the renal abnormalities are more marked than in TTP (Table 33.5; see also Chapter 10) and the underlying pathogenetic mechanisms of the two conditions differ.

Table 33.5 Classification of thrombocytopenic purpura and hemolytic uremic syndrome.

Idiopathic TTP/HUS	Classic adult TTP and childhood non-verotoxin-associated HUS-TTP
Secondary TTP-HUS	Pregnancy-related	TTP, postpartum HUS
	Verotoxin-induced	Escherichia coli and Shigella dysenteriae I
		Childhood HUS
		Epidemic adult TTP-HUS
	Malignant disease	Especially metastatic carcinomas
	Drug-induced	Chemotherapy agents, e.g. mitomycin C, cisplatin, and other drugs
		Immunosuppressive agents, e.g. cyclosporin, quinine, ticlopidine
	Post-marrow/stem cell transplantation	Especially in conjunction with total body irradiation or high-dose (intensive) chemotherapy

HUS, hemolytic uremic syndrome; TTP, thrombocytopenic purpura.

(Modified from George JN, El-Harake M 1995. Thrombocytopenia due to enhanced platelet destruction by non-immunologic mechanisms. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ (eds), Williams Hematology, 5th edn. McGraw-Hill, New York, 1290–1315)

Clinical features of TTP

TTP is characterized by the pentad:

• thrombocytopenia

• microangiopathic hemolysis

• neurologic symptoms and signs (typically fluctuating)

• renal function abnormalities

• fever.

TTP can affect any organ system but it is the involvement of the hematopoietic, renal and central nervous systems that leads to the typical clinical features.

The previously high mortality rate has been reduced through effective treatment and good supportive care. The disorder is sporadic, but appears to be more common in people of black origin and in obese individuals. It may develop in pregnancy and in this setting must be differentiated from pre-eclampsia, eclampsia and the HELLP (hemolysis, elevated liver enzymes and low platelets) syndrome.

TTP typically has a sudden onset, with fever and neurologic symptoms including paralysis, coma, fits and psychiatric disturbance. There is usually purpura and the clinical picture is one of a fluctuating course. The causes of TTP can be subclassified into: 1) congenital; 2) idiopathic; and 3) non-idiopathic.¹⁶

Laboratory investigations

A full blood count and blood film will show anemia (hemoglobin ~ 8–9 g/dl) with polychromasia and other evidence of hemolysis. The film will usually show red-cell fragments and thrombocytopenia (Fig. 33.8). There is usually evidence of hemoglobinemia reflecting the presence of intravascular hemolysis. Lactate dehydrogenase will be elevated. Clotting tests are generally normal although occasionally there may be features of DIC. The direct Coombs test is negative. Renal failure is uncommon but elevated serum creatinine is seen in about a third of cases.

Fig. 33.8 Peripheral blood film from a patient with thrombotic thrombocytopenic purpura (TTP). Note the many fragmented red cells, characteristic of this disorder.

(Figure kindly provided by Dr John Amess, Barts & The London NHS Trust.)

Pathogenesis

Failure to cleave large von Willebrand factor (vWF) multimers is thought to be crucial in the pathogenesis of TTP. Von Willebrand factor is synthesized in vascular endothelial cells and megakaryocytes and assembles into multimers linked by disulfide bonds which are stored in the Weibel–Palade bodies of endothelial cells and α-granules of platelets. A small proportion of these multimers is constitutively secreted by endothelial cells into the circulation, stabilizing factor VIII and mediating both platelet adhesion to sites of vascular injury and platelet aggregation in conditions of high shear. Endothelial cell stimulation causes unusually large vWF multimers to be secreted and these attach to the endothelial cell surface.¹⁶

A key recent discovery to understanding the pathogenesis of TTP has been the recognition of the role of a deficiency of a von Willebrand factor-cleaving protease, termed ADAMTS 13 (an acronym for a disintegrin and metalloprotease with thrombospondin-1-like domains). ADAMTS 13 cleaves the large vWF multimers secreted by endothelial cells to generate the range of multimer sizes that normally circulate in the blood. This function appears to be critical in preventing thrombosis in the microvasculature because hereditary or acquired deficiency of ADAMTS 13, resulting in an inability to cleave newly secreted von Willebrand factor multimers, leads to increased binding of vWF to platelets and to the disseminated platelet thrombi that are characteristic of TTP.

More than 50 mutations of the ADAMTS 13 gene have been described in patients with the rare congenital or familial form of TTP. About half of these patients present during childhood but the remainder present in adulthood when the acute event may be triggered by conditions such as infection or pregnancy.

In contrast, in the more common idiopathic TTP, ADAMTS 13 deficiency is caused by autoantibodies that inhibit its activity or enhance its clearance from the circulation. Mostly the antibodies are IgG.

There are a number of causes of non-idiopathic thrombotic microangiopathy. These include cancer, pregnancy (see below), certain drugs (quinine, mitomycin, ciclosporin, ticlopidine and clopidogrel) and hemopoietic progenitor cell transplantation.

The characteristic histological features include hyaline thrombi in terminal arterioles and capillaries. The thrombi are composed of platelets and are rich in von Willebrand factor but contain little fibrinogen or fibrin. Immunoglobulins and complement are also often present consistent with the autoimmune origin of TTP.

Diagnosis

There is no specific diagnostic test for TTP and in practice for management purposes the diagnosis is based on laboratory confirmation of thrombocytopenia and microangiopathic hemolytic anemia without an apparent alternative cause and with or without the characteristic clinical features described above.

Management

Plasma exchange with cryosupernatant or FFP is now regarded as the treatment of choice for TTP and should be instituted as soon as possible after diagnosis and continued on a daily basis with replacement of 1.0 to 1.5 times the predicted plasma volume of the patient. Plasma exchange should continue until there is normalization of the neurological state, the platelet count has been >150 × 10⁹/l for at least 2 days, the LDH is normal and the hemoglobin rising.¹⁸ The disease is highly variable in its course and it is difficult to predict the clinical outcome at the outset. Patients require intensive care nursing, and may require ventilation. Hemodialysis may be required along with plasma exchange. A short course of high dose corticosteroids has also been recommended along with aspirin when the platelet count has recovered to 50 × 10⁹/l.¹⁸ Platelet transfusions should be avoided except in cases of severe hemorrhage. Remission is achieved when the platelet count remains normal for 30 days after discontinuation of plasma exchange.¹⁹ Patients with severe deficiency of ADAMTS 13 appear to have an increased risk of relapse, most within a year. Rituximab has been used to try to reduce the risk of relapse.^20,²¹

Hemolytic uremic syndrome

The disorder was first described in 1955 by Gasser,²² who reported on five patients (all infants) with acute renal failure, who died of renal cortical necrosis. HUS is classified into D+HUS and D-HUS.¹⁶ D+HUS accounts for >90% of cases and is caused by enteric infection with Shiga toxin-producing bacteria, most commonly Escherichia coli O157:H7 or occasionally Shigella dysenteriae I.²² It can occur at all ages but is seen typically in young children.²²^–²⁴ HUS usually develops 4–6 days after the onset of diarrhea. There is a seasonal incidence, with the highest number of new cases reported in the summer months.^25,²⁶

D-HUS is uncommon and clinically heterogeneous. These individuals do not have an antecedent enteric infection with a Shiga toxin-producing organism and have a relatively poor prognosis with a mortality rate of about 25%.¹⁶

The histopathological features of HUS are different from those of TTP. The kidneys are preferentially involved and the thrombi are composed primarily of fibrin with few platelets and little vWF.¹⁶ Endothelial damage is pronounced in childhood D+HUS. Marked destruction of the renal cortex may occur and glomerular thrombosis is characteristic with an appearance suggesting capillary congestion rather than ischemia.¹⁶ D-HUS has been little studied but the renal lesions appear to differ with more pronounced mesangial involvement and less glomerular thrombosis than in D+HUS.¹⁶

Pathogenesis. Most D+HUS results from E. coli O157 infections caused by food-borne outbreaks from cattle. Unwashed fruit is another source of some outbreaks. Subunits of Shiga toxin produced by the bacteria bind to microvascular endothelial cells particularly in the kidney and on monocytes and platelets. The toxin is subsequently internalized and inhibits protein synthesis leading to cell death. Cytokine release is stimulated further increasing Shiga toxin binding. Resulting endothelial cell damage has prothrombotic consequences that lead to the histopathological changes described above.¹⁶

D-HUS in contrast is associated with complement dysregulation and mutations in one of the complement regulatory proteins (factor H, factor I, membrane cofactor protein (MCP) and factor B) have been described in about 50% of these patients.¹⁶ In some patients an autoantibody against factor H has been recognized and in about 5% of cases mutations that impair thrombomodulin function have been identified.²⁷

Clinical features. Children typically present with diarrhea (often bloody), vomiting and abdominal pain. Because of the large volume of fluid loss, patients are often oliguric or anuric at presentation. Fever, hypertension and fits are also features.

Laboratory findings. There is evidence of acute renal failure and features of microangiopathic hemolysis.²⁸ The blood count will generally show an elevated WBC, most usually neutrophilia; the prognosis is worst for patients with total neutrophil counts exceeding 20 × 10⁹/l.²⁵

Management. Around half of the children affected will require hemodialysis for between 1 and 2 weeks. Unlike in adult TTP, plasma exchange using FFP replacement is not often required in children.

Outlook. Unlike adult TTP, childhood D+HUS is rarely fatal and the mortality is around 3–5%. The outlook is less favorable in D-HUS. In cases with end-stage renal failure that have undergone renal transplantation, the outcome appears to correlate with the cause of the complement dysregulation. Recurrence of HUS has not been seen in transplanted kidneys in patients with MCP mutations whereas the condition did recur following renal transplantation in patients with factor H or factor I mutations, perhaps because these latter two factors are made in the liver. Combined liver and kidney transplants may be successful in some of these cases.¹⁶