Chapter 119: Reactive Thrombocytosis

The upper limit of the normal platelet count in most clinical laboratories is between 350,000/μL (350 × 10⁹/L) and 450,000/μL (450 × 10⁹/L). In a sample of 10,000 healthy individuals 18 to 65 years of age, 1 percent had platelet counts greater than 400,000/μL. Only in eight of these 99 individuals was thrombocytosis confirmed 6 months to 1 year later.¹ Nevertheless, it is clear that thrombocytosis is a feature of several important disorders, including cancer, and that even a high normal platelet count is associated with morbidity and mortality. In a longitudinal study of healthy Norwegian men, a platelet count in the top quartile of the normal range (from 275 × 10⁹/L to 350 × 10⁹/L) was associated with a twofold increase in cardiovascular mortality over a 12-year followup.² Whether the platelet count per se, or an underlying inflammatory condition resulting in both thrombocytosis and accelerated atherogenesis is responsible for these observations is not certain. The causes of thrombocytosis in which the platelet count exceeds the upper limit can be broadly categorized as (1) clonal, including essential thrombocythemia and other myeloproliferative neoplasms, (2) familial, and (3) reactive, or secondary (see Chap. 85, Table 85–1). This chapter focuses on the causes and molecular mechanisms that underlie reactive, or secondary, thrombocytosis; clonal and familial thrombocytosis are discussed in detail in Chap. 85.

The regulation of platelet production is discussed extensively in Chap. 111, but a brief discussion here provides the appropriate background for discussion of reactive thrombocytosis. Thrombopoietin (TPO), the ligand for the megakaryocytic growth factor receptor c-Mpl, is the major humoral regulator of megakaryocyte survival, growth, and development, although, curiously, it does not stimulate the final step in thrombopoiesis: platelet release from megakaryocyte proplatelet processes. Although TPO supports the entire continuum of megakaryocyte development from stem cell to mature megakaryocyte,³ other cytokines including interleukin (IL)-6,⁴ IL-3,^5,6 IL-11,⁷ leukemia inhibitory factor (LIF),^8,9 fibroblast growth factor (FGF)-4,¹⁰ stromal cell-derived factor (SDF)-1,^10,11 interferon (IFN)-γ,¹² and granulocyte-macrophage colony-stimulating factor (GM-CSF)¹³ also affect thrombopoiesis, both in vitro and in vivo. Many of these cytokines act in synergy with other cytokines, including TPO.^11,12,14

The regulation of thrombopoiesis occurs primarily by humoral mechanisms, with the levels of TPO inversely related to platelet counts.^15,16 In contrast, other cytokines shown to affect megakaryopoiesis in vitro do not vary with platelet levels.¹⁷ Despite these important insights, the regulation of TPO blood levels is complex, and incompletely understood. The liver produces approximately half of all the hormone that circulates, based on platelet production in liver specific knockout mice.¹⁸ However, platelet levels do not affect hepatic TPO production; instead, platelets themselves have an important role in regulating plasma levels, as their receptors for TPO (c-mpl) remove it from plasma.¹⁹ Thus, as the platelet count drops, increased free plasma TPO levels stimulate megakaryopoiesis; conversely, as the platelet count rises, depletion of free plasma TPO decreases platelet production. This modulatory mechanism results in the steady-state level of platelet production. However, marrow stromal cells also produce TPO,²⁰ and are responsive to platelet products which serve to down-modulate expression of the hormone.²¹ A third mechanism by which platelets regulate TPO levels occurs through the Ashwell-Morell hepatocyte receptor, whereby their binding of senescent platelets leads to stimulation of hepatocyte signaling pathways and subsequent expression of TPO.²²

THROMBOCYTOSIS IN INFLAMMATORY CONDITIONS

Inflammation is the most common cause of secondary thrombocytosis. In one survey, thrombocytosis was believed secondary to one or more inflammatory conditions in nearly 80 percent of all patients with an elevated platelet count. Table 119–1 lists the clinical conditions associated with reactive thrombocytosis. The most common diagnoses in such patients are inflammatory bowel disease and rheumatoid arthritis,²³ although most conditions in which the erythrocyte sedimentation rate or C-reactive protein is elevated have been reported to cause secondary thrombocytosis. Although several cytokines and lymphokines are elevated in the blood of such patients, the most compelling evidence suggests that IL-6 and IFN-γ are responsible for the thrombocytosis seen in patients with inflammation.

Interleukin-6

IL-6 was cloned by several groups of investigators using a number of distinct assays, including antiviral activity, myeloma cell growth, hepatocyte growth, and immunoglobulin secretion.²⁴ The recombinant protein was later found to affect megakaryocyte growth and differentiation, both in vitro and in vivo.^4,25,26 The IL-6 gene is present on the short arm of human chromosome 7, and encodes a 26-kDa polypeptide produced in almost all tissues from T cells, fibroblasts, macrophages, and stromal cells, and is a key regulator of the inflammatory response.²⁷

IL-6 production is dependent on the presence of IL-1 and tumor necrosis factor (TNF)-α, cytokines produced by lymphocytes and monocytes in response to phagocytosis of microorganisms, the binding of immune complexes, and several other innate immune stimuli. IL-6 production is regulated primarily by transcriptional enhancement; regulatory elements responsible for IL-6 promoter activation include nuclear factor-κB (NFκB), adapter protein (AP)-1, CCAAT/enhancer binding protein (C/EBP) α and C/EBPβ.

Although not critical for steady-state thrombopoiesis, as the combined genetic elimination of c-mpl and the signaling component of the IL-6 receptor (gp130) produces no more severe thrombocytopenia than elimination of c-mpl alone,²⁹ IL-6 contributes to inflammatory thrombopoiesis, primarily by stimulating the hepatic production of TPO.³⁰ Most studies report that patients with inflammation display an increased level of TPO,^31,32 but TPO is not the only cytokine responsible for this effect,³³ especially when corrected for the thrombocytosis, which would normally act to reduce levels of the hormone. Stimulation of hepatocytes with IL-6 results in enhanced production of TPO mRNA and protein.^34,35

INTERFERON-γ

A second inflammatory cytokine that contributes to inflammatory thrombopoiesis is IFN-γ. The interferons are proteins first defined by their ability to induce an antiviral state in mammalian cells. Biochemical fractionation revealed three classes of interferons: IFN-α, a family of 17 distinct but highly homologous molecules; IFN-β, a single molecule more distantly related to the various isoforms of IFN-α; and IFN-γ, a unique molecule that shares functional properties but not structure with the others. IFN-γ exerts the most profound hematologic effects of the three classes of protein, including direct suppression of erythroid colony-forming cell growth and the activation of macrophages to secrete a number of inflammatory cytokines; several comprehensive reviews on IFN-γ have been published.^36,37

IFN-γ is produced by activated T lymphocytes and natural killer (NK) cells in response to T-cell antigen crosslinking and in response to stimulation by the inflammatory mediators TNF-α, IL-12, and IL-15.³⁸ Prominent hematologic effects include activation of macrophages to assume an inflammatory phenotype (e.g., secretion of TNF-α and enhanced tumor cell killing), upregulation of major histocompatibility complex (MHC) class I and class II molecules enhancing antigen recognition responses,³⁷ and inhibition of proliferative responses in stem cells and erythroid progenitors.^39,40 These latter effects accounts for the association of IFN-γ and aplastic anemia⁴¹ are discussed more fully in Chap. 35. However, in stark contrast to the inhibitory effects of IFN-γ on erythropoiesis, the cytokine stimulates megakaryocyte growth and differentiation.⁴² This is likely related to its stimulation of signal transducer and activator of transcription (STAT)-1 in megakaryocytes, as transgenic expression of the transcription factor mimics the effect of the cytokine, and corrects the thrombocytopenia seen in a genetic model system.⁴³ These findings argue that IFN-γ also contributes to the thrombocytosis seen in inflammatory states in humans.

Notwithstanding the above two mechanisms, patients with inflammatory conditions and thrombocytosis might have an additional cause of the elevated platelet count. The evaluation of iron deficiency is often difficult in patients with inflammation, as the most reliable indicator of tissue iron stores, serum ferritin, is an acute-phase reactant, possibly obscuring a diagnosis of iron deficiency in patients with an inflammatory condition. In a recent study of patients with inflammatory bowel disease, thrombocytosis was eliminated in half of the subjects by the administration or iron.⁴⁴

THROMBOCYTOSIS CAUSED BY IRONDEFICIENCY

Although most patients with inflammation-related thrombocytosis display increased production of the hormone, TPO levels in patients with iron deficiency and thrombocytosis are not elevated.⁴⁵ In contrast, erythropoietin (EPO) levels are elevated in patients with iron-deficiency anemia, and are thought by some to be the responsible for the thrombocytosis seen in iron deficiency, at least in part. Consistent with this hypothesis, administration of EPO to animals and humans leads to a modest increase in the platelet count.⁴⁶ Although some have suggested that this is a result of cross-reactivity of EPO on the TPO receptor,⁴⁷ direct EPO- and TPO-receptor binding studies refute this hypothesis.⁴⁸ Rather, megakaryocytic progenitors display EPO receptors, and their binding of the hormone leads to many of the same intracellular biochemical signals as induced by TPO (Chap. 17).

However, several lines of evidence indicate that pathophysiologic mechanisms other than anemia must be responsible, at least in part, for the thrombocytosis seen in patients with iron deficiency. For example, many patients with iron-deficiency anemia do not have thrombocytosis.⁴⁵ Moreover, EPO levels are elevated in nearly all types of anemia, but iron deficiency is the only type of anemia that is regularly associated with thrombocytosis, other than the anemia of chronic inflammation, in which the inflammatory state that causes the anemia by modulation of hepcidin levels (Chap. 37) also causes thrombocytosis (as discussed in “Thrombocytosis in Inflammatory Conditions” above). Thus, although several lines of evidence suggest that enhanced levels of EPO as a consequence of the anemia associated with iron deficiency contribute to this form of reactive thrombocytosis, elevated EPO levels cannot completely account for it.

THERAPEUTIC ERYTHROPOIETIN AND ENHANCED CARDIOVASCULAR MORTALITY

Several reports have linked the use of large doses of EPO or other erythropoiesis-stimulating agents (ESAs) to enhanced cardiovascular mortality,⁴⁹ and in patients with renal insufficiency, to progression to dialysis in patients with renal insufficiency,⁵⁰ although not all studies concur with these landmark results.⁵¹ Although also discussed in Chap. 18, this finding is presented here because evidence is accumulating that the rapid expansion of erythropoiesis caused by pharmacologic levels of EPO often induces functional iron deficiency. If so, because iron deficiency leads to thrombocytosis, the excessive cardiovascular morbidity and mortality associated with the administration of EPO and ESAs to patients is hypothesized to be secondary to the thrombocytosis. Consistent with this view is that even a high normal platelet count was found associated with enhanced cardiovascular morbidity and mortality in a longitudinal study of healthy Norwegian men.² In support of this hypothesis (that the excessive cardiovascular morbidity and mortality is secondary to the thrombocytosis) is the finding that patients with renal insufficiency on high therapeutic doses of EPO (>20,000 U/week) and hemoglobin (Hgb) values in excess of 13 g/dL are more likely to develop functional iron deficiency and thrombocytosis, and that those individuals in whom the platelet count exceeds 300,000/μL display a statistically significantly higher 3-year mortality rate.⁵² An alternate explanation is that EPO directly increases thrombopoiesis independently of iron deficiency and/or enhances the vascular reactivity of platelets. This hypothesis is based on the finding that megakaryocytes and platelets bear EPO receptors,⁵³ and that TPO, which stimulates very similar signaling pathways as EPO in receptor-bearing cells (Chap. 17), primes platelets to enhanced aggregation responses to classic platelet agonists.⁵⁴ Still other researchers have hypothesized that an alternate form of the EPO receptor, made up of the classic EPO receptor and the β subunit of the GM-CSF, IL-3, and IL-5 receptors, is displayed on vascular endothelial cells,⁵⁵ and in that site could mediate enhanced vascular events. Thus, given the widespread use of ESAs in patients with anemia caused by cancer, kidney failure, myelodysplastic syndromes, and many other conditions, verifying these hypotheses or disproving them and establishing new ones appears to be important and a field ripe for new discovery.

The clinical features of secondary thrombocytosis are almost always a result of the underlying disorder provoking the reaction, usually an inflammatory condition or iron-deficiency anemia. It is also highly unusual for the thrombocytosis per se to provoke any untoward symptoms. Although pathologic thrombosis is a major feature of primary thrombocythemia (Chap. 85), it is virtually absent in reactive thrombocytosis, unless provoked by other features of the underlying condition (e.g., vasculitis) or completely unrelated conditions in the patient (e.g., atherosclerotic disease). Whether this is because patients with reactive thrombocytosis do not have as high platelet counts, on average, as patients with primary thrombocythemia⁵⁶; or because they have smaller mean platelet volumes⁵⁶; or are a result of the activated signaling characteristic of the platelets or other blood cells in patients with myeloproliferative diseases; or because of the presence of a mutant Janus kinase (JAK) 2,⁵⁷ or a constitutively active TPO receptor,⁵⁸ is uncertain at this time. Nevertheless, because vascular complications of reactive thrombocytosis are so unlikely to be a consequence of the elevated platelet count, treatment of the thrombocytosis per se is not recommended in reactive thrombocytosis except in very unusual circumstances.