An adequate supply of circulating platelets is essential to maintain vascular integrity and to facilitate thrombus formation at sites of vascular injury. The normal platelet count in humans ranges from 150×10/L to 400×10/L. Given that platelets have a circulating lifespan of around 10 days, and that about one third of platelets are sequestered in the spleen at any time, approximately 100×10 of these small anucleate cells must be released from mature megakaryocytes into the circulation each day in order to maintain a normal platelet count. A constant balance is, therefore, required between thrombopoiesis, and platelet consumption and senescence. Thrombopoietin is the primary humoral regulator of megakaryocyte differentiation and platelet number under steady state conditions.1 It is synthesized in the liver and kidney and mediates its effects through its receptor c-Mpl which is present on megakaryocyte and platelet membranes.1 Levels of thrombopoietin are controlled via binding to, and internalization into, cells expressing the receptor. When platelets and megakaryocytes are decreased in number, less thrombopoietin is removed from plasma, and the thrombopoietin level rises, while when platelet numbers increase, more thrombopoietin is cleared from the plasma and the thrombopoietin level falls again.2 In addition to the thrombopoietin (THPO) and c-Mpl (MPL) genes, a large number of genes encoding membrane glycoproteins, cytoskeletal components and proteins involved in transcription, cell cycle regulation and signaling have been demonstrated to participate in the differentiation of pluripotent stem cells to mature platelet-shedding megakaryocytes.3
Defined as a platelet count less than 150×10/L, thrombocytopenia is usually an acquired disorder. Causes include increased platelet consumption, splenomegaly, drugs or infection-mediated bone marrow suppression, and bone marrow failure. Increasingly, however, inherited forms of thrombocytopenia, caused by mutations in genes encoding proteins involved in the differentiation of megakaryocytes and platelet production, which can result in autosomal dominant, autosomal recessive, and X-linked recessive forms of inherited thrombocytopenia, are being recognized.3
Thrombocytosis, defined as a platelet count exceeding the upper limit of the normal range (>400×10/L), is associated with an increased risk of thrombosis.4 Primary thrombocytosis can be either inherited or acquired and is caused by alterations targeting hematopoietic cells (e.g. MPL, THPO or JAK2 mutations) while secondary thrombocytosis is due to external factors such as chronic inflammation or cancer.4
While much has been done to elucidate the molecular mechanisms controlling megakaryocyte differentiation and platelet production and to identify defects in the genes encoding the relevant proteins in patients with inherited thrombocytopenia, the factors that determine platelet count in the normal population are less well characterized. In a study published in this issue of the journal, Biino et al. addressed this issue by carrying out a cross-sectional investigation of platelet counts among 12,517 inhabitants of ten villages in the Ogliastra region of Eastern Sardinia.5 This sparsely populated, mountainous province in Sardinia is unusual in that it has one of the most genetically homogeneous populations in Europe. The population has remained small and isolated throughout its history. Despite this, sub-populations in the region show a strong demarcation in their genetic pools, possibly due to different founders, to the extent that the small populations have divergent genetic histories even where they inhabit neighboring villages.6 In their study, Biino et al. found that platelet count was higher in women, and that the prevalence of thrombocytopenia was higher among men (4.8%) than among women (3.2%). Interestingly, the distribution of platelet counts varied markedly between villages and the prevalence of thrombocytopenia ranged from 1.5% to 6.8% in the different villages studied. The thrombocytopenia was, in general, mild and asymptomatic and was not associated with other cytopenias or conditions known to be associated with thrombocytopenia. Furthermore, it showed a negative correlation with a mild form of thrombocytosis which ranged in prevalence from 0.9% to 4.5% between villages. Platelet counts were shown to decrease progressively with age, with the consequence that thrombocytopenia was more common among the elderly, while thrombocytosis was more frequent among younger people. Biino et al. concluded that the predispositions to platelet counts above or below the normal range are inherited traits.5
There is considerable evidence that the platelet count is largely determined by genetic factors. Monozygotic twins show a higher level of concordance in platelet count than dizygotic twins.7 Also, differences in platelet count have been described in populations of different ethnic origin that share the same environment.8 Longitudinal studies demonstrate considerable stability of steady state platelet counts. For example, in their analysis of serial platelet counts from 3,789 subjects, Buckley et al. showed that the repeatability of the platelet count was 0.871 in males and 0.849 in females, indicating that heritability of platelet count is high,9 and while Biino et al. found that the heritability of platelet count was 54% in their study, figures of up to 87% have been reported in previous studies.5,9
So what genetic factors are responsible for determining platelet count? Despite the evidence that thrombopoietin and c-Mpl are essential for platelet production, there is little evidence for their genes having effects on the variation in platelet count in the normal population. In a genome-wide association study performed to identify quantitative trait loci for platelet count in inbred mouse strains, genotyping of mice with platelet counts at the extremes of the distribution led to the identification of two quantitative trait loci on chromosomes 1 and 11, neither of which appeared to be associated with known genes that regulate platelet production.10 More interesting, perhaps, was the absence of quantitative trait loci in the region of the Thpo or Mpl genes, or those genes encoding interleukin-11, nuclear factor erythroid-derived 2 (Nf-e2), Gata-1 or Fog-1 all of which are important in platelet production.10
The first quantitative trait locus influencing platelet count in humans was identified by Soranzo et al. who performed a genome-wide association study for mean platelet volume, also a highly heritable trait, which is inversely correlated with platelet count, in the healthy population.11 They identified a single nucleotide polymorphism, rs342293, on chromosome 7q22.3 which was significantly associated with mean platelet volume and platelet count in healthy subjects, though the effect on platelet count was entirely explained by its association with volume. The single nucleotide polymorphism occurred in an intergenic region that did not contain any genes, suggesting that regulation of mean platelet volume and platelet count are probably mediated through cis- or trans-regulatory effects. Interestingly, all six genes in a 1 Mb interval centered on the single nucleotide polymorphism were expressed in megakaryocytes and the platelet transcript levels of one of these, PIK3CG, varied according to genotype for rs342293, while those of a second gene, PRKAR2B, showed a non-significant correlation with genotype.11 Both PIK3CG and PRKAR2B are credible candidates for contributing to mean platelet volume and platelet count. PIK3CG encodes the β-chain of PI3/PI4-kinase which is responsible for the synthesis of phos-phatidylinositol-3,4,5-trisphosphate (PIP3), which is essential for the initiation of megakaryopoiesis and pro-platelet formation.12 PRKAR2B encodes the β-chain of cAMP-dependent protein kinase, which has an antagonistic effect on megakaryopoiesis and proplatelet formation by reducing the release of intracellular calcium through the phosphorylation of ITPR3, the receptor for PIP3.13
More recently, Soranzo et al. detected 12 loci which were reproducibly associated with mean platelet volume in 13,943 samples from six European population-based studies.14 Four of these had been found to be associated with mean platelet volume in earlier studies by the group. Nine of the 12 loci were also associated with platelet count, the mean platelet volume-raising alleles being linked with a decrease in platelet count, though in all nine cases the single nucleotide polymorphisms were found to exert their effects on platelet count through the mean platelet volume. Three further loci which independently influenced platelet count were detected in the same study. An association signal at 6p21.3 (rs210135) centered on the BAK1 gene which encodes a pro-apoptotic protein that regulates steady state platelet lifespan.15 It was suggested that a locus at 9p24.1–p24.3 (rs385893) may regulate the expression of the proximal JAK2 gene which is a key regulator of megakaryocyte maturation.4,14 Two further single nucleotide polymorphisms, rs11065987 and rs11066301, map to a 1.6 Mb region located at 12q24, which contains 15 genes. Further investigation of the region identified ten single nucleotide polymorphisms, which formed a common haplotype spanning the length of the associated interval, all of which displayed significant association with platelet count (Table 1). These include a non-synonymous single nucleotide polymorphism, rs3184504, in the gene SH2B3, which encodes Lnk, an adaptor protein that mediates growth and maturation of megakaryocytes through its role in regulating cross-talk between integrin and cytokine signaling pathways.16 Interestingly, this single nucleotide polymorphism was also associated with risk of coronary artery disease.14 In addition, seven intronic single nucleotide polymorphisms within four genes (ATXN2, NAA25, C12orf51 and PTPN11), one intergenic single nucleotide polymorphism and one single nucleotide polymorphism in the 3′ region of the TRAFD1 gene contributed to the 12q24 haplotype (Table 1).
In addition to genetic factors, there is evidence that gender, age and seasonal factors play a role in determining platelet count. Gender-dependent differences in the distribution of normal platelet counts have been reported in several studies, with higher counts being observed in females than in males, as was observed by Biino et al.5,17 The inter- and intra-individual components of the variance in platelet count are similar in males and females, suggesting that the gender-dependent differences are most likely due to differences in hormone profiles. Megakaryocytes and platelets express steroid hormone receptors.18 Differences in hormonal profiles between subjects might, therefore, be expected to result in different platelet phenotypes. Of interest is the finding that estrogen stimulates differentiation of CD34 cells and modulates expression of estrogen receptors α and β in vitro.19 Furthermore, estradiol has been demonstrated to trigger proplatelet formation in megakaryocytes. Indeed, one of the targets in megakaryocytes for NF-E2, a transcription factor that is essential for proplatelet formation, is the gene for 3β-hydroxysteroid dehydrogenase which is essential for the formation of estradiol.20
An explanation for the age-dependent decline in platelet count observed by Biino et al. and others is still awaited.5 It has been suggested that it may be due to reduced hematopoietic stem cell reserve in aging individuals.8 It is also possible that the age-dependent decline in platelet number reflects epigenetic changes in the megakaryocyte genome, such as hypomethylation of genes that determine platelet count or changes in histone acetylation, which lead to differences in gene expression as we get older. The seasonal factors that in one longitudinal study accounted for 2% of the overall variance in platelet count, and caused a peak in platelet count during autumn and winter months, also remain to be clarified, though it is possible that this reflects the increased prevalence of viral infections during the winter months, since infection has been established to increase platelet count.9 Thus, while considerable progress has been made in identifying the genes that determine platelet count in humans, several challenges remain, not least of which will be to determine the impact of epigenetic programming on platelet count. The value of populations such as those of the province of Ogliastra in facilitating greater understanding of this area cannot be overestimated.
- Martina Daly, PhD is a Senior Lecturer in Molecular Medicine with the Haemostasis Research Group at the University of Sheffield, UK.
- (Related Original Article on page 96)
- Financial and other disclosures provided by the author using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are available with the full text of this paper at www.haematologica.org.
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