Normal Animal Physiology

Much has been learned about the normal physiology of TPO. First, TPO is the only physiologically relevant regulator of platelet production and acts to amplify the basal production rate of megakaryocytes and platelets. TPO is not necessary for megakaryocyte differentiation. When TPO or its receptor have been knocked out by homologous recombination in mice (56-59), the megakaryocyte and platelet mass are reduced to approx 10% of normal, but the animals are healthy and do not spontaneously bleed (Fig. 3). The neutrophil and erythrocyte counts are normal. In animals in which only one of the TPO genes has been deleted, the platelet count is reduced to approx 65% of normal. If treated with other thrombopoietic growth factors such as IL-6, IL-11, and stem cell factor (SCF), such TPO-deficient mice modestly increase their platelet count (60).

Second, TPO affects bone marrow precursor cells of all lineages. In the animals deficient in TPO or c-mpl, Meg-CFC are reduced to <5% of normal as expected, and the myeloid and erythroid precursor cells are also reduced to 20-30% of normal (56,60), but with no change in the neutrophil or erythrocyte counts (Fig. 4). The normal counts in these animals are presumably maintained by the intact feedback mechanisms mediated by G-CSF and EPO. This finding supports the concept that TPO is an important stimulus for the growth of early progenitor cells of all lineages but only affects the late maturation of megakaryocytes and only stimulates platelet production.

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Fig. 3. Mice deficient in thrombopoietin (TPO) or its receptor (MPL) are thrombocytopenic. Platelet counts (+ SD) in normal (+/+) mice were compared with those in TPO or MPL homozygous (-/-) or heterozygous (+/-) deficient mice. (Adapted from refs. 59 and 58.)

Fig. 4. Bone marrow progenitor cells of all lineages are decreased in mice deficient in thrombopoietin (TPO-/-) or its receptor (MPL-/-) compared with normal wild type (WT). Granulocyte-macrophage colony-forming cells (GM-CFC) (GM), erythroid burst-forming cells (E-BFC), and megakaryocyte-CFC (Meg), as well as mixed colonies (Mix-CFC) with erythroid component (E Mix), erythroid and megakaryocytic components (E/M Mix), or megakaryocytic with nonerythroid components (M-Mix) were enumerated (+ SD). (Adapted from ref. 60.)

Fig. 4. Bone marrow progenitor cells of all lineages are decreased in mice deficient in thrombopoietin (TPO-/-) or its receptor (MPL-/-) compared with normal wild type (WT). Granulocyte-macrophage colony-forming cells (GM-CFC) (GM), erythroid burst-forming cells (E-BFC), and megakaryocyte-CFC (Meg), as well as mixed colonies (Mix-CFC) with erythroid component (E Mix), erythroid and megakaryocytic components (E/M Mix), or megakaryocytic with nonerythroid components (M-Mix) were enumerated (+ SD). (Adapted from ref. 60.)

Fig. 5. The physiologic regulation of thrombopoietin levels. The constitutive hepatic production of thrombopoietin (center) is cleared by avid thrombopoietin receptors on platelets, resulting in normal levels when the platelet production is normal (left) and increased amounts when platelet production is reduced (right). The bone marrow megakaryocytes are stimulated as the circulating thrombopoietin concentration increases.

Fig. 5. The physiologic regulation of thrombopoietin levels. The constitutive hepatic production of thrombopoietin (center) is cleared by avid thrombopoietin receptors on platelets, resulting in normal levels when the platelet production is normal (left) and increased amounts when platelet production is reduced (right). The bone marrow megakaryocytes are stimulated as the circulating thrombopoietin concentration increases.

Third, TPO production is constitutive, and the circulating concentrations are directly determined by the circulating platelet mass (Fig. 5). Whereas the production of red blood cells is regulated by a cytochrome P-450 system that senses changes in the hematocrit and alters the rate of transcription of the EPO gene, there is no such sensor of the platelet mass (4,9,45,61-64), rather, TPO mRNA is produced at the same rate in normal and thrombocytopenic individuals (65). No drug or clinical condition has been shown to increase hepatic TPO production. Platelets and megakaryocytes contain high-affinity TPO (c-mpl) receptors that bind and clear TPO from the circulation and thereby directly determine the amount of circulating TPO. When platelet production is decreased, clearance of TPO is reduced and amounts increase.

This type of feedback system is not unusual in hematology. Indeed, both monocyte colony-stimulating factor (M-CSF) and G-CSF are normally regulated primarily by the amount of circulating monocytes and neutrophils, respectively. It appears that only for EPO is there a true sensor of the circulating blood cell mass that in turn alters production of this HGF. Transfusion of platelets into thrombocytopenic animals or humans decreases the amount of plasma TPO (4,9,45,62,66), and similar results have been observed when normal platelets are transfused into c-mpl-deficient mice (48). These findings indicate that TPO is constitutively synthesized in the liver and removed from circulation by binding to the c-mpl receptor on platelets and possibly bone marrow megakaryocytes.

Alternatively, some investigators have suggested that local production of TPO by bone marrow stromal cells may be increased during thrombocytopenia and may stimulate megakaryocyte growth (29). Direct evidence to support the relative contribution of this mechanism to platelet production is lacking, and recent studies of hepatic TPO production make this mechanism unlikely (67). These studies show that reduced hepatic production of TPO may play a major role in thrombocytopenia associated with liver disease. TPO is produced primarily in the liver, and thrombocytopenia in experimental animals seems to be proportional to the extent of liver resection (68). In addition, after transplantation of healthy livers into TPO-/- mice, platelet counts returned toward normal, suggesting that most TPO is produced in the liver (69). An association between low platelet counts (median: 84 x 109/L; range: 26-112 x 109/L) and low levels of TPO (median: <20 pg/mL; range: <20-182 pg/mL) has been reported in patients before orthotopic liver transplantation (70,71). Within 4 d of orthotopic liver transplantation, amounts of TPO increased above normal and were accompanied by increased amounts of reticulated platelets. Fourteen days after transplantation, platelet counts were normal in 14/18 patients (median: 254 x 109/L; range: 70-398 x 109/L), and TPO concentrations returned to normal in 14/18 patients (median: 59 pg/mL; range: <20-639 pg/mL). No appreciable change in spleen size was observed. In multivariate analysis, the increase in TPO was the only variable that correlated with the increase in platelet count.

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