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Thrombopoietin (Tpo), the main regulator of thrombocytopoiesis, is a probable candidate to play a role in the increase in platelet counts that is frequently seen after surgery. In the current study, serial blood samples of patients that underwent major surgery were analysed with respect to Tpo kinetics, platelet turnover and inflammatory cytokines. Platelet Tpo content and plasma Tpo levels rose before platelet counts increased, suggesting that Tpo was indeed responsible for the elevation in platelet counts. In addition, an increase in interleukin 6 (IL-6) levels, but not in IL-11 and tumour necrosis factor alpha levels, was seen before the rise in Tpo concentration. In vitro, IL-6 was shown to enhance Tpo production by the HepG2 liver cell line. Thus, increased Tpo levels after surgery, possibly resulting from enhanced Tpo production under the influence of IL-6 or other inflammatory cytokines, are involved in an enhanced thrombocytopoiesis.
In response to major surgery, reactive thrombocytosis may occur (Pepper & Lindsay, 1960; Robbins & Barnard, 1983; Adams, 1994). Particularly after coronary bypass surgery, reactive thrombocytosis represents a risk factor for thrombotic complications (Schmuziger et al, 1995; Christenson et al, 1996). A maximal rise in platelet count has been observed from d 7 to d 20 post surgery and may encompass a platelet increase of more than 100% (Pepper & Lindsay, 1960; Robbins & Barnard, 1983; Adams, 1994; Schmuziger et al, 1995; Christenson et al, 1996; Cerutti et al, 1999). The exact mechanism leading to the rise in platelet counts is unknown. Thrombopoietin (Tpo) is the main regulator of platelet production (Kaushansky, 1995) and, therefore, is a probable candidate to play a role in the platelet rise. Tpo alone can stimulate both proliferation and differentiation of megakaryocytic cells, which eventually results in the release of platelets in the circulation (Wendling et al, 1994; Choi et al, 1995; Debili et al, 1995a; Kaushansky et al, 1995; Cramer et al, 1997; Gehling et al, 1997; Norol et al, 1998). A potential increase in circulating Tpo after major surgery may arise via different mechanisms. Tpo production might be enhanced or Tpo clearance might be decreased. Alternatively, as we reported previously (Folman et al, 2000), Tpo might be released from activated platelets, which will also result in increased levels of circulating Tpo. Under normal conditions, Tpo is produced constitutively by the liver and, to a lesser extent, by the kidneys and is removed from the circulation by binding to the Tpo receptor, Mpl, which is present on platelets and megakaryocytic progenitors (Debili et al, 1995b; Fielder et al, 1996, 1997; Li et al, 1999). In cases of thrombocytopenia, bone marrow stromal cells have also been reported to produce Tpo (Sungaran et al, 1997; Hirayama et al, 1998).
In the current study, the kinetics of circulating and platelet-associated Tpo and the potential mechanisms underlying Tpo regulation in patients undergoing major surgery were addressed in detail. Serial samples of patients undergoing orthopaedic or cardiac surgery were analysed with respect to platelet count, plasma Tpo concentration and platelet Tpo content. Plasma glycocalicin (GC) concentrations were determined as a measure of platelet turnover. Inflammatory cytokines might play a role in thrombocytopoiesis either directly or indirectly via enhancement of Tpo production. Plasma interleukin 6 (IL-6), IL-11 and tumour necrosis factor alpha (TNFα) were measured as representatives of these cytokines. To investigate whether inflammatory cytokines or other factors could influence Tpo production, a liver cell line, a bone marrow endothelial cell line and primary bone marrow stromal cells were cultured with different cytokines and Tpo production was analysed.
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- Patients and methods
The current study shows that the reactive increase in platelets that is often seen after major surgery is probably caused by an increment in circulating Tpo, the main regulator of thrombocytopoiesis. The increase in platelet Tpo content suggested that the rise in circulating Tpo is first reflected by enhanced Tpo uptake by platelets. Previously, several in vitro studies have shown that platelets bind and internalize Tpo (Fielder et al, 1996, 1997; Broudy et al, 1997; Li et al, 1999). A change in average Tpo content per platelet in vivo, however, has to our knowledge not been reported before.
It is of note that the Tpo uptake per platelet initially increased, as evidenced by the increased platelet Tpo content, but as circulating Tpo levels increased, the platelet Tpo content decreased again. The cause of this decrease is unknown. Possibly, the total receptor expression diminishes as a result of internalization of the Tpo–Mpl complex and subsequent processing. Alternatively, platelets with a high Tpo content might represent aged platelets that leave the circulation.
In accordance with our findings, an elevation in circulating Tpo upon surgery has been reported by Hobisch-Hagen et al (1998) and Cerutti et al (1999). The former group reported that significantly elevated serum Tpo levels (compared with levels before haemodilution) occurred 3 h after surgery. This early increment might be attributed to the measurement of serum instead of plasma Tpo levels. Previously, we have shown that platelets can release Tpo and that serum levels are, on average, 3·4 times higher than plasma Tpo levels (Folman et al, 1997, 2000). In serum, both Tpo released from platelets and circulating Tpo is measured. Cerutti et al (1999) reported elevated plasma Tpo levels with a peak at d 3, which was associated with a rise in IL-6 concentrations, in patients undergoing hip replacement surgery. In our study, IL-6 levels peaked within hours after surgery and preceded the peak in platelet-associated Tpo.
A similar sequence of events (an IL-6 peak followed by an increase in circulating Tpo and platelet counts) was noted in patients with Kawasaki disease and upon induction of endotoxaemia in healthy individuals (Ishiguro et al, 1998; Stohlawetz et al, 1999).
Recently, it was shown that, in healthy individuals who received a bolus injection of recombinant human megakaryocyte growth and development factor (rHu-MGDF), the rise in platelet count, a reflection of enhanced thrombopoiesis, followed a few days after the rise in circulating Tpo (Harker et al, 2000). In line with this observation, we observed a 2–3 d delay before platelet counts started to rise. This suggests that the increased Tpo levels in the surgery patients might indeed have caused the rise in platelet counts.
Although platelet counts decreased immediately after surgery and gradually increased over time, no change in the GC concentration was observed. Previously, GC has been shown to be a marker for platelet turnover and is thought to be enzymatically cleaved from the platelet during destruction (Coller et al, 1984; Steinberg et al, 1987; Beer et al, 1994). It is probable that the follow-up of the patients in this study was too short to detect an increment in GC concentration because the newly produced platelets were still viable at the end of the follow-up. This is in agreement with results from a study in which patients were treated with MGDF (O'Malley et al, 1996). In these patients, GC levels started to rise 3 d after the start of the increase in platelet count.
The exact origin of the increase in circulating Tpo is unknown and might involve several mechanisms. According to the model in which the total platelet and megakaryocyte mass affects the amount of circulating Tpo via receptor-mediated binding (Kuter & Rosenberg, 1995), a decrease in platelet mass might result in increased Tpo levels. In patients undergoing CABG, a decrease in platelet mass was not reflected by a decrease in platelet counts. However, it is plausible that platelets are also consumed in these patients. The absence of a decrease in circulating numbers might result from an enhanced platelet release by the splenic pool or other compartments, thus masking platelet turnover in the periphery.
Another mechanism that might contribute to the Tpo increment is Tpo release by activated platelets. Previously, we and colleagues have shown that Tpo levels are elevated in patients with disseminated intravascular coagulation (Hiyoyama et al, 1997; Kawasugi et al, 1998; Folman et al, 2000) and correlate with markers for thrombin generation (Folman et al, 2000). In those patients, massive platelet activation occurs. Mild induction of endotoxaemia in healthy individuals also leads to an elevation of plasma Tpo levels (Stohlawetz et al, 1999). In analogy, Tpo might also be released by platelets activated upon surgery.
A third mechanism that might be involved is enhanced production of Tpo. To date, no regulation at the mRNA level has been reported for the liver and kidney in response to thrombocytopenia or thrombocytosis (Stoffel et al, 1996). In contrast, Tpo production by BM stromal cells can be induced. In patients with thrombocytopenia as a result of bone marrow aplasia or idiopathic thrombocytopenic purpura, an increased Tpo mRNA concentration in bone marrow was found (Sungaran et al, 1997; Hirayama et al, 1998). It has been reported that platelet α-granular proteins such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), platelet factor 4 (PF4), thrombospondin (TSP) and TGFβ could influence the production of Tpo by BM stromal cells (Sakamaki et al, 1999; Sungaran et al, 2000). Apart from these proteins, enhanced Tpo production might be induced under the influence of cytokines and/or growth factors that are involved in the inflammatory reaction upon surgery. Elevated levels of IL-6 and acute-phase reactants such as C-reactive protein in combination with elevated Tpo levels have been reported in patients with thrombocytosis secondary to inflammation or infection (Cerutti et al, 1997; Uppenkamp et al, 1998; Hsu et al, 1999). In two studies in which blood was drawn at one time-point in each patient, Tpo levels correlated with IL-6 levels and acute-phase reactants (Cerutti et al, 1997; Hsu et al, 1999). To assess whether the production of Tpo could be influenced by inflammatory factors, cell lines or cells potentially involved in Tpo production, i.e. HepG2, BMEC and BM stromal cells, were cultured with IL-1β, IL-6, TNFα, TGFβ and compared with unstimulated cells. Tpo production was not seen for the BMEC and the BM stromal cells. In contrast, Sakamaki et al (1999) showed that TGFβ increased the expression of Tpo mRNA. However, Sungaran et al (2000) also found no upregulation but rather a downregulation of Tpo mRNA in BM stromal cells under the influence of TGFβ. Similar to our findings, Wolber & Jelkman (2000) reported no Tpo production by BM fibroblasts either cultured without growth factors or with IL-1β, IL-6 or TNFα.
HepG2 cells did produce Tpo and a significant increase in Tpo was seen upon stimulation of these cells with IL-6. Analysis of the amount of Tpo mRNA expression showed no evidence that the IL-6-related increase in Tpo production resulted from enhanced Tpo mRNA expression. Although an increase in the amount of Tpo mRNA was noted after 24 h, this did not reach statistical significance. Possibly, peak Tpo mRNA levels were present at an earlier time-point. Alternatively, IL-6 might have increased the stability of Tpo mRNA or induced increased Tpo secretion either directly or by enhancement of cell proliferation, thereby increasing the amount of produced Tpo. In agreement with our findings, Wolber & Jelkman (2000) reported a 1·5-fold increase in Tpo protein production. In their study, a significant twofold increase of the Tpo mRNA content was noted using competitive PCR for quantification in both the HepG2 and the Hep3B cell lines after 24 h of culture with IL-6. In contrast, Hino et al (1995) and Yang et al (1997) observed no enhanced protein production.
In addition to Tpo, IL-6, which is a thrombopoietic factor as well as a proinflammatory factor, might also be directly involved in thrombocytopoiesis. Although to a lesser extent than Tpo, IL-6 is able to support megakaryocytopoiesis (Ishibashi et al, 1989). The increase in IL-6 that was seen after surgery might therefore play a direct role in enhancing thrombocytopoiesis. Apart from IL-6 other thrombopoietic factors such as IL-1, IL-3 and IL-12 might contribute. Further studies should be performed to investigate this.
In conclusion, it is probable that the surgery-related increase in platelet counts is caused by increased platelet production under the influence of Tpo and, possibly, to some extent by IL-6 or other cytokines with thrombopoietic activity. Upon surgery, Tpo levels increased, as was demonstrated by an increased Tpo content per platelet followed by an increased level of circulating Tpo. The exact origin of the Tpo elevation is unknown, but possibly multiple mechanisms are involved, i.e. accumulation as a result of diminished uptake, Tpo release by activated platelets and enhanced production under the influence of IL-6. These findings may contribute to the clarification of the mechanisms underlying the finding that aspirin reduces the risk of pulmonary embolism and deep-vein thrombosis after surgery, as was demonstrated again recently in a large multicentre trial (Pulmonary Embolism Prevention (PEP) Trial Collaborative Group, 2000).