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Effects of anticoagulation on thrombopoietin release during endotoxemia
Effects of anticoagulation on thrombopoietin release during endotoxemia PETRA JILMA-STOHLAWETZ, CLAUDIA C. FOLMAN, ALBERT E. G. KR. VON DEM BORNE, THOMAS PERNERSTORFER, URSULA HOLLENSTEIN, MAARTEN KNECHTELSDORFER, HANS-GEORG EICHLER, and BERND JILMA VIENNA, AUSTRIA, and AMSTERDAM, THE NETHERLANDS
Several lines of evidence suggest that coagulation may induce the release of thrombopoietin (TPO) into plasma and that TPO levels are higher in disseminated intravascular coagulation. Therefore we set out to illuminate the mechanism of TPO release in the setting of experimental endotoxemia, which induces activation of coagulation and platelets. Endotoxin (lipopolysachharide [LPS], 2 ng/kg) was infused into a total of 54 healthy men in two subsequent studies. Volunteers received infusions of unfractionated heparin, low-molecular-weight heparin, lepirudin, or placebo in a randomized, placebo-controlled fashion after bolus injection of LPS. TPO levels increased on average by 27% to 38% in all groups at 6 hours (P < .05 vs baseline), although all active drugs effectively blocked coagulation. Platelet counts dropped by about 15% at 1 hour after LPS infusion, recovered after 2 days, and exceeded baseline values by 8% to 18% after 7 days (P < .001 vs baseline for all groups). Yet lepirudin blunted the LPS-induced increase in circulating P-selectin by one half (P < .005 vs placebo), whereas both heparins did not diminish the increase in this platelet or endothelial activation marker as compared with placebo. Endotoxemia enhances TPO plasma levels independent of the degree of coagulation induction, which eventually results in increased platelet numbers. Of potential clinical interest is the observation that the direct thrombin inhibitor lepirudin, in contrast to heparins, mitigated LPS-induced platelet activation. (J Lab Clin Med 2001;137:64-9)
Abbreviations: ANOVA = analysis of variance; cP-selectin = circulating P-selectin; CRP = C-reactive protein; IL = interleukin; LPS = lipopolysaccharide; LMWH = low-molecular-weight heparin; TPO = thrombopoietin; UFH = unfractionated heparin
From the Department of Clinical Pharmacology, The Adhesion Research Group Elaborating Therapeutics, and the Department of Blood Group Serology and Transfusion Medicine, Division of Transfusion Medicine, Vienna University School of Medicine; the Department of Hematology, Division of Internal Medicine, Academic Medical Center, University of Amsterdam; and the Central Laboratory of Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam. Supported by grants from the Austrian Science Foundation and the Jubiläumsfonds der österreichischen Nationalbank. Submitted for publication June 6, 2000; revision submitted September 6, 2000; accepted September 7, 2000. Reprint requests: Bernd Jilma, MD, Department of Clinical Pharmacology, The Adhesion Research Group Elaborating Therapeutics, Vienna University, Währinger Gürtel 18-20, A-1090 Wien, Austria. Copyright © 2001 Mosby, Inc. 0022-2143/2001 $35.00 + 0 5/1/111468 doi:10.1067/mlc.2001.111468 64
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latelet production is a self-regulating process: the induction of thrombocytopenia results in an increase in TPO,1 the key regulator of thrombopoiesis. TPO, which is mainly produced in the liver,2 enhances the size, number, and ploidy of mega-karyocytes and hence the number of peripheral platelets.3,4 In addition to TPO, interleukins such as IL-6, IL-3, and IL-11 stimulate thrombopoiesis.5 In particular, elevated IL-6 generation has been implicated in secondary thrombocytosis.6-8 However, recent publications have also shown elevated TPO concentrations in reactive thrombocytosis.9,10 Further, clinical studies have demonstrated elevated TPO levels in patients with septicemia and disseminated intravascular coagulation,11,12 and TPO levels have
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been correlated with coagulation markers.12 Recently it was shown that TPO levels increase after endotoxin infusion into healthy volunteers,13 which represents an established model of systemic inflammation.14,15 Furthermore, platelet activation was shown to induce TPO release from isolated platelets,12 and TPO levels in serum are threefold higher than those in plasma, which is likely due to the release of TPO during the coagulation process.16 Thus it has been assumed that coagulation induces TPO release into plasma. However, this concept has never been verified by experimental data in vivo. Hence we hypothesized that activation of platelets during coagulation might cause the early increase in TPO levels during endotoxemia. We assessed the effects of anticoagulation on TPO release during endotoxemia. We analyzed TPO levels, soluble P-selectin (a marker of platelet activation),17,18 and glycocalicin levels—as a marker of platelet mass—from frozen samples retained from two studies.19,20 METHODS Study design and study subjects. Data are derived from two independent placebo-controlled studies19,20 that were conducted in a total of 54 healthy male subjects in a randomized, double-blind fashion in parallel groups. Blinding was maintained for the analysis of the current study. The studies were approved by the Institutional Ethics Committee, and written informed consent was obtained from all participants before enrollment in the study. Study subjects. Subject characteristics are presented in detail elsewhere.19,20 In brief, the mean age of the healthy volunteers was 28 ± 4 years, and the body mass index averaged 23.5 ± 1.8 kg/m2. Study protocol. Volunteers were admitted to the study ward at 8 AM after an overnight fast. Throughout the entire study period, subjects were confined to bedrest and kept fasting for 8.5 hours after LPS infusion. Participants in the trial received 500 mg acetaminophen (Paracetamol; Genericon Pharma, Lannach, Austria), which alleviates subjective LPS-related symptoms without compromising the systemic host response to LPS.19 Thirty minutes thereafter, all subjects (n = 54) received an intravenous bolus of 2 ng/kg LPS (National Reference Endotoxin, Escherichia coli; USP Convention Inc, Rockville, MD). Ten minutes after LPS infusion, study subjects in the lepirudin group (n = 12) received 0.1 mg/kg recombinant hirudin (ie, lepirudin [Refludan, Hoechst Marion Roussel]) followed by a continuous infusion of lepirudin at a rate of 0.1 mg/kg/h for 5 hours. Volunteers in the UFH group (n = 10) received 80 IU/kg UFH (Heparin Immuno; Immuno) followed by a continuous UFH infusion at a rate of 18 IU/kg/h for 6 hours. Study subjects assigned to the LMWH group (n = 10) received 40 IU/kg Fragmin (Dalteparin; Pharmacia & Upjohn, Stockholm, Sweden) followed by continuous infusion of 15 IU/kg/h for
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6 hours. Identical volumes of saline solution were given in the placebo groups (n = 12 and n = 10 in the lepirudin and heparin trials, respectively). Sampling and analysis. Blood samples were collected by venipuncture into citrated vacuum blood collection tubes (final concentration 0.13 mmol/L sodium citrate; Vacutainer; Becton Dickinson, San Jose, CA) before drug administration and thereafter as indicated in the figures by repeated venipuncture (except platelet counts, which were obtained from an indwelling venous line) on the contralateral arm from where LPS had been administered. Plasma samples were processed immediately by centrifugation at 2000g at 4°C for 15 minutes and stored at –80°C before analysis as duplicates. All samples were analyzed by enzyme immunoassays, and samples from individual subjects were run in the same assay. Assays were as follows: TPO plasma level (TPO assay; CLB, Amsterdam) 16; glycocalicin level, a marker of platelet mass, (glycocalicin assay; CLB, Amsterdam) 21; soluble P-selectin (R&D Systems, Oxon, England).22,23 The prothrombin fragment F1+2 assay was purchased from Behringwerke (Enzygnost F1+2).24 Plasma levels of IL-6 were measured when maximal levels were expected25 (Quantikine HS; R&D Systems).26 CRP levels were determined by nephelometry (Tinaquant CRP; Boehringer Mannheim/ Hitachi)26 at 24 hours. Platelet counts were obtained with a cell counter (Sysmex). Data analysis. Data from the placebo groups of the two trials were not pooled, because baseline TPO levels were measured on two occasions and were somewhat different between trials. Therefore between-study comparisons are strictly not valid. Data are expressed in the text as the mean and the 95% confidence interval. Non-parametric statistics were applied. All statistical comparisons within groups were done with the Friedman ANOVA and the Wilcoxon signed rank test for post-hoc comparisons. To test changes in end points between groups for statistical significance, the Kruskal-Wallis ANOVA was used, and post-hoc comparisons were performed by the Mann-Whitney test. A two-tailed P value of ≤.05 was considered significant. RESULTS TPO levels Heparin trial. TPO levels increased by 32% to 37% at 6 hours after LPS infusion (Fig 1; P < .05 vs baseline in all groups). Thereafter, TPO levels decreased in the UFH and placebo group, while the peak values were observed at 24 hiurs in the LMWH group, which could have been due to chance only. Interestingly, TPO levels were still 5% to 9% above baseline levels after 7 days. No differences between the groups were observed (P > .05). Lepirudin trial. TPO plasma levels increased by 27% and 38% in the lepirudin and placebo group, respectively, after 6 hours (Fig 1; P > .05 vs baseline).
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Fig 1. Percent increase in plasma levels of thrombopoietin (left) and circulating P-selectin (right) after an intravenous injection of 2 ng/kg LPS (P < .001, Friedman ANOVA vs baseline for both parameters and all groups). Top, Subjects received placebo () or lepirudin (■) as a 5-hour intravenous infusion. Bottom, Subjects received placebo (), unfractionated heparin (■), or low-molecular-weight heparin () as a 6-hour intravenous infusion starting 10 minutes after LPS infusion. None of the anticoagulants prevented an increase in TPO levels, but lepirudin blunted the increase in the platelet activation marker cP-selectin. *P < .05 versus baseline. **P < .01 versus baseline. °P < .05 between treatment groups.
Platelet counts Heparin trial. Platelet counts dropped by about 13% at 2 hours after LPS infusion (P = .005 vs baseline in all groups). Platelet counts recovered after 24 to 48 hours and were 15% to 18% higher than baseline values 7 days after LPS infusion (P = .005 in all three groups; P > .05 between groups). Lepirudin trial. Platelet counts dropped on average by 16% to 17% in both groups (P = .002 vs baseline). Again, platelet counts recovered after 24 to 48 hours, and platelet counts increased 16% and 10% over baseline in the placebo and lepirudin groups at 7 days, respectively (P < .005 vs baseline in both groups; P > .05 between groups). Glycocalicin levels Lepirudin trial. The increase in platelet mass was further corroborated by similar changes in glycocalicin levels, which were determined only in the lepirudin trial. Glycocalicin levels did not change acutely but increased by 9% to 18% at 3 and 7 days after LPS infusion (P < .05 vs baseline in both groups).
Plasma levels of soluble P-selectin Heparin trial. Soluble P-selectin levels increased by a maximum of 49% to 66% at 6 hours after LPS infusion (Fig 1; P < .01 vs baseline in all groups; P > .05 between groups) and declined thereafter. Hirudin trial. Soluble P-selectin levels increased by a maximum of 58% (CI: 33% to 84%) in the placebo group 6 hours after LPS infusion (Fig 1). In contrast, soluble P-selectin levels increased by only 28% (CI: 5% to 53%; P = .04 vs placebo) at 6 hours after lepirudin infusion. This difference between groups was even more pronounced at 24 hours (P = .015 between groups). Plasma levels of IL-6, CRP, and prothrombin fragment
IL-6 levels increased more than 500-fold in all groups at 3 hours (range: 65 to 4480 ng/L; P > .05 between groups), and serum levels of CRP increased from below 0.5 mg/dL to 3.2 mg/dL (CI: 2.2 to 4.2; P < .001) at 24 hours (P > .05 between groups for both parameters). The time course of F1+2 levels has been described in detail previously.19,20 For clarity, nevertheless, baseline Heparin and lepirudin trial.
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Table I. Baseline values of outcome parameters in healthy subjects undergoing endotoxemia Heparin trial
Platelet counts (109/L) Thrombopoietin (AU/mL) Glycocalicin (AU/mL) cP-selectin (ng/mL) Prothrombin fragment F1+2 (ng/mL) Interleukin-6 (pg/mL)
Lepirudin trial
Placebo
Heparin
LMWH
Lepirudin
Placebo
203 (186-219) 7.9 (6.9-8.9) ND — 36 (26-46) 0.63 (0.45-0.81) 1.5 (1.0-2.0)
207 (184-230) 6.2 (5.2-7.3) ND — 28 (21-35) 0.71 (0.53-0.89) 1.4 (0.8-2.1)
221 (207-235) 7.1 (5.3-9.9) ND — 30 (25-36) 0.66 (0.43-0.89) 1.9 (0.2-3.6)
225 (199-250) 12.8 (10.0-15.6) 430 (358-501) 36 (31-41) 0.56 (0.46-0.65) 1.7 (0.5-2.9)
241 (199-284) 13.3 (9.9-16.8) 381 (329-433) 33 (28-39) 0.55 (0.43-0.66) 1.3 (0.6-2.0)
Values are expressed as mean and 95% confidence interval. AU, Arbitrary units; ND, not done.
values are provided in Table I. As expected, baseline F1+2 levels did not change during the first hour after LPS administration. At 4 hours after LPS infusion, F1+2 levels increased more than 10-fold, to 8.5 ng/mL (CI: 3.4 to 13.8) and to 7.3 ng/mL (CI: 3.4 to 11.2) in the two placebo groups of the heparin and the hirudin trials, respectively. This increase was completely blunted by unfractionated heparin (0.68 ng/mL; CI: 0.47 to 0.89) and inhibited by LMWH (1.1 ng/mL; CI: 0.7 to 1.6) and lepirudin (1.9 ng/mL; CI: 1.3 to 2.5). DISCUSSION
The current study aimed to clarify whether platelet activation during coagulation might cause the early increase in TPO levels during endotoxemia. We hypothesized that inhibition of coagulation would abrogate the increase in TPO levels. In contrast to our study hypothesis, the infusion of heparin, LMWH, or lepirudin had no effects on the LPS-induced increase in TPO as compared with placebo (Fig 1). This was unexpected, since all three anticoagulants effectively inhibited coagulation.19,20 Hence the activation of coagulation does not appear to contribute to the release of TPO during lowgrade endotoxemia, although a minor contribution cannot be excluded. Thus we have to ask why TPO levels increase during endotoxemia. First, the observed decrease in circulating platelets could in part account for the increase in plasma TPO levels, because platelets bind and internalize TPO by specific receptors.27 Thus, as TPO levels inversely correlate with platelet counts, a decreased clearance of TPO could cause the increase in TPO during endotoxemia. Yet we previously observed a similar decrease in platelet counts after a two-fold higher dose
of LPS,13 whereas TPO levels increased on average by 55% in that previous trial, as compared with 33% in the current study (Fig 1). Therefore factors other than the drop in platelets appear to contribute to the dose-dependent increase in TPO levels after LPS challenge. Platelet activation during endotoxemia might induce the release of TPO from platelets, because stimulation of isolated platelets with strong activators leads to the release of TPO into the supernatant.12 As expected from our previous trial,28 the platelet or endothelial marker cP-selectin17,18,29 increased substantially after LPS infusion in the placebo groups. An exciting finding is that lepirudin, in contrast to both heparins, blunted the increase in cP-selectin (Fig 1). P-selectin is stored in the α-granules of platelets and is released into the plasma after activation of platelets22,30 but not after degranulation of the endothelial storage pool of P-selectin in human beings.29 Hence our results support the concept that the direct thrombin inhibitor lepirudin may have anti-platelet properties in the setting of systemic coagulation in human beings. This could be of clinical relevance, because lethal lung injury in septic patients has been associated with high cP-selectin levels.31 Yet although lepirudin inhibited the increase in cP-selectin after LPS infusion, it was not able to blunt the increase in TPO levels. Thus, activated platelets do not seem to be the only source of increased TPO levels during experimental endotoxemia. An alternative explanation could be provided by the observation that IL-6 stimulates thrombopoietin gene expression and secretion.32 As LPS infusion increases IL-6 plasma levels by three orders of magnitude, this might stimulate TPO secretion from the liver. Hyperexpression of TPO could also explain why TPO levels
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remain elevated for 7 days after intravenous LPS (Fig 1), whereas it is unlikely that this elevation is due to coagulation and decreased clearance, because LPSinduced coagulation normalizes 24 hours after LPS infusion,33 and platelet counts recover by day 2 (Fig 1). Finally, platelet counts increased on day 7, a finding that was reflected in a concomitant increase in glycocalicin levels. These data further confirm that plasma glycocalicin level may serve as a stable surrogate marker of platelet mass34 or turnover,35 even in systemic inflammatory diseases such as septicemia. In conclusion, low-grade endotoxemia induces a rapid fall in platelet count and an enhancement of TPO plasma level independent of anticoagulation. Although lepirudin inhibited the release of soluble P-selectin into circulation, it did not mitigate TPO release into plasma. However, further studies in animals are needed to corroborate these findings in experimental sepsis or other models of coagulation. REFERENCES
1. Hou M, Andersson PO, Stockelberg D, Mellqvist UH, Ridell B, Wadenvik H. Plasma thrombopoietin levels in thrombocytopenic states: implication for a regulatory role of bone marrow megakaryocytes. Br J Haematol 1998;101:420-4. 2. Siemensma NP, Bathal PS, Penington DG. The effect of massive liver resection on platelet kinetics in the rat. J Lab Clin Med 1975;86:817-33. 3. de Sauvage FJ, Villeval JL, Shivdasani RA. Regulation of megakaryocytopoiesis and platelet production: lessons from animal models. J Lab Clin Med 1998;131:496-501. 4. Jackson CW. New insights into the regulation of megakaryocyte differentiation and platelet production derived from cloning of thrombopoietin and its receptor: surprises and unresolved questions as thrombopoietin proceeds through clinical trials. J Lab Clin Med 1998;131:480-4. 5. Burstein SA. Cytokines, platelet production and hemostasis. Platelets 1997;8:93-104. 6. Hollen CW, Henthorn J, Koziol JA, Burstein SA. Elevated serum interleukin-6 levels in patients with reactive thrombocytosis. Br J Haematol 1991;79:286-90. 7. Gastl G, Plante M, Finstad CL, et al. High IL-6 levels in ascitic fluid correlate with reactive thrombocytosis in patients with epithelial ovarian cancer. Br J Haematol 1993;83:43341. 8. Ishiguro A, Ishikita T, Shimbo T, et al. Elevation of serum thrombopoietin precedes thrombocytosis in Kawasaki disease. Thromb Haemost 1998;79:1096-100. 9. Cerutti A, Custodi P, Duranti M, Noris P, Balduini CL. Thrombopoietin levels in patients with primary and reactive thrombocytosis. Br J Haematol 1997;99:281-4. 10. Hsu HC, Tsai WH, Jiang ML, et al. Circulating levels of thrombopoietic and inflammatory cytokines in patients with clonal and reactive thrombocytosis. J Lab Clin Med 1999; 134:392-7. 11. Hiyoyama K, Wada H, Shimura T, et al. Increased serum levels of thrombopoietin in patients with thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura, or disseminated intravascular coagulation. Blood Coagul Fibrinolysis 1997;8:345-9.
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12. Folman CC, Linthorst GE, van MJ, et al. Platelets release thrombopoietin (Tpo) upon activation: another regulatory loop in thrombocytopoiesis? Thromb Haemost 2000;83:92330. 13. Stohlawetz P, Folman CC, von dem Borne AEGK, et al. Effects of endotoxemia on thrombopoiesis in men. Thromb Haemost 1999;81:613-7. 14. Martich GD, Boujoukos AJ, Suffredini AF. Response of man to endotoxin. Immunobiology 1993;187:403-16. 15. Suffredini AF, Hochstein HD, McMahon FG. Dose-related inflammatory effects of intravenous endotoxin in humans: evaluation of a new clinical lot of Escherichia coli O:113 endotoxin. J Infect Dis 1999;179:1278-82. 16. Folman CC, von dem Borne AE, Rensink IH, et al. Sensitive measurement of thrombopoietin by a monoclonal antibody based sandwich enzyme-linked immunosorbent assay. Thromb Haemost 1997;78:1262-7. 17. Blann AD, Lip GY. Hypothesis: is soluble P-selectin a new marker of platelet activation? Atherosclerosis 1997;128:1358. 18. Fijnheer R, Frijns CJ, Korteweg J, et al. The origin of Pselectin as a circulating plasma protein. Thromb Haemost 1997;77:1081-5. 19. Pernerstorfer T, Hollenstein U, Hansen JB, et al. Heparin blunts endotoxin-induced coagulation activation. Circulation 1999:2485-90. 20. Pernerstorfer T, Hollenstein U, Hansen JB, et al. Lepirudin blunts endotoxin-induced coagulation activation. Blood 2000;95:1729-34. 21. Porcelijn J, Folman CC, Bossers B, et al. The diagnostic value of thrombopoietin level measurements in thrombocytopenia. Thromb Haemost 1998;79:1101-5. 22. Stohlawetz P, Hergovich N, Stiegler G, et al. Differential induction of P-selectin by two cell separators during plateletpheresis and the effect of gender on the release of soluble Pselectin. Transfusion 1998;38:24-30. 23. Stohlawetz P, Horvath M, Pernerstorfer T, et al. Effects of nitric oxide on platelet activation during plateletpheresis and in vivo tracking of biotinylated platelets in humans. Transfusion 1999;39:506-14. 24. Stohlawetz P, Kapiotis S, Seidl D, et al. Safety issues of plateletpheresis: comparison of the effects of two cell separators on the activation of coagulation, fibrinolysis, neutrophils and on leukocyte-platelet aggregates. Transfusion 1999;39:420-7. 25. Suffredini AF, Reda D, Banks SM, Tropea M, Agosti JM, Miller R. Effects of recombinant dimeric TNF receptor on human inflammatory responses following intravenous endotoxin administration. J Immunol 1995;155:5038-45. 26. Jilma B, Dirnberger E, Loscher I, et al. Menstrual cycle-associated changes in blood levels of interleukin-6, α1 acid glycoprotein, and C-reactive protein. J Lab Clin Med 1997;130: 69-75. 27. Li J, Xia Y, Kuter DJ. Interaction of thrombopoietin with the platelet c-mpl receptor in plasma: binding, internalization, stability and pharmacokinetics. Br J Haematol 1999;106: 345-56. 28. Jilma B, Blann AD, Pernerstorfer T, et al. Regulation of adhesion molecules during human endotoxemia: no acute effects of aspirin. Am J Respir Crit Care Med 1999;159:857-63. 29. Jilma B, Eichler HG, Vondrovec B, et al. Effects of desmopressin on circulating P-selectin. Br J Haematol 1996;93: 432-6. 30. Stohlawetz PJ, Dzirlo L, Hergovich N, et al. Effects of
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erythropoietin on platelet reactivity and thrombopoiesis in humans. Blood 2000;95:2983-9. 31. Sakamaki F, Ishizaka A, Handa M, et al. Soluble form of Pselectin in plasma is elevated in acute lung injury. Am J Respir Crit Care Med 1995;151:1821-6. 32. Wolber EM, Jelkmann W. Interleukin-6 increases thrombopoietin production in human hepatoma cells HepG2 and Hep3B. J Interferon Cytokine Res 2000;20:499-506. 33. van der Poll T, Coyle SM, Levi M, et al. Effect of a recom-
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binant dimeric tumor necrosis factor receptor on inflammatory responses to intravenous endotoxin in normal humans. Blood 1997;89:3727-34. 34. Steinberg MH, Kelton JG, Coller BS. Plasma glycocalicin: an aid in the classification of thrombocytopenic disorders. N Engl J Med 1987;317:1037-42. 35. Williams SB, Sano M, Smith N, et al. Glycocalicin levels in the plasma of HIV+ patients: an indicator of platelet turnover. J Lab Clin Med 1998;132:303-7.
Several lines of evidence suggest that coagulation may induce the release of thrombopoietin (TPO) into plasma and that TPO levels are higher in disseminated intravascular coagulation. Therefore we set out to illuminate the mechanism of TPO release in the setting of experimental endotoxemia, which induces activation of coagulation and platelets. Endotoxin (lipopolysachharide [LPS], 2 ng/kg) was infused into a total of 54 healthy men in two subsequent studies. Volunteers received infusions of unfractionated heparin, low-molecular-weight heparin, lepirudin, or placebo in a randomized, placebo-controlled fashion after bolus injection of LPS. TPO levels increased on average by 27% to 38% in all groups at 6 hours (P < .05 vs baseline), although all active drugs effectively blocked coagulation. Platelet counts dropped by about 15% at 1 hour after LPS infusion, recovered after 2 days, and exceeded baseline values by 8% to 18% after 7 days (P < .001 vs baseline for all groups). Yet lepirudin blunted the LPS-induced increase in circulating P-selectin by one half (P < .005 vs placebo), whereas both heparins did not diminish the increase in this platelet or endothelial activation marker as compared with placebo. Endotoxemia enhances TPO plasma levels independent of the degree of coagulation induction, which eventually results in increased platelet numbers. Of potential clinical interest is the observation that the direct thrombin inhibitor lepirudin, in contrast to heparins, mitigated LPS-induced platelet activation. (J Lab Clin Med 2001;137:64-9)
Abbreviations: ANOVA = analysis of variance; cP-selectin = circulating P-selectin; CRP = C-reactive protein; IL = interleukin; LPS = lipopolysaccharide; LMWH = low-molecular-weight heparin; TPO = thrombopoietin; UFH = unfractionated heparin
From the Department of Clinical Pharmacology, The Adhesion Research Group Elaborating Therapeutics, and the Department of Blood Group Serology and Transfusion Medicine, Division of Transfusion Medicine, Vienna University School of Medicine; the Department of Hematology, Division of Internal Medicine, Academic Medical Center, University of Amsterdam; and the Central Laboratory of Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam. Supported by grants from the Austrian Science Foundation and the Jubiläumsfonds der österreichischen Nationalbank. Submitted for publication June 6, 2000; revision submitted September 6, 2000; accepted September 7, 2000. Reprint requests: Bernd Jilma, MD, Department of Clinical Pharmacology, The Adhesion Research Group Elaborating Therapeutics, Vienna University, Währinger Gürtel 18-20, A-1090 Wien, Austria. Copyright © 2001 Mosby, Inc. 0022-2143/2001 $35.00 + 0 5/1/111468 doi:10.1067/mlc.2001.111468 64
P
latelet production is a self-regulating process: the induction of thrombocytopenia results in an increase in TPO,1 the key regulator of thrombopoiesis. TPO, which is mainly produced in the liver,2 enhances the size, number, and ploidy of mega-karyocytes and hence the number of peripheral platelets.3,4 In addition to TPO, interleukins such as IL-6, IL-3, and IL-11 stimulate thrombopoiesis.5 In particular, elevated IL-6 generation has been implicated in secondary thrombocytosis.6-8 However, recent publications have also shown elevated TPO concentrations in reactive thrombocytosis.9,10 Further, clinical studies have demonstrated elevated TPO levels in patients with septicemia and disseminated intravascular coagulation,11,12 and TPO levels have
J Lab Clin Med Volume 137, Number 1
been correlated with coagulation markers.12 Recently it was shown that TPO levels increase after endotoxin infusion into healthy volunteers,13 which represents an established model of systemic inflammation.14,15 Furthermore, platelet activation was shown to induce TPO release from isolated platelets,12 and TPO levels in serum are threefold higher than those in plasma, which is likely due to the release of TPO during the coagulation process.16 Thus it has been assumed that coagulation induces TPO release into plasma. However, this concept has never been verified by experimental data in vivo. Hence we hypothesized that activation of platelets during coagulation might cause the early increase in TPO levels during endotoxemia. We assessed the effects of anticoagulation on TPO release during endotoxemia. We analyzed TPO levels, soluble P-selectin (a marker of platelet activation),17,18 and glycocalicin levels—as a marker of platelet mass—from frozen samples retained from two studies.19,20 METHODS Study design and study subjects. Data are derived from two independent placebo-controlled studies19,20 that were conducted in a total of 54 healthy male subjects in a randomized, double-blind fashion in parallel groups. Blinding was maintained for the analysis of the current study. The studies were approved by the Institutional Ethics Committee, and written informed consent was obtained from all participants before enrollment in the study. Study subjects. Subject characteristics are presented in detail elsewhere.19,20 In brief, the mean age of the healthy volunteers was 28 ± 4 years, and the body mass index averaged 23.5 ± 1.8 kg/m2. Study protocol. Volunteers were admitted to the study ward at 8 AM after an overnight fast. Throughout the entire study period, subjects were confined to bedrest and kept fasting for 8.5 hours after LPS infusion. Participants in the trial received 500 mg acetaminophen (Paracetamol; Genericon Pharma, Lannach, Austria), which alleviates subjective LPS-related symptoms without compromising the systemic host response to LPS.19 Thirty minutes thereafter, all subjects (n = 54) received an intravenous bolus of 2 ng/kg LPS (National Reference Endotoxin, Escherichia coli; USP Convention Inc, Rockville, MD). Ten minutes after LPS infusion, study subjects in the lepirudin group (n = 12) received 0.1 mg/kg recombinant hirudin (ie, lepirudin [Refludan, Hoechst Marion Roussel]) followed by a continuous infusion of lepirudin at a rate of 0.1 mg/kg/h for 5 hours. Volunteers in the UFH group (n = 10) received 80 IU/kg UFH (Heparin Immuno; Immuno) followed by a continuous UFH infusion at a rate of 18 IU/kg/h for 6 hours. Study subjects assigned to the LMWH group (n = 10) received 40 IU/kg Fragmin (Dalteparin; Pharmacia & Upjohn, Stockholm, Sweden) followed by continuous infusion of 15 IU/kg/h for
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6 hours. Identical volumes of saline solution were given in the placebo groups (n = 12 and n = 10 in the lepirudin and heparin trials, respectively). Sampling and analysis. Blood samples were collected by venipuncture into citrated vacuum blood collection tubes (final concentration 0.13 mmol/L sodium citrate; Vacutainer; Becton Dickinson, San Jose, CA) before drug administration and thereafter as indicated in the figures by repeated venipuncture (except platelet counts, which were obtained from an indwelling venous line) on the contralateral arm from where LPS had been administered. Plasma samples were processed immediately by centrifugation at 2000g at 4°C for 15 minutes and stored at –80°C before analysis as duplicates. All samples were analyzed by enzyme immunoassays, and samples from individual subjects were run in the same assay. Assays were as follows: TPO plasma level (TPO assay; CLB, Amsterdam) 16; glycocalicin level, a marker of platelet mass, (glycocalicin assay; CLB, Amsterdam) 21; soluble P-selectin (R&D Systems, Oxon, England).22,23 The prothrombin fragment F1+2 assay was purchased from Behringwerke (Enzygnost F1+2).24 Plasma levels of IL-6 were measured when maximal levels were expected25 (Quantikine HS; R&D Systems).26 CRP levels were determined by nephelometry (Tinaquant CRP; Boehringer Mannheim/ Hitachi)26 at 24 hours. Platelet counts were obtained with a cell counter (Sysmex). Data analysis. Data from the placebo groups of the two trials were not pooled, because baseline TPO levels were measured on two occasions and were somewhat different between trials. Therefore between-study comparisons are strictly not valid. Data are expressed in the text as the mean and the 95% confidence interval. Non-parametric statistics were applied. All statistical comparisons within groups were done with the Friedman ANOVA and the Wilcoxon signed rank test for post-hoc comparisons. To test changes in end points between groups for statistical significance, the Kruskal-Wallis ANOVA was used, and post-hoc comparisons were performed by the Mann-Whitney test. A two-tailed P value of ≤.05 was considered significant. RESULTS TPO levels Heparin trial. TPO levels increased by 32% to 37% at 6 hours after LPS infusion (Fig 1; P < .05 vs baseline in all groups). Thereafter, TPO levels decreased in the UFH and placebo group, while the peak values were observed at 24 hiurs in the LMWH group, which could have been due to chance only. Interestingly, TPO levels were still 5% to 9% above baseline levels after 7 days. No differences between the groups were observed (P > .05). Lepirudin trial. TPO plasma levels increased by 27% and 38% in the lepirudin and placebo group, respectively, after 6 hours (Fig 1; P > .05 vs baseline).
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Fig 1. Percent increase in plasma levels of thrombopoietin (left) and circulating P-selectin (right) after an intravenous injection of 2 ng/kg LPS (P < .001, Friedman ANOVA vs baseline for both parameters and all groups). Top, Subjects received placebo () or lepirudin (■) as a 5-hour intravenous infusion. Bottom, Subjects received placebo (), unfractionated heparin (■), or low-molecular-weight heparin () as a 6-hour intravenous infusion starting 10 minutes after LPS infusion. None of the anticoagulants prevented an increase in TPO levels, but lepirudin blunted the increase in the platelet activation marker cP-selectin. *P < .05 versus baseline. **P < .01 versus baseline. °P < .05 between treatment groups.
Platelet counts Heparin trial. Platelet counts dropped by about 13% at 2 hours after LPS infusion (P = .005 vs baseline in all groups). Platelet counts recovered after 24 to 48 hours and were 15% to 18% higher than baseline values 7 days after LPS infusion (P = .005 in all three groups; P > .05 between groups). Lepirudin trial. Platelet counts dropped on average by 16% to 17% in both groups (P = .002 vs baseline). Again, platelet counts recovered after 24 to 48 hours, and platelet counts increased 16% and 10% over baseline in the placebo and lepirudin groups at 7 days, respectively (P < .005 vs baseline in both groups; P > .05 between groups). Glycocalicin levels Lepirudin trial. The increase in platelet mass was further corroborated by similar changes in glycocalicin levels, which were determined only in the lepirudin trial. Glycocalicin levels did not change acutely but increased by 9% to 18% at 3 and 7 days after LPS infusion (P < .05 vs baseline in both groups).
Plasma levels of soluble P-selectin Heparin trial. Soluble P-selectin levels increased by a maximum of 49% to 66% at 6 hours after LPS infusion (Fig 1; P < .01 vs baseline in all groups; P > .05 between groups) and declined thereafter. Hirudin trial. Soluble P-selectin levels increased by a maximum of 58% (CI: 33% to 84%) in the placebo group 6 hours after LPS infusion (Fig 1). In contrast, soluble P-selectin levels increased by only 28% (CI: 5% to 53%; P = .04 vs placebo) at 6 hours after lepirudin infusion. This difference between groups was even more pronounced at 24 hours (P = .015 between groups). Plasma levels of IL-6, CRP, and prothrombin fragment
IL-6 levels increased more than 500-fold in all groups at 3 hours (range: 65 to 4480 ng/L; P > .05 between groups), and serum levels of CRP increased from below 0.5 mg/dL to 3.2 mg/dL (CI: 2.2 to 4.2; P < .001) at 24 hours (P > .05 between groups for both parameters). The time course of F1+2 levels has been described in detail previously.19,20 For clarity, nevertheless, baseline Heparin and lepirudin trial.
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Table I. Baseline values of outcome parameters in healthy subjects undergoing endotoxemia Heparin trial
Platelet counts (109/L) Thrombopoietin (AU/mL) Glycocalicin (AU/mL) cP-selectin (ng/mL) Prothrombin fragment F1+2 (ng/mL) Interleukin-6 (pg/mL)
Lepirudin trial
Placebo
Heparin
LMWH
Lepirudin
Placebo
203 (186-219) 7.9 (6.9-8.9) ND — 36 (26-46) 0.63 (0.45-0.81) 1.5 (1.0-2.0)
207 (184-230) 6.2 (5.2-7.3) ND — 28 (21-35) 0.71 (0.53-0.89) 1.4 (0.8-2.1)
221 (207-235) 7.1 (5.3-9.9) ND — 30 (25-36) 0.66 (0.43-0.89) 1.9 (0.2-3.6)
225 (199-250) 12.8 (10.0-15.6) 430 (358-501) 36 (31-41) 0.56 (0.46-0.65) 1.7 (0.5-2.9)
241 (199-284) 13.3 (9.9-16.8) 381 (329-433) 33 (28-39) 0.55 (0.43-0.66) 1.3 (0.6-2.0)
Values are expressed as mean and 95% confidence interval. AU, Arbitrary units; ND, not done.
values are provided in Table I. As expected, baseline F1+2 levels did not change during the first hour after LPS administration. At 4 hours after LPS infusion, F1+2 levels increased more than 10-fold, to 8.5 ng/mL (CI: 3.4 to 13.8) and to 7.3 ng/mL (CI: 3.4 to 11.2) in the two placebo groups of the heparin and the hirudin trials, respectively. This increase was completely blunted by unfractionated heparin (0.68 ng/mL; CI: 0.47 to 0.89) and inhibited by LMWH (1.1 ng/mL; CI: 0.7 to 1.6) and lepirudin (1.9 ng/mL; CI: 1.3 to 2.5). DISCUSSION
The current study aimed to clarify whether platelet activation during coagulation might cause the early increase in TPO levels during endotoxemia. We hypothesized that inhibition of coagulation would abrogate the increase in TPO levels. In contrast to our study hypothesis, the infusion of heparin, LMWH, or lepirudin had no effects on the LPS-induced increase in TPO as compared with placebo (Fig 1). This was unexpected, since all three anticoagulants effectively inhibited coagulation.19,20 Hence the activation of coagulation does not appear to contribute to the release of TPO during lowgrade endotoxemia, although a minor contribution cannot be excluded. Thus we have to ask why TPO levels increase during endotoxemia. First, the observed decrease in circulating platelets could in part account for the increase in plasma TPO levels, because platelets bind and internalize TPO by specific receptors.27 Thus, as TPO levels inversely correlate with platelet counts, a decreased clearance of TPO could cause the increase in TPO during endotoxemia. Yet we previously observed a similar decrease in platelet counts after a two-fold higher dose
of LPS,13 whereas TPO levels increased on average by 55% in that previous trial, as compared with 33% in the current study (Fig 1). Therefore factors other than the drop in platelets appear to contribute to the dose-dependent increase in TPO levels after LPS challenge. Platelet activation during endotoxemia might induce the release of TPO from platelets, because stimulation of isolated platelets with strong activators leads to the release of TPO into the supernatant.12 As expected from our previous trial,28 the platelet or endothelial marker cP-selectin17,18,29 increased substantially after LPS infusion in the placebo groups. An exciting finding is that lepirudin, in contrast to both heparins, blunted the increase in cP-selectin (Fig 1). P-selectin is stored in the α-granules of platelets and is released into the plasma after activation of platelets22,30 but not after degranulation of the endothelial storage pool of P-selectin in human beings.29 Hence our results support the concept that the direct thrombin inhibitor lepirudin may have anti-platelet properties in the setting of systemic coagulation in human beings. This could be of clinical relevance, because lethal lung injury in septic patients has been associated with high cP-selectin levels.31 Yet although lepirudin inhibited the increase in cP-selectin after LPS infusion, it was not able to blunt the increase in TPO levels. Thus, activated platelets do not seem to be the only source of increased TPO levels during experimental endotoxemia. An alternative explanation could be provided by the observation that IL-6 stimulates thrombopoietin gene expression and secretion.32 As LPS infusion increases IL-6 plasma levels by three orders of magnitude, this might stimulate TPO secretion from the liver. Hyperexpression of TPO could also explain why TPO levels
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remain elevated for 7 days after intravenous LPS (Fig 1), whereas it is unlikely that this elevation is due to coagulation and decreased clearance, because LPSinduced coagulation normalizes 24 hours after LPS infusion,33 and platelet counts recover by day 2 (Fig 1). Finally, platelet counts increased on day 7, a finding that was reflected in a concomitant increase in glycocalicin levels. These data further confirm that plasma glycocalicin level may serve as a stable surrogate marker of platelet mass34 or turnover,35 even in systemic inflammatory diseases such as septicemia. In conclusion, low-grade endotoxemia induces a rapid fall in platelet count and an enhancement of TPO plasma level independent of anticoagulation. Although lepirudin inhibited the release of soluble P-selectin into circulation, it did not mitigate TPO release into plasma. However, further studies in animals are needed to corroborate these findings in experimental sepsis or other models of coagulation. REFERENCES
1. Hou M, Andersson PO, Stockelberg D, Mellqvist UH, Ridell B, Wadenvik H. Plasma thrombopoietin levels in thrombocytopenic states: implication for a regulatory role of bone marrow megakaryocytes. Br J Haematol 1998;101:420-4. 2. Siemensma NP, Bathal PS, Penington DG. The effect of massive liver resection on platelet kinetics in the rat. J Lab Clin Med 1975;86:817-33. 3. de Sauvage FJ, Villeval JL, Shivdasani RA. Regulation of megakaryocytopoiesis and platelet production: lessons from animal models. J Lab Clin Med 1998;131:496-501. 4. Jackson CW. New insights into the regulation of megakaryocyte differentiation and platelet production derived from cloning of thrombopoietin and its receptor: surprises and unresolved questions as thrombopoietin proceeds through clinical trials. J Lab Clin Med 1998;131:480-4. 5. Burstein SA. Cytokines, platelet production and hemostasis. Platelets 1997;8:93-104. 6. Hollen CW, Henthorn J, Koziol JA, Burstein SA. Elevated serum interleukin-6 levels in patients with reactive thrombocytosis. Br J Haematol 1991;79:286-90. 7. Gastl G, Plante M, Finstad CL, et al. High IL-6 levels in ascitic fluid correlate with reactive thrombocytosis in patients with epithelial ovarian cancer. Br J Haematol 1993;83:43341. 8. Ishiguro A, Ishikita T, Shimbo T, et al. Elevation of serum thrombopoietin precedes thrombocytosis in Kawasaki disease. Thromb Haemost 1998;79:1096-100. 9. Cerutti A, Custodi P, Duranti M, Noris P, Balduini CL. Thrombopoietin levels in patients with primary and reactive thrombocytosis. Br J Haematol 1997;99:281-4. 10. Hsu HC, Tsai WH, Jiang ML, et al. Circulating levels of thrombopoietic and inflammatory cytokines in patients with clonal and reactive thrombocytosis. J Lab Clin Med 1999; 134:392-7. 11. Hiyoyama K, Wada H, Shimura T, et al. Increased serum levels of thrombopoietin in patients with thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura, or disseminated intravascular coagulation. Blood Coagul Fibrinolysis 1997;8:345-9.
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12. Folman CC, Linthorst GE, van MJ, et al. Platelets release thrombopoietin (Tpo) upon activation: another regulatory loop in thrombocytopoiesis? Thromb Haemost 2000;83:92330. 13. Stohlawetz P, Folman CC, von dem Borne AEGK, et al. Effects of endotoxemia on thrombopoiesis in men. Thromb Haemost 1999;81:613-7. 14. Martich GD, Boujoukos AJ, Suffredini AF. Response of man to endotoxin. Immunobiology 1993;187:403-16. 15. Suffredini AF, Hochstein HD, McMahon FG. Dose-related inflammatory effects of intravenous endotoxin in humans: evaluation of a new clinical lot of Escherichia coli O:113 endotoxin. J Infect Dis 1999;179:1278-82. 16. Folman CC, von dem Borne AE, Rensink IH, et al. Sensitive measurement of thrombopoietin by a monoclonal antibody based sandwich enzyme-linked immunosorbent assay. Thromb Haemost 1997;78:1262-7. 17. Blann AD, Lip GY. Hypothesis: is soluble P-selectin a new marker of platelet activation? Atherosclerosis 1997;128:1358. 18. Fijnheer R, Frijns CJ, Korteweg J, et al. The origin of Pselectin as a circulating plasma protein. Thromb Haemost 1997;77:1081-5. 19. Pernerstorfer T, Hollenstein U, Hansen JB, et al. Heparin blunts endotoxin-induced coagulation activation. Circulation 1999:2485-90. 20. Pernerstorfer T, Hollenstein U, Hansen JB, et al. Lepirudin blunts endotoxin-induced coagulation activation. Blood 2000;95:1729-34. 21. Porcelijn J, Folman CC, Bossers B, et al. The diagnostic value of thrombopoietin level measurements in thrombocytopenia. Thromb Haemost 1998;79:1101-5. 22. Stohlawetz P, Hergovich N, Stiegler G, et al. Differential induction of P-selectin by two cell separators during plateletpheresis and the effect of gender on the release of soluble Pselectin. Transfusion 1998;38:24-30. 23. Stohlawetz P, Horvath M, Pernerstorfer T, et al. Effects of nitric oxide on platelet activation during plateletpheresis and in vivo tracking of biotinylated platelets in humans. Transfusion 1999;39:506-14. 24. Stohlawetz P, Kapiotis S, Seidl D, et al. Safety issues of plateletpheresis: comparison of the effects of two cell separators on the activation of coagulation, fibrinolysis, neutrophils and on leukocyte-platelet aggregates. Transfusion 1999;39:420-7. 25. Suffredini AF, Reda D, Banks SM, Tropea M, Agosti JM, Miller R. Effects of recombinant dimeric TNF receptor on human inflammatory responses following intravenous endotoxin administration. J Immunol 1995;155:5038-45. 26. Jilma B, Dirnberger E, Loscher I, et al. Menstrual cycle-associated changes in blood levels of interleukin-6, α1 acid glycoprotein, and C-reactive protein. J Lab Clin Med 1997;130: 69-75. 27. Li J, Xia Y, Kuter DJ. Interaction of thrombopoietin with the platelet c-mpl receptor in plasma: binding, internalization, stability and pharmacokinetics. Br J Haematol 1999;106: 345-56. 28. Jilma B, Blann AD, Pernerstorfer T, et al. Regulation of adhesion molecules during human endotoxemia: no acute effects of aspirin. Am J Respir Crit Care Med 1999;159:857-63. 29. Jilma B, Eichler HG, Vondrovec B, et al. Effects of desmopressin on circulating P-selectin. Br J Haematol 1996;93: 432-6. 30. Stohlawetz PJ, Dzirlo L, Hergovich N, et al. Effects of
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erythropoietin on platelet reactivity and thrombopoiesis in humans. Blood 2000;95:2983-9. 31. Sakamaki F, Ishizaka A, Handa M, et al. Soluble form of Pselectin in plasma is elevated in acute lung injury. Am J Respir Crit Care Med 1995;151:1821-6. 32. Wolber EM, Jelkmann W. Interleukin-6 increases thrombopoietin production in human hepatoma cells HepG2 and Hep3B. J Interferon Cytokine Res 2000;20:499-506. 33. van der Poll T, Coyle SM, Levi M, et al. Effect of a recom-
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binant dimeric tumor necrosis factor receptor on inflammatory responses to intravenous endotoxin in normal humans. Blood 1997;89:3727-34. 34. Steinberg MH, Kelton JG, Coller BS. Plasma glycocalicin: an aid in the classification of thrombocytopenic disorders. N Engl J Med 1987;317:1037-42. 35. Williams SB, Sano M, Smith N, et al. Glycocalicin levels in the plasma of HIV+ patients: an indicator of platelet turnover. J Lab Clin Med 1998;132:303-7.