In orthotopic liver transplantation bleeding problems are well-recognized and prevalent early post-operative complications. Thrombocytopaenia is found in about 30-64% of cirrhotic patients . However, the contribution of thrombocytopaenia to bleeding is unclear . Massive blood loss results in hypovolaemia requiring the transfusion of blood components, and crystalloids or hydroxyethyl starch (HES) for volume substitution. HES solutions are widely used for plasma volume expansion, although they compromise coagulation , and especially platelet function both in in vitro experiments  and after in vivo infusion [5-7]. HES of varying molecular weights inhibit platelet function by reducing the availability of glycoprotein (GP) IIb/IIIa (CD41), which is the functional receptor for fibrinogen on the platelet surface . Platelet activation leads to an increase in the number of GPIIb/IIIa complexes expressed on the platelet surface, and transforms GPIIb/IIIa complexes to a state that is able to bind to fibrinogen or von Willebrand factor , which is the prerequisite for platelet aggregation.
Like GPIIb/IIIa, the adhesion molecule P-selectin (CD62P), which is synthesized by megakaryocytes and incorporated in the platelet α-granules, plays a central role in platelet activation. After stimulation of platelets, P-selectin is rapidly transported to the cell surface during fusion of the α-granule membrane with the plasma membrane. P-selectin is involved in the adhesion of activated platelets to endothelium, leucocytes, monocytes and granulocytes. Findings from previous studies have shown that platelet activation is a potent stimulus for platelet-neutrophil complex formation and neutrophil function [10,11]. The complex formation is mediated by platelet P-selectin and leucocyte β2 integrins, and plays a prominent role in haemostasis and inflammation [12,13].
The present prospective study was performed to assess the availability of platelet GPIIb/IIIa following infusion of HES in the case of thrombocytopaenia associated with liver transplantation. Also we evaluated the P-selectin expression and the effects on the aggregation of platelets to leucocytes.
After written informed consent from our local Ethics Committee, 12 patients (7 male, mean age 47 ± 14.8 yr) were included in the study. All patients underwent full size orthotopic liver transplantation for end-stage liver disease (viral cirrhosis) and were on days 3-5 of postoperative thrombocytopaenia after transplantation (platelet count 50-70 GL−1). All patients received a bolus infusion of HES 6% 10 mL kg−1, of medium molecular weight (200 kDa/0.5; Hemohes, Braun, Germany), over a period of 30 min.
The immunosuppression protocol consists of bolus methylprednisolone intraoperatively, which was gradually tapered down in the following days. Furthermore, interleukin 2 (IL-2) receptor antibody (Basiliximab; Novartis, Basel, Switzerland) was given 6 h postoperatively, and at the postoperative day 4; and cyclosporin (Novartis) was administered intravenously (i.v.) starting on postoperative day 1. In cases of hepatitis C, only one single dose of corticosteroids was given intraoperatively, and mycophenolate mofetil (MMF; Roche, Grenzach-Wyhlen, Germany) was added on postoperative day 1.
The activation of platelets was evaluated by measuring the GPIIb/IIIa and P-selectin expression on the cell surface under unstimulated and thrombin receptor activating peptide-6 (TRAP-6; Bachem, Heidelberg, Germany) or adenosine diphosphate (Sigma Chemicals, Deisenhofen, Germany) activated conditions. Platelets were identified using a phycoerythin labelled monoclonal antibody against GPIIb/IIIa (CD41, clone P2; Beckman-Coulter, Krefeld, Germany). P-selectin expression was analysed using a fluorescein isothiocyanate labelled monoclonal antibody against P-selectin (CD62P, clone CLB/Thromb/6; Beckman-Coulter). Effects on the aggregation of platelets to leucocytes (CD45+/CD41+) were also evaluated under baseline and TRAP-6 stimulated conditions. The main leucocyte subpopulations were detected and gated according to the specific forward and side scatter signals of lymphocytes, monocytes and granulocytes.
Briefly, venous blood samples (3 mL) were collected in sodium citrate disposable blood sampling tubes (0.3 mL, 0.106 mol L−1, Monovette®; Sarstedt, Nümbrecht, Germany) before, 20 and 120 min after the termination of HES infusion. Sodium citrate was chosen, because other anticoagulants are known to alter the physiological behaviour of platelets . To avoid ex vivo platelet activation, blood samples were processed within 45 min after drawing . All samples were taken, processed and measured by the same investigator. To minimize the formation of platelet aggregates, the platelet concentration was adjusted to 20 000 μL−1 with 37°C prewarmed phosphate-buffered saline (pH 7.2, without Ca2+ and MgCl2, GIBCO BRL, Eggenstein, Germany) and bovine serum albumin 0.1% (Boehringer, Mannheim, Germany). The first tube was used for isotype controls, and to evaluate cellular autofluorescence. For platelet identification and GPIIb/IIIa activation, the other tubes were stained with 6 μL anti-GPIIb/IIIa phycoerythin conjugated monoclonal antibody. For detection of platelet activation and platelet-leucocyte complexes under basal conditions, 6 μL fluorescein isothiocyanate labelled monoclonal antibody against P-selectin or staining leucocytes (CD45, clone KC56; Beckman-Coulter), respectively, was added. Samples were incubated at 37°C for 10 min in the dark. To evaluate platelet reactivity, tubes were activated with 4 μL (100 μmol) TRAP-6 or 10 μmol adenosine diphosphate for 10 min at 37°C before staining with the monoclonal antibodies. After addition of phosphate-buffered saline 2 mL with 1% bovine serum albumin, transferring the samples onto ice stopped the activation. For each sample a minimum of 25 000 platelets were analysed.
Since numeric data showed a Gaussian distribution (Kolmogorov-Smirnov test) and skewness was |γ| ± 0.4, the paired two-tailed t-test was used for statistical analysis of the flow cytometric data (SPSS/PC® V 10.0 software package; SPSS, Munich, Germany). When these conditions were not met, the Wilcoxon signed rank sum test was used to compare the intragroup data. Results are expressed as mean and standard error of mean (mean ± SEM %), and P ≤ 0.05 was considered significant.
In all patients the median platelet count was 57 GL−1 with a range of 52-69 GL−1 before infusion of medium molecular weight HES.
Under TRAP-6 or adenosine diphosphate stimulation, the P-selectin expression showed a transient enhancement after 20 min and decreased 120 min after HES infusion. However, only by the response to TRAP-6 activation the expression of P-selectin decreased significantly to 89.1 ± 4.2% (P = 0.029, 120 min vs. before; P = 0.046, 120 min vs. 20 min). Following adenosine diphosphate stimulation there were no significant differences (P = 0.072, 120 min vs. before; P = 0.077, 120 min vs. 20 min) (Fig. 1).
Both without stimulation and under TRAP-6 or adenosine diphosphate stimulation the GPIIb/IIIa expression decreased in the first 20 min and remained at this level in the observed period. However, this effect was not shown to be significant (Fig. 2).
Next the percentages of CD 45+/CD 41+ monocytes and granulocytes complexes as a measure for platelet aggregation to the leucocyte subpopulations were also evaluated under TRAP-6 stimulated conditions. Compared to pre-infusion values the percentages of monocyte-platelet complexes decreased significantly after 20 min (81.1 ± 7.8%; P = 0.047) and 2 h (73.3 ± 4.1%; P = 0.001). In contrast, granulocytes-platelet complexes decreased significantly in the first 20 min (85.2 ± 2.9%; P = 0.001) after which they started to rise to the initial values (Fig. 3).
Our study deals with the effect of HES solution on platelet receptor expression in patients after full size orthotopic liver transplantation. HES solutions are highly effective volume expanders and are used for peri- and postoperative volume replacement. Yet, HES has been reported to compromise haemostasis by decreasing platelet count due to dilution, interference with plasmatic coagulation components and by direct platelet inhibiting effects [4,5,16-18]. However, the platelet dysfunction induced by HES solutions is mainly observed with volumes greater than 30 mL kg−1 day−1 and with the high molecular weight HES. Bleeding tendency after liver transplantation remains a critical point despite refined surgical skills and improved peri- and postoperative management. Especially following liver transplantation, patients with progressive liver cirrhosis are known to have an impaired coagulation system with cellular and plasmatic dysfunction. Diminished platelet aggregation, reduced adhesiveness and impaired procoagulative properties of platelets are well described and are strictly related to the severity of cirrhosis [19-22]. For our investigation we chose postoperative days 3-5, because a previous study showed that platelet counts decreased in patients during liver transplantation, and remained lower than before liver transplantation for the next 6 days .
Our data demonstrated a slight but significant change of P-selectin expression following administration of HES (Fig. 1). Corresponding to these results, a significant reduction in the formation of platelet-monocyte aggregates, and an n.s. reduction in the formation of platelet-neutrophil complexes were observed. P-selectin is involved in the adhesion of activated platelets to endothelium, monocytes and granulocytes. For this reason, the observed reduction of leucocyte-platelet complexes is likely to be caused by the decreased P-selectin expression. Kozeck-Langenecker's working group observed no significant change in P-selectin expression immediately after the infusion of HES (200 kDa/0.6) in patients with normal platelet counts undergoing minor elective surgery [4,7]. The difference in our results may thus be explained by the different points of time for P-selectin expression analysis, the underlying diseases or the platelet counts of patients. The latter explanations are supported by the study of Laffi and colleagues , who reported the levels of platelet P-selectin expression after ex vivo stimulation to be significantly lower in liver cirrhosis compared to healthy controls. Since P-selectin is responsible for the formation of platelet-leucocyte aggregates, and for the adhesion to sinusoidal endothelial cells after graft reperfusion [24,25], our data suggest that the effect of HES infusion thus may have a beneficial effect on microvascular graft perfusion in transplantation.
With regard to GPIIb/IIIa we observed no significant difference in the expression before and after administration of HES. By contrast, previous studies [5,8] described a reduced GPIIb/IIIa expression after 6% HES 200 kDa/0.6 infusion. In the former study , an unspecific coating effect of HES 200 kDa/0.6 was excluded because platelet membrane expression of GPIb was unaltered. In our study, too, an unspecific coating effect was rather unlikely since, following TRAP-6 stimulation, there was no significant difference in GPIIb/IIIa expression before and after HES infusion. Therefore, we conclude that HES reduces P-selectin expression or receptor availability at the platelet surface by more 'specific' mechanisms, e.g. conformational changes.
There are some possible explanations for these discrepancies with the results of the previous studies: (a) in our study all patients were thrombocytopaenic and received orthotopic liver transplantation because of viral cirrhosis and, whereas in the other studies, all patients underwent minor surgery without having known platelet disorders. Diminished platelet aggregation, reduced adhesiveness and impaired procoagulative properties of platelets are well described and are strictly related to the severity of cirrhosis [19-22]; (b) previous studies [5,8] assessed the expression of GPIIb/IIIa by the binding of a monoclonal antibody (PAC-1®; Becton Dickinson, San Jose, CA, USA), which specifically recognizes activation transformed, but not unactivated platelet GPIIb/IIIa complexes. In contrast to PAC-1®, the antiCD41 monoclonal antibody (clone P2) employed in our study reacts both with unactivated and activated GPIIb in the intact complex with GPIIIa. For this reason, it cannot be excluded, that HES leads to a reduction of the availability of only the activated GPIIb on the platelet surface, which could not be detected with the antibody employed in our study. This reduction of availability of activated GPIIb/IIIa might be caused by a modification of the platelet cytoplasmatic membrane structure by HES 200 kDa, resulting in conformational activation of the GPIIb/IIIa complex.
Taken together, our data demonstrate that infusion of HES 200 kDa/0.5 in clinically relevant doses does not alter GPIIb/IIIa expression in thrombocytopaenic patients with pre-existing platelet dysfunction after orthotopic liver transplantation. Accordingly, HES infusion may have a beneficial effect on microvascular graft perfusion through the resulting haemodilution and reduced P-selectin expression with subsequent reduced leucocyte-platelet complexes and endothelial adhesion.
The excellent technical assistance of Birgitt Haarmeijer is gratefully acknowledged.
1. Bashour FN, Teran JC, Mullen KD. Prevalence of peripheral blood cytopenias (hypersplenism) in patients with nonalcoholic chronic liver disease. Am J Gastroenterol
2. Ozier Y, Steib A, Ickx B, et al.
Haemostatic disorders during liver transplantation
. Eur J Anaesthesiol
3. Treib J, Baron JF, Grauer MT, Strauss RG. An international view of hydroxyethyl starches. Intens Care Med
4. Jamnicki M, Bombeli T, Seifert B, et al.
Low- and medium-molecular-weight hydroxyethyl starches: comparison of their effect on blood coagulation. Anesthesiology
5. Stögermüller B, Stark J, Willschke H, Felfernig M, Hoerauf K, Kozek-Langenecker SA. The effect of hydroxyethyl starch 200 kD on platelet function. Anesth Analg
6. Boldt J, Knothe C, Zickmann B, Andres P, Dapper F, Hempelmann G. Influence of different intravascular volume therapies on platelet function in patients undergoing cardiopulmonary bypass. Anesth Analg
7. Turkan H, Ural AU, Beyan C, Yalcin A. Effects of hydroxyethyl starch on blood coagulation profile. Eur J Anaesthesiol
8. Franz A, Braunlich P, Gamsjager T, Felfernig M, Gustorff B, Kozek-Langenecker SA. The effects of hydroxyethyl starches of varying molecular weights on platelet function. Anesth Analg
9. Liu YK, Nemoto A, Feng Y, Uemura T. The binding ability to matrix proteins and the inhibitory effects on cell adhesion of synthetic peptides derived from a conserved sequence of integrins. J Biochem
10. Peters MJ, Heyderman RS, Hatch DJ, Klein NJ. Investigation of platelet-neutrophil interactions in whole blood by flow cytometry. J Immunol Meth
11. Peters MJ, Dixon G, Kotowicz KT, Hatch DJ, Heyderman RS, Klein NJ. Circulating platelet-neutrophil complexes represent a subpopulation of activated neutrophils primed for adhesion, phagocytosis and intracellular killing. Br J Haematol
12. Gawaz M, Dickfeld T, Bogner C, Fateh-Moghadam S, Neumann FJ. Platelet function in septic multiple organ dysfunction syndrome. Intensive Care Med
13. Evangelista V, Manarini S, Rotondo S, et al.
Platelet/polymorphonuclear leucocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18. Blood
14. Golanski J, Pietrucha T, Baj Z, Greger J, Watala C. Molecular insights into anticoagulant-induced spontaneous activation of platelets in whole blood: various anticoagulants are not equal. Thromb Res
15. Shattil SJ, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood
16. Stump DC, Strauss RG, Henriksen RA, Petersen RE, Saunders R. Effects of hydroxyethyl starch on blood coagulation, particularly factor VIII. Transfusion
17. Blaicher AM, Reiter WJ, Blaicher W, et al.
The effect of hydroxyethyl starch on platelet aggregation in vitro. Anesth Analg
18. de Jonge E, Levi M, Buller HR, Berends F, Kesecioglu J. Decreased circulating levels of von Willebrand factor after intravenous administration of a rapidly degradable hydroxyethyl starch (HES 200/0.5/6) in healthy human subjects. Intensive Care Med
19. Aoki Y, Hirai K, Tanikawa K. Mechanism of thrombocytopenia in liver cirrhosis: kinetics of indium-111 tropolone labelled platelets. Eur J Nucl Med
20. Peck-Radosavljevic M, Wichlas M, Zacherl J, et al.
Thrombopoietin induces rapid resolution of thrombocytopenia after orthotopic liver transplantation
through increased platelet production. Blood
21. Martin 3rd TG, Somberg KA, Meng YG, et al.
Thrombopoietin levels in patients with cirrhosis before and after orthotopic liver transplantation
. Ann Intern Med
22. Escolar G, Cases A, Vinas M, et al.
Evaluation of acquired platelet dysfunctions in uremic and cirrhotic patients using the platelet function analyzer (PFA-100): influence of hematocrit elevation. Haematologica
23. Laffi G, Cinotti S, Filimberti E, et al.
Defective aggregation in cirrhosis is independent of in vivo
platelet activation. J Hepatol
24. Upadhya GA, Strasberg SM. Platelet adherence to isolated rat hepatic sinusoidal endothelial cells after cold preservation. Transplantation
25. Yadav SS, Howell DN, Steeber DA, Harland RC, Tedder TF, Clavien PA. P-selectin mediates reperfusion injury through neutrophil and platelet sequestration in the warm ischemic mouse liver. Hepatology