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Anesth Analg 2003;97:680-683 © 2003 International Anesthesia Research Society
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Abstract |
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Hydroxyethyl starch (HES) solutions impair platelet function by reducing the availability of the fibrinogen receptor. This effect is not mediated by intracellular signal transduction pathways. Also, an unspecific coating of platelets by HES macromolecules may be responsible for its antiplatelet effects. To test this hypothesis, we investigated the binding of fluorochrome-coupled HES to the surface of human platelets using whole blood flow cytometry. Citrated whole blood from 8 volunteers was incubated (5 min, 22°C, in the dark) with fluorescein isothiocyanate (FITC)-coupled HES (200-kDa molecular weight, 0.5 degree of substitution, 0.042 molar ratio of FITC-conjugation) resulting in 0%, 1%, 3%, 5%, 10%, 20%, and 40% hemodilution. The percentage of platelets binding FITC-HES was determined using a FACSCaliburTM flow cytometer and CellQuestProTM software. The percentage of FITC-positive platelets increased in a concentration-dependent manner reaching statistical significance at 10% hemodilution. Binding was independent of fibrinogen receptor blockade. The present experiments clearly demonstrate that extracellular binding of HES to the platelet surface is, at least in part, responsible for the antiplatelet effects of HES by blocking the access of ligands to the platelet fibrinogen receptor.
IMPLICATIONS: Hydroxyethyl starch solutions are widely used for fluid replacement in patients undergoing surgery but may compromise blood coagulation. The present study demonstrates that one of the mechanisms for this unwanted side effect is related to the binding of hydroxyethyl starch to the outer surface of blood platelets.
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Introduction |
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TopAbstractIntroductionMethodsDiscussionReferences |
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Hydroxyethyl starch (HES) solutions are widely used for intravascular volume expansion but may compromise platelet function as measured by platelet aggregometry (1,2), the Platelet Function Analyzer (3,4), thromboelastography (5), and flow cytometry (3,4). HES solutions reduce the availability of the functional receptor for fibrinogen on the platelet surface (glycoprotein IIb-IIIa) depending on structural characteristics of HES molecules, such as mean molecular weight and the degree of substitution (3,4). Recently, we have demonstrated that HES does not inhibit platelets by interfering with calcium-dependent intracellular signal transduction pathways (6). Previous authors hypothesized that coating of platelet surface structures by HES macromolecules may be responsible for its antiplatelet effects. Therefore, in the present study, we investigated the extracellular binding of HES to the platelet surface of individual cells by means of whole blood flow cytometry.
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Methods |
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TopAbstractIntroductionMethodsDiscussionReferences |
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Fluorescein Isothiocyanate (FITC)-Coupled HES
HES molecules with a mean molecular weight of 200 kDa and a 0.5% degree of substitution were conjugated with the fluorescent dye FITC resulting in 4.2% of HES glucose moieties coupled to FITC fluorochromes (kindly provided by Fresenius Kabi Austria, Linz, Austria). The FITC-coupled HES lyophilisate was dissolved in phosphate-buffered saline free of Ca2+, Mg2+, and NaHCO3 (Na2HPO4 1.15 g/L, NaCl 8 g/L, KH2PO4 0.2 g/L, KCl 0.2 g/L, pH 7.3; Dulbecco’s, Life Technologies, Paisley, UK) to achieve a concentration of 6% w/v.
Blood Samples
After IRB approval and informed consent, blood from 8 healthy adult male volunteers was withdrawn into VacuetteTM tubes (Greiner, Kremsmünster, Austria) containing 3.8% trisodium citrate (9:1 v/v) from an antecubital vein by venipuncture without stasis using a 21-gauge butterfly needle. The first 3 mL was always discarded. All participants denied taking any medication within the previous 14 days. Citrate was used as an anticoagulant because of its negligible intrinsic effects on platelets (7). All samples were processed within 3 min in polystyrene round-bottom tubes (5 mL) (FalconTM; Becton Dickinson, Franklin Lakes, NJ). Clinically relevant hemodilutions (1%, 3%, 5%, 10%, 20%, and 40% v/v) were achieved by incubating aliquots of citrated whole blood of each donor with appropriate volumes of FITC-coupled HES. As controls, additional aliquots were diluted with saline (Fresenius), and one aliquot remained undiluted. All incubations were performed at room temperature in the dark for 5 min.
Flow Cytometry
Thereafter, the blood samples were diluted (1:5) with phosphate-buffered saline to prevent contact between individual platelets, and were analyzed by flow cytometry using a FACSCaliburTM flow cytometer and CellQuest ProTM software (Becton Dickinson Immunocytometry Systems, San Jose, CA) immediately after incubation. A gate was set around the platelet population identified by forward and side scatter characteristics. Quantum fluorescence microbeads (Calibrite BeadsTM; Becton Dickinson Immunocytometry Systems) were used for the standardization of instrument settings on the day of experiment.
To prove the platelet nature of the gated events, additional samples were double-stained with FITC-coupled HES and saturating concentrations of a phycoerythrin-coupled antihuman platelet glycoprotein IIIa monoclonal antibody (anti-CD61; Diaclone Research, Besançon, France) reacting only with platelets. To examine the fibrinogen receptor glycoprotein IIb-IIIa as a potential binding site for HES, aliquots were incubated for 5 min with a receptor antagonist (abciximab antibody-fragment, ReoPro®; Centocor, Leiden, The Netherlands) at a final concentration of 150 µg/mL before a 20% hemodilution was achieved by the addition of HES (8).
Data were tested for normal distribution using the Kolmogorov-Smirnov test. The effect of in vitro hemodilution was assessed by using analysis of variance for repeated measures. Post hoc comparisons between undiluted controls and hemodilutions were made by a paired t-test. The level of significance was adjusted according to Bonferroni’s correction. Data were expressed as mean ± SD. P < 0.05 was considered statistically significant.
Results
The platelet population was identified by forward and side scatter characteristics in the whole blood flow cytometric assay (Fig. 1). The binding of the specific platelet marker anti-CD61 proved that the gated events binding FITC-coupled HES exclusively represented platelets.
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The percentage of FITC-positive platelets increased in a concentration-dependent manner and reached statistical significance at a 10% HES hemodilution when compared with the undiluted control (Fig. 2). Clinically relevant hemodilutions of 10%, 20%, and 40% resulted in 11% ± 1%, 46% ± 6%, and 78% ± 4% of platelets binding FITC-HES molecules, respectively.
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As shown in Figure 3, fibrinogen receptor blockade using the abciximab antibody-fragment had no significant effect on the percentage of FITC-positive platelets at a 20% hemodilution with FITC-coupled HES (46% ± 6% without abciximab antibody-fragment versus 48% ± 4% with abciximab antibody-fragment).
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Discussion |
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TopAbstractIntroductionMethodsDiscussionReferences |
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The present experiments prove, for the first time, that the repeatedly postulated binding of HES molecules to the platelet surface indeed occurs. The use of flow cytometry permitted us to visualize the binding of HES molecules to platelets in their physiologic milieu of whole blood. Moreover, the percentage of platelets binding fluorochrome-coupled HES increased in a concentration-dependent manner at clinically relevant degrees of hemodilution (Fig. 2).
HES solutions reduce the availability of the functional receptor for fibrinogen on the platelet surface (glycoprotein IIb-IIIa) depending on structural characteristics of HES molecules such as mean molecular weight and the degree of substitution (3,4). Interference of HES with calcium-dependent intracellular signal transduction pathways has recently been excluded as a mechanism of HES-induced platelet dysfunction (6): Using Fluo-3 as the calcium-sensitive probe for flow cytometric analysis, the agonist-induced increase in the cytoplasmic calcium concentration of platelets was similar in the presence of different HES solutions at various degrees of hemodilution when compared with saline and undiluted control samples. The exclusion of a signal-transduction-related mechanism together with the present results indicate that the known inhibiting effect of HES on platelet function is caused by the interference of HES with extracellular sites. HES macromolecules may thus impair the access of ligands to their binding sites on the platelet surface or may inhibit the conformational activation of platelet receptors upon subsequent stimulation. The access to activated glycoprotein IIb-IIIa is especially reduced in the presence of HES (3,4). Platelet aggregometry, bleeding times, Platelet Function Analyzer-closure times, as well as thromboelastographic variables assessing platelet function are all dependent on the interaction of platelet membrane glycoprotein IIb-IIIa receptors and glycoprotein Ib with their ligands such as fibrinogen and von Willebrand factor (9,10). As shown by the binding of fluorochrome-coupled HES to platelets preincubated with the glycoprotein IIb-IIIa receptor antagonist abciximab antibody-fragment, HES binding could not be prevented by a receptor blockade (Fig. 3). These results suggest a binding mechanism that is not mediated by the specific fibrinogen receptor binding site. Further investigations, however, are needed to elucidate the exact mechanism(s) of HES binding.
HES solutions are widely used for hemodilution and plasma volume expansion. Despite its hemodynamic and rheological benefits, unwanted side effects of HES on blood coagulation are well documented. HES solutions compromise the plasmatic coagulation cascade (11) as well as platelet function (1–5). Plasmatic abnormalities, such as a decrease in von Willebrand factor (12,13), fibrinogen levels (14), and thrombin generation (15), may contribute to a decreased platelet responsiveness after HES administration. The present investigation extends the list of potential mechanisms of HES-induced platelet dysfunctions: HES macromolecules bind to human platelets and thus impair the access of ligands to the platelet fibrinogen receptor.
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Acknowledgments |
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This work was supported by the Department of Anesthesiology and General Intensive Care, University of Vienna, Vienna, Austria and in part by Österreichische Nationalbank Jubiläumsfonds No. 9583.
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References |
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- Türkan H, Ural A, Beyan C, et al. Effects of hydroxyethyl starch on blood coagulation profile. Eur J Anaesth 1999; 16: 156–9.[Web of Science][Medline]
- Stögermüller B, Stark J, Willschke H, et al. The effects of hydroxyethyl starch 200 kD on platelet function. Anesth Analg 2000; 91: 823–7.
[Abstract/Free Full Text] - Franz A, Bräunlich P, Gamsjäger T, et al. The effects of hydroxyethyl starches of varying molecular weight on platelet function. Anesth Analg 2001; 92: 1402–7.
[Abstract/Free Full Text] - Felfernig M, Franz A, Braeunlich P, et al. The effects of hydroxyethyl starch solutions on thromboelastography in preoperative male patients. Acta Anaesth Scand 2003; 47: 70–3.[Web of Science][Medline]
- Gamsjäger T, Gustorff B, Kozek-Langenecker S. Effects of hydroxyethyl starches on intracellular calcium in platelets. Anesth Analg 2002; 95: 866–9.
[Abstract/Free Full Text] - Golanski J, Pietrucha T, Baj Z, et al. Molecular insight into anticoagulant-induced spontaneous activation of platelets in whole blood: various anticoagulants are not equal. Thromb Res 1996; 83: 199–215.[Web of Science][Medline]
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[Free Full Text] - Kundu S, Heilmann E, Sio R, et al. Description of an in vitro platelet function analyzer—PFA-100. Semin Thromb Hemost 1995; 21: 106–12.[Web of Science][Medline]
- Jamnicki M, Bombeli T, Seifert B, et al. Low- and medium-molecular-weight hydroxyethyl starches: comparison of their effects on blood coagulation. Anesthesiology 2000; 93: 1231–7.[Web of Science][Medline]
- Conroy J, Fishman R, Reeves S, et al. The effects of desmopressin and 6% hydroxyethyl starch on factor VIII:C. Anesth Analg 1996; 83: 804–7.[Abstract]
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[Abstract/Free Full Text] - Engelmann L, Pilz U, Gundelach K, et al. Akute elementare Effekte der kolloidalen Plasmaersatzmittel Infukoll HES 10%, Gelafusal-N und Infukoll M40 (Dextran) bei Patienten mit Sepsis. J Anaesth Intensivmed 1997; 3: 39–46.
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