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Anesth Analg 2002;95:866-869 © 2002 International Anesthesia Research Society
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Abstract |
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Hydroxyethyl starch (HES) solutions impair platelet function. To determine whether this effect is achieved through interference of HES with intracellular activation processes, in which calcium is the key second messenger, we evaluated the agonist-induced increase of the cytoplasmic calcium concentration in the presence of HES of different molecular weights. Aliquots of citrated whole blood of 12 volunteers were incubated in vitr. with saline, HES 450 (molecular weight in kilodalton), HES 130, and HES 70, resulting in 20% hemodilution. An undiluted sample served as control. The samples were stained with Fluo-3 as the calcium-sensitive fluorescent probe with subsequent flow cytometric analysis. After determination of a baseline, platelets were activated with thrombin receptor activator peptide 6. Platelet activation with thrombin receptor activator peptide 6 resulted in a fast increase in fluorescence (approximately eightfold), representing intracellular calcium mobilization. None of the tested HES solutions exerted a statistically significant effect on the cytoplasmic calcium concentration compared with samples that were incubated with saline or that remained undiluted. These results indicate that the known inhibiting effect of HES on platelets is not achieved through interference with intracellular activation processes.
IMPLICATIONS: Hydroxyethyl starch does not exert its known inhibitory effect on platelet function by interfering with intracellular activation processes.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Hydroxyethyl starch (HES) solutions are among the substances most widely used for perioperative volume replacement and, in addition, are used to improve the rheologic properties of blood under conditions of impaired arterial perfusion (1,2). However, HES has been reported to compromise overall platelet function as measured by platelet aggregometry (3,4) and by using the Platelet Function Analyzer (PFA-100TM; Dade-Behring, Miami, FL) (5,6). Recent investigations were able to specify that certain HES solutions (depending on the molecular weight of the starch, or possibly on the degree of substitution) reduce the availability of activated fibrinogen binding sites (glycoprotein IIb–IIIa) on the platelet surface (5,6). However, it is unclear whether this effect is caused by interference of HES with intracellular activation processes leading to reduced platelet reactivity.
Therefore, we tested the potential mechanism that HES interferes negatively with intracellular activation processes, in which calcium is the key second messenger (7). We measured the agonist-induced increase of the cytoplasmic calcium concentration after in vitr. incubation with different HES solutions, with Fluo-3 as the calcium sensitive probe. This substance can be loaded into living cells and, upon contact with free calcium, exhibits greatly increased fluorescence, which can be measured by flow cytometry (8).
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Methods |
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Blood samples were obtained from 12 healthy adult volunteers. All participants gave written informed consent and denied taking any medication within the previous 14 days. The study was approved by the IRB.
Blood was drawn by venipuncture from an antecubital vein after applying only minimal stasis by using a 21-gauge butterfly needle. After the first 2 mL of blood was discarded, 4 mL was collected into VacuetteTM tubes (Greiner, Kremsmünster, Austria) containing 3.8% sodium citrate (9:1 vol/vol). The following preparative steps were performed in polystyrene round-bottom tubes (5 mL) (FalconTM; Becton Dickinson, Franklin Lakes, NJ).
Clinically relevant hemodilutions of 20% were achieved by incubating 3 aliquots of citrated whole blood of each participant with appropriate volumes of HES 450/0.7–0.8 (mean molecular weight in kilodalton/degree of substitution; PlasmasterilTM 6%), HES 130/0.38–0.45 (VoluvenTM 6%), and HES 70/0.5–0.55 (ExpafusinTM 6%), respectively, all of which were obtained from Fresenius Pharma Austria (Graz, Austria). To serve as controls, 1 additional aliquot was incubated with saline (Fresenius) and 1 aliquot remained undiluted. Incubations took place at room temperature for 5 min.
To clarify whether platelet function might be affected by HES only at higher degrees of hemodilution, aliquots of citrated whole blood of 5 participants were diluted with 20%, 40%, and 60% of HES 450/0.7–0.8, which, among the different HES preparations, has the greatest impact on platelets (6). An undiluted aliquot and appropriate dilutions with saline served as controls.
The calcium-sensitive probe Fluo-3 (Molecular Probes, Eugene, OR) was dissolved in the detergent PluronicTM (Molecular Probes; 10% in dimethyl sulfoxide) at a stock concentration of 1 mmol/L. The stock solution was stored at -20°C until the experiments were performed. Immediately before use, appropriate aliquots of Fluo-3 were diluted 1:10 in phosphate-buffered saline (PBS) (Dulbecco’s, without calcium, magnesium, and sodium bicarbonate; Life Technologies, Paisley, UK) to facilitate the handling of the otherwise very small volumes.
Aliquots of the HES-incubated blood were diluted 1:10 with PBS to minimize cellular interaction during the following procedures (9). They were incubated with Fluo-3 at a final concentration of 5 µmol/L for 15 min at 37°C (8). After the incubation, the Fluo-3-loaded blood samples were diluted 1:15 in PBS to achieve a degree of dilution appropriate for flow cytometric analysis. In each experiment, an additional sample of Fluo-3-loaded blood of the undiluted control was prepared in PBS for the instrument setup.
To determine the increase of the cytoplasmic free calcium concentration upon stimulation, all measurements were performed on a FACSCaliburTM flow cytometer by using CellQuest 3.1TM software (Becton Dickinson Immunocytometry Systems, San Jose, CA). In the instrument setup, the amplification of the detector suitable for Fluo-3 fluorescence (FL1) was set to give a baseline value of approximately 2 arbitrary units. A gate was set around the platelet population, which was identified by forward and side scatter characteristics. The fluorescence analysis was performed on the basis of this gate by using a dotplot with time on the x-axis and Fluo-3 fluorescence on the y-axis. Fifteen consecutive regions in this dotplot allowed the time-dependent interpretation of the data (Fig. 1).
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After determination of a baseline for approximately 10 s, the acquisition was briefly paused for the addition of the platelet agonist thrombin receptor activator peptide 6 (TRAP-6) (SFLLRN; Bachem, Bubendorf, Switzerland) at a final concentration of 30 µmol/L and immediately resumed afterward (manipulation time approximately 10 s). Data were acquired for a further 40 s.
Because the use of Fluo-3 is an approach to the evaluation of platelet function in whole blood that was developed only recently (8), proof that this method is able to detect decreased platelet reactivity seemed to be necessary. This was achieved by incubating Fluo-3-loaded samples of 9 of the 12 subjects (preparation as described above) with the platelet inhibitor prostaglandin (PG) E1 (MinprogTM, diluted in saline; Pharmacia & Upjohn, Vienna, Austria) at a final concentration of 450 ng/mL for 1 min immediately before the baseline reading with subsequent TRAP-6 activation.
Data were expressed as means ± SD. The increase from baseline to maximal Fluo-3 fluorescence was calculated as the quotient between these two values. Data were tested for normal distribution by using the Kolmogorov-Smirnov test. Analysis of variance was used to assess the differences in the Fluo-3 fluorescence increase between the groups. Multiple comparisons were calculated with Tukey’s post hoc test. A P value < 0.05 was considered statistically significant.
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Results |
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Activation of the samples with TRAP-6 at a final concentration of 30 µmol/L resulted in a marked increase in fluorescence intensity (approximately eightfold), corresponding to an increase in the cytoplasmic free calcium concentration. Maximal values were reached within seconds after the addition of the platelet activator, followed by a progressive decrease of fluorescence until the end of the measurement (Fig. 1). Incubation of samples of 9 of the 12 subjects with PGE1 immediately before the measurements reduced the increase in fluorescence upon stimulation by approximately 65% (P < 0.05, Fig. 2), indicating that the method used in this investigation is able to detect decreased platelet reactivity.
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Twenty percent dilution of citrated whole blood of 12 subjects with HES solutions of different compositions (HES 450/0.7–0.8, HES 130/0.38–0.45, and HES 70/0.5–0.55) had no significant effect on the intracellular calcium concentration compared with the dilution with saline and the undiluted control sample (Fig. 2).
Serial dilutions of citrated whole blood with 20%, 40%, and 60% of HES 450/0.7–0.8 and saline, respectively, revealed no differences at the same degrees of dilution, although there was an overall tendency to a reduced fluorescence increase at higher degrees of dilution, reaching statistical significance with 60% saline when compared with the undiluted control (P < 0.05, Fig. 3).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the current study, we examined the influence of different types of HES (HES 450/0.7–0.8, HES 130/0.38–0.45, and HES 70/0.50–0.55) on platelet activation in vitr. by assessing the level of intracellular calcium, which acts as a key second messenger in intracellular signal transduction (7). Clinically relevant hemodilutions of 20% had no effect on intracellular calcium (Fig. 2). In the subset of experiments using serial dilutions of 20%, 40%, and 60% with HES 450/0.7–0.8 and saline, respectively, a tendency to reduced fluorescence increases occurred at higher degrees of dilution with a statistically significant difference only between 60% saline and the undiluted control. No differences between HES 450 and saline at the same degrees of dilution were observed, indicating an unspecific dilutional effect (Fig. 3).
These results demonstrate that the known inhibiting effect of HES on platelet function, which has been established using platelet aggregometry (3,4) and the Platelet Function Analyzer (5,6), is not caused by interference with calcium-dependent intracellular activation processes. Interference of HES with signal transduction pathways working independent from calcium cannot be excluded; however, such mechanisms have only a minor role in platelet activation (7).
The exclusion of an intracellular pathomechanism of HES-induced platelet dysfunction together with our previous observation that HES impaired the availability of platelet fibrinogen receptors (5,6) suggest an extracellular mechanism. HES molecules may coat the platelets, leading to limited access of ligands to their binding sites on the platelet surface. Because the exogenous stimulus of TRAP-6 (interacting with the thrombin receptor) readily induced platelet activation in all groups, the putative coating apparently does not affect the receptors responsible for transmitting the activation signal to the interior of the cells. Similarly, HES had no effect on the expression of P-selectin (CD62P) (5,6), which is transferred to the platelet surface through the activation-dependent secretion of 
-granules, suggesting undisturbed intracellular signal transduction. To obtain insight into the interaction of HES molecules with platelet surface structures, further experimental studies are warranted.
The flow cytometric measurement of Fluo-3 fluorescence is a novel approach for evaluating platelet function in whole blood (8). In a set of experiments with PGE1, we proved that this method is able to detect pharmacologic inhibition of platelet activation (Fig. 2). PGE1 is a potent inhibitor of platelet activation acting via an increase in intracellular adenosine 3',5'-cyclic monophosphate levels and is clinically used in the antithrombotic (antiplatelet) strategy during extracorporeal circulation (10). As expected, incubation of samples with PGE1 diminished the increase in intracellular calcium significantly.
A limitation of this investigation is that contact between platelets and HES took place only in vitro; thus, initial metabolization steps after administration in vivo, which might modify the properties of HES with regard to its influence on platelets, were eliminated. In addition, the preparative procedures required considerable dilution of the samples for flow cytometric analysis. However, inhibiting effects of HES on platelets in vitr. have been confirmed in a previous study and are compatible with in vivo findings (5).
In contrast to another investigation using Fluo-3 (8), we used TRAP-6 instead of adenosine diphosphate for platelet activation. Pilot studies revealed that adenosine diphosphate induces a considerably faster increase in the intracellular calcium concentration. Maximal fluorescence values were repeatedly reached within the time necessary to resume data acquisition, potentially leading to erroneous data because it was not possible to standardize exactly the manipulation time of the samples.
In conclusion, this investigation indicates that the known inhibiting effect of HES on platelets is not achieved through interference with intracellular activation processes. These findings support the notion that HES impairs platelet reactivity only by interacting with surface structures of the platelets, especially the (activated) fibrinogen receptor, but further investigations are necessary to elucidate the exact mechanisms.
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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.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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- Warren BB, Durieux ME. Hydroxyethyl starch: safe or not? Anesth Analg 1997; 84: 206–12.[Web of Science][Medline]
- Treib J, Haass A, Pindur G. Coagulation disorders caused by hydroxyethyl starch. Thromb Haemost 1997; 78: 974–83.[Web of Science][Medline]
- Boldt J, Knothe C, Zickmann B, et al. Influence of different intravascular volume therapies on platelet function in patients undergoing cardiopulmonary bypass. Anesth Analg 1993; 76: 1185–90.[Abstract]
- Türkan H, Ural AU, Beyan C, Yalcin A. Effects of hydroxyethyl starch on blood coagulation profile. Eur J Anaesthesiol 1999; 16: 156–9.[Web of Science][Medline]
- Stögermüller B, Stark J, Willschke H, et al. The effect 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 weights on platelet function. Anesth Analg 2001; 92: 1402–7.
[Abstract/Free Full Text] - Sargeant P, Sage SO. Calcium signalling in platelets and other nonexcitable cells. Pharmacol Ther 1994; 64: 395–443.[Medline]
- do-Ceu-Monteiro M, Sansonetty F, Goncalves MJ, O’Connor JE. Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo-3 and CD41. Cytometry 1999; 35: 302–10.[Web of Science][Medline]
- Schmitz G, Rothe G, Ruf A, et al. European working group on clinical cell analysis: consensus protocol for the flow cytometric characterisation of platelet function. Thromb Haemost 1998; 79: 885–96.[Web of Science][Medline]
- Kozek-Langenecker SA. Anticoagulation with prostaglandins during extracorporeal circulation. Wien Klin Wochenschr 1999; 111: 129–40.[Medline]
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