Hydroxyethyl starch (HES) solutions are widely used for intravascular volume expansion, but they may compromise platelet function, as measured by platelet aggregometry (1), the Platelet Function Analyzer (PFA) (2,3), thromboelastography (4), and flow cytometry (2,3) both in in vitro experiments (2,5) and in vivo after infusion (1,2). Structural characteristics of HES molecules modulate their influence on platelets (6,7). We observed that high molecular weight HES 450 kd, middle molecular weight HES 200 kd, and low molecular weight HES 70 kd impaired platelet function, whereas low molecular weight HES 130 kd had no significant effect (3). Similarly, Konrad et al. (8) documented that HES 70 kd affected clot retraction in the Sonoclot Analyzer (Sienco Inc, Wheat Ridge, CO) more than HES 130 kd had. Together, these findings indicate that not only the molecular weight, but also the degree of substitution of HES molecules that varies considerably between various HES solutions determines their effects on platelet function.
Recently, a novel high molecular weight HES preparation (550 kd) with a high degree of substitution (0.7) has become commercially available in the United States (Hextend®, Biotime®-Inc, Berkeley, CA). Animal and human studies documented beneficial effects of Hextend® in volume resuscitation (9–12). We hypothesized that the antiplatelet effect of Hextend® is more pronounced than any other HES preparation tested because of the unfavorable structural characteristics with a high molecular weight and a high degree of substitution. In the present study, whole blood flow cytometry was used to evaluate platelet surface-receptor expression on individual cells after in vitro hemodilution of whole blood with Hextend®, high molecular weight HES 600 kd (6%Hetastarch-Baxter®, Deerfield, IL), and medium molecular weight HES 200 kd (Elohäst® 6%, Fresenius Kabi Austria GmbH, Graz, Austria). Because we gained surprising results in pilot experiments, we further investigated the effect of the solvent of HES in Hextend®.
After IRB approval and informed consent, blood from 7 healthy adult male volunteers was withdrawn into Vacuette™ tubes (Greiner, Kremsmünster, Austria) containing 3.8% trisodium citrate (9:1 vol/vol) from an antecubital vein by venipuncture without stasis using a 21-gauge butterfly needle. The first 3 mL were always discarded. All participants denied taking any medication within the previous 14 days. All samples were processed within 3 min in polystyrene round-bottom tubes (5 mL) (Falcon™; Becton Dickinson, Franklin Lakes, NJ).
Blood was pipetted into four polypropylene tubes: 800 μL of blood was incubated with 200 μL (20% hemodilution) of either HES 550/0.7 (Hextend®), HES 600/0.7 (6%Hetastarch-Baxter®), HES 200/0.6–0.66 (Elohäst® 6%), and the solvent of HES 550 in Hextend® containing the following concentration of electrolytes (mmol/L): sodium chloride 115, sodium lactate anhydrous 28, calcium chloride dihydrate 2.5, potassium chloride 3, and magnesium chloride hexahydrate 0.4. The pH value of the solution is approximately 5.9, the calculated osmolarity is 307 mOsm, the content of dextrose hydrous is 5 mmol/L, and HES 550 is 60 g/L (13). All other HES preparations investigated are commercially available in 0.9% saline. Additional aliquots were hemodiluted (20%) with HES 600 plus calcium (2.5 mmol/L). One aliquot of citrated whole blood remained undiluted. All incubations were performed at room temperature for 5 min. Thereafter, flow cytometric analysis was performed, as described previously (14). In brief, blood was diluted (1:5) in phosphate-buffered saline (100 mM of sodium phosphate, pH value of 7.3, and 0.145 M of NaCl) to inhibit contact between individual platelets. Each sample was further divided into two aliquots for fluorescent staining. To evaluate the extent of platelet reactivity, one aliquot was incubated with the strong platelet agonist thrombin receptor activator peptide 6 (TRAP; 25 μM; SFLLRN, Bachem AG, Bubendorf, Switzerland), and the other aliquot was stained without platelet stimulation. Platelet activation transforms platelet membrane glycoprotein (GP) IIb–IIIa complexes to a conformational state that is competent for binding fibrinogen (15). P-selectin, which is expressed on the surface of activated platelets as the internal α-granule membrane, becomes integrated into the cytoplasmatic membrane (16) and serves as a marker for platelet secretion and activation. To determine the expression of activated GP IIb–IIIa complex and P-selectin, aliquots were incubated with a fluorescein isothiocyanate-conjugated activation-dependent anti-human platelet GP IIb–IIIa monoclonal antibody, PAC-1™ (Becton Dickinson Immunocytometry Systems, San Jose, CA), and a phycoerythrin-conjugated monoclonal antibody against human platelet P-selectin (anti-CD62P; Pharmingen, San Diego, CA). After 30 min of incubation with saturating concentrations of monoclonal antibodies at room temperature in the dark, samples were fixed in 1% paraformaldehyde (pH value of 7.3) at 4°C. In each experiment, cellular autofluorescence was determined, and isotype and compensation control were performed. Fluorescence was measured with a FACS-Calibur™ flow cytometer and analyzed with CellQuest-Pro™ software (Becton Dickinson Immunocytometry Systems). Quantum fluorescence microbeads (Calibrite Beads™; Becton Dickinson Immunocytometry Systems) were used each day for standardization of instrument settings.
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 correction. Data were expressed as mean ± SD. P < 0.05 was considered statistically significant.
Figure 1 shows the effect of 20%in vitro hemodilution with HES 550, its solvent, HES 600, and HES 200 on the binding of PAC-1 to nonstimulated and TRAP-activated platelets. A gate was set around the platelet population identified by forward and side scatter characteristics of the flow cytometric analysis. The percentage of gated platelets binding PAC-1 increased significantly in the presence of HES 550 and its solvent in both nonstimulated and TRAP-activated samples (P < 0.05). PAC-1 binding decreased in the presence of HES 600 and HES 200 in TRAP-activated samples (P < 0.05).
The addition of calcium (2.5 mmol/L) to HES 600 reversed its decreasing effect on PAC-1 binding to both nonstimulated and TRAP-activated platelets.
The binding of the phycoerythrin-conjugated monoclonal antibody against human platelet P-selectin anti-CD62P was not significantly changed by any dilution (data not shown).
The present experiments disproved our hypothesis that the platelet-inhibiting effect of Hextend® is more pronounced than the effect of other synthetic colloidal solutions containing HES macromolecules of lower molecular weight and degree of substitution. As shown in Figure 1, Hextend® significantly increased platelet reactivity. This study reports for the first time a platelet-stimulating effect of a HES preparation. To investigate the underlying mechanism of this surprising result, we further investigated the effect of the solvent of HES in Hextend®, and we found an answer to the unexpected stimulating effects of Hextend® on platelets. The incubation of whole blood with the electrolyte solution present in the solvent of Hextend® resulted in a comparable increase in platelet activation markers (Fig. 1). Thus, it seems that the activation of platelets observed in the present in vitro experiments is not a genuine effect of HES 550 but rather mediated by the solvent in the commercially available solution. The large concentration of calcium in the crystalloidal solvent in Hextend® may be responsible for the platelet-activating effect. We speculate that HES 550 inhibits platelets like other high molecular weight HES preparations, but this effect is surpassed by the activating effect of its solvent. The observation that the addition of calcium (2.5 mmol/L) to HES 600 reversed its decreasing effect on PAC-1 binding to platelets to undiluted control values further supports this hypothesis. The antagonism of unwanted side effects of the colloid by the addition of calcium to the solvent has already been performed in the commercially available preparation of urea-linked gelatin, Hemaccel® (Hoescht MarionRoussel, Australia). We recently reported that incubation with Hemaccel® significantly increased the expression of activated GP IIb–IIIa on platelet surface (17).
Wilkes et al. (18) reported that PFA closure times were prolonged after IV infusion of 10 mL/kg of Hextend®, which results approximately in a 16% degree of hemodilution. This study indicates a platelet-inhibiting effect of Hextend®, which is in contrast to our experiments. We have no explanation for the discrepancy, but it may be related to the sensitivity of the PFA to hemodilution per se. In previous experiments, we found that even saline hemodilution prolongs PFA closure times, although it has no platelet-inhibiting effect on the cellular level (2,3). We used the method of flow cytometry in the present study because this technique is increasingly used for characterization of cellular abnormalities of platelets and permits assessment of platelet reactivity in a physiological manner (19).
Confirming our previous results (2,3), HES 200 had a significant platelet-inhibiting effect. Interestingly, there was no significant difference between the degree of platelet inhibition between HES 600 and HES 200. We have demonstrated that HES compromises platelet contribution to hemostasis by reducing the availability of the functional receptor for fibrinogen on the platelet surface (GP IIb–IIIa) depending on structural characteristics of HES molecules, such as mean molecular weight and the degree of substitution (2,3). HES does not inhibit platelets by interfering with calcium-dependent intracellular signal transduction pathways (20). We visualized extracellular binding of HES macromolecules to the platelet surface by means of flow cytometry (21). This coating phenomenon may be responsible for the antiplatelet effects of HES by blocking the access of endogenous ligands to the GP IIb–IIIa receptor. In addition to direct effects, plasmatic abnormalities, such as decrease in fibrinogen levels (22), thrombin generation (23), and Von-Willebrand factor (24) may contribute to decreased platelet responsiveness after HES administration. In an open-label study in 21 healthy volunteers, Wilkes et al. (12) investigated the effect of an IV infusion of Hextend® at 10 mL/kg over 20 minutes. The authors found no changes in prothrombin time, and factor XII were found, whereas activated partial thromboplastin time increased, and thrombin time, factors VIII, IX, XI, Von-Willebrand factor antigen, and fibrinogen decreased.
Whereas Boldt et al. (25) reported a prolongation in thromboelastographic r-time in patients undergoing major abdominal surgery and receiving Hextend® when compared with low molecular weight HES or Ringer’s lactated solution, Gan et al. (11) observed an activation of plasmatic coagulation, as indicated by a shortening in thromboelastographic r-time. R-time represents the time from the initiation of the test until the onset of clot formation. Confirming these data, a shortening in thromboelastographic r-time was observed after Hextend® resuscitation in a hemorrhage model in rabbits (10). The content of electrolytes, especially calcium, in the solvent of Hextend® may act as an activator of the plasmatic coagulation cascade by recalcifying the citrated whole blood sample.
Blood loss was larger in cardiac surgical patients receiving high versus middle molecular weight HES (1). The difference in platelet-inhibiting effects between older high and middle molecular weight HES solutions may explain the difference in bleeding diathesis. Although extrapolations from experimental studies to the clinical situation should be made with care, the present data may indicate that the injection of calcium salts during infusion of older HES preparations may counteract the antiplatelet effect.
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