Anesth Analg 2004;99:393-398
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000123491.08697.CA
ANESTHETIC PHARMACOLOGY
Propofol Inhibits Human Platelet Aggregation Induced by Proinflammatory Lipid Mediators
Olivier Fourcade, MD PhD*,
Marie-Françoise Simon, PhD
,
Lawrence Litt, MD PhD
,
Kamran Samii, MD*, and
Hugues Chap, MD PhD
Department of Anesthesia, Purpan Hospital, University of Toulouse, Toulouse, France;
Institut National de la Santé et de la Recherche Médicale, Unit 326, Purpan Hospital, Toulouse, France; and
Department of Anesthesia, University of California, San Francisco, California
Address correspondence and reprint requests to Olivier Fourcade, MD, PhD, Service d’Anesthésie-Réanimation, Hôpital Purpan, Place du Docteur Baylac, 31059 Toulouse Cedex, France. Address e-mail to fourcade.o{at}chu-toulouse.fr
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Abstract
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Lysophosphatidic acid (LPA), platelet-activating factor (PAF), and thromboxane A2 are proinflammatory lipid mediators that activate surface receptors on platelets, producing increased intracellular calcium, which is necessary for aggregation. We investigated propofol’s effect on platelet aggregation and intracellular calcium mobilization caused by these three agonists. Platelets from human volunteers were incubated in buffers containing LPA (1 µM), U46619 (thromboxane A2 analog; 1 µM), or PAF (10 nM). Propofol emulsion or 2,6-diisopropylphenol (propofol without fat emulsion) dissolved in ethanol was added to achieve concentrations of propofol used clinically: 5 or 10 µg/mL. After 2 min, aggregation or intracellular calcium concentrations were measured with optical techniques. Propofol emulsion and propofol in ethanol produced similar inhibition of platelet aggregation induced by LPA, PAF, and U46619 in a dose-dependent fashion. LPA, PAF, and U46619 each caused significant increases in intracellular calcium that were not modified by propofol. Because propofol does not significantly alter intracellular calcium increases caused by receptor activation, inhibition appears to act distal to platelet receptors, inositol phosphate 3, and phospholipase C. Because the three lipid mediators play a key role in inflammation, their inhibition by propofol might be clinically important.
IMPLICATIONS: Propofol inhibited the platelet aggregation induced by three proinflammatory lipid mediators (lysophosphatidic acid, platelet-activating factor, and thromboxane A2). The absence of inhibition of intracellular calcium increase induced by these agonists suggests that propofol acts distal to platelet receptors.
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Introduction
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Lysophosphatidic acid (LPA) (1), platelet-activating factor (PAF) (2), and thromboxane A2 (TXA2) (3) are proinflammatory lipid mediators that have multiple biological effects. TXA2 induces vasoconstriction and bronchoconstriction and stimulates chemotactism, leukocyte adhesion, and degranulation. It also plays a key role in the induction of the secondary phase of platelet aggregation. LPA induces vasoconstriction and bronchoconstriction, whereas PAF stimulates leukocytes and macrophages. These three lipid mediators are secreted by activated platelets and induce their aggregation.
Volatile and local anesthetics have been reported to inhibit LPA (4,5). Rossi et al. (6) showed, in a Xenopus oocyte model, that propofol inhibits an LPA receptor response. However, the effects of propofol on platelet aggregation induced by LPA and PAF have not been described. In contrast, inhibitory effects of propofol on platelet aggregation induced by adenosine diphosphate (ADP) and epinephrine have been described (7–9), as have inhibitory effects for volatile anesthetics such as sevoflurane and halothane (10–12). The inhibition induced by propofol was not explained by the fat emulsion that is used in commercial preparations as propofol’s carrier (8,9). This effect was partial, dose dependent, and observable both in vitro and in vivo (8). However, during continuous infusion of propofol for general anesthesia, bleeding time was unchanged (8), and no alteration of platelet function has been observed in vivo (13). The mechanisms by which propofol inhibits platelet aggregation are not fully understood. The binding of agonists such as ADP or epinephrine to their platelet receptors induces activation of phospholipase C (PLC). This enzyme hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing inositol 1,4,5 triphosphate (IP3). This compound leads to intracellular Ca2+ increases and triggers platelet response. To enhance this primary aggregation, the binding of these agonists to platelet receptors induces cytosolic phospholipase A2 activation to release arachidonic acid. Arachidonic acid is finally converted by type 1 cyclooxygenase to TXA2, which plays a key role in the induction of secondary aggregation. Aoki et al. (8) attributed the inhibition to absent increases in intracellular calcium when stimulated with ADP, because many platelet processes require increased cytosolic calcium. Hirakata et al. (7) found that in response to epinephrine or ADP, propofol inhibits only secondary aggregation without altering primary aggregation, and they suggested this was due to altered function of TXA2 (12).
Human platelets contain specific cell-surface receptors for LPA, PAF, and TXA2. We hypothesize that propofol’s interaction with these receptors provides a mechanism for propofol’s alterations of platelet aggregation. The aim of this study was to test this hypothesis, realizing that intracellular calcium measurements might help to clarify at least one mechanism of platelet inhibition induced by propofol.
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Methods
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This study was approved by our institutional committee on human research, and informed consent was obtained from all participating subjects. Human platelets were isolated from whole blood obtained from voluntary donors and prepared as described by Ardlie et al. (14). Briefly, 20 mL of venous blood was put into siliconized citrated tubes (1:10 volume of 3.8% sodium citrate; Vacutainer®) and centrifuged at 150g for 10 min. Platelet-rich plasma (PRP) was aspirated, acidified by ACD (sodium citrate, 85 mM; citric acid, 65 mM; glucose, 111 mM; 1 vol ACD per 5 vol PRP), and centrifuged for 10 min at 1500g at room temperature. The supernatant (platelet-poor plasma) was discarded, and the platelet pellet was recovered and suspended in physiological buffer (Buffer A: NaCl, 140 mM; KCl, 2.7 mM; MgCl2, 1 mM; CaCl2, 1 mM; glucose, 5 mM; HEPES, 10 mM, pH 7.4) to restore the initial volume of PRP. This technique restores the initial physiological platelet count.
Platelet aggregation was measured by optical transmission of a light beam (photometry by Dual Aggregometer; Stopwatch-Log Corp.) after preincubation with propofol in ethanol or propofol emulsion and a physiologic concentration of one of the lipid agonists. Measurements were performed at 37°C with 300 µL of platelet suspension. First we measured the maximal aggregation induced by LPA (18:1 oleoyl LPA; 1 µM), 9,11-dideoxy-11
, 9 epoxymethanoprostaglandin F2 (U46619, a stable analog mimetic of TXA2; 1 µM), and PAF (ß-acetyl-O-hexadecyl L- phosphatidylcholine; 10 nM). Then we measured aggregations induced by a 2-min preincubation of platelets with added 5 and 10 µg/mL propofol emulsion (with lipid carrier) and with 5 and 10 µg/mL of propofol (2,6-diisopropylphenol in ethanol). Propofol’s inhibition of the platelet aggregation triggered by LPA and PAF was measured by using stepwise increments in the propofol emulsion concentration (1, 3, 6, 12, and 20 µg/mL) to determine the dose dependency of propofol’s effects. Control experiments with ethanol alone and with Intralipid® alone (propofol lipid vehicle at the same concentrations as in the propofol emulsion) were performed with each agonist. The agonists and propofol without lipid carrier were used as ethanolic solutions. Propofol emulsion and Intralipid® were prepared just before their use as mixtures with Buffer A.
PRP, obtained as described above, was incubated with Fura-2-AM. (0.5 µM) at 37°C for 20 min before adding ACD and centrifuging as described previously. Platelets were then recovered and suspended in Buffer A such that the initial volume of PRP was restored without calcium. Physiologic platelet counts were recorded. Intracellular calcium concentrations were measured by fluorometry (15) (JY3 spectrofluorometer, Jobin Yvon®; excitation, 340 nm; emission, 500 nm; 37°C). Each measurement was done with 600 µL of platelet suspension. The intracellular calcium concentration was calculated by using 224 nM as the Kd for the Fura-2/Ca2+ complex. Intracellular calcium concentrations and platelet aggregation were not measured simultaneously with the same platelets.
Platelet intracellular calcium concentration was measured before and after stimulation by LPA (1 µM), PAF (10 nM), and U46619 (1 µM). Each agonist was used with and without a 2-min incubation with 10 µg/mL of propofol (2,6-diisopropylphenol in ethanol). Intracellular platelet calcium concentration variations induced by propofol in ethanol alone (10 µg/mL) or by propofol emulsion (with lipid carrier) (10 µg/mL) were measured as control.
Fura-2; 18:1 oleoyl LPA; U46619; phosphatidylcholine; ß-acetyl-O-hexadecyl L- phosphatidylcholine; and 2,6-diisopropylphenol were obtained from Sigma France. The propofol emulsion (Diprivan®), obtained from Zeneca France, was used in its commercial form for clinical use in a 10% Intralipid® emulsion. Twenty percent Intralipid, obtained from Kabi France, was used in its commercial form for clinical use.
Results are expressed as means ± SEM and were calculated from at least three different reproducible experiments. Measurements of platelet aggregation (maximum point on the aggregation curve) were expressed as the percentage of maximal aggregation obtained when each agonist was used alone. Measurements of intracellular calcium were expressed in nanomoles of calcium. Intracellular free calcium concentrations (nM) were compared by using Student’s t-test; P < 0.05 was considered significant. The percentage of variation of intracellular free calcium concentration, starting from the volume determined before stimulation taken as 100%, was indicated.
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Results
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Propofol emulsion inhibited platelet aggregation induced by the three lipid agonists LPA, PAF, and U46619 (Table 1). Inhibition was significant at the smallest dose of 5 µg/mL of propofol emulsion and increased at 10 µg/mL, suggesting a dose-dependent inhibitory effect. Intralipid® caused only a minimal inhibition of the three agonists at a concentration corresponding to that found in 20 µg/mL of propofol emulsion (Table 1). No inhibition was observed at smaller concentrations of Intralipid®, and ethanol (3 and 6 µL) had no effect. Figure 1 shows the same results, with representative aggregation curves of this inhibition with LPA (Fig. 1a), PAF (Fig. 1b), and U46619 (Fig. 1c).
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Table 1. Effects of Propofol, With or Without a Lipid Carrier, on Platelet Aggregation Induced by LPA, PAF, and U46619
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Figure 1. Effects of propofol emulsion on platelet aggregation induced by lysophosphatidic acid (LPA) 1 µM (a), platelet-activating factor (PAF) 10 nM (b), and U46619 1 µM (c). Representative curves (light transmission versus time after starting incubation) are shown for incubations consisting of each agonist alone (C = control) or with each agonist and Intralipid (IL) at a concentration corresponding to that found in 20 µg/mL of propofol emulsion, Diprivan 5 µg/mL (D5), or Diprivan 10 µg/mL (D10). The first 2 min of all tracings is not shown because there was no aggregation during this time. The time scale (1 min) is shown on the graphs.
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We confirmed that inhibition of the platelet aggregation induced by LPA (Fig. 2a) and PAF (Fig. 2b) was dose dependent at increasing concentrations of propofol emulsion. Inhibition was significant at 1 µg/mL and increased up to 20 µg/mL; this last dose caused complete inhibition of the aggregation induced by PAF.
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Figure 2. a, Dose-response inhibition of propofol emulsion on lysophosphatidic acid (LPA)-induced platelet aggregation. Data are means ± SEM of the percentage of aggregation induced by LPA (1 µM) after 2 min of incubation with different concentrations of propofol emulsion. Data are from three experiments. b, Dose-response inhibition of propofol emulsion on platelet-activating factor (PAF)-induced platelet aggregation. Data are means ± SEM of the percentage of aggregation induced by PAF (10 nM) after 2 min of incubation with different concentrations of propofol emulsion. Data are from three experiments.
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Propofol alone (2,6-diisopropylphenol in ethanol without its lipidic vehicle) inhibited platelet aggregation induced by the three agonists, indicating that the observed effect was due to the phenolic compound and not the vehicle (Table 1). Inhibition was significant at 5 µg/mL and further increased at 10 µg/mL, confirming a dose-dependent effect of propofol.
Table 2 shows that LPA, PAF, and U46619 induced significant intracellular calcium increases when compared with basal calcium in platelets (323% ± 59%, 531% ± 59%, and 410% ± 143%, respectively). The calcium increase induced by these 3 lipid agonists was not modified and remained significant in the presence of propofol at a concentration of 10 µg/mL (344% ± 56%, 680% ± 309%, and 348% ± 103%). As control, calcium increases for 10 µg/mL propofol (n = 16) and 10 µg/mL propofol emulsion (n = 8) were compared. Each elicited small but significant increases in intracellular calcium (137% ± 8% and 144% ± 13%, respectively). No significant difference in calcium mobilization was found between propofol alone and propofol emulsion.
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Discussion
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Our data demonstrate that propofol emulsion (propofol in lipid vehicle) and propofol alone (2,6-diisopropyl-phenol in ethanol without lipid emulsion) each inhibited the platelet aggregation induced by the three proinflammatory lipid mediators LPA, PAF, and TXA2 analog. The greatest inhibition of these agonists was obtained with 10 µg/mL. Because inhibition by propofol of platelet aggregation triggered by LPA and PAF had never been described (contrary to TXA2), we investigated the dose dependency of this inhibition by using step-increasing concentrations of propofol emulsion, and we confirmed an effective inhibition at 1 µg/mL that increased with doses up to 20 µg/mL of propofol. The propofol concentrations used in this study are comparable to those found in clinical practice, which range from 12 to 20 µg/mL during anesthetic induction and are close to 5 µg/mL during the maintenance of anesthesia (16,17). Platelets were isolated from human blood and were kept at their physiological count by the preparation. Each lipid agonist of the aggregation was at physiologic concentration. The results obtained with propofol alone in ethanol solution confirmed that inhibition is due to the anesthetic in propofol emulsion, and this is the first result obtained in which the active agent (2,6-diisopropylphenol) was used without a lipid vehicle. In addition, the lipid emulsion without anesthetic had no effect on optical reading, because aggregation curves were not modified by Intralipid®. The inhibition of platelet aggregation observed was purely a dose-dependent effect and was neither a persistent nor a cumulative effect of propofol, because the inhibition induced by each concentration of propofol was measured with independent assays. The time course for propofol’s effects on platelet aggregation was dependent on the concentration used. Aggregation induced by the agonists alone was irreversible; however, aggregation could be reversible if a stronger inhibition was produced by a larger concentration of propofol. Our findings of dose-dependent inhibition by propofol of platelet aggregation caused by inflammatory mediators resemble those of Aoki et al. (8), who showed an in vitro and in vivo dose-dependent inhibition of platelet aggregation induced by ADP.
The second result of this study, that propofol did not modify intracellular calcium increases induced by LPA, PAF, and U46619, is significant because intracellular calcium increases are common early in the process of platelet activation. LPA (1), PAF (18), and TXA2 (19) interact with specific cell-surface G-protein receptors and, like ADP, initiate the same steps of intracellular signaling that lead to platelet aggregation (Fig. 3).
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Figure 3. Interaction of lysophosphatidic acid (LPA), platelet-activating factor (PAF), and thromboxane A2 (TXA2) with specific cell-surface G-protein platelet receptors. Activation of phospholipase C (PLC) producing 1,4,5 inositol triphosphate (IP3), leading to intracellular Ca2+ increases, and triggering cell response via protein kinase C (PKC) activation is shown. The hypothesis of platelet response inhibition by propofol, without altering intracellular calcium responses, suggests that inhibition is distal to the PLC and IP3 systems.
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Although propofol emulsion and propofol alone cause measurable increases in intracellular calcium, as previously described in human glial cells (20), these increases were not significant enough to explain the persistent large calcium increase induced by the lipid mediators in propofol in our study or to induce platelet aggregation. Hirakata et al. (7) also showed an enhancing effect of propofol in platelets; this came via increased IP3 generation. In that study, the authors also confirmed that propofol does not alter TXA2 affinity for its receptor. In any event, large increases in intracellular calcium come from activated PLC, which produces IP3 (causing the release of calcium from intracellular stores) and also causes the activation of protein kinase C. The intracellular calcium increases that we found are consistent with such PLC activity being caused by the activation of lipid agonist receptors. Rossi et al. (6), using Xenopus oocytes to model the postsynaptic membrane, showed that propofol’s inhibition of a transfected LPA receptor resulted in the blockage of an oocyte membrane chloride channel. Although there are several LPA receptor subtypes (21), the Rossi et al. study suggests that propofol’s interference in our platelet study might also arise from interaction with an LPA receptor.
Although propofol inhibited platelet aggregation without altering intracellular calcium responses, its mechanism of inhibition seems to be distal to the PLC and IP3 systems, illustrating that intracellular calcium increases, although necessary for certain types of platelet aggregation, might not be sufficient. We also note that pathways might be available that do not require intracellular calcium mobilization. It is interesting to note that -tocopherol, another liposoluble molecule that includes a phenol group, inhibits platelet aggregation by inhibition of protein kinase C (22). Propofol causes increased cyclic guanosine monophosphate (cGMP) in endothelial cells (23). If there were a mechanism for propofol to similarly increase cGMP levels in platelets, that could at least partially explain some of propofol’s inhibition of platelet aggregation.
In summary, propofol inhibits both platelet aggregation and intracellular calcium increases in response to thrombin or ADP (8). In contrast, in our study, in which activation was by lipid mediators, propofol inhibited aggregation without modifying intracellular calcium responses. Because platelets clearly exhibit different types of activation in response to different stimuli, it is important to also investigate in vivo platelet responses to propofol for different stimuli, without assuming that what propofol does in one situation is easily generalized to others. The three lipid mediators used may have multiple biological effects, in particular during inflammation, and their inhibition by propofol could be important.
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Acknowledgments
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Supported by Institut National de la Santé et de la Recherche Médicale Unité 326, Hôpital Purpan, Toulouse, France.
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Accepted for publication February 6, 2004.
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Abstract
Introduction
