In Vivo and In Vitro Studies of the Inhibitory Effect of Propofol on Human Platelet Aggregation 

This study was approved by our institutional committee on human research, and informed consent was obtained from all participants.

Blood samples collected from the antecubital vein were anticoagulated with a 1:10 volume of 3.8% sodium tricitrate solution and then centrifuged at 800 rpm for 10 min. The upper layer was collected as platelet-rich plasma (PRP). Platelet-poor plasma (PPP) was obtained by centrifuging the residual blood sample at 3,000 rpm for 10 min. The platelets in sample solution were counted using a Coulter counter (model MD II; Coulter Electronics, Hialeah, FL) and adjusted to 2 x 10⁵cells/micro liter.

Platelet aggregation was assessed by the turbidimetric technique described by Born and Cross [20]using an aggregometer (Hema Tracer 601; Nikoh Bioscience, Tokyo, Japan). Platelet-rich plasma was placed in a silicon-coated glass cuvette and warmed to 37 [degree sign] Celsius for 5 min. Because the fat emulsion contained in Diprivan or Intralipos (The Green Cross Company, Osaka, Japan) causes turbidity in both PRP and PPP, platelet aggregation cannot be assessed in terms of the increase in optical density. [18,21]Thus we determined the minimal adenosine diphosphate (ADP) concentration required to induce irreversible aggregation (the ADP threshold [10,17]). Briefly, five distinct concentrations of ADP (1–5 micro Meter, in 1-micro Meter increments, final concentration) were prepared and added to the samples. The minimal concentration that induce irreversible aggregation was determined as the ADP threshold. An increase in the ADP threshold indicates inhibition of platelet aggregation.

To investigate the effect of Diprivan on hemostasis, template bleeding time was measured in 0.5 min increment in 9 patients before and during the administration of Diprivan using Simplate (Organon Teknica, Durham, NC).

The propofol concentration was measured based on the method of Pavan et al. [22]with some modifications. After aggregation studies were completed, the residual blood samples were centrifuged at 3,500 rpm for 10 min and the supernatants were collected. The samples were mixed with acetonitrile-60% perchloric acid (2:1 vol/vol) and then centrifuged at 3,500 rpm for 10 min. The propofol concentration in the supernatants was measured by high-performance liquid chromatography, using a solvent delivery pump (Hitachi L-6000, Tokyo, Japan) equipped with an autosampler (Hitachi AS-200), an ultraviolet detecter (Hitachi L-7400), and a chromatointegrator (Hitachi D-2500). Chromatographic separation was done on a 250 x 4.6-mm column packed with 5 micro meter Hypersil ODS (GL-Pack, GL Sciences, Tokyo, Japan). Acetonitrile-double distilled water (2:1) adjusted to a pH of 4.0 using acetic acid was eluted at a flow rate of 1.5 ml/min. A 20-micro liter sample of standard solution of propofol was injected, with thymol (10 micro gram/ml) used as the internal standard. Absorbance was measured at 270 nm.

Triglyceride is considered a reliable marker of fat emulsion based on the results of previous reports [16,17,23–25]and our pilot study (data not shown). The triglyceride concentration was measured by the lipoprotein lipase-glycerol kinase-glycerol-3-phosphate oxidase method, [26]with some modifications. The absorbance of the red quinone dye that is synthesized through these reactions was measured at 540 nm and 660 nm using the Hitachi Autoanalyzer 736.

In Vivo Study: Part 1. The effect of propofol-induced anesthesia was investigated in 10 patients (five men and five women; ages 34–64 yr; weight, 56.5 +/- 9.3 kg) scheduled for minor surgery of the face and neck region. All patients were classified as American Society of Anesthesiologists physical status I and had no hematologic abnormalities. Patients receiving medications that may affect platelet function were excluded. Patients were premedicated with intramuscular injections of atropine sulfate (0.01 mg/kg) and hydroxyzine (1 mg/kg). Thirty minutes later, 2 mg/kg propofol (0.2 ml/kg Diprivan) was administered intravenously to induce general anesthesia. Tracheal intubation was facilitated by administration of 0.1 mg/kg vecuronium bromide. Thereafter, anesthesia was maintained by a continuous infusion of propofol at a rate of 5–8 mg [center dot] kg sup -1 [center dot] h sup -1, together with appropriate doses of vecuronium and fentanyl. No inhalation anesthetics, including nitrous oxide, were used during the procedure.

Blood samples were collected at three points: before the induction of anesthesia (control), 5 min after the bolus injection of Diprivan, and 2 h after the end of Diprivan infusion. The platelet aggregation test was performed and plasma concentrations of propofol and triglyceride were measured.

In Vivo Study: Part 2. The effect of the fat emulsion on platelet aggregation was examined in 10 healthy volunteers (five men and five women; ages 25–34 yr; weight, 59.7 +/- 10.6 kg). After an overnight fast, 0.2 ml/kg of a 10% Intralipos solution, which is the solvent used in Diprivan, [27]was injected intravenously. Blood samples were collected before and 5 min after the bolus injection, and platelet aggregation and the triglyceride concentration were measured.

In Vitro Study: Part 1. Platelet-rich plasma obtained from 10 healthy volunteers (six men and four women; aged 22–63 yr) was prepared as follows:(1) 5 micro liter autologous PPP was added to 1 ml PRP (control group);(2) 5 micro liter Diprivan was added to 1 ml PRP (Diprivan group);(3) 5 micro liter 10% Intralipos solution was added to 1 ml PRP (Intralipos group); and (4) 5 micro liter autologous PPP saturated with propofol was added to 1 ml PRP (propofol group). In the propofol group, 100 micro liter propofol was vortexed with autologous PPP and centrifuged at 3,500 rpm for 20 min. The clear lower layer was aspirated and added to PRP. After platelet aggregation tests were performed, the propofol concentration was measured by high-performance liquid chromatography. The propofol concentration was also measured in the samples from the Diprivan group. The triglyceride concentration was measured in the Intralipos group.

In Vitro Study: Part 2. To investigate the effects of different propofol concentrations on platelet aggregation, we obtained PRP from eight healthy volunteers and the samples were divided into four 1-ml aliquots (for the control and for three different concentrations of propofol). Autologous PPP (5 micro liter), which was adjusted to contain three distinct concentrations of propofol, respectively, was added to these samples of PRP except for the control. In the control samples, the same volume of autologous PPP was used. After the ADP thresholds were determined for each control sample, platelet aggregation of the sample was assessed and classified into “Inhibition” meaning that the increase of ADP threshold was recognized compared with the control value, and “No inhibition,” meaning no change in the value of the ADP threshold.

Propofol concentrations were determined by high-performance liquid chromatography.

PRP obtained from six healthy volunteers and anticoagulated with 1:5 volume of acid-citrate-dextrose was incubated with 3 mmol fura-2 acetoxymethyl ester for 15 min at 37 [degree sign] Celsius and then centrifuged at 1,200 rpm for 15 min in the presence of 0.3 U/ml apyrase. The washed platelets loaded with fura-2 were obtained by discarding the supernatant and resuspended in Tyrode-Hepes buffer, pH 7.4. [28]Platelet counts in samples were adjusted to 10⁵cells/micro liter using a Coulter Counter model MD II. Propofol was saturated in Tyrode-Hepes buffer and added to the samples, the concentration of which was determined by high-performance liquid chromatography after the procedure. The intracellular calcium concentration ([Ca²+]ⁱ) and platelet aggregation were measured simultaneously using a calcium analyzer (CAF-110; JASCO, Tokyo, Japan) with repeated alternating excitation wavelengths of 340 and 380 nm and measuring emission at 505 nm. [29]The extracellular calcium concentration was adjusted to 1 mM by adding CaCl²solution and was stirred for 5 min at 37 [degree sign] Celsius. After the basal fluorescence intensity was recorded, the platelets were stimulated by 0.02 U/ml thrombin and the change of fluorescence intensity was monitored. The relative ratio of fluorescence intensity at 340 nm to that at 380 nm (R340:380)[30,31]was recorded at the peak and at the plateau phases. The [Ca²+]ⁱat each point was calculated according to the equation of Tsien et al., [32]using the Kd of fura-2 and Ca²+ as 224 nM. Data are expressed as the rise in [Ca sup 2+]ⁱfrom basal levels.

Ni²+(final concentration of 1 mM) was added to the samples instead of CaCl²to block the influx of extracellular calcium and to demonstrate calcium release from the intracellular storage site. [33,34] 

To investigate the influx of calcium into the cell, the entry of Mn²+ was evaluated using a quenching technique. [33,34]MnCl sub 2 was added to the samples to a final concentration of 1 mM, and fura-2 fluorescence was monitored at the calcium insensitive excitation wavelength, 360 nm. Mn entry, which represents calcium influx, was measured as the rate of fluorescence decrease after stimulation by thrombin. Data were expressed as a percentage of the initial fluorescence.

Adenosine diphosphate and acetonitrile were obtained from Wako Pure Chemical Industries, Osaka, Japan; propofol from Tokyo Kasei Kogyo, Tokyo, Japan; thymol from Sigma Chemical Company, St. Louis, MO; Apyrase from Sigma Chemical; and fura-2 AM from DOJI-NDO Laboratories, Kumamoto, Japan. Thrombin was donated by MOCHIDA Pharmaceutical, Tokyo, Japan. Diprivan and Intralipos were donated by Zeneka Yakuhin K.K., Osaka, Japan and The Green Cross Company, Osaka, Japan, respectively. All other compounds were purchased from commercial sources.

Values are the means +/- SD. Friedman two-way analysis of variance by ranks was adopted for the analysis of the data of ADP thresholds in part 1 of the in vivo study. Two-way analysis of variance followed by Bonferroni correction with repeated measures was adopted to analyze the difference in the triglyceride concentration between the data in part 1 of the in vivo study. Unpaired t tests were used to analyze differences in triglyceride concentrations in parts 1 and 2 of the in vivo studies. The Kruskal-Wallis test was used to analyze the ADP thresholds in part 1 of the in vitro study. One-way analysis of variance followed by Bonferroni correction was used to analyze the differences in triglyceride concentrations between the data of the in vivo studies and that in part 1 of the in vitro study. To analyze the results of template bleeding time, the Wilcoxon signed rank test was used. Paired t tests were used to analyze the data in the studies of intracellular calcium concentration. Probability values < 0.05 were considered significant.

The average operative time and the average duration of the propofol infusion were 135 +/- 35.7 and 160.7 +/- 38.5 min, respectively. The total dose of infused propofol was 1,166.3 +/- 279 mg, and the average rate of continuous infusion of propofol was 6.1 +/- 1.8 mg [center dot] kg sup -1 [center dot] h sup -1.

The ADP threshold for PRP increased significantly after the bolus injection of propofol (P < 0.05, n = 10), indicating inhibition of platelet aggregation. Two hours after surgery, it was not significantly different from the control value (Table 1). Typical aggregation curves are shown in Figure 1. The ADP threshold increased in nine of 10 patients after propofol injection.

The triglyceride concentration increased significantly after the induction of anesthesia (P < 0.05) but was not significantly different from the control value 2 h after surgery (Table 1).

The propofol concentration decreased after operation compared with the concentration after the bolus injection of propofol (Table 1). In the patient who showed no inhibition of platelet aggregation, the propofol concentration was 2.44 micro gram/ml.

Injection of Intralipos did not alter the ADP threshold in any of the participants.

The triglyceride concentration before injection of Intralipos (63.4 +/- 32.1 mg/dl) was not significantly different from the control value in part 1. After injection of Intralipos, the triglyceride concentration increased to 115.6 +/- 56.2 mg/dl, which was not significantly different from the value after the bolus injection of Diprivan in part 1.

The ADP threshold was significantly higher in the Diprivan group (P < 0.05) and in the propofol group (P < 0.01) compared with the control group. There was no significant difference in the ADP threshold between the control group and the Intralipos group (Table 2).

The triglyceride concentrations in the control group were not significantly different from the control values in parts 1 and 2 of the in vivo study. The triglyceride concentration in the Intralipos group did not differ significantly from the in vivo values after injections of Diprivan and Intralipos.

The propofol concentration was significantly higher in the “inhibition” group (5.81 +/- 2.73 micro gram/ml) compared with the “no inhibition” group (2.08 +/- 1.14 micro gram/ml;Figure 2). The propofol concentration at which platelet aggregation was inhibited in part 1 of the in vivo study (2.91 +/- 0.67 micro gram/ml) was in the zone of overlap of the inhibition and no inhibition groups. The propofol concentration at which platelet aggregation was reversed 2 h after surgery (0.78 +/- 0.35 micro gram/ml) corresponded to values in the no inhibition group.

The values of template bleeding time were not significantly different before and during infusion of Diprivan (Table 3).

The mean value of the change in [Ca²+]ⁱat the peak induced by thrombin was 801.8 +/- 245.9 nM in controls and 275.1 +/- 96.8 nM in samples treated with propofol; these values were significantly different (P < 0.01). The mean value of the change in [Ca sup 2+]^Iat the plateau was 166.7 +/- 43.4 nM in controls and 87.6 +/- 26.7 nM in samples treated with propofol; these values were also significantly different (P < 0.05). However, the simultaneously determined aggregation curves showed no significant difference in the maximum aggregation rate (82 +/- 8.3% in control and 75.2 +/- 18.1% in samples treated with propofol, respectively). When Ni²+ was added to the sample, the mean value of the change in [Ca²+]ⁱat the peak was 316.8 +/- 140.4 nM in controls and 136.9 +/- 66.4 nM in samples with propofol. These values were also significantly different (P <0.01), suggesting the decreased discharge of [Ca²+]ⁱ. The mean concentration of propofol in washed platelet samples was 17.36 +/- 1.41 micro gram/ml. Figure 3shows typical patterns of aggregation and change in the R³⁴⁰:380 value (indicating [Ca²+]ⁱ). Figure 4shows the mean value of Mn²+ entry, expressed as the percentage decrease of fluorescence at 360 nm. After stimulation by thrombin, fluorescence decreased rapidly during 20–30 s and thereafter declined gradually during the procedure. The fast phase expresses the Mn sup 2+ entry (which reflects Ca²+ influx) caused by the stimulation of thrombin, and the second phase is attributed to the passive leakage into the cell. [35]In both phases, the decrease of the fluorescence was significantly less with propofol, suggesting inhibition in Ca²+ influx.

A bolus injection of Diprivan inhibited platelet aggregation in vivo, as indicated by the increased ADP threshold. This inhibitory effect was reversed 2 h after operation. Concentrations of propofol and triglyceride increased after the bolus injection of Diprivan and returned to the control level 2 h after the end of surgery. Intralipos did not inhibit platelet aggregation but increased the triglyceride concentration to a similar extent as Diprivan did, suggesting that the inhibitory effect of Diprivan was induced by propofol and not by the fat emulsion. Bleeding time was not prolonged during anesthesia with Diprivan.

In vitro findings confirmed that platelet aggregation was inhibited by propofol and Diprivan but not by the fat emulsion. Propofol was saturated in autologous plasma and the concentration was confirmed by high-performance liquid chromatography, making it possible to examine the separate effects of propofol and its fat solvent on platelet aggregation. The concentration of propofol in part 1 was somewhat higher than the concentration just after the bolus injection of 2 mg/kg propofol (< 10 micro gram/ml). [36]The results presented in part 2 showed that propofol inhibited platelet aggregation even at concentrations < 10 micro gram/ml.

Hansen et al. [12]reported that Diprivan did not inhibit platelet aggregation in vitro. However, they did not measure triglyceride or propofol concentrations. Glen et al. [15]observed no propofol-induced inhibition of platelet aggregation in rats, but their results cannot be extrapolated to humans because there is an interspecies difference in the response of platelets to ADP. [37]Turkan et al. [14]observed no inhibition of platelet aggregation during Diprivan-induced anesthesia in the presence of 20 micro Meter ADP, but the lack of inhibition in their study may be due to the high dose of ADP. We determined the minimal concentration of ADP required to trigger aggregation in the present study. This method also made it possible to evaluate aggregation when conventional parameters of the aggregation rate were not useful because the fat emulsion caused turbidity in samples.

Fat emulsion has been found to inhibit platelets in studies using high-dose [17]or prolonged infusion (> 6 h) of Intralipos. [16,19]Prolonged infusion of the fat emulsion was found to induce inhibition of platelet functions by the phagocytosis of lipid particles in platelet cytoplasm. [17,18]Therefore, when Diprivan is infused for a prolonged period, the fat emulsion may cause marked platelet inhibition.

Although ADP was classified as a relatively weak agonist, [38]a typical biphasic (primary and secondary) aggregation curve can be observed when platelets were stimulated with ADP. In our study, the primary aggregation was inhibited by propofol. When the aggregation reaction begins, platelets initially show an elevation of [Ca²+] sub i and aggregation. [39]Therefore, investigation of the change in [Ca²+]ⁱwas thought to be worthwhile.

The measurements of [Ca²+]ⁱrevealed that propofol inhibited the increase in [Ca²+]ⁱinduced by thrombin, a strong agonist for aggregating platelets. [38]When Ni²+ was added to the extracellular fluid, it was reported to inhibit the calcium influx by blocking the plasma membrane divalent cation channel and making it possible to observe the release of calcium from the storage site (discharge). [33,34]On the other hand, Mn²+ can pass through the calcium channel on the plasma membrane but binds to fura-2 and quenches its fluorescence. By using these divalent ions rather than Ca²+, two distinct pathways, the influx and the discharge of calcium, can be estimated individually. [34]From our results obtained using these divalent cations, propofol was found to inhibit both the influx and the discharge of calcium, supporting the inhibitory effect of propofol on platelets. Platelet aggregation induced by thrombin was not inhibited, even in the higher concentration of propofol, compared with our in vivo and in vitro studies using ADP.

In conclusion, during general anesthesia using Diprivan, platelet aggregation was inhibited, but the fat solvent did not inhibit platelet aggregation, suggesting that propofol itself was responsible for the inhibitory effect. The findings with intracellular calcium using a fluorescent dye, fura-2, also supported the inhibitory effect of propofol. When thrombin, a strong agonist, is used, platelet aggregation is not inhibited. Bleeding time is not prolonged. These findings support the fact that there is no clinical report referring to the hemorrhagic complications, although Diprivan is used in the anesthetic management of patients having surgery.