Mechanisms Involved in the Antiplatelet Activity of Midazolam in Human Platelets

Collagen (type I, bovine achilles tendon), adenosine 5′-diphosphate, midazolam, arachidonic acid, EDTA, luciferin-luciferase, Dowex-1 (100–200 mesh; X⁸, chloride form), prostaglandin E¹, trichloroacetic acid, EGTA, bovine serum albumin, acrylamide, sodium pyruvate, β-NADH, diphenylhexatriene, apyrase, heparin, thrombin, and myoinositol were purchased from Sigma Chemical Co. (St. Louis, MO). Fura 2-AM and fluorescein isothiocyanate (FITC) were purchased from Molecular Probe Inc. (Eugene, OR). Trimeresurus flavoviridis  venom was purchased from Latoxan (Rosans, France). Myo-2-[³H] inositol was purchased from Amersham (Buckinghamshire, United Kingdom). Thromboxane B²enzyme immunoassay kits were purchased from Cayman Co. (Ann Arbor, MI).

Human platelet suspensions were prepared as previously described. 9In this study, human volunteers gave informed consent. In brief, blood was collected from healthy human volunteers who had taken no medicine during the preceding 2 weeks and was mixed with acid/citrate/glucose (9:1, vol/vol). After centrifugation at 120 g  for 10 min at room temperature, the supernatant (platelet-rich plasma) was supplemented with prostaglandin E¹(0.5 μm) and heparin (6.4 IU/ml), then incubated for 10 min at 37°C and centrifuged at 500 g  for 10 min. The washed platelets were finally suspended in Tyrode solution containing bovine serum albumin (3.5 mg/ml) and adjusted to approximately 4.5 × 10⁸platelets/ml. The final concentration of Ca²⁺in Tyrode solution was 1 mm.

The turbidimetric method used a Lumi-Aggregometer (Payton, Canada) as described previously. 10In brief, platelet suspensions (0.4 ml) were prewarmed at 37°C for 2 min, and then midazolam was added for 3 min before the addition of platelet-aggregation inducers. The reaction was allowed to proceed for at least 6 min, and the extent of aggregation was expressed as a percentage of the control (in the absence of midazolam). The degree of aggregation was expressed in light-transmission units. While measuring adenosine triphosphate (ATP) release, 20 μl of a luciferin–luciferase mixture was added 1 min before the addition of agonists, and ATP release was compared with that of the control.

Triflavin, a specific fibrinogen receptor (glycoprotein IIb–IIIa complex) antagonist, was prepared as previously described. 11Fluorescence-conjugated triflavin was also prepared as previously described. 12The final concentration of FITC-conjugated triflavin was adjusted to 1 mg/ml. Human platelet suspensions were prepared as described above. Aliquots of platelet suspensions (4.5 × 10⁸/ml) were preincubated with midazolam (15 and 26 μm) for 3 min, followed by the addition of 2 μl of FITC–triflavin. The suspensions were then incubated for another 5 min, and the volume was adjusted to 1 ml/tube with Tyrode solution. The suspensions were then assayed for fluorescein-labeled platelets with a flow cytometer (FACScan System; Becton Dickinson, San Jose, CA). Data were collected from 50,000 platelets per experimental group. All experiments were repeated at least five times to ensure reproducibility.

The method was conducted as previously described. 13Briefly, citrated human platelet-rich plasma was centrifuged, and the pellets were suspended in Tyrode solution containing [³H]-inositol (75 μCi/ml). Platelets were incubated for 2 h followed by centrifugation, and finally resuspended in Ca²⁺-free Tyrode solution (5 × 10⁸platelets/ml). Midazolam was preincubated with 1 ml of loaded platelets at room temperature for 3 min, and collagen (2 μg/ml) was then added to trigger aggregation. The reaction was stopped after 6 min, and the samples were centrifuged at 1,000 g  for 4 min. The inositol phosphates of the supernatants were separated in a Dowex-1 anion exchange column. Only [³H]-inositol monophosphate (IP) was measured as an index of total inositol phosphate formation.

Citrated whole blood was centrifuged at 120 g  for 10 min. The supernatant was protected from light and incubated with Fura 2-AM (5 μm) at 37°C for 1 h. Human platelet suspensions were then prepared as described above. Finally, the external Ca²⁺concentration of the platelet suspensions was adjusted to 1 mm. The increase in intracellular Ca²⁺concentration ([Ca²⁺]ⁱ) was measured using a fluorescence spectrophotometer (CAF 110; Jasco, Tokyo, Japan) with excitation wavelengths of 340 and 380 nm and an emission wavelength of 500 nm, respectively. The [Ca²⁺]ⁱwas calculated from the fluorescence measured using 224 nm as the Ca²⁺-Fura 2 dissociation constant. 14 

The intensity of fluorescence in human platelets was measured as described previously. 15Briefly, platelets (4.5 × 10⁸/ml) were mixed with nine volumes of sodium–potassium-Tris medium. A solution of diphenylhexatriene in dimethyl formamide was added to the suspension at a final concentration of 0.5 μm. Platelets were preincubated with various concentrations of midazolam (15 and 26 μm) for 3 min followed by the addition of diphenylhexatriene at 37°C for another 6 min. The relative fluorescence intensity of platelets was measured in a fluorescence spectrophotometer (Hitachi F4500; Hitachi, Tokyo, Japan) at 37°C.

Washed human platelet suspensions (4.5 × 10⁸/ml) were preincubated for 3 min in the presence or absence of midazolam (6–26 μm) before the addition of collagen (2 μg/ml). Six minutes after the addition of the agonist, 2 mm EDTA and 50 μm indomethacin were added to the reaction suspensions. The vials were then centrifuged for 3 min at 14,000 rpm. The thromboxane B²concentrations of the supernatants were measured using an enzyme immunoassay kit according to the instructions of the manufacturer.

Lactate dehydrogenase was measured according to previously described methods. 16Platelets (4.5 × 10⁸/ml) were preincubated with various concentrations of midazolam for either 10 or 30 min, followed by centrifugation at 14,000 rpm for 5 min. An aliquot of supernatant was incubated with phosphate buffer containing 0.2 mg β-NADH for 20 min at room temperature. Thereafter, 100 μl of pyruvate solution was added, and the absorbance wavelength was read at 334 nm using an ultraviolet-visible recording spectrophotometer (UV-160; Shimazu, Kyoto, Japan). A maximal value of lactate dehydrogenase was constructed from sonicated platelets.

Washed human platelets (2 × 10⁹/ml) were incubated for 60 min at 37°C with phosphorus-32 (0.5 mCi/ml). Platelet suspensions were next washed twice with Tris-saline buffer. The [³²P]-labeled platelets were preincubated with midazolam (26 μm) in an aggregometer at 37°C for 3 min, then collagen (2 μg/ml) was added for 1 min to trigger protein kinase C activation. Activation was terminated by the addition of Laemmli sample buffer and analyzed by electrophoresis (12.5%, wt/vol) as described previously. 17The gels were dried, and the relative intensities of the radioactive bands were analyzed using a Bio-imaging analyzer system (FAL2000; Fuji, Tokyo, Japan), expressed as photostimulated luminescence per squared millimeters.

Experimental results are expressed as the mean ± SD and are accompanied by the number of observations. Data were assessed by Student t  test, and P < 0.05 was considered statistically significant.

Midazolam dose-dependently inhibited platelet aggregation stimulated by collagen (2 μg/ml), arachidonic acid (100 μm), and thrombin (0.5 U/ml) in human platelet suspensions (figs. 1A and B). Furthermore, midazolam also inhibited the ATP-release reaction when stimulated by agonists (i.e. , collagen;fig. 1A). The IC⁵⁰values of midazolam for platelet aggregation induced by collagen, arachidonic acid, and thrombin were estimated to be approximately 16.1, 23, and 9.7 μm, respectively. In addition, midazolam (26 and 50 μm) did not significantly induce platelet aggregation in the absence of agonists (data not shown). Furthermore, platelets were preincubated with midazolam (26 μm) or normal saline for 10 min followed by two washes with Tyrode solution, and we found that there are no significant differences between the aggregation curves of both platelet preparations stimulated by collagen (2 μg/ml), indicating that the effect of midazolam on inhibition of platelet aggregation occurs in a reversible manner (data not shown). These results also indicate that midazolam significantly inhibits agonist-induced human platelet aggregation.

Triflavin is an Arg-Gly-Asp–containing antiplatelet peptide, purified from Trimeresurus flavoviridis  snake venom. 11,18Triflavin inhibits platelet aggregation through direct interference with fibrinogen binding to the glycoprotein IIb–IIIa complex. 11There is now a multitude of evidence suggesting that the binding of fibrinogen to the glycoprotein IIb–IIIa complex is the final common pathway for agonist-induced platelet aggregation. Therefore, we decided to further evaluate whether midazolam binds directly to the platelet glycoprotein IIb–IIIa complex, leading to inhibition of platelet aggregation induced by agonists.

In this study, the relative intensity of fluorescence of FITC–triflavin (2 μg/ml) bound directly to collagen (2 μg/ml)-activated platelets was 89.2 ± 10.7 (fig. 2A), and it was markedly reduced in the presence of 5 mm EDTA (negative control, 6.6 ± 2.5;fig. 2B). At a concentration of 15 μm, midazolam did not significantly inhibit FITC–triflavin binding to glycoprotein IIb–IIIa in platelet suspensions (90.6 ± 17.4), or even at a higher concentration (26 μm; 88.3 ± 14.5;figs. 2C and D), indicating that the mechanism of midazolam's inhibitory effect on platelet aggregation does not involve binding to the glycoprotein IIb–IIIa complex.

Phosphoinositide breakdown occurs in platelets activated by many different agonists. 19In this study, we found that collagen (2 μg/ml) induces the rapid formation of radioactive IP, IP², and IP³in human platelets loaded with [³H]-inositol. We only measured [³H]-IP formation as an index of total inositol phosphate formation. As shown in figure 3, the addition of collagen (2 μg/ml) resulted in an increase of IP formation of approximately 2.1-fold compared with that in resting platelets ([1.6 ± 0.6 vs.  3.4 ± 0.4]× 10⁻³cpm). In the presence of midazolam (15 and 26 μm), the radioactivity of IP formation in collagen-stimulated human platelets markedly decreased. These results indicate that midazolam interferes with phosphoinositide breakdown in human platelets stimulated by collagen.

Free cytoplasmic Ca²⁺concentrations in human platelets were measured by the Fura 2-AM loading method. As shown in figure 4, collagen (2 μg/ml) evoked an increase of [Ca²⁺]ⁱfrom 36.6 ± 5.0 to 201.6 ± 36.6 nm. This collagen-evoked increase in [Ca²⁺]ⁱwas markedly inhibited in the presence of midazolam (15 μm, 74.6 ± 8.4%; 26 μm, 89.5 ± 12.6%; n = 4). This suggests that midazolam exerts an inhibitory effect on [Ca²⁺]ⁱmobilization in human platelets stimulated by collagen.

As shown in table 1, resting platelets produced relatively little thromboxane B²compared with collagen-activated platelets. Prostaglandin E¹(10 μm) inhibited thromboxane B²formation in collagen-activated platelets by 81% (data not shown). Furthermore, results obtained using various concentrations of midazolam indicated that midazolam (6–26 μm) dose-dependently inhibited thromboxane B²formation in platelets stimulated by collagen (2 μg/ml;table 1). These results suggest that midazolam exerts an inhibitory effect on thromboxane A²formation.

Platelet membrane fluidity was measured in diphenylhexatriene-labeled human platelets. Measurements using the fluorescent probe technique demonstrated that midazolam is capable of direct interaction with platelet membranes (fig. 5). Addition of midazolam (15 and 26 μm) to platelet preparations for 3 min resulted in a dose-dependent decrease in the diphenylhexatriene-related fluorescence intensity. This result implies that the inhibitory effect of midazolam on platelet aggregation may be a result, at least in part, of the results of its effects on platelet membrane fluidity.

In this study, midazolam (26 μm) treatment of human platelets for 10 min did not significantly increase lactate dehydrogenase activity compared with resting platelets (resting platelets, 35.6 ± 5.6 units vs.  midazolam-treated platelets, 37.5 ± 9.8 units; n = 4), even when the incubation time of midazolam with platelets was prolonged to 30 min (34.6 ± 8.6 units; n = 4; data not shown). This result indicates that although midazolam (15 and 26 μm) significantly changes the fluidity of platelet membranes, by itself, it does not affect platelet permeabilization or induce platelet cytolysis under this range of concentrations.

Stimulation of platelets with a number of different agonists induces activation of protein kinase C, which then phosphorylates proteins of Mr  40,000–47,000 in addition to other proteins. 20In this study, phosphorylation experiments were conducted to examine the role of midazolam in activation of protein kinase C in human platelets. When collagen (2 μg/ml) was added to human platelets prelabeled with ³²PO⁴for 2 min, a protein with an apparent Mr  of 47,000 (P47) was predominately phosphorylated as compared with resting platelets (figs. 6A and B). On the other hand, midazolam (26 μm) markedly inhibited the phosphorylation of P47 in human platelets stimulated by collagen (2 μg/ml) when preincubated with midazolam for 3 min. In this study, the extent of radioactivity in P47 was expressed as a relative detection density (photostimulated luminescence per squared millimeters) of the radioactive bands.

The principal objective of this study was to describe the detailed mechanisms involved in the inhibition of agonist-induced human platelet aggregation by midazolam. Midazolam is a familiar agent commonly used to provide sedation and for behavioral emergencies. 1The results demonstrate that midazolam exerts inhibitory activity on human platelet aggregation, a finding that has not been described previously. This inhibitory effect of midazolam was demonstrable with the use of various agonists: collagen, arachidonic acid, and thrombin. The inhibition was directly proportional to the concentration of midazolam used. Ramoska et al.  21demonstrated adequate sedation with effective intravenous doses of 1–3 mg midazolam. Harper et al.  22reported that intravenous doses of 0.3 mg/kg may be required when midazolam is used for sedation in surgical patients. In this study, midazolam was used at concentrations (approximately 6–26 μm) that inhibited platelet aggregation induced by agonists. These results indicate that the concentrations of midazolam used to inhibit platelet aggregation in vitro  are reasonably close to those of blood concentrations obtained during midazolam-induced sedation in vivo . 23In this study, both platelet aggregation and the ATP-release reaction induced by these agonists (i.e. , collagen) appeared to be affected by the presence of midazolam. Therefore, this partly infers that midazolam may affect Ca²⁺release from intracellular Ca²⁺-storage sites (i.e. , dense tubular systems or dense bodies;fig. 4), and this is in accord with the concept that intracellular Ca²⁺release is responsible for the ATP-release reaction. 24 

Although the action mechanisms of various platelet aggregation agonists, such as collagen, arachidonic acid, and thrombin, differ (fig. 7), midazolam significantly inhibited platelet aggregation stimulated by all of them. This implies that midazolam may block a common step shared by these inducers. These results also indicate that the site of action of midazolam is not at the receptor level of individual agonists. Platelet membrane plays a dominant role in regulating and executing platelet functions, particularly platelet aggregation. Triflavin acts by binding to the glycoprotein IIb–IIIa complex on the platelet surface membrane, resulting in interference with the interaction of fibrinogen with its specific receptor. 11,12,18In this study, we found that midazolam did not significantly inhibit FITC-triflavin binding to the glycoprotein IIb–IIIa complex, indicating that the antiplatelet effect of midazolam might not be directly caused by interference with the binding of fibrinogen to its specific receptor on the platelet membrane. Conformational changes in the plasma membrane or changes in membrane fluidity represent a generally accepted mechanism for the antiplatelet effect of numerous drugs, including local anesthetics, chlorpromazine, and β blockers. Therefore, we wondered whether midazolam might also inhibit platelet aggregation by influencing membrane fluidity. To test this hypothesis, the fluorescent probe, diphenylhexatriene, was used to label biologic membranes. In this study, midazolam (15 and 26 μm) dose-dependently decreased the diphenylhexatriene relative fluorescence intensity of platelet membranes. These findings suggest that changes in platelet membrane fluidity may be the primary mechanism responsible for the antiplatelet effect of midazolam in vitro .

Furthermore, stimulation of platelets by agonists (i.e. , collagen) results in phospholipase C–catalyzed hydrolysis of the minor plasma membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, with concomitant formation of inositol 1,4,5-trisphosphate and diacylglycerol (fig. 7). 25There is strong evidence that inositol 1,4,5-trisphosphate induces the release of Ca²⁺from intracellular stores. 26Diacylglycerol activates protein kinase C, inducing protein phosphorylation and a release reaction (fig. 7). In this study, phosphoinositide breakdown of collagen-activated platelets was inhibited by midazolam, suggesting that inhibition of platelet aggregation by midazolam is related to inhibition of phosphoinositide breakdown. Thromboxane A²is an important mediator of the release reaction and aggregation of platelets. 27Collagen-induced formation of thromboxane B², a stable metabolite of thromboxane A², was markedly inhibited by midazolam (6–26 μm;table 1). It has been demonstrated that phosphoinositide breakdown can induce thromboxane A²formation via  free arachidonic acid release by diglyceride lipase or by endogenous phospholipase A²from membrane phospholipids (fig. 7). 28Thus, it seems likely that thromboxane B²formation plays a role in the mediation of the inhibitory effect of midazolam on human platelets.

Furthermore, in this study, activation of protein kinase C in human platelets occurred in response to collagen. The important finding in our studies is that midazolam inhibits collagen-induced activation of protein kinase C. In addition, it has been reported that increased nitric oxide–cyclic guanosine monophosphate formation negatively affects agonist-induced protein kinase C activation. 29However, midazolam (15 and 26 μm) did not significantly increase the concentration of nitric oxide or cyclic guanosine monophosphate in human platelets (data not shown). Therefore, based on the aforementioned observations, we suggest that the inhibitory effect of midazolam in collagen-induced activation of protein kinase C may be a result, at least in part, of its involvement in changing platelet membrane fluidity. This change in membrane fluidity influences the activation of phospholipase C, which is incorporated into plasma membranes, resulting in inhibition of phosphoinositide breakdown and subsequent inhibition of the activation of protein kinase C.

In conclusion, the observations of this study suggest that midazolam inhibits agonist-induced human platelet aggregation. This inhibitory effect may involve the following mechanisms: the antiplatelet activity of midazolam may be caused by the induction of conformational changes in platelet membrane, with a resulting influence on membrane fluidity followed by inhibition of the activation of phospholipase C and subsequent inhibition of phosphoinositide breakdown and thromboxane A²formation, thereby leading to inhibition of both intracellular Ca²⁺mobilization and phosphorylation of P47.