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© 2003 by the American Institute of Ultrasound in Medicine
J Ultrasound Med 22:365-373 • 0278-4297

In Vitro Platelet Activation by an Echo Contrast Agent

Kouichirou Shigeta, MD, Nobuyuki Taniguchi, MD, PhD, Kiyoka Omoto, MD, PhD, Seiji Madoiwa, MD, PhD, Yoichi Sakata, MD, PhD, Masaki Mori, MD, PhD, Kiyohiko Hatake, MD, PhD and Kouichi Itoh, MD, PhD

Departments of Clinical Laboratory Medicine (K.S., N.T., K.O., K.I.) and Hematology (M.M., K.H.) and Division of Cell and Molecular Medicine, Center for Molecular Medicine (S.M., Y.S.), Jichi Medical School, Tochigi-ken, Japan.

Address correspondence and reprint requests to Kouichirou Shigeta, MD, Department of Clinical Laboratory Medicine, Jichi Medical School, 3311-1 Yakusiji, Minamikawachi-machi, Kawachi-gun, Tochigi-ken 329-0498, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Objective. We investigated whether an ultrasonic echo contrast agent containing microbubbles (Levovist [SH U 508A]; Schering AG, Berlin, Germany) could in routine use activate platelets. Methods. Levovist and its main component, galactose, were mixed with separate samples of whole blood (1.5–75 mg/mL) from 5 healthy volunteers to form a 1-mL suspension sample. After in vitro exposure to ultrasound emitted from a commercial ultrasonic scanner at a pulse frequency of 3.5 MHz with a mechanical index of 1.9 and an exposure duration of 5 minutes, 5 µL of the sample was incubated for 20 minutes with the fluorescein isothiocyanate-labeled CD61 antibody, which is a platelet-specific antigen, and the phycoerythrin-labeled CD62P (P-selectin) antibody, an activation-specific antigen, both on the platelet surface. After more than 30 minutes of fixing in 1% paraformaldehyde, flow cytometric analysis was performed. Results. The percentage of CD62P-expressing platelets increased according to the concentrations of Levovist and galactose, which showed almost equal effects. Ultrasound exposure did not enhance the effect except at the highest concentration of Levovist (75 mg/mL). Conclusions. In vitro, a galactose-based echo contrast agent could not activate the platelets at its routine concentration.

Key Words: CD62P • echo contrast agents • in vitro • platelet activation • ultrasound

Abbreviations: ADP, adenosine diphosphate • FITC, fluorescein isothiocyanate • GLUT, glucose transporter • MCV, mean cellular volume • MI, mechanical index • MPV, mean platelet volume • PE, phycoerythrin • RBC, red blood cell


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Echo contrast agents, which consist of microbubbles of galactose, albumin, and other substances, have been developed to improve the sonographic imaging of tissues, and their action as cavitation nuclei has also been studied.1–4 Pulsed ultrasound in the 1- to 10-MHz frequency range can, through resonance, expand and collapse microbubbles violently enough to damage formed elements of the blood and the tissues of the internal organs, a process known as transient acoustic cavitation or inertial cavitation.5 Cavitation-related bioeffects can include hemolysis,6 blood vessel damage in organs,7,8 and DNA damage in cultured cells.9

The platelets play important roles not only in hemostasis but also when activated in the formation of atherosclerotic lesions in blood vessels. Platelets circulate in their resting state, in which they are spherical and are activated by coagulation factors such as thrombin, thromboxane A2, and adenosine diphosphate (ADP) or by mechanical forces such as shear stress.10 Activated platelets extend pseudopodia and release their granules, which contain cytokines and coagulation factors. They contribute to blood coagulation and to the migration and proliferation of smooth muscle cells and monocytes.11

There are many reports of the action of ultrasound on platelets. High-intensity focused ultrasound causes activation, aggregation, and adhesion of platelets.12 Platelets were induced by ultrasound to form aggregates around gas-filled pores in membranes immersed in platelet-rich plasma.13 The platelet-specific protein ß-thromboglobulin was released by ultrasound at therapeutic intensities.14 Furthermore, the action of ultrasound on the microbubbles in echo contrast agents can cause platelet lysis.15 Previous study conditions for cavitation were rather specialized: low counts of formed elements (e.g., blood with 5% hematocrit or platelet-rich plasma alone), longer pulse durations, and lower frequencies than used clinically. The bioeffects of platelet activation have not been studied under routine sonographic diagnostic conditions. The purpose of our study was to determine whether the inclusion of microbubbles in echo contrast agents could affect the activation of platelets. We examined their interaction in diagnostic use in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Basic Study
Blood Collection and In Vitro Agonist Stimulation of Platelets
Three healthy men, 30, 34, and 38 years of age, who were nonsmokers and had not taken any antithrombotic drugs for at least 10 days, were examined after informed consent was obtained. Freshly donated blood was treated with 1 mL of 0.13-mol sodium citrate (pH 5.0) per 10 mL of blood to prevent coagulation. The citrated whole blood was incubated with ADP (A-9665; Sigma-Aldrich Corp, St Louis, MO) at concentrations of 0.1 to 1000 µmol/L for 5 minutes at room temperature without agitation to confirm platelet reactivity and analyzed using a flow cytometer.

Cell Counts After Mixing With Contrast Agent or Galactose
The contrast agent Levovist (SH U 508A; Schering AG, Berlin, Germany) at a quantity of 2.5 g was dissolved in 7 mL of distilled water so that the final concentration was 300 mg/mL, and the solution was used within 15 minutes. In addition, 2.5 g of D(+)-galactose (Kanto Chemical Co, Inc, Tokyo, Japan) was mixed with 7 mL of distilled water to give a final concentration of 300 mg/mL. The fresh Levovist and galactose solutions were carefully mixed with citrated whole blood to yield concentrations of 1.5, 15, and 75 mg/mL. The number of blood cells per microliter (referred to below as blood cell counts) of each sample, the mean cellular volume (MCV) of red blood cells (RBCs), and the mean platelet volume (MPV) were determined in each sample with a hematologic analyzing system (STKS-Retic; Beckman Coulter, Inc, Fullerton, CA) before and after being mixed with Levovist or galactose for 5, 15, and 30 minutes.

Changing Duration After Ultrasound Exposure and Mechanical Index
Samples (1 mL) of Levovist at a concentration of 75 mg/mL were placed in 2-mL polyester centrifuge tubes (8 mm in internal diameter, 0.25 mm in wall thickness; Beckman Coulter, Inc), which were reported to be sufficiently acoustically transparent at frequencies of 1 to 5 MHz.14 The loaded tubes were placed 5 cm from the transducer in a container filled with degassed water at room temperature and moved manually up and down through the imaging plane to help ensure exposure of the cell suspension. The container was made of an acryl board 4 mm in thickness and measured 15 x 15 x 10 cm.

The ultrasonic pulses with a center frequency of 3.5 MHz were focused on the sample from the diagnostic scanner (Acuson Sequoia 512; Siemens Medical Solutions, Mountain View, CA) with a model 5V2c sector-type transducer (whose focus was set at a depth of 5 cm). The exposure conditions were B-mode, a mechanical index (MI) of 1.9, a pulse duration of 0.84 µsec (which covers 3 cycles), and a pulse repetition cycle of 2870 Hz. We recognize that the use of the MI was limited as an exposure parameter but was necessary, because measurements of acoustic output from the systems were otherwise unavailable to us. The samples containing Levovist at a concentration of 75 mg/mL were exposed to ultrasound for 5 minutes, and the CD62P (P-selectin) expression rates were determined immediately and after 5 and 10 minutes with a flow cytometer to ascertain time dependency.

The samples with Levovist at 75 mg/mL were exposed to ultrasound (MI, 0 [sham], 0.5, 1.0, 1.5, and 1.9) for 5 minutes, and CD62P expression was determined with the flow cytometer to study the dependency on the ultrasonic power.

Main Study Procedure
Blood Collection and Levovist, Galactose, Ultrasound, and ADP Exposure
Five healthy men (23–38 years old; mean, 30.4 years), 3 of whom also participated in the basic study, were examined under the same conditions as in the basic study after giving their informed consent. Blood was collected as citrated whole blood, and fresh Levovist solution was mixed in carefully to make up samples of 1 mL. Final concentrations of 1.5, 15, 30, 45, and 75 mg/mL were obtained by adding 5, 50, 100, 150, and 250 µL of fresh Levovist solution to 995, 950, 900, 850, and 750 µL of citrated whole blood, respectively. Each sample was sham exposed or exposed (MI, 1.9) to the scanner for 5 minutes, and then each sample was incubated with or without ADP at 10 µmol/L for 5 minutes, and the CD62P expression was analyzed by flow cytometry (Fig. 1Go). Galactose was mixed with citrated whole blood at concentrations of 1.5, 15, 30, 45, and 75 mg/mL without addition of ADP or exposed to ultrasound, and the CD62P expression was analyzed by flow cytometry.



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  		Figure 1. Experiments in the main study. Citrated whole blood was mixed with Levovist and galactose at concentrations of 0, 1.5, 15, 30, 45, and 75 mg/mL, exposed to ultrasound (US), and incubated with ADP. The blood cells in each sample were counted and analyzed on a flow cytometer.

 
Antibodies
CD61 is a marker of platelet glycoprotein IIIa, which is found on both normal (resting) and activated platelets. We used murine antihuman monoclonal fluorescein isothiocyanate (FITC)-conjugated CD61 (Becton, Dickinson and Company, San Jose, CA) to identify all platelets. CD62P is found on the external membrane of activated platelets after {alpha} granule secretion. Murine antihuman monoclonal phycoerythrin (PE)-conjugated CD62P (Becton, Dickinson and Company) was used to detect platelet activation. Isotype controls were run in parallel with all monoclonal antibodies: FITCconjugated immunoglobulin G1 (BD34041; Becton, Dickinson and Company) and PE-conjugated immunoglobulin G1 (BD34013; Becton, Dickinson and Company).

Preparation of Samples for Flow Cytometry
Each sample of 5 µL was transferred to a tube containing a mixture of 10 µL of FITC-conjugated CD61 antibody and 10 µL of PE-conjugated CD62 antibody and was incubated for 20 minutes in the dark at room temperature. Then 1 mL of ice-cold 1% paraformaldehyde was added to the tube, which was placed in the dark at 4°C for more than 30 minutes, and the samples were analyzed on a flow cytometer. For the flow cytometric analysis, samples must be placed in the dark in an ice-cold situation.

Flow Cytometric Analysis
Flow cytometric analysis was performed on a FACScan (Becton, Dickinson and Company) equipped with an argon ion laser and CellQuest software (Becton, Dickinson and Company). Cytometer performance was verified with FITC and PE calibration beads (Becton, Dickinson and Company; and Flow Cytometry Standards Corporation, Research Triangle Park, NC). Forward light scatter and 2 fluorescent signals were determined for each cell, and 4000 platelet events were inserted into the list mode data files. The platelet population was logic gated by a forward scatter versus side scatter dot plot gate and by an FITC-conjugated CD61 versus side scatter dot plot gate. CD62P-positive activated platelets were identified by fluorescein intensity greater than that of the appropriate isotype control staining sample. The frequencies of CD62P-positive platelets were expressed as percentages of the total platelet population.

Statistical Analysis
Data are represented as mean ± SD unless otherwise stated. Statistical differences were determined by the paired Student t test. P < .05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Basic Study
When the platelets were activated with ADP, the platelet activation rate assessed from CD62P expression increased as the concentration of ADP increased, reaching about 60% at a maximal ADP concentration of 1000 µmol/L. We used ADP at 10 µmol/L in the main study (discussed below) because the expression rate was intermediate, which was appropriate for this study (Fig. 2Go).



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  		Figure 2. Effect of ADP on CD62P expression on platelets in vitro. Citrated whole blood was mixed with the ADP solutions at concentrations of 0.1, 1, 10, 100, 500, and 1000 µmol/L for 5 minutes. Bars represent actual percentages of CD62P-expressing platelets.

 
After the Levovist sample (75 mg/mL) was exposed to ultrasound (MI, 1.9) for 5 minutes, there was no difference in the CD62P expression rate immediately (0 minutes) and after 5 and 10 minutes. We measured the CD62P expression immediately after exposure to ultrasound in the main study using Levovist (Fig. 3Go).



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  		Figure 3. CD62P expression on platelets after ultrasound exposure. Citrated whole blood was mixed with Levovist at 75 mg/mL and exposed to ultrasound for 5 minutes, and CD62P was measured immediately (0) and after 5 and 10 minutes. Bars represent actual percentages of CD62P-expressing platelets.

 
The expression rate increased with the power of the ultrasound (MI) but had almost the same values at MIs of 1.5 and 1.9 (Fig. 4Go).



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  		Figure 4. CD62P expression on platelets after changes in MI. Citrated whole blood was mixed with Levovist at 75 mg/mL and exposed to ultrasound for 5 minutes, and the MI was varied between 0 and 1.9. 0 indicates no exposure to ultrasound. Bars represent actual percentages of CD62P-expressing platelets.

 
Blood cell counts after mixing with Levovist concentrations of 1.5, 15, and 75 mg/mL are presented in Table 1Go. Red blood cell counts did not change significantly in these procedures; however, the platelet counts decreased slightly at 30 minutes in Levovist at a concentration 15 mg/mL and significantly at 5, 15, and 30 minutes at 75 mg/mL. The MCV decreased at 30 minutes in Levovist at 1.5 mg/mL and increased significantly at 5, 15, and 30 minutes at 75 mg/mL. The MPV increased significantly at 30 minutes in Levovist at 1.5 mg/mL, at 15 and 30 minutes at 15 mg/mL, and at 5, 15, and 30 minutes at 75 mg/mL. The effects of galactose on RBC and platelets were almost the same as those at the same concentration of Levovist (75 mg/mL; Table 1Go).


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  		Table 1. Blood Cell Counts and Volumes After Mixing Blood With Levovist and Galactose
 
Main Study
CD62P expression increased with the concentration of the Levovist (Fig. 5Go). The maximal percentage of CD62P expression, 55%, was seen at a Levovist concentration of 75 mg/mL and was significantly greater than that in the control. At concentrations between 1.5 and 45 mg/mL, there was no significant difference from the control. When the expression rate after ultrasound exposure was compared with that without exposure, CD62P expression was significantly increased in the exposed group only at the highest concentration (75 mg/mL). The highest concentration was approximately 5 times clinical levels.



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  		Figure 5. Effect of Levovist concentration and ultrasound exposure on CD62P expression on platelets. Citrated whole blood was mixed with Levovist at concentrations of 1.5, 15, 30, 45, and 75 mg/mL. Open and filled bars represent the nonuse and use of ultrasound, respectively, and show actual percentages of CD62P expression. Asterisks indicate significant differences (P < .05).

 
In the study in which the ADP solution at a concentration of 10 µm/L was added, the CD62P expression rate increased at all concentrations both with and without ultrasound exposure. A significant synergistic effect compared with the control (Fig. 6Go) was observed at concentrations of Levovist from 30 to 75 mg/mL, but no significant difference was observed at concentrations of 1.5 and 15 mg/mL. When results with ultrasound exposure were compared with those with no exposure, no significant difference in CD62P expression was observed at any concentration.



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  		Figure 6. Synergistic effect of ADP on Levovist-induced CD62P expression on platelets. Citrated whole blood was mixed with ADP solution at 10 µmol/L plus Levovist at 1.5, 15, 30, 45, and 75 mg/mL. Open and filled bars represent the nonuse and use of ultrasound, respectively, and show actual percentages of CD62P expression. Asterisks indicate significant differences compared with control.

 
The CD62P expression rate also increased according to the concentration of galactose, but it was almost the same as that in the Levovist experiment. The difference was greater at the highest concentrations of Levovist and galactose (Fig. 7Go)



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  		Figure 7. Effect of concentrations of Levovist and galactose on CD62P expression on platelets. Citrated whole blood was mixed with Levovist (filled squares) and galactose (open squares) at 1.5, 15, 30, 45, and 75 mg/mL. Each square represents the actual percentage of CD62P expression.

 
A representative pair of contour plots of platelets is presented in Figure 8Go. The dots in the top right quadrant of each contour plot represent CD61-positive platelets that coexpress the activation marker CD62P. The results from the Levovist sample at 75 mg/mL exposed to ultrasound are on the right, showing an expression rate of 88.2%, and those of the Levovist sample at 75 mg/mL without exposure are on the left, with an expression rate of 80.2%.



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  		Figure 8. Flow cytometric dot plot of platelets without (A) and with (B) ultrasound exposure in samples containing Levovist. Citrated whole blood was mixed with Levovist at 75 mg/mL and exposed to ultrasound (MI, 1.9). The platelet population is expressed as a dot plot resulting from the gating of FITC-conjugated CD61 versus PE-conjugated CD62P. The number under each contour plot represents the percentage of CD61-positive platelets that coexpress the activation marker CD62P.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study showed that galactose-based echo contrast agents could not activate platelets at their clinical concentrations. If microbubbles collapse violently, mechanical effects may arise through an erosion process, through the growth of intracellular bubbles, or through acoustic microstreams; also, a rising temperature could affect the cells.16 Platelets have a diameter of less than half that of RBCs, so they can be expected to be more resistant to the shear forces that accompany violent bubble collapse.17 Moreover, damage to the platelet membrane does not result in platelet lysis but may affect platelet function.18,19

Recently, several monoclonal antibodies that recognize platelet surface antigens have been developed.20–23 Activated platelets express activation- specific antigens on the platelet surface, for example, PAC-1, and CD62P. PAC-1 is used for the detection of activation of glycoprotein IIb-IIIa, which binds to fibrinogen, and CD62P is used for detection of {alpha} granule degranulation. CD62P has higher thresholds for ADP than does PAC-124 and changes the expression rate in a dose-dependent manner, so it was better as a marker for platelet activation in this study. Antibodies for these specific antigens on the platelet surface can be used for detecting activated platelets by flow cytometry.25 Flow cytometry is likely to be considerably more useful for studies of platelet reactivity than assays of biochemical markers of activation, including platelet factor 4 and ß-thromboglobulin.26 Using this method, several investigators have found platelet activation in patients who underwent cardiopulmonary bypass surgery or who had coronary artery disease and other diseases.27–29 Therefore, we used the anti-CD62P antibody to detect activated platelets by flow cytometry.

In this study we investigated whether a microbubble echo contrast agent exposed to ultrasound could activate platelets in a routine clinical situation. Although a dose-dependent increase in CD62P expression in response to Levovist and galactose was shown between concentrations of 1.5 and 15 mg/mL, which are thought to be practical for clinical use,6 CD62P expression increased slightly but not significantly. Levovist and galactose showed the potential to activate platelets at the high concentration (75 mg/mL, approximately 5 times clinical levels). Ultrasound showed no enhancement effects except at the high concentration (75 mg/mL).

The presence of ADP enhanced the effect, suggesting that the destruction of platelets in vivo could lead to enhanced platelets. Adenosine diphosphate is contained in the dense body of the platelet, is excreted after platelet activation, and can itself activate platelets. CD62P expression increased significantly compared with the control at Levovist concentrations higher than 30 mg/mL but did not increase at concentrations for practical use.

At the maximal concentration of Levovist, CD62P expression increased with the MI but reached a plateau at MIs of greater than 1.5. This may indicate that the platelet activation mechanism of ultrasound has a threshold resulting from a cavitationlike effect.30

In the blood cell counts after the addition and mixing of Levovist or galactose, the RBC count showed no change, but the platelet count decreased with increasing concentrations of either of the 2 solutes. Both the MCV of RBCs and the MPV increased with Levovist and galactose concentrations. Levovist consists of 99.9% galactose and 0.1% palmitic acid, and the results were almost the same for both Levovist and galactose. Red blood cells and platelets express the glucose transporter (GLUT) isoforms GLUT131 and GLUT3,32 respectively. GLUT1 and GLUT3 are known to transport not only glucose but also galactose into RBCs and platelets. Transported galactose is changed predominately into galactitol by aldose reductase because galactose is a better substrate for aldose reductase than glucose, and the stored galactitol in the cells can cause cellular damage via processes such as osmotic dysregulation,33,34 which may be one reason for the activation of platelets.

Although Levovist could activate platelets at the high concentration, to our knowledge, no hemostatic complication in the use of Levovist has been reported. Considering that the maximal clinical concentration is about 15 mg/mL,6 it may not be possible to activate platelets under normal conditions. Moreover, ultrasound is attenuated in the human body, and that effect occurs far less in vitro, so it is probably not powerful enough to cause damage in vivo. We performed this study in vitro, but now an in vivo study of the possibility of the platelet activation is necessary to ensure the safety of contrast-enhanced sonography.


   Footnotes
 
Received April 2, 2002, from the Departments of Clinical Laboratory Medicine (K.S., N.T., K.O., K.I.) and Hematology (M.M., K.H.) and Division of Cell and Molecular Medicine, Center for Molecular Medicine (S.M., Y.S.), Jichi Medical School, Tochigi-ken, Japan. Revision requested May 8, 2002. Revised manuscript accepted for publication December 5, 2002.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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