Hypercholesterolemia is associated with both hypercoagulability and enhanced platelet activation (Labiós et al., 2005). It has been increasingly recognized that the beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) inhibitors (statins) on hypercholesterolemia-associated diseases such as acute coronary syndrome, stroke, and atherosclerotic lesions (Vaughan et al., 2000) are attributable not only to their low-density lipoprotein-lowering effect but also to additional mechanisms of action (Cannon et al., 2005). These “pleiotropic” effects include the stabilization of arterial plaques, normalization of endothelial functions, anti-inflammatory effects, and inhibition of platelet thrombus formation (Endres and Laufs, 2004; Futterman and Lemberg, 2004). In platelets, changes in the membrane cholesterol/phospholipid ratio (Hochgraf et al., 1995), enhanced expression of thromboxane A₂ (Notarbartolo et al., 1995) and α₂-adrenergic receptors (Takemoto and Liao, 2001), and an enhanced intracellular calcium level have been proposed to be associated with hypercholesterolemia. In simvastatin-treated patients with hypercholesterolemia, a reduction in thromboxane A₂ biosynthesis and diminished platelet aggregability have been reported (Notarbartolo et al., 1995). Likewise, treatment with pravastatin resulted in the reduction of intracellular Ca²⁺ levels in platelets (Sang et al., 1995). On a molecular level, pleiotropic effects of statins are attributed to the inhibition of HMGCR in nonhepatic structures such as endothelial cells (Li et al., 2002). This inhibition results in diminished biosynthesis of mevalonate and, finally, in reduction of prenylation processes, essential for proper sorting and function of several cell membrane-associated proteins. Among such proteins are key players in cellular signaling including γ-subunits of heterotrimeric G proteins or small G proteins of the ras or rho classes (Mühlhäuser et al., 2006).

If modification of platelet function by statins proceeds via HMGCR inhibition, uptake of these drugs into platelets is a prerequisite for such drug action. In liver, the prime target organ for statins, uptake of these compounds is influenced by the expression of transport proteins of the organic anion-transporting polypeptide (OATP) family, mainly OATP1B1. However, expression of this protein is restricted to liver (König et al., 2000). In contrast, OATP2B1 has been localized in many other tissues (König et al., 2006). OATP2B1 has been shown recently to transport atorvastatin (Grube et al., 2006) and rosuvastatin (Ho et al., 2006) as high-affinity substrates, whereas other statins are transported with lower affinity.

Therefore, we characterized expression of OATPs in human platelets. Proceeding from the finding that OATP2B1 is highly abundant in these blood cells as indicated by immunoblotting we analyzed subcellular localization in the plasma membrane and the respective transport function. Furthermore, we could identify the OATP2B1 protein with a very sensitive LC-MS/MS/MS method in platelet membranes. A second prerequisite for an antithrombotic action of statins is the presence of the target structure, i.e., HMGCR. Here, we demonstrate the expression of HMGCR in platelets. To study the link between the inhibition of HMGCR and platelet aggregation, we examined in addition modification of the intracellular calcium signaling by atorvastatin, resulting from inhibition of mevalonate production by platelet HMGCR.

Materials and Methods

Reagents and Antibodies. The mouse monoclonal antibodies anti-Gi27 and anti-Gi5 against the β-subunit of the glycoprotein Ib (GpIb, CD42Ib) receptor and the GPIIb/IIIa (epitope formed by both subunits) receptor, respectively, were kindly provided by Dr. Santoso (Department of Immunology and Transfusion Medicine, Justus-Liebig-University, Gießen, Germany). The mouse monoclonal CD62P antibody against P-selectin was obtained from Beckman Coulter (Krefeld, Germany), and the mouse monoclonal antibodies against LAMP2 and integrin β3 (CD61) as well as the goat polyclonal antibodies to GpIb and P-selectin were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The rabbit polyclonal antibodies against platelet HMGCR were obtained from Millipore Corporation (Billerica, MA) and Santa Cruz Biotechnology Inc. The super paramagnetic and secondary antibody-coated Dynabead M-280 sheep anti-rabbit IgG and pan anti-mouse IgG and the magnet were obtained from Invitrogen (Karlsruhe, Germany).

The anti-OATP2B1-r antibody was raised in rabbits against the 15 amino acids at the carboxyl terminus of the deduced OATP2B1 sequence (LLVSGPGKKPEDSRV) as described previously (Grube et al., 2006). The anti-OATP2B1-g antibody was purchased from Santa Cruz Biotechnology Inc. This affinity-purified goat polyclonal antiserum was raised against a peptide mapping within an internal region of OATP2B1.

Preparation of Platelet-Rich Plasma and Subcellular Fractions. Platelet-rich plasma (PRP) and platelet subcellular fractions were prepared as described previously (Greinacher et al., 1991; Jedlitschky et al., 2004; Niessen et al., 2007).

Immunoblot Analysis. Protein concentrations of subcellular fractions were analyzed using the bicinchoninic acid method (Smith et al., 1985); subsequently, 50 μg of each fraction was loaded on a 7.5% SDS-polyacrylamide gel after denaturation at 95°C for 10 min. Immunoblotting to polyvinylidene difluoride membranes was performed using a tank blotting system (Bio-Rad, Hercules, CA). The membrane was blocked with 5% dry milk in Tris-buffered saline containing 0.05% Tween 20 and 1% BSA (TBST). Primary antibodies for Western blotting were diluted in TBST to the following final concentrations: polyclonal rabbit and goat anti-OATP2B1 and polyclonal rabbit anti-HMGCR, 1:1000. Secondary horseradish peroxidase-conjugated goat anti-rabbit as well as horse anti-goat IgG antibody (Vector Laboratories, Burlingame, CA) were used at a 1:2000 dilution. Signals were detected on an X-ray film using an enhanced chemiluminescence detection system (GE Healthcare, Piscataway, NJ). Results were quantified by densitometric analysis (ImageQuant 5.0 program), and the relative optical densities of the specific bands were calculated.

Immunofluorescence Microscopy. Coverslips incubated in acetone (5 min) and washed with double-deionized water were covered with 40 μlof human collagen type I (1 mg/ml; Sigma-Aldrich, Munich, Germany), incubated (1.5 h, 37°C), washed twice with 1 ml of PBS (all PBS wash steps were 10 min), incubated with 20 μl of platelet suspension (1 × 10¹¹/l, 30 min, room temperature), washed three times with 1 ml of PBS (pH 7.3), and fixed by formaldehyde in PBS (1%, 30 min, room temperature). After additional washes with PBS, platelets were permeabilized with 1% saponin in PBS (30 min, room temperature) and blocked using 20% human serum in PBS (15 min). Antibody staining was carried out using the primary antibodies at the following dilutions: goat polyclonal antibodies to GpIb and HMGCR 1:100 and rabbit polyclonal antibody to OATP2B1 1:50. The respective secondary antibodies, either conjugated to Alexa Fluor488 or Alexa Fluor568, were used at a dilution of 1:250 or 1:50, respectively. Fluorescence micrographs were taken with a confocal laser scanning microscope (Chromaphor Analysen Technik, Duisburg, Germany) with a 100× oil-immersion objective. A charge-coupled device camera and Improvision software from VisiTech International (Sunderland, UK) were used for analysis.

For detection of transporter localization in megakaryocytes, air-dried bone marrow aspirates (kindly provided by Professor Doelken, Department of Hematology and Oncology, University of Greifswald) were fixed for 10 min in ethanol at –20°C. Afterward they were washed in PBS and incubated for 5 min with 0.01% Triton X-100 for permeabilization. Cells were blocked with 5% fetal calf serum, washed again, and incubated with the primary antibody overnight. After subsequent washing steps and blocking with 5% fetal calf serum for 30 min, slides were incubated with Alexa Fluor secondary antibodies (Invitrogen, Breda, The Netherlands) for 1 h. After the washing steps, slides were incubated with TOTO-3-iodide (Invitrogen) in a 1:1000 dilution with Dako fluorescent mounting medium (Dako North America, Inc., Carpinteria, CA) for nuclei staining.

LC-MS/MS/MS Detection of OATP2B1. The protein expression of OATP2B1 was examined by the LC-MS/MS/MS method as described previously (Kamiie et al., 2008) with slight modifications as follows; for detection, VLLQTLR was selected as a tryptic peptide fragment specific for OATP2B1 in humans by checking against the protein database of the National Center for Biotechnology Information. Platelet membranes were prepared and suspended in 1 mM Tris-sucrose buffer, and the samples were S-carbamoylmethylated and precipitated with a mixture of methanol and chloroform. The precipitates were dissolved in 6 M urea, diluted with 100 mM Tris-HCl (pH 8.0), and digested with N-tosyl-l-phenylalanine chloromethyl ketone-treated trypsin (Promega, Madison, WI) at an enzyme/substrate ratio of 1:100 at 37°C for 16 h. The tryptic digests were acidified with formic acid, and 100 fmol of the stable isotope-labeled peptides specific for human OATP2B1 (VLLQTL*R) were spiked into the digest as an internal standard to confirm the expression of OATP2B1. Synthesized nonlabeled target peptide (VLLQTLR, 0.100 or 10.0 fmol) was mixed with 50 fmol of isotope-labeled peptides and analyzed by an HPLC system (20A Prominence; Shimadzu, Kyoto, Japan) connected to an electrospray ionization-triple quadrupole mass spectrometer, in which the third quadrupole acts as a linear ion trap (AB SCIEX QTRAP 5500; Applied Biosystems, Foster City, CA). HPLC was performed with a C18 column (Zorbax SB-C18 0.5 mm i.d. × 150 mm, 5 μm particles). Linear gradients of 1 to 45% acetonitrile in 0.1% formic acid were applied to elute the peptides at a flow rate of 50 μl/min for 60 min. The mass spectrometer was set up to run MS/MS/MS experiments for peptide detection by using the dynamic fill time mode. The signal for each peptide was detected by specific m/z channels consisting of three mass filters combined with sequential peptide breakages: 421.8/372.4/517.3 for the authentic peptide and 425.5/376.0/524.3 for the stable isotope-labeled peptide [m/z for precursor ion (VLLQTLR, z = 2), MS2 product ion (LLQTLR, z = 2), and MS3 product ion (LQTLR, z = 1), respectively]. The precursor ions were selected by the first mass filter, then the MS2 product ions were selected by the second mass filter from the ions produced from the precursor ions by collision, and, after the second collision, the MS3 product ions were selected by the third mass filter from ions produced from the MS2 product ions. Individual signal peaks are identified on the basis of equal retention times of MS/MS/MS product ions derived from the authentic peptide and stable isotope-labeled peptide with Analyst software version 1.5 (Applied Biosystems).

Megakaryocyte Cultures. CD34⁺ stem cells were isolated from human umbilical cord blood and were differentiated into megakaryocytes that produce proplatelets using methods that were described previously (Denis et al., 2005).

Preparation of Platelet, Megakaryocyte, and Human Liver RNA and RT-PCR. Human platelets were isolated from healthy volunteers; residual leukocytes were removed from the washed preparations by CD45⁺ bead selection as described previously (Schwertz et al., 2006). Platelets were resuspended in M199 serum-free culture medium at a concentration of 1 × 10⁹ cells/ml. Platelets were left quiescent or allowed to adhere to immobilized human fibrinogen (Calbiochem, Darmstadt, Germany) in the presence of thrombin (0.05 U/ml; Sigma-Aldrich). For RNA isolation, platelets and differentiated megakaryocytes were lysed in TRIzol reagent, and the RNA was isolated according to the manufacturer's protocol. RNA preparation of human liver was performed with a NucleoSpin RNA II Kit (Machery-Nagel, Düren, Germany) according to the manufacturer's protocol. The concentrations of RNA were measured using a nanodrop photospectrometer (PEQLAB Biotechnologie GmbH, Erlangen, Germany). cDNA was prepared using 500 ng of each RNA sample and a Reverse Transcriptase Core kit (Eurogentec, Seraing, Belgium) according to the manufacturer's protocol. TaqMan real-time PCRs for OATP2B1, HMGCR, and OATP1B1 were performed with 20 ng of reverse-transcribed RNA, respectively, on an ABI Prism 7900 HT system using specific gene expression assays (OATP1B1 Hs00272374_m1, OATP2B1 Hs00200670_m1, and HMGCR Hs00168352_m1; Applied Biosystems, Darmstadt, Germany). For each respective assay a “no template sample” served as a negative control. mRNA expression values of human platelets, megakaryocytes, and hepatic control samples were determined.

After amplified segments were run on a 3% agarose gel with Tris-acetate-EDTA buffer (pH 8) at 70 V for 45 min. The gel was stained with a 0.05-μg/ml ethidium bromide solution and visualized under ultraviolet light.

Transport Studies. Transport studies were performed with PRP. PRP (500,000 platelets/ml) was incubated with [³H]estrone sulfate (5 μCi/ml, specific activity: 50 Ci/mmol; Hartmann Analytic, Braunschweig, Germany) in the presence or absence of atorvastatin (100 μM) for 30 min. After addition of 111 μl/ml adenine-citrate-dextrose anticoagulant, 0.1 μM PGE₁, and 5 μl/ml 1000 U/ml apyrase, PRP was centrifuged (7 min, 650g). The supernatant was discarded, and platelets were resuspended in washing buffer (0.9% NaCl, 0.05% EDTA, and 0.2% BSA, pH 7.2) containing 50 μM unlabeled E1S and 0.1 μM PGE_l. PRP was incubated for 15 min at 37°C. Then the platelets were centrifuged again, the supernatant was discarded, platelets were lysed using 0.2% SDS, and 100 μl of each sample was mixed with a scintillation cocktail (Rotiszint; Roth, Karlsruhe, Germany) and measured in a scintillation beta-counter (type 1409; LKB-Wallac, Turku, Finland).

Direct uptake of atorvastatin and its inhibition with unlabeled E3S were investigated in a similar manner. Atorvastatin uptake was determined by LC-tandem mass spectrometry (LC-MS/MS) measurement of atorvastatin in the lysate as described previously. Rates of transport are given in nanograms per milliliter of PRP. All transport studies were performed at 37 and 4°C as controls for unspecific binding. PRP (500,000 platelets/ml) was incubated with atorvastatin (5 and 20 μM) in the presence or absence of E1S (100 μM) for 20 min. After addition of 111 μl/ml adenine-citrate-dextrose anticoagulant, 0.1 μM PGE₁, and 5 μl/ml 1000 U/ml apyrase, PRP was centrifuged (7 min, 650g). The supernatant was discarded, and platelets were resuspended in washing buffer (0.9% NaCl, 0.05% EDTA, and 0.2% BSA, pH 7.2) containing 0.1 μM PGE_l. PRP was incubated for 15 min at 37°C. Then, the platelets were centrifuged again, the supernatant was discarded, and platelets were lysed using a buffer containing 0.2% SDS and 5 mM EDTA.

Quantitative Assay for Atorvastatin. Concentrations of atorvastatin in cell homogenates were quantified via a validated LC-MS/MS method by using an Agilent 1100 series HPLC system (Agilent Technologies, Waldbronn, Germany) coupled with a PerkinElmerSciex API 4000 mass spectrometer (Applied Biosystems, Darmstadt, Germany). In brief, 100 μl of cell lysate sample were mixed with 350 μl of ice-cold acetonitrile, 50 μl of formic acid (1% v/v), and 25 μl of internal standard solution (50 μg/ml simvastatin acid; SynFine Research, Richmond Hill, ON, Canada) and centrifuged for 5 min at 10.900 rpm. Then, 100 μl of the resulting supernatant was transferred into vials of which 5 μl was injected into the chromatographic system. Chromatography was performed using the following step-gradient elution (flow rate, 200 μl/min): 0 to 0.5 min, 50% A/50% B; 0.5 to 4 min, 20% A/80% B; and 4 to 10 min, 50% A/50% B (A, 0.1% formic acid, pH 3.5; B, acetonitrile) using an XTerra MS column (C18, 2.1 × 100 mm, particle size 3.5 μm; Waters, Milford, MA) temporized at 40°C. In the positive multiple reaction monitoring mode, the monitored mass/charge ratio transition for atorvastatin was 559.2 to 440.2. The method was validated between 1 and 1500 ng/ml. All chromatograms were evaluated with Analyst 1.4 software (Applied Biosystems), applying the internal standard method by use of peak area ratios for calculation of the calibration curves (linear regression, 1/x weighting).

During drug analysis, within-day and between-day precision and accuracy of all calibrators and quality control samples for atorvastatin were less than 15%, fulfilling the requirements of the current bioanalytical guideline from the Food and Drug Administration (http://www.fda.gov/cder/guidance/4252fnl.htm).

Measurement of Intracellular Calcium Concentrations ([Ca²⁺]_i). Measurements of [Ca²⁺]_i were performed with the calcium-sensitive fluorescent dye Fura2 (Sigma-Aldrich) (Grynkiewicz et al., 1985). To investigate for effects of atorvastatin, PRP was preincubated with different concentrations of atorvastatin alone, with atorvastatin and the competing OATP2B1-substrate estrone sulfate (E1S), with the prenylation inhibitor N-acetyl-S-farnesyl-l-cysteine (AFC) (Axxora, Lörrach, Germany). Furthermore, we add mevalonate, a cholesterol precursor, downstream of HMGCR, to the incubations with atorvastatin or the solvent DMSO for 6 h. As a control statin, the active form of the prodrug lovastatin (lovastatin acid) was tested in the same manner. Subsequently, the membrane-permeant Fura2-acetoxymethylester was added (final concentration 5 μM), and the platelet suspension was further incubated at 37°C for 45 min under light protection. PGE₁ (0.1 μM) (Calbiochem) was added to the suspension, and platelets were harvested by centrifugation (900g, 15 min), washed once in resuspension buffer (10 mM HEPES, 145 mM NaCl, 5 mM KCl, and 5.5 mM glucose, pH 7.4), and finally resuspended in HEPES buffer (140 mM NaCl, 5 mM KCl, 5 mM KH₂PO₄, 1 mM MgSO₄, 10 mM HEPES, and 5 mM glucose, pH 7.4) and kept at room temperature and under light protection until use. Fura2 fluorescence was recorded in platelets (2-ml aliquots) at room temperature with gentle stirring using a PerkinElmer LS50-B spectrofluorimeter equipped with a fast filter device at excitation wavelengths of 340 and 380 nm and emission wavelengths of 510 nm essentially as described previously (Rosskopf et al., 2003). Fluorescence intensity ratios were calibrated in terms of [Ca²⁺]_i by the digitonin-EGTA method and [Ca²⁺]_i was calculated using the equation of Grynkiewicz et al. (1985): where K_d = 224 nM, the dissociation constant of the Fura2 and Ca²⁺ complex, R is the measured fluorescence ratio of 340/380, R_max is the maximal ratio of fluorescence when the cells were permeated by 0.2 mg/ml digitonin, allowing Ca²⁺ to saturate all intracellular Fura2, R_min is the minimal ratio of fluorescence after chelation of Ca²⁺ by addition of 10 mM EGTA, and Sf2/Sb2 is the ratio of the fluorescence at 380 nm of free Fura2 and of Fura2 saturated by Ca²⁺.

After establishment of a baseline recording, platelets were stimulated with 1 U/ml thrombin (thrombin, bovine; Sigma-Aldrich). The maximum rise in [Ca²⁺]_i for each platelet control preparation [pretreated with the solvent DMSO (0.1%) only], i.e., the difference between maximum and baseline [Ca²⁺]_i was determined. All results are means ± S.D. from three independent experiments with measurements each performed as triplicates/quadruplicates. To compare different platelet preparations, the mean maximum thrombin-induced rise in [Ca²⁺]_i in control platelets was defined as 100%.

Statistical Methods. Values are mean ± S.D. or S.E.M. as indicated in the figure legends. Student's t test was used for comparison of the uptake data. The differences were considered significant at P ≤ 0.05. The IC₅₀ value was calculated with GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, CA).

Results

Protein Detection of OATP2B1 in Human Platelets. Expression of OATP2B1 was analyzed by immunoblotting of crude membrane fractions of human platelets and human liver using OATP2B1 antibodies directed against two different epitopes of the OATP2B1 protein. Two bands of approximately 84 and 60 kDa, representing the glycosylated and unglycosylated protein, respectively, were detected in liver and in platelets with higher intensity in liver with both antibodies (Fig. 1A). OATP2B1 glycosylation has been demonstrated (Tamai et al., 2000; Hänggi et al., 2006), and we have obtained similar results in a peptide-N^–-(N-acetyl-β-glucosaminyl)asparagine amidase assay with platelet OATP2B1 as described before for the recombinant protein (figure not shown). As a measure for the expression level of OATP2B1 in platelets compared with liver, immunoblot results were quantified by densitometric analysis (ImageQuant 5.0), and the relative optical density of the specific bands was calculated. The expression level of OATP2B1 in liver was 2.1-fold higher with the anti-OATP2B1-r antibody and 4.5-fold higher with the use of the anti-OATP2B1-g antibody compared with the expression level in platelets. In addition, variations in expression levels in a sample collection of 12 different subjects (6 male and 6 female healthy blood donors) were investigated, and a low variability in interindividual OATP2B1 protein expression was detected (<20% difference in optical density of the specific band; data not shown).

The platelet crude membranes were further subjected to subcellular separation with immunomagnetic beads. Three fractions enriched in plasma membrane, α-granules, and dense granules were precipitated by the fraction-specific antibodies, Gi5, P-selectin, and LAMP2, respectively (Fig. 1C), and OATP2B1 expression in these fractions was analyzed. The plasma membrane fractions exhibited the highest amount of OATP2B1. OATP2B1 could not be detected in dense and alpha granules. As a control, anti-OATP2B1-r was also used as precipitating antibody.

LC-MS/MS/MS Detection of OATP2B1. The expression of OATP2B1 protein in the platelet membrane was examined by MS/MS/MS analysis. As shown in Fig. 2A, a significant peak was observed at 28.9 min in the trypsin-digested platelet membrane at the m/z channel for VLLQTLR (421.8/372.4/517.3), which is a tryptic peptide specific for OATP2B1 (Fig. 2A). At the same retention time, a peak of stable isotope-labeled peptide was also detected at the m/z channel (425.5/376.0/524.3) for the labeled peptide, VLLQTL*R (Fig. 2B). The peak of 10.0 fmol of authentic peptide and that of stable isotope-labeled peptide were detected at the retention time within a 0.1 min difference (30.1 and 30.0 min, respectively) (Fig. 2, C and D), whereas the peak was not observed for 0.1 fmol of authentic peptide at the retention time when that of stable isotope-labeled peptide was detected (Fig. 2, E and F). These results suggest expression of OATP2B1 protein in the human platelet membrane.

OATP1B1 and OATP2B1 mRNA Detection in Platelets and Megakaryocytes. RNA was isolated from human platelets, differentiated megakaryocytes, and human liver. Using quantitative real-time RT-PCR and separation of the amplified segments on an agarose gel, significant amounts of OATP2B1 (Fig. 1B, top panel) transcripts could be detected in platelet RNA before (Fig. 1B, lane 5) and after a 2-h thrombin stimulation (Fig. 1B, lane 6) in RNA from purified megakaryocytes (Fig. 1B, lane 4) as well as in human liver RNA (Fig. 1B, lane 3) as a positive control. The C_T values amounted to 32.0 ± 1.6 and 33.4 ± 3.2 before and after a 2-h thrombin stimulation in platelets, 28.1 ± 0.4 in megakaryocytes, and 27.9 ± 0.25 in the liver control sample (mean ± S.D., n = 4; 20 ng cDNA/sample). The expression of liver-specific uptake transporter OATP1B1 (Fig. 1B, bottom panel), also a candidate for the atorvastatin uptake, could not be detected in platelet (Fig. 1B, bottom panel, lanes 5 and 6)- or in megakaryocyte-derived RNA (Fig. 1B, bottom panel, lane 4). Human liver served as a positive control (Fig. 1B, bottom panel, lane 3) in this experiment. For each respective assay a no template sample served as a negative control (Fig. 1B, top and bottom panels, lane 7) (C_T undetermined after 45 cycles).

Immunolocalization of OATP2B1 in Human Platelets and Megakaryocytes. The subcellular localization of OATP2B1 in human platelets was further investigated using immunofluorescence microscopy, revealing positive staining mainly in the plasma membrane (Fig. 3A). Control staining with the preimmune serum (Fig. 3D) indicated the specificity of the signal. Furthermore, to confirm the subcellular localization, OATP2B1 was costained with glycoprotein Ib, a marker for plasma membrane (Fig. 3B), demonstrating a complete overlay of both signals (Fig. 3C). In addition, OATP2B1 expression was investigated in hematopoietic progenitor cells by immunostaining of human bone marrow samples and could be localized in cells staining positive for CD61, a marker for megakaryocytes (Fig. 3, E–H).

Detection of HMGCR in Platelets. Expression of HMGCR, the primary target of atorvastatin, was analyzed by immunoblot analysis as shown in Fig. 4A. The anti-HMGCR antibody detected a strong band around 45 kDa in human platelet lysates and liver as positive controls. HMGCR transcripts were detected by real-time RT-PCR in RNA from purified platelets with C_T values of 30.9 ± 1.5 and 33.0 ± 0.8, before and after a 2-h thrombin stimulation, respectively, and in RNA from purified megakaryocytes with a C_T value of 30.7. In comparison, RNA from human liver revealed a C_T value of 27.1. Separation of the fragments obtained on an agarose gel is shown in Fig. 4B. For each respective assay a no template sample served as a negative control (Fig. 4B, line 7) (C_T undetermined after 45 cycles). The subcellular localization of HMGCR in human platelets was further investigated using immunofluorescence microscopy. As shown in Fig. 4C, the anti-HMGCR antibody revealed a positive intracellular staining. To confirm the subcellular localization of HGMCR, double-label experiments were performed using costaining with glycoprotein Ib as a marker for plasma membrane. The overlay image in Fig. 4C clearly indicates no colocalization with plasma membrane (GpIb). In addition, no colocalization was observed with markers for dense (LAMP2) or alpha (P-selectin) granule structures (data not shown), suggesting a cytosolic localization.

E1S Transport into Platelets and Its Inhibition by Atorvastatin. The transport function of OATP2B1 was tested in isolated human platelets. PRP was incubated with tritium-labeled E1S, and cellular accumulation was determined. In addition, inhibition of uptake by atorvastatin was examined. Platelets were incubated with [³H]E1S (10 μM) in the presence and absence of 100 μM atorvastatin at 37°C. Platelet-associated radioactivity at 4°C was subtracted from the uptake values at 37°C to obtain rates of diffusion-independent transport. The coincubation with atorvastatin showed a significant inhibition (p ≤ 0.05) of E1S uptake (74 ± 14.53% of control) in comparison with the control (100 ± 18.6%) (Fig. 5A).

Direct Atorvastatin Uptake Measurement by LC-MS/MS. The interaction of atorvastatin with OATP2B1 was further characterized by measurement (LC-MS/MS) of direct atorvastatin uptake into the platelets (Fig. 5B). In the experimental setup, platelets were incubated with 5 and 20 μM atorvastatin in the presence or absence of 100 μM E1S for 20 min. Platelet-associated atorvastatin uptake values at 37 and at 4°C were plotted to obtain rates of diffusion-independent transport. At 5 μM atorvastatin uptake of 700.2 ± 106.2 ng/ml PRP at 37°C was observed, compared with 68.9 ± 10.2 ng/ml at 4°C). The respective values for 20 μM atorvastatin were 900.2 ± 87.8 ng/ml PRP at 37°C and 136.3 ± 3.6 ng/ml PRP at 4°C. The coincubation with atorvastatin (5 and 20 μM) and E1S (100 μM) showed significant inhibition of atorvastatin uptake [35.0 ± 8.23 ng/ml at 5 μM atorvastatin (p ≤ 0.001; 37°C) and 56.0 ± 11.1 ng/ml at 20 μM atorvastatin (p ≤ 0.0001; 37°C)].

Influence of Atorvastatin on Intracellular Calcium Mobilization. To further examine whether atorvastatin treatment affects platelet functions we analyzed thrombin-induced calcium mobilization in platelets preincubated with atorvastatin or its solvent DMSO. Calcium mobilization was strongly inhibited by atorvastatin in a concentration-dependent manner (Fig. 6A). Calcium mobilization in atorvastatin-treated platelets was potently reduced upon stimulation with the strong agonist thrombin to 37.3 ± 6.7% of controls in platelets pretreated with 15 μM atorvastatin, the highest concentration used in these experiments. The calculated IC₅₀ value of atorvastatin on intracellular calcium mobilizing was 8.04 μM. Furthermore, we studied the effect of lovastatin acid, the hydrolysate of the prodrug lovastatin. At a concentration of 15 μM a maximal inhibition to 78 ± 8% of control was observed.

To demonstrate that atorvastatin-mediated inhibition of Ca²⁺ mobilization is related to OATP2B1-mediated atorvastatin uptake, we performed competition experiments with the well characterized OATP2B1 substrate estrone sulfate. Figure 6B depicts results from a representative experiment. Pretreatment with 10 μM atorvastatin resulted in diminished [Ca²⁺] increases after thrombin stimulation of only 44.6 ± 11.6% of control (n = 3; p ≤ 0.01). However, coincubation with 10 μM E1S attenuated the atorvastatin-mediated inhibition and [Ca²⁺] increases were quantified at 69.0 ± 6.7% of control (n = 3; p ≤ 0.05). Sole incubation with 10 μM E1S had no significant effect on thrombin-stimulated [Ca²⁺] signals compared with control platelets. With lovastatin as well as with lovastatin acid no significant effects were observed (104.6 ± 8.1% of control and 76.6 ± 10.6% of control, respectively; n = 3; p ≤ 0.01). We used AFC (10 μM) in control experiments and observed significantly (p = 0.01) decreased thrombin-induced Ca²⁺ mobilization to 51.0 ± 9.3% of control (n = 3) in the presence of AFC (Fig. 6B). Furthermore, we studied the effect of adding mevalonate, which is a precursor to cholesterol and hydrophobic prenyl moieties, alone and in the presence of atorvastatin (Fig. 6C). Mevalonate (0.5 mM) reversed the inhibitory effects of atorvastatin on [Ca²⁺] signals induced by thrombin (44.6 ± 11.6%) to 99.0 ± 10.1% of control.

Discussion

The present study identifies OATP2B1 as a candidate for carrier-mediated transport of statins in human platelets. This assumption is based on the following observations. Immunoblotting and immunofluorescence microscopy showed the high abundance of OATP2B1 in platelets and its predominant localization in the platelet plasma membrane, as indicated by colocalization with GpIb, which is specifically concentrated in plasma membranes. Furthermore, the tryptic peptide fragment (VLLQTLR) specific for OATP2B1 was detected in a tryptic digest of platelet membrane proteins using a very sensitive LC-MS/MS/MS method (Fig. 2). As described under Materials and Methods, we detected the specific tryptic peptides by three different m/z values of peptide ions, which included those of precursor ion, MS2 product ion, and MS3 product ion produced by sequential collisions. This procedure enabled us to confirm the sequence information of the peptide fragment detected. We also demonstrated expression of OATP2B1 mRNA in human platelets and megakaryocytes, using real-time RT-PCR. Protein synthesis by anucleated platelets was reported two decades ago (Kieffer et al., 1987), but only recently was de novo synthesis of several polypeptides by human platelets convincingly demonstrated (Lindemann et al., 2001). Denis et al. (2005) found constitutive pre-mRNA in human platelets, and critical factors were able to splice it upon activation, generating mature mRNA and new protein. In this context, the detection and splicing of tissue factor-mRNA have been demonstrated in platelets (Schwertz et al., 2006), and we could also detect OATP2B1 but not OATP1B1 expression in megakaryocytes on the mRNA as well as protein level.

The role of OATP2B1 in platelet plasma membranes is supported by functional studies, demonstrating an active E1S uptake into platelets, which is significantly reduced by atorvastatin. Furthermore, we could demonstrate active transport of atorvastatin into platelets using a LC/MS/MS detection method. We have chosen atorvastatin as a model statin for functional analysis because it has been described as a high-affinity substrate for OATP2B1 (Grube et al., 2006). In addition, it can achieve high systemic concentrations (Cilla et al., 1996). Maximum plasma concentrations occur over a range of 30 min to 6 h. The elimination half-life varies from 15 to 58 h, so atorvastatin can accumulate in blood (Lins et al., 2003), whereas most of the other statins have markedly shorter elimination half-lives. The half-maximal inhibition of HMGCR by atorvastatin (IC₅₀ between 40 and 100 nM) is significantly lower than that of pravastatin and simvastatin (IC₅₀ between 100 and 300 nM), for example, indicating that a sufficient concentration of atorvastatin can be reached in platelets, provided that there is an uptake mechanism (Jones et al., 1998). The differences in HMGCR inhibition hamper the direct comparison of several statins in the Ca²⁺ release assay, with regard to the evaluation of uptake. However, we used lovastatin as a control compound. The IC₅₀ value for lovastatin is relatively similar to that of atorvastatin. However, in contrast to atorvastatin, lovastatin did not seem to be a good substrate for OATP2B1 (Grube et al., 2006). Therefore, we investigated the effect of lovastatin and its active metabolite, lovastatin acid, on the Ca²⁺ release in platelets. As expected, we could observe no significant effect of both compounds on the platelet Ca²⁺ release, probably due to the low uptake. A contribution of other transport proteins to the observed E1S transport cannot be excluded. However, as for the statin transport, the major transporter in liver, OATP1B1, seems to be predominantly expressed only in this tissue (König et al., 2000). The present study indicates a lack of OATP1B1 expression in human platelets (Fig. 1B).

Agonist-induced Ca²⁺ mobilization is a key step in platelet signal transduction ultimately leading to platelet aggregation. Here, we provide evidence that uptake of atorvastatin into platelets is accompanied by a strong reduction in thrombin-induced Ca²⁺ mobilization. Competition experiments with E1S support the notion that the uptake of atorvastatin by OATP2B1 is actually involved in this mechanism. Statin-mediated effects on cellular signaling are commonly explained by an altered prenylation of signal proteins, for example, γ-subunits of heterotrimeric G proteins (Mühlhäuser et al., 2006) and membrane receptors (O'Meara and Kinsella, 2004). AFC interferes with the prenylation process and has been demonstrated to inhibit agonist-mediated signaling in human platelets, including Ca²⁺ signaling (Rosado and Sage, 2000). Of note, the effects we detected for atorvastatin were of a magnitude similar to those generated by this specific inhibitor of the posttranslational prenylation process. Furthermore, we studied the effect of adding mevalonate, a precursor to cholesterol and hydrophobic prenyl moieties. Mevalonate reversed the inhibitory effects of atorvastatin on [Ca²⁺] signals induced by thrombin, indicating that the inhibitory mechanism includes inhibition of HMGCR. The concept of atorvastatin absorption by OATP2B1 followed by inhibition of protein prenylation as devised above requires the imperative presence of autonomous mevalonate production by HMGCR in platelets as a prime target for the statin. In this study, we demonstrated actual HMGCR expression in human platelets on the mRNA and on the protein level.

Available evidence supports the notion that atorvastatin affects platelet function, thrombus formation, and progress of atherosclerosis in part independently of its cholesterol-lowering effects. In recent comprehensive randomized trials, atorvastatin was shown to reduce the risk of stroke with or without a history of coronary heart disease (Hess et al., 2000; Farmer, 2007), and these effects could not be explained only by the lipid-lowering effects. It was also shown that atorvastatin delays thrombus formation in arterial vessels exposed to oxidative stress independently of its effects on plasma cholesterol levels (Gaddam et al., 2002). Our findings are of interest with respect to some observations in the Stroke Prevention by Aggressive Reduction in Cholesterol Levels trial (Amarenco et al., 2006). Here, high-dose atorvastatin was demonstrated to be effective in the secondary prophylaxis of stroke (Amarenco et al., 2006). However, effects on survival were in part diminished by an increase in hemorrhagic strokes, particularly in a small group of patients who already had had a preceding hemorrhagic stroke, potentially mediated by atorvastatin-induced inhibition of platelet aggregation. Besides statins, drugs and metabolites such as dehydroepiandrosterone-3-sulfate, fexofenadine, or estrone sulfate, which are described as OATP2B1 substrates, could be taken up into platelets by this transporter. For example, dehydroepiandrosterone administration improves platelet superoxide dismutase activity, which protects cells against oxidative damage (Bednarek-Tupikowska et al., 2000), whereas treatment with estrone sulfate leads to an inhibitory effect on platelet-induced aggregation and secretion (Blache et al., 1995).

Against this background, our findings of abundant expression of OATP2B1 in platelet membranes together with its function as a high-affinity atorvastatin transporter (Grube et al., 2006) as well as of the expression of HMGCR in platelets cytosol may contribute to an improved understanding of drug action of statins.