ABSTRACT:Statins are widely used to treat dyslipidemia. Effects of statins in addition to low-density lipoprotein lowering include altered platelet aggregation, requiring drug uptake into platelets. Possible candidates for mediating intraplatelet accumulation of statins include members of the organic anion-transporting polypeptide family such as OATP2B1 (SLCO2B1), a high-affinity uptake transporter for atorvastatin. Therefore, we analyzed OATP expression, localization, and function in human platelets. OATP2B1, but not OATP1B1, was detected in platelets and megakaryocytes on transcript and protein levels. Protein localization was almost exclusively confined to the plasma membrane. Moreover, we could demonstrate significant inhibition of estrone sulfate uptake into platelets by atorvastatin as well as direct transport of atorvastatin into platelets using a liquid chromatography-tandem mass spectrometry method. As a consequence of OATP2B1-mediated uptake of atorvastatin, we observed significant atorvastatin-mediated reduction of thrombininduced Ca 2؉ mobilization in platelets (37.3 ؎ 6.7% of control at 15 M atorvastatin), mechanistically explainable by reduced lipid modification of signal proteins. This effect was reversed by addition of mevalonate. Finally, we demonstrated expression of HMGCoA reductase, the primary target of atorvastatin, in platelet cytosol. In conclusion, OATP2B1 is an uptake transporter expressed in platelets and is involved in statin-mediated alteration of platelet aggregation.
Several types of peripheral blood cells express ABC transporters. ABCC4 (MRP4) and ABCC5 (MRP5) localize to different cellular sites and fulfill lineage-specific functions such as mediator storage in platelets' dense granules. All mature blood cells originate from the same precursor and specific functionalities arise during differentiation. To characterize this process, expression, localization and function of MRP4 and MRP5 were assessed in differentiating human CD341 progenitors and leukemia cell lines using real time polymerase chain reaction (PCR), immunofluorescence microscopy and cell viability assays. Median MRP4 mRNA copy numbers were significantly enhanced by megakaryocytic differentiation from 7.9 3 10 3 to 5.8 3 10 4 copies per nanograms of total RNA (p < 0.05) in CD341 progenitors and in M-07e cells (MRP4 mRNA/18S rRNA ratios: 5.4 6 3.8 3 10 24 vs. 2.7 6 0.9 3 10 23 for native and differentiated cells, respectively, p < 0.05), and MRP4 protein was localized to granular structures and to the plasma membrane both in differentiated progenitors and bone marrow megakaryocytes. In contrast, expression of MRP4 decreased during maturation to leukocytes (MRP4 mRNA/18S rRNA ratios: 5.2 3 10 23 for native vs. 3.5 3 10 23 for CD341 cells in the presence of G-CSF, p < 0.05) and was significantly reduced in mature monocytes and granulocytes compared with progenitors (MRP4 mRNA/18S rRNA ratios: 8.1 6 5.4 3 10 25 and 2.8 6 1.6 3 10 24 vs. 1.2 6 0.7 3 10 23, respectively, p < 0.05). Expression of MRP5 was not significantly altered under all differentiation conditions. These results indicate that MRP4 expression is differentially regulated during hematopoiesis. The increase of MRP4 together with its specific localization during differentiation toward megakaryocytes supports the concept of platelet specific functions whereas decreased transporter expression in leukocyte differentiation may have implications for chemotherapy. ' 2008 Wiley-Liss, Inc.Key words: drug transporters; hematopoietic system; differentiation; megakaryocytes; monocytes ATP binding cassette (ABC) drug transporters are expressed in a range of mature peripheral blood cells and fulfill different functions. In lymphocytes, several studies have indicated substantial variability of P-glycoprotein (P-gp, ABCB1) expression 1-3 and a relationship between P-gp and IL-2 release.4,5 In addition to P-gp and BCRP (ABCG2), members of the C-branch of ABC transporters termed MRPs, which mediate the unidirectional transport of amphiphilic anions are expressed in peripheral blood cells. 6 The role of MRP1 has been investigated in detail in various populations of human lymphocytes.7 Inhibition of MRP1 abrogated the secretion of interferon-g, interleukin (IL)-10, IL-2 and tumor necrosis factor (TNF)-a, and reduced T-cell activation, indicating a functional role of MRP1-associated transport activity for T-cell function.8 MRP1 has also been detected in human erythrocytes, 9 playing a role in the maintenance of lipid asymmetry 10 and the efflux of reduced and oxidized glu...
We have identified the ATP-binding cassette (ABC) transporter ABCC4 as an active constituent of mediator-storing granules in human platelets. In addition to multidrug resistance protein 4, other ABC-type transport proteins may contribute to platelet secretory function as well as determine intended or adverse effects of drugs. Here, we provide a comprehensive expression profiling of ABC transporters in human platelets based on a novel screening approach by combining the TaqMan low-density array RNA screening platform with a recently developed liquid chromatography/mass spectrometry (MS)/MS method for the simultaneous detection of membrane proteins. Transcripts of 25 ABC transporters were detected and showed differential expression compared with megakaryocytic progenitor cells. On the protein level ABCA7, ABCB4, ABCC1, ABCC3 and ABCC4 were identified by liquid chromatography/MS/MS and localized by immunofluorescence microscopy. Their functions may be related to glutathione and lipid homeostasis, secretion of lipid mediators, cell protection as well as drug transport.
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