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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.

Functional analysis of intracellular structures requires isolation and purification of these cellular compartments. With regard to platelet function both delta and alpha granules are relevant target structures. However, the availability of sufficient purification protocols for these structures is rather limited and restricted to density gradient centrifugation. Because this method is time-consuming and the resulting products are often of limited purity, we designed a new purification method based on immunolabeling followed by magnetic sorting. We directly compared this new method with the conventional method of ultracentrifugation. We were able to get highly purified subcellular fractions of human platelets using several antibodies against specific markers for dense granules (LAMP2), alpha granules (P-selectin) and the plasma membrane (GPIIb/IIIa) in combination with antibody-coated magnetic beads. In the respective fractions the marker proteins used for isolation as well as further independent, structure specific markers (for example MRP4 for dense granules, von Willebrand factor (vWF) for alpha granules and protein disulfide isomerase, PDI and GPIb beta, for plasma membrane) could be detected by Western blotting. The method describes purification of membranal structures of human platelets such as the plasma membrane and both types of granules. Therefore, studies requiring highly purified material (e.g. identification of specific transporters and receptors) will benefit from these results.