title: Subfractionation and purification of intracellular granule-structures of human platelets: an improved method based on magnetic sorting.
abstract: 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.
authors: Research Center of Pharmacology and Experimental Therapeutics, Ernst-Moritz-Arndt-University, Greifswald, Germany.
authors: Juliane
authors: J
authors: Niessen
authors: Gabriele
authors: G
authors: Jedlitschky
authors: Markus
authors: M
authors: Grube
authors: Sandra
authors: S
authors: Bien
authors: Ulrike
authors: U
authors: Strobel
authors: Christoph A
authors: CA
authors: Ritter
authors: Andreas
authors: A
authors: Greinacher
authors: Heyo K
authors: HK
authors: Kroemer
mesh_terms: D001792:Blood Platelets / Q000648:ultrastructure*; D006801:Humans; D018189:Immunomagnetic Separation; D002479:Inclusion Bodies / Q000648:ultrastructure; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins / Q000302:isolation & purification*; D019007:P-Selectin / Q000302:isolation & purification*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000302:isolation & purification*; D014461:Ultracentrifugation
chemical_list: C497811:LAMP2 protein, human; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins; D019007:P-Selectin; D019039:Platelet Glycoprotein GPIIb-IIIa Complex