The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage.
Platelet aggregation is initiated by the release of mediators as adenosine diphosphate (ADP) stored in platelet granules. Possible candidates for transport proteins mediating accumulation of these mediators in granules include multidrug resistance protein 4 (MRP4, ABCC4), a transport pump for cyclic nucleotides and nucleotide analogs. We investigated the expression of MRP4 in human platelets by immunoblotting, detecting a strong signal at 170 kDa. Immunofluorescence microscopy using 2 MRP4-specific antibodies revealed staining mainly in intracellular structures, which largely colocalized with the accumulation of mepacrine as marker for delta-granules and to a lower extent at the plasma membrane. Furthermore, an altered distribution of MRP4 was observed in platelets from a patient with Hermansky-Pudlak syndrome with defective delta-granules. Adenosine triphosphate (ATP)-dependent cyclic guanosine monophosphate (cGMP) transport codistributed with MRP4 detection in subcellular fractions, with highest activities in the dense granule and plasma membrane fractions. This transport was inhibited by dipyramidole, indomethacin, and MK571 with median inhibitory concentration (IC(50)) values of 12, 22, and 43 microM, and by ibuprofen. Transport studies with [(3)H]ADP indicated the presence of an orthovanadate-sensitive ADP transporting system, inhibited by dipyramidole, MK571, and cyclic nucleotides. The results indicate a function of MRP4 in platelet mediator storage and inhibition of MRP4 may represent a novel mechanism for inhibition of platelet function by some anti-inflammatory drugs.
Jedlitschky
Gabriele
G
Department of Pharmacology, Peter Holtz Research Center of Pharmacology and Experimental Therapeutics, Greifswald, Germany.
Tirschmann
Konstanze
K
Lubenow
Lena E
LE
Nieuwenhuis
Hendrik K
HK
Akkerman
Jan W N
JW
Greinacher
Andreas
A
Kroemer
Heyo K
HK
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001692:Biological Transport; D001792:Blood Platelets / Q000737:chemistry* / Q000648:ultrastructure; D006152:Cyclic GMP / Q000378:metabolism; D022861:Hermanski-Pudlak Syndrome / Q000097:blood; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000502:physiology*; D022142:Secretory Vesicles / Q000737:chemistry* / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006152:Cyclic GMP; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D014638:Vanadates; C073492:ABCC4 protein, human