Multidrug resistance protein 4 (MRP4/ABCC4) regulates thrombus formation in vitro and in vivo.
The multidrug resistance protein 4 (MRP4) is a member of the ABCC subfamily of the adenosine triphosphate-binding cassette transporters that remove cyclic nucleotides from platelets and uptake ADP into dense granule in platelets. However, whether MRP4 directly involves platelet activation remains unclear. Thus, the aim of our study was to determine the detailed mechanisms underlying the regulation of MRP4 in platelet activation. Our results revealed that the MRP4 inhibitor MK571 inhibited collagen-induced platelet aggregation which was partially reversed by the PKA inhibitor H89, but not by the adenylyl cyclase (AC) inhibitor SQ22536 and the guanylyl cyclase (GC) inhibitor ODQ, suggesting that MK571 can prevent collagen-induced aggregation via a route independent of cyclic nucleotide production. In the present study, we found that MK571 inhibited collagen-induced ATP release and calcium mobilization. The phosphorylation of protein kinase C, JNK, and Akt was also inhibited by MK571, and electron spin resonance experiment showed that MK571 significantly reduced hydroxyl radical formation. Moreover, MK571 delayed platelet plug formation in vitro by a PFA-100 device, and delayed thrombus formation in mesenteric venules of mice irradiated by fluorescein sodium. However, previous studies have reported that MK571 also blocks MRP1 and leukotriene D4 (LTD4) receptor. Therefore, whether MK571 inhibits platelet activation through MRP1 or LTD4 receptor needs to be considered and further defined. In conclusion, in addition to blocking the transport of cyclic nucleotides, MRP4 inhibition may prevent thrombus formation in vitro and in vivo. Our findings also support the idea that MRP4 may represent a potential target for the development of novel therapeutic interventions for the treatment of thromboembolic disorders.
School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; Department of Neurology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.
Li-Ming
LM
Lien
Department of Cardiology, Chi-Mei Medical Center, Tainan City, Taiwan; Department of Pharmacy, Chia Nan University of Pharmacy & Science, Tainan City, Taiwan; Department of Pharmacology, and Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan.
Zhih-Cherng
ZC
Chen
Division of Pulmonary Medicine, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan; School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan.
Chi-Li
CL
Chung
Department of Pharmacology, and Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan.
Ting-Lin
TL
Yen
Department of Neurology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; College of Medicine, Fu-Jen Catholic University, Taipei, Taiwan.
Hou-Chang
HC
Chiu
Department of Pharmacology, and Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan.
Duen-Suey
DS
Chou
School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan.
Shih-Yi
SY
Huang
Department of Pharmacology, and Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan.
Joen-Rong
JR
Sheu
Department of Pharmacology, and Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan. Electronic address: d119096013@tmu.edu.tw.
Wan-Jung
WJ
Lu
Department of Pharmacology, and Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan; Central Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, 95 Wen-Chang Rd., Taipei 111, Taiwan. Electronic address: d102092002@tmu.edu.tw.
Kuan-Hung
KH
Lin
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D017665:Hydroxyl Radical / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D051379:Mice; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D011422:Propionates / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011804:Quinolines / Q000494:pharmacology; D013927:Thrombosis / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002118:Calcium; D048049:Extracellular Signal-Regulated MAP Kinases; D017665:Hydroxyl Radical; D048031:JNK Mitogen-Activated Protein Kinases; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011422:Propionates; D051057:Proto-Oncogene Proteins c-akt; D011804:Quinolines; D048051:p38 Mitogen-Activated Protein Kinases; C073492:ABCC4 protein, human; C553975:p37 protein, human; C059141:verlukast
Cyclic nucleotide; MRP4; Platelet activation; Thrombus formation