Specific inhibition of the transporter MRP4/ABCC4 affects multiple signaling pathways and thrombus formation in human platelets.
Specific inhibition of the transporter MRP4/ABCC4 affects multiple signaling pathways and thrombus formation in human platelets.
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DOI：
10.3324/haematol.2021.279761
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发表时间：
2022-09-01
期刊：
影响因子：
10.1
通讯作者：
Jedlitschky G
中科院分区：
文献类型：
--
作者：
Wolf R;Grammbauer S;Palankar R;Tolksdorf C;Moritz E;Böhm A;Hasan M;Hafkemeyer A;Greinacher A;Tzvetkov MV;Rauch BH;Jedlitschky G
多药耐药蛋白4（MRP 4）在血小板中高度表达，几条证据表明对血小板功能有影响。MRP 4代表环核苷酸以及某些脂质介质的转运蛋白。本研究的目的是全面表征MRP 4的短时间特异性药理学抑制对血小板信号通路的影响。在分离的膜囊泡中的转运试验显示，选择性MRP 4抑制剂Ceefourin-1对MRP 4介导的环核苷酸、血栓烷（Tx）B2和荧光素（FITC）标记的鞘氨醇-1-磷酸（S1 P）转运具有浓度依赖性抑制作用。在人血小板的离体聚集测定研究中，当ADP或胶原分别用作活化剂时，Ceefourin-1显著抑制血小板聚集约30-50%。Ceefourin-1显著降低了ADP诱导的整合素aIIbb 3的活化，这通过FITC-纤维蛋白原的结合（在50 mM Ceefourin-1下降低约50%）来指示，并且减少了钙内流。此外，与Ceefourin-1预孵育显著增加PGE 1和西那西呱诱导的血管舒张剂刺激的磷蛋白（VASP）磷酸化，分别表明细胞溶质cAMP和cGMP浓度增加。从活化的人血小板释放TxB 2也减弱。最后，在流动室模型中，选择性MRP 4抑制显著减少血栓覆盖的总面积和平均血栓尺寸约40%。总之，在流动条件下，选择性MRP 4抑制导致血小板粘附和血栓形成减少。基于抑制环核苷酸和血栓烷转运以及可能的其他机制，这一发现在机制上得到了整联蛋白aIIbb 3活化抑制、VASP磷酸化升高和钙内流减少的支持。
The multidrug resistance protein 4 (MRP4) is highly expressed in platelets and several lines of evidence point to an impact on platelet function. MRP4 represents a transporter for cyclic nucleotides as well as for certain lipid mediators. The aim of the present study was to comprehensively characterize the effect of a short-time specific pharmacological inhibition of MRP4 on signaling pathways in platelets. Transport assays in isolated membrane vesicles showed a concentration-dependent inhibition of MRP4-mediated transport of cyclic nucleotides, thromboxane (Tx)B2 and fluorescein (FITC)-labeled sphingosine-1-phosphate (S1P) by the selective MRP4 inhibitor Ceefourin-1. In ex vivo aggregometry studies in human platelets, Ceefourin-1 significantly inhibited platelet aggregation by about 30-50% when ADP or collagen was used as activating agents, respectively. Ceefourin-1 significantly lowered the ADP-induced activation of integrin aIIbb3, indicated by binding of FITC-fibrinogen (about 50% reduction at 50 mM Ceefourin-1), and reduced calcium influx. Furthermore, pre-incubation with Ceefourin-1 significantly increased PGE1- and cinaciguat-induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation, indicating increased cytosolic cAMP as well as cGMP concentrations, respectively. The release of TxB2 from activated human platelets was also attenuated. Finally, selective MRP4 inhibition significantly reduced both the total area covered by thrombi and the average thrombus size by about 40% in a flow chamber model. In conclusion, selective MRP4 inhibition causes reduced platelet adhesion and thrombus formation under flow conditions. This finding is mechanistically supported by inhibition of integrin aIIbb3 activation, elevated VASP phosphorylation and reduced calcium influx, based on inhibited cyclic nucleotide and thromboxane transport as well as possible further mechanisms.
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