© Schattauer 2014 Thrombosis and Haemostasis 111.5/2014 892 Platelets and Blood Cells Identification of CalDAG-GEFI as an intracellular target for the vicinal dithiol binding agent phenylarsine oxide in human platelets Chih-Yun Kuo 1 ; Hui-Chun Wang 1 ; Po-Hsiung Kung 1 ; Chi-Yu Lu 2 ; Chieh-Yu Liao 1 ; Ming-Tsang Wu 3 ; Chin-Chung Wu 1 1 Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan; 2 Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 3 Department of Family Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan Summary CalDAG-GEFI, a guanine nucleotide exchange factor activating Rap1, is known to play a key role in Ca 2+ -dependent glycoprotein (GP)IIb/IIIa activation and platelet aggregation. Although inhibition of CalDAG- GEFI could be a potential strategy for antiplatelet therapy, no inhibitor of this protein has been identified. In the present study, phenylarsine oxide (PAO), a vicinal dithiol blocker, potently prevented Rap1 acti- vation in thrombin-stimulated human platelets without significantly inhibiting intracellular Ca 2+ mobilisation and protein kinase C acti- vation. PAO also prevented the Ca 2+ ionophore-induced Rap1 acti- vation and platelet aggregation, which are dependent on CalDAG- GEFI. In the biotin-streptavidin pull-down assay, CalDAG-GEFI was ef- ficiently pull-downed by streptavidin beads from the lysates of biotin- conjugated PAO-treated platelets, suggesting that PAO binds to intra- cellular CalDAG-GEFI with high affinity. The above effects of PAO were reversed by a vicinal dithiol compound 2,3-dimercaptopropanol. In ad- dition, CalDAG-GEFI formed disulfide-linked oligomers in platelets treated with the thiol-oxidant diamide, indicating that CalDAG-GEFI contains redox-sensitive thiols. In a purified recombinant protein sys- tem, PAO directly inhibited CalDAG-GEFI-stimulated GTP binding to Rap1. Using CalDAG-GEFI and Rap1-overexpressed human embryonic kidney 293T cells, we further confirmed that PAO abolished Ca 2+ -me- diated Rap1 activation. Taken together, these results have demon- strated that CalDAG-GEFI is one of the targets of action of PAO, and propose an important role of vicinal cysteines for the functions of Cal- DAG-GEFI. Keywords CalDAG-GEFI, Rap1, platelet aggregation, phenylarsine oxide, vicinal dithiol Correspondence to: Chin-Chung Wu Graduate Institute of Natural Products Kaohsiung Medical University Kaohsiung City, Taiwan Tel.: +886 7 3211101 E-mail: ccwu@kmu.edu.tw Received: July 31, 2013 Accepted after major revision: November 22, 2013 Prepublished online: December 19, 2013 doi:10.1160/TH13-07-0629 Thromb Haemost 2014; 111: 892–901 Introduction Platelet-mediated arterial thrombotic diseases, such as myocardial infarction and ischaemic stroke, are the major causes of death around the world (1, 2). Although current antiplatelet drugs, in- cluding aspirin, the ADP receptor antagonist clopidogrel, and gly- coprotein (GP)IIb/IIIa antagonists, reduce cardiovascular events, their efficacy is relatively modest and their use is limited by the risk of bleeding complications (3, 4). More effective and safer anti- platelet agents are still needed. Growing research on the molecular mechanisms of platelet ac- tivation and aggregation has revealed certain signalling molecules as novel targets for antiplatelet therapy. Upon platelet stimulation by agonists, several signalling pathways, such as phospholipase C (PLC), phospholipase A 2 (PLA 2 ), phosphoinositide 3-kinase (PI3K), and tyrosine kinases, are activated and involved in the in- duction and/or maintenance of platelet aggregation (5, 6). Among them, the PLC signalling pathway appears to play a critical role. Upon activation, PLC hydrolyses phosphatidylinositol 4,5-bisp- hosphate to inositol 1,4,5-trisphosphate, which contributes to Ca 2+ release from internal stores, and diacylglycerol (DAG), which acti- vates protein kinase C (PKC). The intracellular Ca 2+ and PKC act synergistically to induce activation of GPIIb/IIIa, which is the fi- brinogen receptor responsible for the final step of platelet aggre- gation. Recently, a model has been developed to integrate the underlying molecular mechanism for GPIIb/IIIa activation (7-9). In this model, Ca 2+ activates calcium-diacylglycerol guanine nu- cleotide exchange factor-I (CalDAG-GEFI), which stimulates the small GTPase Rap1 by enhancing the exchange of GDP for GTP on Rap1 (10). On the other hand, PKC is also able to activate Rap1 independent of CalDAG-GEFI (11). Rap1-GTP subsequently in- teracts with Rap1-interacting adaptor molecule (RIAM) and re- cruits talin to GPIIb/IIIa, thus triggers activation of GPIIb/IIIa (12). Mouse genetic studies have suggested that CalDAG-GEFI may be as a potential target for developing of antiplatelet agents. Cal- DAG-GEFI-deficient mice show defects in platelet aggregation and protection against arterial thrombus formation (13). More- over, CalDAG-GEFI-deficient mice lost significantly less blood than clopidogrel-treated wild-type mice (14). 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