title: Identification of CalDAG-GEFI as an intracellular target for the vicinal dithiol binding agent phenylarsine oxide in human platelets.
abstract: CalDAG-GEFI, a guanine nucleotide exchange factor activating Rap1, is known to play a key role in Ca2+-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 activation in thrombin-stimulated human platelets without significantly inhibiting intracellular Ca2+ mobilisation and protein kinase C activation. PAO also prevented the Ca2+ ionophore-induced Rap1 activation and platelet aggregation, which are dependent on CalDAG-GEFI. In the biotin-streptavidin pull-down assay, CalDAG-GEFI was efficiently pull-downed by streptavidin beads from the lysates of biotin-conjugated PAO-treated platelets, suggesting that PAO binds to intracellular CalDAG-GEFI with high affinity. The above effects of PAO were reversed by a vicinal dithiol compound 2,3-dimercaptopropanol. In addition, 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 system, 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 Ca2+-mediated Rap1 activation. Taken together, these results have demonstrated that CalDAG-GEFI is one of the targets of action of PAO, and propose an important role of vicinal cysteines for the functions of CalDAG-GEFI.
authors.forename: Chih-Yun
authors.initials: CY
authors.lastname: Kuo
authors.forename: Hui-Chun
authors.initials: HC
authors.lastname: Wang
authors.forename: Po-Hsiung
authors.initials: PH
authors.lastname: Kung
authors.forename: Chi-Yu
authors.initials: CY
authors.lastname: Lu
authors.forename: Chieh-Yu
authors.initials: CY
authors.lastname: Liao
authors.forename: Ming-Tsang
authors.initials: MT
authors.lastname: Wu
authors.affiliation: Chin-Chung Wu, Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung City, Taiwan, Tel.: +886 7 3211101, E-mail: ccwu@kmu.edu.tw.
authors.forename: Chin-Chung
authors.initials: CC
authors.lastname: Wu
mesh_terms: D001152:Arsenicals / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects* / Q000502:physiology; D003958:Diamide / Q000494:pharmacology; D004112:Dimercaprol / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000302:isolation & purification / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D058990:Molecular Targeted Therapy; D010084:Oxidation-Reduction / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D013917:Thrombin / Q000378:metabolism; D013927:Thrombosis / Q000188:drug therapy*; D014050:Toluene / Q000031:analogs & derivatives / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D001152:Arsenicals; D020662:Guanine Nucleotide Exchange Factors; C415483:RASGRP2 protein, human; D004112:Dimercaprol; C029341:oxophenylarsine; D003958:Diamide; D014050:Toluene; D013917:Thrombin; D020827:rap1 GTP-Binding Proteins; C004848:dithiol
keywords: CalDAG-GEFI; Rap1; phenylarsine oxide; platelet aggregation; vicinal dithiol