Identification of CalDAG-GEFI as an intracellular target for the vicinal dithiol binding agent phenylarsine oxide in human platelets

Chih-Yun Kuo; Hui-Chun Wang; Po-Hsiung Kung; Chi-Yu Lu; Chieh-Yu Liao; Ming-Tsang Wu; Chin-Chung Wu

doi:10.1160/TH13-07-0629

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2014; 111(05): 892-901
DOI: 10.1160/TH13-07-0629

Platelets and Blood Cells

Schattauer GmbH

Identification of CalDAG-GEFI as an intracellular target for the vicinal dithiol binding agent phenylarsine oxide in human platelets

Chih-Yun Kuo

¹Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan

,

Hui-Chun Wang

¹Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan

,

Po-Hsiung Kung

¹Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan

,

Chi-Yu Lu

²Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

,

Chieh-Yu Liao

¹Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan

,

Ming-Tsang Wu

³Department of Family Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan

,

Chin-Chung Wu

¹Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan

› Author Affiliations

Abstract

Summary

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.

Keywords

CalDAG-GEFI - Rap1 - platelet aggregation - phenylarsine oxide - vicinal dithiol

Full Text

References

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