title: CLL2-1, a chemical derivative of orchid 1,4-phenanthrenequinones, inhibits human platelet aggregation through thiol modification of calcium-diacylglycerol guanine nucleotide exchange factor-I (CalDAG-GEFI).
abstract: CalDAG-GEFI is a guanine nucleotide exchange factor, which actives small GTPase Rap1 and plays an important role in platelet aggregation. Our previous study has shown that CalDAG-GEFI contains redox-sensitive thiols, and its function can be inhibited by thiol modification. In the present study, the effect of CLL2-1, a 1,4-phenanthrenequinone, on CalDAG-GEFI and platelet functions was investigated. In human platelets, CLL2-1 prevented platelet aggregation caused by various stimulators. Flow cytometric analysis revealed that CLL2-1 inhibited GPIIb/IIIa activation and P-selectin secretion. Moreover, CLL2-1 prevented Rap1 activation caused by thrombin, the Ca(2+) ionophore A23187, and the diacylglycerol mimetic phorbol 12-myristate 13-acetate, while only slightly inhibited thrombin-induced increases in [Ca(2+)]i and did not inhibit protein kinase C activation. Western blots after reducing SDS-PAGE showed that treatment of either platelets or platelet lysates with CLL2-1 led to a decrease of monomeric CalDAG-GEFI and appearance of cross-linked oligomers of CalDAG-GEFI, and these effects were inhibited by pretreatment of platelets or lysates with thiol reducing agents prior to the addition of CLL2-1, indicating thiol modification of CalDAG-GEFI by CLL2-1. Furthermore, the thiol reducing agents also prevented the inhibitory effect of CLL2-1 on Rap1 activation, GPIIb/IIIa activation, and platelet aggregation. In CalDAG-GEFI-overexpressing human embryonic kidney 293T cells, CLL2-1 also inhibited CalDAG-GEFI-mediated Rap1 activation. Taken together, our results suggest that the antiplatelet effect of CLL2-1 is due to, at least in part, inhibition of CalDAG-GEFI-mediated Rap1 activation, and provide the basis for development of novel antiplatelet drugs.
authors.affiliation: Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Chieh-Yu
authors.initials: CY
authors.lastname: Liao
authors.affiliation: School of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan.
authors.forename: Chia-Lin
authors.initials: CL
authors.lastname: Lee
authors.affiliation: Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Hui-Chun
authors.initials: HC
authors.lastname: Wang
authors.affiliation: Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Shih-Shin
authors.initials: SS
authors.lastname: Liang
authors.affiliation: Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Po-Hsiung
authors.initials: PH
authors.lastname: Kung
authors.affiliation: School of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan.
authors.forename: Yang-Chang
authors.initials: YC
authors.lastname: Wu
authors.affiliation: Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Fang-Rong
authors.initials: FR
authors.lastname: Chang
authors.affiliation: Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, Kaohsiung, Taiwan. Electronic address: ccwu@kmu.edu.tw.
authors.forename: Chin-Chung
authors.initials: CC
authors.lastname: Wu
mesh_terms: D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D005434:Flow Cytometry; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D010616:Phenanthrenes / Q000737:chemistry* / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D011809:Quinones / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: C000601637:5,6,7-trimethoxy-1,4-phenanthrenequinone; D020662:Guanine Nucleotide Exchange Factors; D010616:Phenanthrenes; D010975:Platelet Aggregation Inhibitors; D011809:Quinones; C415483:RASGRP2 protein, human; D013438:Sulfhydryl Compounds; C105959:1,4-phenanthrenequinone; D002118:Calcium
keywords: 1,4-Phenanthrenequinone; CLL2-1; CalDAG-GEFI; Platelet aggregation; Thiol modification