Regulation of platelet dense granule secretion by the Ral GTPase-exocyst pathway.
Non-hydrolyzable GTP analogues, such as guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp), induce granule secretion from permeabilized platelets in the absence of increased intracellular Ca(2+). Here, we show that the GppNHp-induced dense granule secretion from permeabilized platelets occurred concomitantly with the activation of small GTPase Ral. This secretion was inhibited by the addition of GTP-Ral-binding domain (RBD) of Sec5, which is a component of the exocyst complex known to function as a tethering factor at the plasma membrane for vesicles. We generated an antibody against Sec5-RBD, which abolished the interaction between GTP-Ral and the exocyst complex in vitro. The addition of this antibody inhibited the GppNHp-induced secretion. These data indicate that Ral mediates the GppNHp-induced dense granule secretion from permeabilized platelets through interaction with its effector, the exocyst complex. Furthermore, GppNHp enhanced the Ca(2+) sensitivity of dense granule secretion from permeabilized platelets, and this enhancement was inhibited by Sec5-RBD. In intact platelets, the association between Ral and the exocyst complex was induced by thrombin stimulation with a time course similar to that of dense granule secretion and Ral activation. Taken together, our results suggest that the Ral-exocyst pathway participates in the regulation of platelet dense granule secretion by enhancing the Ca(2+) sensitivity of the secretion.
Kawato
Mitsunori
M
Department of Cardiovascular Medicine, Kyoto University, Kyoto, 606-8507, Japan.
Shirakawa
Ryutaro
R
Department of Cardiovascular Medicine, Kyoto University, Kyoto, 606-8507, Japan.
Kondo
Hirokazu
H
Department of Cardiovascular Medicine, Kyoto University, Kyoto, 606-8507, Japan.
Higashi
Tomohito
T
Department of Cardiovascular Medicine, Kyoto University, Kyoto, 606-8507, Japan.
Ikeda
Tomoyuki
T
Department of Cardiovascular Medicine, Kyoto University, Kyoto, 606-8507, Japan.
Okawa
Katsuya
K
Frontier Technology Center, Graduate School of Medicine, Kyoto University, Kyoto, 606-8507, Japan.
Fukai
Shuya
S
Department of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 226-8501, Japan; Life Science Division, Synchrotron Radiation Research Organization, University of Tokyo, Tokyo, 113-0032, Japan.
Nureki
Osamu
O
Department of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 226-8501, Japan.
Kita
Toru
T
Department of Cardiovascular Medicine, Kyoto University, Kyoto, 606-8507, Japan.
Horiuchi
Hisanori
H
Department of Cardiovascular Medicine, Kyoto University, Kyoto, 606-8507, Japan. Electronic address: horiuchi@kuhp.kyoto-u.ac.jp.
D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005089:Exocytosis / Q000187:drug effects; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology*; D006801:Humans; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism; D020725:ral GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D013917:Thrombin; D020725:ral GTP-Binding Proteins; D002118:Calcium