Activation of integrin alpha IIb beta 3 in the glycoprotein Ib-high population of a megakaryocytic cell line, CMK, by inside-out signaling.
Affinity/avidity state of integrin alpha IIb beta 3 is regulated by intracellular inside-out signaling. Although several megakaryocytic cell lines have been established, soluble ligand binding to alpha IIb beta 3 expressed in these cells by cellular agonists has not been demonstrated. We have re-examined agonist-induced alpha IIb beta 3 activation on megakaryocytic cell lines with a marker of the late stage of megakaryocytic differentiation, glycoprotein Ib (GPIb). Activation of alpha IIb beta 3 was assessed by PAC1 and soluble fibrinogen binding to the cells. We found that alpha IIb beta 3 expressed in CMK cells with high GPIb expression was activated by a phorbor ester, phorbol myristate acetate (PMA). Although the population of the GPIbhigh cells was <0.5% of the total cells, incubation with a nucleoside analog, ribavirin, efficiently increased the PMA-reactive GPIbhigh cells. Not only PMA but also a calcium ionophore, A23187, induced alpha IIb beta 3 activation, and PMA and A23187 had an additive effect on alpha IIb beta 3 activation. Ligand binding to the activated alpha IIb beta 3 in the GPIbhigh CMK cells is totally abolished by an alpha IIb beta 3-specific antagonist, and inhibited by wortmannin, cytochalasin-D and prostaglandin E1, and the effects of these inhibitors on alpha IIb beta 3 activation in the GPIbhigh CMK cells were compatible with those in platelets. We have also demonstrated that the ribavirin-treated CMK cells express PKC-alpha, -beta, -delta and -theta, and suggested that PKC-alpha and/or -beta appear to be responsible for PMA-induced activation of alpha IIb beta 3 in CMK cells.
Kashiwagi
H
H
Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University and Department of Blood Transfusion, Osaka University Hospital, Suita, Osaka, Japan. kashi@hp-blood.med.osaka-u.ac.jp
Shiraga
M
M
Honda
S
S
Kosugi
S
S
Kamae
T
T
Kato
H
H
Kurata
Y
Y
Tomiyama
Y
Y
D000527:Alprostadil / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D015638:Cytochalasin D / Q000494:pharmacology; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008533:Megakaryocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D012254:Ribavirin / Q000494:pharmacology; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D007476:Ionophores; D007527:Isoenzymes; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D015638:Cytochalasin D; D000001:Calcimycin; D012254:Ribavirin; D011493:Protein Kinase C; D000527:Alprostadil; D013755:Tetradecanoylphorbol Acetate; D000077191:Wortmannin