Phorbol ester stimulates calcium sequestration in saponized human platelets

• Published in: The Journal of biological chemistry Vol. 262; no. 33; pp. 16048 - 16054
• Main Authors: Yoshida, K, Nachmias, V T
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25-11-1987; American Society for Biochemistry and Molecular Biology
• Subjects: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Diphosphate - pharmacology; Alprostadil - pharmacology; Benzofurans; Biological and medical sciences; Biological Transport, Active - drug effects; Blood Platelets - drug effects; Blood Platelets - metabolism; Calcimycin - pharmacology; Calcium - blood; Calcium Radioisotopes; Cell physiology; Chelating Agents; Fundamental and applied biological sciences. Psychology; Fura-2; Humans; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol Phosphates - pharmacology; Isoquinolines - pharmacology; Kinetics; Membrane and intracellular transports; Molecular and cellular biology; Piperazines - pharmacology; Protein Kinase C - antagonists & inhibitors; Tetradecanoylphorbol Acetate - pharmacology; Thrombin - physiology; Human; Agonist; GTP; Protein kinase C; Calcium; Enzyme; Cyclic AMP; Phospholipid; Ion transport; ADP; Uptake; Saponin; Binding protein; Platelet; Radioimmunoassay; Inhibitor
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)47694-6

When platelets are activated by agonists, calcium (Ca2+) is released from an intracellular storage site. Recent studies using fura-2 show that, after thrombin stimulation, the rise in free calcium is transient and returns to base-line levels in 2-3 min, while the transient following ADP stimulation lasts only 15-20 s. We reported previously that the phorbol ester 12,13-phorbol myristate acetate (PMA), added at nanomolar levels after thrombin, immediately accelerated the rate of return of calcium to the base line severalfold (Yoshida, K., Dubyak, G., and Nachmias, V.T. (1986) FEBS Lett. 206, 273-278). In the present study, we used both intact and saponized platelets to determine whether this is due to stimulation of calcium sequestration. Using fura-2 and intact platelets, we found 1) that PMA stimulated the restoration of free Ca2+ levels after ADP as well as after thrombin, and 2) that H-7, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), slowed the return of Ca2+ to baseline levels. Using saponized platelets, we also found 3) that pretreatment of platelets with PMA before saponin treatment increased the ATP-dependent 45Ca2+ uptake 2-fold, with a half-maximal effect at 5 nm; 4) that most of the Ca2+ released by ionomycin or by myoinositol 1,4,5-trisphosphate; and 5) that a GTP-binding protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), decreased basal or PMA-stimulated 45Ca2+ uptake in saponin-treated platelets. Our data suggest that activation of protein kinase C stimulates the sequestration of Ca2+ independently of cAMP or myoinositol 1,4,5-trisphosphate.