Secreted Dense Granule Adenine Nucleotides Promote Calcium Influx and the Maintenance of Elevated Cytosolic Calcium Levels in Stimulated Human Platelets

Summary

Evidence that secreted dense granule adenine nucleotides mediate part of the agonist-induced cytosolic calcium ([Ca²⁺]_i) responses in human platelets was obtained from comparisons of fura-2-loaded platelets from normal subjects and from patients with a form of platelet storage pool deficiency (SPD) in which the secretory dense granules and their contents are virtually absent. SPD platelets had normal initial [Ca²⁺]_i in creases induced by thrombin and the endoperoxide analog U46619, but a significantly enhanced decay of elevated [Ca²⁺]_i levels following the initial increases. With thrombin, this enhanced [Ca²⁺]_i decay was associated with decreased Ca²⁺ influx, as measured by Mn²⁺ quench of fura-2 fluorescence. Addition of micromolar concentrations of ADP, alone or together with ATP, after stimulation reversed the enhanced [Ca²⁺]_I decay and increased Mn²⁺ quench in SPD platelets, but had no effect on these responses in normal platelets, while addition of 100-fold higher concentrations of ATP or apyrase before stimulation increased [Ca²⁺]_I decay and decreased Mn²⁺ quench in normal platelets, but had little effect in SPD platelets. ATP and α,β-methylene ATP, a specific agonist for P2X1 receptors, at micromolar concentrations also increased Mn²⁺ quench, but to lesser extents than did ADP, in SPD platelets isolated and loaded with fura-2 in the presence of apyrase. Similar effects of ADP and excess ATP were seen in U46619-stimulated platelets, but decreased Ca²⁺ influx could not be measured directly in SPD platelets, presumably due to the very transient influx response seen with U46619. These results suggest that secreted dense granule ADP and ATP contribute to the maintenance of elevated [Ca²⁺]_i levels, but not to the initial [Ca²⁺]_i increases, in stimulated human platelets, most likely via a nucleotide-specific component of Ca²⁺ influx which may be mediated by interactions with both P2X₁ and P2Y₁ purinoceptors.

• References

• 1 Heemskerk JWM, Sage SO. Calcium signalling in platelets and other cells. Platelets 1994; 5: 295-316.
• 2 Meldolesi J, Clementi E, Fasolato C, Zacchetti D, Pozzan T. Ca²⁺ influx following receptor activation. Trends in Pharmaceut Sci 1991; 12: 282-92.
• 3 Zwaal RFA, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 1997; 89: 1121-32.
• 4 Ozaki Y, Yatomi Y, Wakasugi S, Shirasawa Y, Saito H, Kume S. Thrombin-induced calcium oscillation in human platelets and MEG-01, a megakaryoblastic leukemia cell line. Biochem Biophys Res Commun 1992; 183: 864-71.
• 5 Heemskerk JWM, Vis P, Feijge MAH, Hoyland J, Mason WT, Sage SO. Roles of phospholipase C and Ca²⁺-ATPase in calcium responses of single, fibrinogen-bound platelets. J Biol Chem 1993; 268: 356-63.
• 6 Ariyoshi H, Salzman EW. Spatial distribution and temporal change in cytosolic pH and [Ca²⁺] in resting and activated single human platelets. Cell Calcium 1995; 17: 317-26.
• 7 Sage SO. Calcium entry mechanisms in human platelets. Experimental Physiology 1997; 82: 807-23.
• 8 Holmsen H, Weiss HJ. Secretable storage pools in platelets. Ann Rev Med 1979; 30: 119-34.
• 9 Leon C, Hechler B, Vial C, Leray C, Cazenave J-P, Gachet C. The P2Y1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells. FEBS Letters 1997; 403: 26-30.
• 10 MacKenzie AB, Mahaut-Smith MP, Sage SO. Activation of receptor-operated cation channels via P2X1 not P2T purinoceptors in human platelets. J Biol Chem 1996; 271: 2879-81.
• 11 Vial C, Hechler B, Leon C, Cazenave J-P, Gachet C. Presence of P2X1 purinoceptors in human platelets and megakaryoblastic cell lines. Thromb Haemost 1997; 78: 1500-4.
• 12 Weiss HJ, Witte LD, Kaplan KL, Lages BA, Chernoff A, Nossel HL, Goodman DS, Baumgartner HR. Heterogeneity in storage pool deficiency: studies on granule-bound substances in 18 patients including variants deficient in α-granules, platelet factor 4, β-thromboglobulin, and platelet-derived growth factor. Blood 1979; 54: 1296-319.
• 13 Weiss HJ, Lages B, Vicic W, Tsung LY, White JG. Heterogeneous abnormalities of platelet dense granule ultrastructure in 20 patients with congenital storage pool deficiency. Brit J Haematol 1993; 83: 282-95.
• 14 Lages B, Weiss HJ. Enhanced increases in cytosolic Ca²⁺ in ADP-stimulated platelets from patients with delta storage pool deficiency – a possible indicator of interactions between granule-bound ADP and the membrane ADP receptor. Thromb Haemost 1997; 77: 376-82.
• 15 Weiss HJ, Lages B. Platelet prothrombinase activity and intracellular calcium responses in patients with storage pool deficiency, glycoprotein IIb-IIIa deficiency, or impaired platelet coagulant activity – a comparison with Scott syndrome. Blood 1997; 89: 1599-611.
• 16 Molnar J, Lorand L. Studies on apyrases. Arch Biochem Biophys 1961; 93: 353-63.
• 17 Lages B, Weiss HJ. Evidence for a role of glycoprotein IIb-IIIa, distinct from its ability to support aggregation, in platelet activation by ionophores in the presence of extracellular divalent cations. Blood 1994; 83: 2549-59.
• 18 Sage SO, Merritt JE, Hallam TJ, Rink TJ. Receptor-mediated calcium entry in fura-2-loaded human platelets stimulated with ADP and thrombin. Dual wavelength studies with Mn²⁺ . Biochem J 1989; 258: 923-6.
• 19 Hallam TJ, Jacob R, Merritt JE. Evidence that agonists stimulate bivalent cation influx into human endothelial cells. Biochem J 1988; 255: 179-84.
• 20 Rink TJ, Pozzan T. Using quin2 in cell suspensions. Cell Calcium 1985; 6: 133-44.
• 21 Lages B, Holmsen H, Weiss HJ, Dangelmaier C. Thrombin and ionophore A23187-induced dense granule secretion in storage pool deficient platelets: evidence for impaired nucleotide storage as the primary dense granule defect. Blood 1983; 61: 154-62.
• 22 Lages B, Scrutton MC, Holmsen H. Secretion by gel-filtered human platelets: response of platelet Ca²⁺, Mg², and K⁺ to secretory agents. J Lab Clin Med 1977; 90: 873-82.
• 23 Surprenant A, Buell G, North RA. P2x receptors bring new structure to ligand-gated ion channels. Trends in Neurosci 1995; 18: 224-9.
• 24 Mahaut-Smith MP, Sage SO, Rink TJ. Rapid ADP-evoked currents in human platelets recorded with the nystatin permeabilized patch technique. J Biol Chem 1992; 267: 3060-5.
• 25 Holmsen H. Prostaglandin endoperoxide-thromboxane synthesis and dense granule secretion as positive feedback loops in the propagation of platelet responses during the “basic platelet reaction”. Thromb Haemost 1977; 38: 1030-41.
• 26 Kroll MH, Schafer I. A. Biochemical mechanisms of platelet activation. Blood 1989; 74: 1181-95.
• 27 Hourani SM, Hall DA. Receptors for ADP on human blood platelets. Trends in Pharmaceut Sci 1994; 15: 103-8.
• 28 Coade SB, Pearson JD. Metabolism of adenine nucleotides in human blood. Circulation Res 1989; 65: 531-7.
• 29 Flodgaard H, Klenow H. Abundant amounts of diadenosine 5’, 5’”-P¹, P⁴-tetraphosphate are present and releasable, but metabolically inactive, in human platelets. Biochem J 1982; 208: 737-42.
• 30 Sage SO, MacKenzie AB, Jenner S, Mahaut-Smith MP. Purinoceptor-evoked calcium signalling in human platelets. Prostaglandins Leukotrienes Essent Fatty Acids 1997; 57: 435-8.
• 31 Daniel JL, Dangelmaier C, Jin J, Ashby B, Smith JB, Kunapuli SP. Molecular basis for ADP-induced platelet activation I. Evidence for three distinct ADP receptors on human platelets. J Biol Chem 1998; 273: 2024-9.