Regulation of Platelet Function by Cytosolic Free Calcium and Cyclic AMP

Bushfield, Mark (1986) Regulation of Platelet Function by Cytosolic Free Calcium and Cyclic AMP. PhD thesis, University of Glasgow.

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Human blood platelet responsiveness can be modulated by a variety of stimulatory and inhibitory agonists. These agents are recognised by specific receptors which presumably are situated on the outer surface of the platelet plasma membrane. Receptors have been characterised for a number of endogenous compounds including adrenaline, ADP, vasopressin, platelet-activating factor, PGI2, PGD2 and adenosine. Platelets are electrically non-excitable, hence, there must exist transduction processes which link events at the cell surface (i. e. agonist-receptor interaction) to the key intracellular reactions which control cellular responsiveness. In platelets, as in many other cell types, it has been established that inositol phospholipid hydrolysis serves as one of the transduction processes that link stimulatory receptor occupancy to cellular activation. Phospholipase C-catalysed hydrolysis of phosphatidylinositol 4,5-bisphosphate yields 1,2-diacylglycerol, an activator of protein kinase C, and inositol (1,4,5) trisphosphate which can mobilise calcium ions from intracellular stores. Inositol phospholipid hydrolysis may also be involved in the agonist-induced influx of calcium ions which also occurs in platelets. Inhibition of platelet reactivity is associated with agonist-induced stimulation of adenylate cyclase activity and increased intracellular levels of cyclic AMP. Cyclic AMP, via activation of cyclic AMP-dependent protein kinase, can modify a variety of platelet intracellular reactions. In vivo platelets may be exposed to combinations of stimulatory and/or inhibitory agonists. Hence, the phenomena of synergism and desensitization, which have been demonstrated in vitro, may occur in vivo and may be of major importance in haemostasis and in the pathogenesis of occlusive vascular disease. This study examined the effects of agonists, alone and in combination, on platelet responsiveness and the putative underlying transduction processes of inositol phospholipid hydrolysis, elevation of cytosolic free calcium concentration [Ca2+]i) and cyclic AMP concentration. Platelet responsiveness was monitored by measuring (a) shape change; (b) aggregation in whole blood (by a single platelet counting method); (c) aggregation in platelet-rich plasma (by two methods: turbidometrically and by counting single platelets); (d) aggregation in gel-filtered platelets and (e) release of dense granule constituents. Stimulation of inositol phospholipid metabolism was assessed as [32P]-Ptd OH formation in platelets prelabelled with [32P] -Pi. Changes in [Ca2+] i were measured using the fluorescent calcium-indicator dye Quin 2 and cyclic AMP levels were measured by radioimmunoassay. 1. In platelet-rich plasma, the single platelet counting method provided a much more sensitive index of aggregation than did the measurement of changes in light transmission in an aggregometer. 2. PAF and vasopressin induced concentration-dependent platelet activation, [32P]-Ptd OH formation and elevation of [Ca2+] ADP-induced platelet activation was associated with elevation of (Ca2+]i but not [32P]-Ptd OH formation. Adrenaline also caused platelet activation but had no direct effect on [Ca2+]i or [32P]-Ptd OH levels. 3. Adrenaline potentiated ADP-, vasopressin- and PAF-induced platelet activation and elevation of Agonist (vasopressin)-induced [32P]-Ptd OH formation was also potentiated. 4. Potentiation of platelet activation by adrenaline may be mediated by enhanced inositol phospholipid hydrolysis and elevation of [Ca2+]i. 5. PGI2, PGE1, 6-keto-PGE1, PGD2 and adenosine induced concentration-dependent elevations in platelet cyclic AMP content. 6. PAF-induced functional platelet responses and transduction processes show differential sensitivity to inhibition by PGD2 and PGI2. A doubling in the platelet cyclic AMP content resulted in abolition of PAF-induced aggregation and ATP secretion, whereas maximal inhibition of shape change, [32P]-Ptd OH formation and elevation of [Ca2+]i required a greater than ten-fold elevation in cyclic AMP. This differential sensitivity of the various responses to inhibition by cyclic AMP suggests that the mechanisms underlying inhibition of PAF-induced aggregation and ATP secretion differ from those underlying shape change. Thus a major component of cyclic AMP-dependent inhibition of PAF-induced aggregation and ATP secretion is mediated by suppression of certain components of the activation process that occur distal to the formation of diacylglycerol or elevation of 7. Inhibitors of adenylate cyclase (adrenaline, ADP) but not vasopressin or PAF, prevented elevation of cyclic AMP content and inhibition of platelet responsiveness and transduction processes by adenylate cyclase stimulants (PGI2, PGD2) but not by other means (e. g. inhibition of phosphodiesterase activity). 8. Activation of protein kinase C by PMA directly inhibited PGD2- but not PGI2-, PGE1-, 6-keto-PGE1- or adenosine-induced cyclic AMP formation. PMA had no effect on the inhibition of cyclic AMP formation by adrenaline.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Pharmacology
Date of Award: 1986
Depositing User: Enlighten Team
Unique ID: glathesis:1986-77421
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 11:53
Last Modified: 14 Jan 2020 11:53

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