Cytosolic Free Calcium and Platelet Responses to Putative Lipid Mediators of Platelet Activation

Shaw, Angus M (1985) Cytosolic Free Calcium and Platelet Responses to Putative Lipid Mediators of Platelet Activation. PhD thesis, University of Glasgow.

Abstract

"Primary" platelet aggregation is usually reversible and is mediated solely by an exogenous agonist. "Secondary" platelet aggregation is irreversible and is initiated by an exogenous mediator but propagated by endogenous mediators synthesised (eg. TXA2 , PGG2 , PGH2 ) and/or secreted (eg. ADP, 5HT) by activated platelets. Activated platelets also synthesis two phospholipids l-0-alkyl-2-acetyl-glycerol-3-phosphorylcholine (PAF; PAF acether) and lysophosphatidic acid (LPA) which are potent inducers of platelet activation and hence may also serve as endogenous mediators of human platelet activation. These various agonists interact with specific recognition sites or receptors in/on the platelet. Agonist-receptor interaction influences the intracellular concentrations of biochemical intermediaries. These so-called "second messenger" molecules (eg. Ca++, cAMP, 1,2-diacylglycerol) influence the rates of those very biochemical reactions that govern the cellular response. At the commencement of this study no systematic examination of the effects and/or mechanism(s) of action on platelets of putative lipids mediators of platelet adivation (PAF, LPA and TXA2 ) had been conducted. Conseguently my initial studies were concerned with assessing:- 1. The effects of PAF, LPA, TXA2 (using U44069, a TXA2 mimetic) on platelet aggregation, degranulation and TXB2 biosynthesis. 2. The role of endogenous arachidonate metabolites and ADP in the above responses. Thereafter, using the fluorescent Ca++ indicator dye quin2, I examined the role, the sources and the pharmacology of elevated cytosolic free calcium concentration ([Ca++]i) in agonist-induced platelet activation. In platelet rich plasma PAF (10 - 100nM), U44069 (10 - 100nM) and LPA (1 - 30 muM) induced "primary" aggregation whereas higher concentrations elicited "secondary" aggregation, degranulation and (except for U44069) TXB2 biosynthesis. Agents : (aspirin, dazoxiben, trimethoguinol and 13-azaprostanoic acid) that inhibit the formation or action of PGG2, PGH2 and TXA2 impaired PAF- and LPA-induced aggregation and degranulation. In this respect the thromboxane synthetase inhibitor dazoxiben was much less effective than inhibitors of cyclooxygenase or endoperoxide/TXA2 receptor antagonists. It was concluded that PAF and LPA-induced secondary aggregation and degranulation depend upon an intact cyclooxygenase but not thromboxane synthetase. The ADP receptor antagonist (beta-gamma methylene ATP) suppressed "secondary" aggregation but not degranulation or TXB2 formation elicited by PAF or LPA. Conseguently, endogenous ADP is important in mediating phospholipid-induced secondary aggregation. Resting [Ca++]i in quin2 loaded platelets was estimated to be 90 + 3 nM The platelet stimulatory agonists PAF, LPA, U44069, thrombin, ADP, 3HT and vasopressin but not adrenaline induced a concentration-dependent elevation of Ca++ i to around 500nM. Chelation of extracellular Ca++ using EGTA markedly attenuated the elevation of [Ca++]i induced by all agonists, indicating that the major source of elevated Ca++ i derives via influx of external Ca++. Moreover the effects of PAF and ADP on 5HT release were markedly attenuated by removal of external Ca++ whereas the effects of thrombin were only slightly reduced. This suggests that for PAF and ADP elevated [Ca++]i is an important mediator/determinant of 5HT release, but is less important for the effect of thrombin. Depolarization of the platelet trans-membrane potential using high [formula removed] failed to elevate [Ca++]i or alter the extent of the elevation of [Ca++]i inducedby agonists. In addition, the dihydropyridine Ca++ channel agonist BAY K8644 failed to elevate i or alter responses to agonists. It is concluded that Ca++ influx across the plasma membrane occurs via channels that open as a consequence of agonist-receptor interaction - receptor operated channels (ROCs) rather than as a consequence of changes in membrane potential - voltage operated channels (VOCs). (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Pharmacology
• Date of Award: 1985
• Depositing User: Enlighten Team
• Unique ID: glathesis:1985-77361
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jan 2020 09:10
• Last Modified: 14 Jan 2020 09:10
• URI: https://theses.gla.ac.uk/id/eprint/77361