2018IFTIKHARTPhD.pdf (5.68 MB)
A study of Ca2+ signalling during platelet-human erythroleukemia cell interactions and the heterogeneity of platelet store-operated Ca2+ entry in healthy donors
thesisposted on 2019-02-14, 09:59 authored by Tayyaba Iftikhar
Growing evidence indicates a role for platelets in cancer metastasis. This study has investigated communication between human platelets and the human megakaryocytic/erythroleukemic cell line (HEL) at the level of the ubiquitous second messenger, intracellular Ca2+ ([Ca2+]i). In mixed cell suspensions, platelets amplified [Ca2+]i responses to both thrombin and Pam3CSK4 (TLR1/2 agonist). Both agonists evoked ATP release from platelets, but not HEL cells, over a time course similar to the enhanced [Ca2+]i increase. HEL cells showed robust [Ca2+]i responses to ADP and ATP and expressed multiple P2 receptor subtypes (P2X4>P2Y11>>P2X1>P2X7>P2X6> P2Y12>P2Y13> P2Y2>P2Y1). The interaction mechanism proved difficult to study with thrombin due to direct Ca2+ responses in both cell types. However, Pam3CSK4 evoked a direct response only in platelets alone. ThromboxaneA2 dependent ATP/ADP secretion was required for the platelet-dependent HEL response to Pam3CSK4 as this was blocked by either apyrase or aspirin. The role of specific P2 receptors could not be confirmed due to poor antagonist specificity. At the single cell level, Pam3CSK4-stimulated platelets evoked HEL Ca2+ responses that resembled responses to ATP more than ADP. Morphological responses were also observed in HEL cells, but did not correlate with the pattern of [Ca2+]i increases. In a further set of experiments, platelet store operated Ca2+ entry (SOCE) in healthy individuals was assessed with a view to studying its role in cancer. Marked inter-donor heterogeneity of SOCE was observed following activation by thapsigargin that reflected variable Ca2+ release due in part to a cAMP-dependent pathway. Less SOCEheterogeneity was observed with cyclopiazonic acid, which may therefore be a preferable pharmacological tool for studies of this major platelet Ca2+ entry pathway. In conclusion, this study has identified a role for Ca2+ signalling between platelets and myeloid leukemic cells that depends upon ATP/ADP secretion and thromboxaneA2 release and may play a role in metastasis.
Supervisor(s)Mahaut-Smith, Martyn; Willars, Gary
Date of award2019-01-18
Author affiliationDepartment of Molecular and Cell Biology
Awarding institutionUniversity of Leicester