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Title: Molecular mechanisms of thrombin-evoked calcium entry in human platelets
Author: Harper, M. T.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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The mechanisms responsible for thrombin-evoked Ca2+ entry in human platelets were investigated. Previous studies have identified several Ca2+ entry pathways in platelets. Store-operated Ca2+ entry (SOCE) is activated by a decrease in the Ca2+ content of the intracellular Ca2+ stores following Ca2+ release. In addition, non-capacitative cation entry (NCCE) may be activated independently of store depletion. However, the contribution of these pathways to the Ca2+ entry evoked by physiological agonists, such as thrombin, has not been previously clearly defined. Conflicting models for SOCE activation have been proposed, and the de novo conformational coupling model and the CIF-iPLA2 model were further investigated. The data presented here do not support a necessary role for iPLA2 in SOCE activation, but rather suggest that iPLA2 was necessary for phospholipid remodelling in resting cells. The data do however support a role for InsP3 in SOCE, as proposed by the de novo conformational coupling model. One pathway regulating de novo conformational coupling requires actin polymerisation and pp60src activation. PAR-1-dependent pp60src activation is here shown to be dependent on increased [Ca2+]i, cPKC and actin polymerisation. However, PAR-1-dependent Ca2+ entry was independent of actin polymerisation and cPKC, and so pp60src is unlikely to be involved. ERK activation is required for the remaining SOCE, and ERK was required for approximately 30% of PAR-1-dependent Ca2+ entry. Combined inhibition of ERK and actin polymerisation had no further effect. ERK inhibition had little effect on PAR-1-dependent platelet aggregation. Therefore, these data suggest that SOCE plays only a small role in PAR-1-dependent Ca2+ entry, and that store-independent Ca2+ entry pathways are likely to play a major role in thrombin-evoked platelet activation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available