Molecular mechanisms of thrombin-evoked calcium entry in human platelets

• Main Author: Harper, M. T.
• Published: University of Cambridge 2007
• Subjects: 571.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603728

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 <i>de novo </i>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 <i>de novo </i>conformational coupling model. One pathway regulating <i>de novo </i>conformational coupling requires actin polymerisation and pp60^src activation. PAR-1-dependent pp60^src 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 pp60^srcis 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.