Molecular mechanisms of thrombin-evoked calcium entry in human platelets

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 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^src 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.