Question:

Thrombin and CRP activate pore forming channel protein Orai1 resulting in store operated Ca²⁺ entry (SOCE) with Ca²⁺ dependent release of platelet granules, activation of integrin α_IIbβ₃, adhesion, aggregation and thrombus formation. Platelets lack nuclei and are thus unable to modify protein abundance by transcriptional regulation. Nevertheless, they still contain pre-mRNA and mRNA and are thus able to express proteins by stimulation of translation and to translocate them onto the cell membrane. Both mechanisms are sensitive to phosphoinositide-3-kinase (PI3K). The present study explored whether thrombin and CRP modify Orai1 protein abundance and cell surface expression.

Methods & Results:

Platelets contain according to RT-PCR pre-mRNA and mRNA encoding Orai1. Activation with thrombin (0.1 U/ml) result in a significant decline of pre-mRNA and an increase of mRNA, which is, according to Western blotting and FACS, paralleled by a marked and statistically significant increase of Orai1 protein abundance and surface expression. The increase of Orai1 is shown to be insensitive to the inhibition of transcription with actinomycin (4 µg/ml), but is significantly blunted by inhibition of translation with puromycin (100 µM) or by inhibition of PI3K with wortmannin (100 nM) or LY294002 (25 µM). The increase of surface expression is additionally sensitive to inhibition of both PI3K or protein kinase C (PKC).

Conclusions:

Activation of platelets stimulates the expression of Orai1 protein abundance and surface expression, thus augmenting the power of Ca²⁺ signaling.