1,25(OH) ₂ vitamin D ₃ ‐dependent inhibition of platelet Ca ²⁺ signaling and thrombus formation in klotho‐deficient mice

Platelets are activated by increased cytosolic Ca 2+ concentration ([Ca 2+ ] i ) following store‐operated calcium entry (SOCE) accomplished by calcium‐release‐activated calcium (CRAC) channel moiety Orai1 and its regulator STIM1. In other cells, Ca 2+ transport is regulated by 1,25(OH) 2 vitamin D 3 [1,25(OH) 2 D 3 ]. 1,25(OH) 2 D 3 formation is inhibited by klotho and excessive in klotho‐deficient mice (kl/kl ). The present study explored the effect of klotho deficiency on platelet Ca 2+ signaling and activation. Platelets and megakaryocytes isolated from WT and kl/kl ‐mice were analyzed by RT‐PCR, Western blotting, confocal microscopy, Fura‐2‐fluorescence, patch clamp, flow cytometry, aggregometry, and flow chamber. STIM1/Orai1 transcript and protein levels, SOCE, agonist‐induced [Ca 2+ ] i increase, activation‐dependent degranulation, integrin α IIb β 3 activation and aggregation, and thrombus formation were significantly blunted in kl/kl platelets (by 27–90%). STIM1/Orai1 transcript and protein levels, as well as CRAC currents, were significantly reduced in kl/kl megakaryocytes (by 38–73%) and 1,25(OH) 2 D 3 ‐treated WT megakaryocytes. Nuclear NF‐κB subunit p50/p65 abundance was significantly reduced in kl/kl ‐megakaryocytes (by 51–76%). Transfection with p50/p65 significantly increased STIM1/Orai1 transcript and protein levels in megakaryocytic MEG‐01 cells (by 46–97%). Low‐vitamin D diet (LVD) of kl/kl mice normalized plasma 1,25(OH) 2 D 3 concentration and function of platelets and megakaryocytes. Klotho deficiency inhibits platelet Ca 2+ signaling and activation, an effect at least partially due to 1,25(OH) 2 D 3 ‐dependent down‐regulation of NF‐κB activity and STIM1/Orai1 expression in megakaryocytes.—Borst, O., Münzer, P., Schmid, E., Schmidt, E.‐M., Russo, A., Walker, B., Yang, W., Leibrock, C., Szteyn, K., Schmidt, S., Elvers, M., Faggio, C., Shumilina, E., Kuro‐o, M., Gawaz, M., Lang, F. 1,25(OH) 2 vitamin D 3 ‐dependent inhibition of platelet Ca 2+ signaling and thrombus formation in klotho‐deficient mice. FASEB J . 28, 2108–2119 (2014). www.fasebj.org