1,25(OH)₂ vitamin D₃-dependent inhibition of platelet Ca²⁺ signaling and thrombus formation in klotho-deficient mice

Platelets are activated by increased cytosolic Ca²⁺ concentration ([Ca²⁺]_i) following store-operated calcium entry (SOCE) accomplished by calciumrelease-activated calcium (CRAC) channel moiety Orai1 and its regulator STIM1. In other cells, Ca²⁺ transport is regulated by 1,25(OH)₂ vitamin D₃ [1,25(OH) ₂D₃]. 1,25(OH)₂D₃ formation is inhibited by klotho and excessive in klotho-deficient mice (kl/kl). The present study explored the effect of klotho deficiency on platelet Ca²⁺ 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 [Ca²⁺]_i increase, activation-dependent degranulation, integrin α_IIbβ₃ 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) ₂D₃-treated WT megakaryocytes. Nuclear NF-kB 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)₂D₃ concentration and function of platelets and megakaryocytes. Klotho deficiency inhibits platelet Ca²⁺ signaling and activation, an effect at least partially due to 1,25(OH)₂D₃-dependent down-regulation of NF-kB activity and STIM1/Orai1 expression in megakaryocytes.