Question:

Platelets are activated by increase of cytosolic Ca²⁺-concentration ([Ca²⁺]_i) following stimulation of store operated Ca²⁺ entry (SOCE), which is accomplished by the pore forming unit Orai1 and its regulator STIM1. In other cell types Ca²⁺-transport across cell membranes is regulated by dihydroxyvitamin D3 (1,25(OH)₂D₃). Formation of 1,25(OH)₂D₃ is inhibited by anti-aging protein klotho. Thus, 1,25(OH)₂D₃ plasma levels are excessive in klotho-deficient mice (kl/kl), which suffer from severe vascular calcification. The present study explored whether klotho deficiency impacts on [Ca²⁺]_i and function of platelets.

Methods and Results:

SOCE and agonist-induced [Ca²⁺]_i increase were significantly blunted in platelets from kl/kl mice. Similarly, degranulation, integrin α_IIbβ₃ activation, aggregation and in vitro thrombus formation were significantly impaired in platelets from kl/kl mice. Low vitamin D diet (LVD) abrogated the impaired Ca²⁺-dependent activation of platelets from kl/kl mice. Platelet and megakaryocyte transcript levels and membrane protein abundance of STIM1 and Orai1 were significantly reduced in platelets from kl/kl mice and significantly decreased by treatment with 1,25(OH)₂D₃ in wildtype megakaryocytes. Nuclear abundance of NF-?B subunits p50 and p65 were significantly lower in megakaryocytes derived from kl/kl mice than in megakaryocytes from wildtype littermates or from klotho-deficient mice treated with LVD. Transfection of MEG-01 cells with p50/p65 mimicked the 1,25(OH)₂D₃-induced decrease of STIM1 and Orai1 expression on mRNA and protein level.

Conclusions:

In conclusion, klotho-deficiency decreases SOCE in platelets leading to impaired thrombus formation. The effect is at least in part due to 1,25(OH)₂D₃-induced impairment of NF-?B-dependent STIM1 and Orai1 expression in megakaryocytes.