SARAF overexpression impairs thrombin-induced Ca2+ homeostasis in neonatal platelets

Abstract

Neonatal platelets present a reduced response to the platelet agonist, thrombin (Thr), thus resulting in a deficient Thr-induced aggregation. These alterations are more pronounced in premature newborns. Here, our aim was to uncover the causes underneath the impaired Ca(2+) homeostasis described in neonatal platelets. Both Ca(2+) mobilization and Ca(2+) influx in response to Thr are decreased in neonatal platelets compared to maternal and control woman platelets. In neonatal platelets, we observed impaired Ca(2+) mobilization in response to the PAR-1 agonist (SFLLRN) or by blocking SERCA3 function with tert-butylhydroquinone. Regarding SOCE, the STIM1 regulatory protein, SARAF, was found overexpressed in neonatal platelets, promoting an increase in STIM1/SARAF interaction even under resting conditions. Additionally, higher interaction between SARAF and PDCD61/ALG2 was also observed, reducing SARAF ubiquitination and prolonging its half-life. These results were reproduced by overexpressing SARAF in MEG01 and DAMI cells. Finally, we also observed that pannexin 1 permeability is enhanced in response to Thr in control woman and maternal platelets, but not in neonatal platelets, hence, leading to the deregulation of the Ca(2+) entry found in neonatal platelets. Summarizing, we show that in neonatal platelets both Ca(2+) accumulation in the intracellular stores and Thr-evoked Ca(2+) entry through either capacitative channels or non-selective channels are altered in neonatal platelets, contributing to deregulated Ca(2+) homeostasis in neonatal platelets and leading to the altered aggregation observed in these subjects.