Alejandro Berna-Erro

Intracellular Ca²⁺ homeostasis is essential to regulate the molecular mechanisms underlying platelet physiology and aggregation. Store-operated Ca²⁺ entry (SOCE) is the main mechanism of extracellular Ca²⁺ influx in platelets and it has been involved in platelet aggregation. We reported the function of the uncharacterized protein EFCAB13 as a positive regulator of SOCE in megakaryoblastic cells and platelets from either mothers and their newborns. Alternatively, MEG-01 and HEK293 cells were used as sourrogated of platelets to perform changes in the expression of EFCAB13, which was ensured by immunoprecipitations and Western Blotting techniques. Once EFCAB13 was genetically modifies and to achive its role in Ca²⁺ homeostasis we used fluorescence microscope under single cells configuration. Finally, MEG-01 maturation was assay using flow-cytometry and specific fluorescent antibodies or cell cycle dies. Intracellular distribution and protein interaction was also evaluated by confocal microscopy. EFCAB13 is underexpressed in neonatal platelets as compared with adults, indicating an age-dependent expression. Silencing of EFCAB13 expression reduced the colocalization of STIM1 with Orai1 and, subsequently, impairs SOCE in megakaryoblastic MEG-01 cells stimulated with thapsigargin (TG). Coimmunoprecipitation experiments showed that EFCAB13 interacts with Orai1, and colocalization between both proteins increases during TG-evoked SOCE. Indeed, EFCAB13 localized nearby the ER during TG-evoked SOCE activation. Finally, PMA-induced MEG-01 maturation was altered in EFCAB13-silenced cells. Altogether, we conclude that EFCAB13 binds to Orai1 to stabilize and regulate the STIM1/Orai1 complex formation, which is necessary to support SOCE in adults. However, its function is less important in neonatal platelets. This regulatory function may also be important for megakaryocytic physiology during maturation. Therefore, EFCAB13 could be an interesting target for specific treatment of maternal hemostatic disorders during pregnancy. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Please, contact pcr@unex.es or alejandrobe@unex.es for more details and information. This study followed the Helsinki's Declaration guidelines and the mandatory of the Ethical committees of the University of Extremadura and the Junta of Extremadura Health Service. P.C.R., and A.B.-E. contributed to conceptualization. E.F. and M.P.G. contributed to blood sample collection, and managing. P.C.R., A.B-E., R.T-M., and M.A.C-P. contributed to investigation. P.C.R. and A.B-E. contributed to validation. P.C.R. and A.B-E. contributed to supervision. A.B-E. contributed to writing the original manuscript. P.C.R., M.P.G., R.T-M., and F.F-M. contributed to manuscript editing. P.C.R. contributed to funding acquisition. P.C.R. contributed to project administration. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Received: 07 April 2025 Accepted: 25 August 2025 Article published online: 19 September 2025 © 2025. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany

Summary: 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.

Hypoxia is considered to be a stressful physiological condition, which may occur during labor and the later stages of pregnancy as a result of, among other reasons, an aged placenta. Therefore, when gestation or labor is prolonged, low oxygen supply to the tissues may last for minutes, and newborns may present breathing problems and may require resuscitation maneuvers. As a result, poor oxygen supply to tissues and to circulating cells may last for longer periods of time, leading to life-threatening conditions. In contrast to the well-known platelet activation that occurs after reperfusion of the tissues due to an ischemia/reperfusion episode, platelet alterations in response to reduced oxygen exposition following labor have been less frequently investigated. Newborns overcome temporal hypoxic conditions by changing their organ functions or by adaptation of the intracellular molecular pathways. In the present review, we aim to analyze the main platelet modifications that appear at the protein level during hypoxia in order to highlight new platelet markers linked to complications arising from temporal hypoxic conditions during labor. Thus, we demonstrate that hypoxia modifies the expression and activity of hypoxic-response proteins (HRPs), including hypoxia-induced factor (HIF-1), endoplasmic reticulum oxidase 1 (Ero1), and carbonic anhydrase (CIX). Finally, we provide updates on research related to the regulation of platelet function due to HRP activation, as well as the role of HRPs in intracellular Ca²⁺ homeostasis.

The STIM family of proteins plays a crucial role in a plethora of cellular functions through the regulation of store-operated Ca²⁺ entry (SOCE) and, thus, intracellular calcium homeostasis. The two members of the mammalian STIM family, STIM1 and STIM2, are transmembrane proteins that act as Ca²⁺ sensors in the endoplasmic reticulum (ER) and, upon Ca²⁺ store discharge, interact with and activate the Orai/CRACs in the plasma membrane. Dysregulation of Ca²⁺ signaling leads to the pathogenesis of a variety of human diseases, including neurodegenerative disorders, cardiovascular diseases, cancer, and immune disorders. Therefore, understanding the mechanisms underlying Ca²⁺ signaling pathways is crucial for developing therapeutic strategies targeting these diseases. This review focuses on several rare conditions associated with STIM1 mutations that lead to either gain- or loss-of-function, characterized by myopathy, hematological and immunological disorders, among others, and due to abnormal activation of CRACs. In addition, we summarize the current evidence concerning STIM2 allele duplication and deletion associated with language, intellectual, and developmental delay, recurrent pulmonary infections, microcephaly, facial dimorphism, limb anomalies, hypogonadism, and congenital heart defects.

Background: Altered intracellular Ca ²⁺ homeostasis in neonatal platelets has been previously reported. This study aims to examine the changes in the Ca ²⁺ entry through the store-operated calcium entry (SOCE) mechanism in neonatal platelets. Methods: Human platelets from either control women, mothers, and neonates were isolated and, following, were fixed after being treated as required. Platelet samples were analyzed by Western blotting, qRT-PCR, and MALDITOF/TOF. Ca ²⁺ homeostasis was also determined. Culture cells were used as surrogated of platelets to overexpress the proteins of interest to reproduce the alterations observed in platelets. Results: Altered TG (thapsigargin)-evoked SOCE, alternative molecular weight form of STIM1 (stromal interaction molecule 1; s-STIM1 [short STIM1 isoform (478 aa)], around 60 kDa) and overexpression of SARAF (SOCE-associated regulatory factor) were found in neonatal platelets as compared to maternal and control women platelets. s-STIM1 may result due to CAPN1 (calpain1)-dependent processing, as confirmed in platelets and MEG01 cells by using calpeptin and overexpressing CAPN1, respectively. In HEK293 (STIM1 and STIM2 [stromal interaction molecule 2] double knockout) cells transfected either with c-STIM1 (canonical STIM1 [685 aa]), s-STIM1 (478), STIM1B (540), and CAPN1 overexpression plasmids, we found s-STIM1 and c-STIM1, except in cells overexpressing s-STIM1 (478) that lacked CAPN1 target residues. These results and the in silico analysis, lead us to conclude that STIM1 is cleaved at Q496 by CAPN1. Ca ²⁺ imaging analysis and coimmunoprecipitation assay using MEG01 and HEK293 cells overexpressing SARAF together with s-STIM1 (478) reported a reduced slow Ca ²⁺ –dependent inactivation, so reproducing the Ca ²⁺ -homeostasis pattern observed in neonatal platelets. Conclusions: CAPN1 may cleave STIM1 in neonatal platelets, hence, impairing SARAF coupling after SOCE activation. s-STIM1 may avoid slow Ca ²⁺ –dependent inactivation and, subsequently, results in an enhanced TG-evoked SOCE as observed in neonatal platelets.

N-linked glycosylation is a post-translational modification that affects protein function, structure, and interaction with other proteins. The store-operated Ca²⁺ entry (SOCE) core proteins, Orai1 and STIM1, exhibit N-glycosylation consensus motifs. Abnormal SOCE has been associated to a number of disorders, including cancer, and alterations in Orai1 glycosylation have been related to cancer invasiveness and metastasis. Here we show that treatment of non-tumoral breast epithelial cells with tunicamycin attenuates SOCE. Meanwhile, tunicamycin was without effect on SOCE in luminal MCF7 and triple negative breast cancer (TNBC) MDA-MB-231 cells. Ca²⁺ imaging experiments revealed that expression of the glycosylation-deficient Orai1 mutant (Orai1N223A) did not alter SOCE in MCF10A, MCF7 and MDA-MB-231 cells. However, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) significantly attenuated SOCE in MCF10A cells but was without effect in SOCE in MCF7 and MDA-MB-231 cells. In non-tumoral cells impairment of STIM1 N-linked glycosylation attenuated thapsigargin (TG)-induced caspase-3 activation while in breast cancer cells, which exhibit a smaller caspase-3 activity in response to TG, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) was without effect on TG-evoked caspase-3 activation. Summarizing, STIM1 N-linked glycosylation is essential for full SOCE activation in non-tumoral breast epithelial cells; by contrast, SOCE in breast cancer MCF7 and MDA-MB-231 cells is insensitive to Orai1 and STIM1 N-linked glycosylation, and this event might participate in the development of apoptosis resistance.

Orai1, the first identified member of the Orai protein family, is ubiquitously expressed in the animal kingdom. Orai1 was initially characterized as the channel responsible for the store-operated calcium entry (SOCE), a major mechanism that allows cytosolic calcium concentration increments upon receptor-mediated IP₃ generation, which results in intracellular Ca²⁺ store depletion. Furthermore, current evidence supports that abnormal Orai1 expression or function underlies several disorders. Orai1 is, together with STIM1, the key element of SOCE, conducting the Ca²⁺ release-activated Ca²⁺ (CRAC) current and, in association with TRPC1, the store-operated Ca²⁺ (SOC) current. Additionally, Orai1 is involved in non-capacitative pathways, as the arachidonate-regulated or LTC4-regulated Ca²⁺ channel (ARC/LRC), store-independent Ca²⁺ influx activated by the secretory pathway Ca²⁺-ATPase (SPCA2) and the small conductance Ca²⁺-activated K⁺ channel 3 (SK3). Furthermore, Orai1 possesses two variants, Orai1α and Orai1β, the latter lacking 63 amino acids in the N-terminus as compared to the full-length Orai1α form, which confers distinct features to each variant. Here, we review the current knowledge about the differences between Orai1α and Orai1β, the implications of the Ca²⁺ signals triggered by each variant, and their downstream modulatory effect within the cell.

Background/Aims: STIM1 and Orai1 are the key components of store-operated Ca2+ entry (SOCE). Among the proteins involved in the regulation of SOCE, SARAF prevents spontaneous activation of SOCE and modulates STIM1 function. Methods: Cytosolic Ca2+ mobilization was estimated in fura-2-loaded cells using an epifluorescence inverted microscope. STIM1 interaction with Orai1, EFHB (EF-hand domain family member B, also known as CFAP21) and SARAF was detected by immunoprecipitation followed by Western blotting using specific antibodies. The involvement of EFHB in the translocation of NFAT to the nucleus was detected by confocal microscopy. Results: Here, we report the identification of EFHB as a new SOCE regulator. EFHB interacts with STIM1 upon store depletion and dissociates through a Ca2+-dependent mechanism. RNAi-mediated silencing as well as overexpression studies revealed that EFHB plays a relevant role in the interaction of STIM1 and Orai1 upon store depletion, the activation of SOCE and NFAT translocation from the cytosol to the nucleus. Silencing EFHB expression abolished the dissociation of SARAF from STIM1, which indicates that EFHB might play an important role in the dynamic interaction between both proteins, which is relevant for the activation of Orai1 channels upon Ca2+ store depletion and their subsequent modulation via slow Ca2+-dependent inactivation. Conclusion: Our results indicate that EFHB is a new SOCE regulator that modulates STIM1-SARAF interaction.

Cytosolic Ca²⁺ plays an important role in cellular biology, and since its identification as a second messenger, a number of techniques and methods to analyze the changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_c) induced by physiological agonists have been developed. Changes in [Ca²⁺]_c might be determined in single cells or in cell populations. Measurement in single cells allows to determine changes in [Ca²⁺]_c at a subcellular level but often results in heterogeneous responses among cells. Determination of intracellular Ca²⁺ mobilization at the cell population level reduces this heterogeneity and allows [Ca²⁺]_c measurements in small cells that load little amounts of indicator. Here, we describe the measurement of agonist-evoked changes in [Ca²⁺]_c associated with Ca²⁺ influx in cell populations.