EFCAB13 is a Novel Age-Dependent Promotor of SOCE in Blood Platelets and Megakaryoblastic MEG-01 Cells

Abstract

Background

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.

Material and Methods

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.

Results

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.

Conclusion

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.

Keywords

EFCAB13 - STIM1 - Orai1 - SOCE - platelets

• References

• 1 López E, Salido GM, Rosado JA, Berna-Erro A. Unraveling STIM2 function. J Physiol Biochem 2012; 68 (04) 619-633
• 2 Redondo PC, Berna-Erro A, Dionisio N, Rosado JA. Fluorescence-based measurements of the CRAC channel activity in cell populations. Methods Mol Biol 2018; 1843: 69-82
• 3 Pick T, Gamayun I, Tinschert R, Cavalié A. Kinetics of the thapsigargin-induced Ca²⁺ mobilisation: a quantitative analysis in the HEK-293 cell line. Front Physiol 2023; 14: 1127545
• 4 Berna-Erro A, Sanchez-Collado J, Nieto-Felipe J. et al. The Ca²⁺ sensor STIM in human diseases. Biomolecules 2023; 13 (09) 1284
• 5 Nagy M, Mastenbroek TG, Mattheij NJA. et al. Variable impairment of platelet functions in patients with severe, genetically linked immune deficiencies. Haematologica 2018; 103 (03) 540-549
• 6 Berlansky S, Humer C, Sallinger M, Frischauf I. More than just simple interaction between STIM and Orai proteins: CRAC channel function enabled by a network of interactions with regulatory proteins. Int J Mol Sci 2021; 22 (01) 471
• 7 Di Buduo CA, Abbonante V, Marty C. et al. Defective interaction of mutant calreticulin and SOCE in megakaryocytes from patients with myeloproliferative neoplasms. Blood 2020; 135 (02) 133-144
• 8 Di Buduo CA, Moccia F, Battiston M. et al. The importance of calcium in the regulation of megakaryocyte function. Haematologica 2014; 99 (04) 769-778
• 9 Mammadova-Bach E, Nagy M, Heemskerk JWM, Nieswandt B, Braun A. Store-operated calcium entry in thrombosis and thrombo-inflammation. Cell Calcium 2019; 77: 39-48
• 10 Caparrós-Pérez E, Teruel-Montoya R, López-Andreo MJ. et al. Comprehensive comparison of neonate and adult human platelet transcriptomes. PLoS One 2017; 12 (08) e0183042
• 11 Ohmori T, Kashiwakura Y, Ishiwata A, Madoiwa S, Mimuro J, Sakata Y. Silencing of a targeted protein in in vivo platelets using a lentiviral vector delivering short hairpin RNA sequence. Arterioscler Thromb Vasc Biol 2007; 27 (10) 2266-2272
• 12 Di Buduo CA, Balduini A, Moccia F. Pathophysiological significance of store-operated calcium entry in megakaryocyte function: opening new paths for understanding the role of calcium in thrombopoiesis. Int J Mol Sci 2016; 17 (12) 2055
• 13 Borst O, Schmidt EM, Münzer P. et al. The serum- and glucocorticoid-inducible kinase 1 (SGK1) influences platelet calcium signaling and function by regulation of Orai1 expression in megakaryocytes. Blood 2012; 119 (01) 251-261
• 14 Luik RM, Wu MM, Buchanan J, Lewis RS. The elementary unit of store-operated Ca2+ entry: local activation of CRAC channels by STIM1 at ER-plasma membrane junctions. J Cell Biol 2006; 174 (06) 815-825
• 15 Hearn JI, Green TN, Chopra M. et al. N-Methyl-D-aspartate receptor hypofunction in Meg-01 cells reveals a role for intracellular calcium homeostasis in balancing megakaryocytic-erythroid differentiation. Thromb Haemost 2020; 120 (04) 671-686
• 16 Isakari Y, Harada Y, Ishikawa D. et al. Efficient gene expression in megakaryocytic cell line using nucleofection. Int J Pharm 2007; 338 (1–2): 157-164
• 17 Emrich SM, Yoast RE, Xin P. et al. Omnitemporal choreographies of all five STIM/Orai and IP₃Rs underlie the complexity of mammalian Ca²⁺ signaling. Cell Rep 2021; 34 (09) 108760
• 18 Nieto-Felipe J, Macias-Díaz A, Jimenez-Velarde V. et al. Feedback modulation of Orai1α and Orai1β protein content mediated by STIM proteins. J Cell Physiol 2025; 240 (01) e31450
• 19 Long MW, Smolen JE, Szczepanski P, Boxer LA. Role of phorbol diesters in in vitro murine megakaryocyte colony formation. J Clin Invest 1984; 74 (05) 1686-1692
• 20 Lopez JJ, Palazzo A, Chaabane C. et al. Crucial role for endoplasmic reticulum stress during megakaryocyte maturation. Arterioscler Thromb Vasc Biol 2013; 33 (12) 2750-2758
• 21 Lilue J, Doran AG, Fiddes IT. et al. Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci. Nat Genet 2018; 50 (11) 1574-1583
• 22 Dean B, Parkin GM, Gibbons AS. Associations between catechol-O-methyltransferase (COMT) genotypes at rs4818 and rs4680 and gene expression in human dorsolateral prefrontal cortex. Exp Brain Res 2020; 238 (02) 477-486
• 23 Clarelli F, Barizzone N, Mangano E. et al; PROGEMUS, PROGRESSO. Contribution of rare and low-frequency variants to multiple sclerosis susceptibility in the Italian continental population. Front Genet 2022; 12: 800262
• 24 Laitinen VH, Rantapero T, Fischer D. et al; PRACTICAL Consortium. Fine-mapping the 2q37 and 17q11.2-q22 loci for novel genes and sequence variants associated with a genetic predisposition to prostate cancer. Int J Cancer 2015; 136 (10) 2316-2327
• 25 Natanzon Y, Earp M, Cunningham JM. et al. Genomic analysis using regularized regression in high-grade serous ovarian cancer. Cancer Inform 2018; 17: 1176935118755341
• 26 Braun A, Varga-Szabo D, Kleinschnitz C. et al. Orai1 (CRACM1) is the platelet SOC channel and essential for pathological thrombus formation. Blood 2009; 113 (09) 2056-2063
• 27 Gilio K, van Kruchten R, Braun A. et al. Roles of platelet STIM1 and Orai1 in glycoprotein VI- and thrombin-dependent procoagulant activity and thrombus formation. J Biol Chem 2010; 285 (31) 23629-23638
• 28 Varga-Szabo D, Braun A, Kleinschnitz C. et al. The calcium sensor STIM1 is an essential mediator of arterial thrombosis and ischemic brain infarction. J Exp Med 2008; 205 (07) 1583-1591
• 29 Bhuria V, Franz T, Baldauf C. et al. Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry. Cell Commun Signal 2024; 22 (01) 186