CAPN1 (Calpain1)-Dependent Cleavage of STIM1 (Stromal Interaction Molecule 1) Results in an Enhanced SOCE (Store-Operated Calcium Entry) in Human Neonatal Platelets

Abstract

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 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 [TMEM66]) were found in neonatal platelet 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, result in an enhanced thapsigargin-evoked SOCE as observed in neonatal platelets.

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

“…Given that calcium is pumped in the AGs by the activity of SERCA3 and the acidic nature of these granules, we further analysed the possible alteration in the acidic nature of these granules using LysoSensor-green, a pH-sensitive dye that is directed to the acidic compartments and, thus, is widely used to stain lysosomes. [45][46][47] Our results demonstrated an alteration in neonatal platelet AGs as the amount of LysoSensor-green accumulated in these AGs was reduced in neonatal platelets compared with platelets from maternal and control women (see confocal images in Figure S3C; Blue: DTS stained with ER-Tracker, Green: AGs stained with LysoSensor-green). As a result, the subsequent addition of GPN to platelets caused a rapid reduction in LysoSensor-green fluorescence in maternal platelets; meanwhile, fluorescence in neonatal platelets unchanged (Figure S3B; Video S1).…”

“…As reported in the literature, TG at very low concentration (10 nM) depletes DTS from adult platelets because SERCA2b is found in DTS membranes; meanwhile, 20 μM of TBHQ blocks SERCA3 function and, subsequently, depletes Ca 2+ stored in the AGs, where the SERCA3 isoform has been localized. 24,45,46 As shown in Figure 2A, TBHQ (20 μM) was unable to mobilize Ca 2+ from AGs in neonatal platelets compared with platelets from maternal or control woman (p < 0.05, n = 4); similarly, Ca 2+ entry due to emptying of AGs was also significantly reduced by 80% ± 10% compared with adult platelets (p < 0.001; n = 2-4). In addition, preincubation of neonatal platelets for 5 min with TBHQ (20 μM) in a Ca 2+ -free HBS medium (100 μM of EGTA was added) and, subsequent, stimulation with Thr (0.1 U/mL) presented a significant reduction of 20% ± 8% in the Ca 2+ mobilization compared with adult platelets [p < 0.05; compare black-bars in the graph bars of the Figure 2B(…”

“…We analysed possible alterations in the molecular mechanisms underlying Thr-induced Ca 2+ entry in neonatal platelets. In fact, different Ca 2+ stores may regulate different Ca 2+ entry pathways, since both types of platelet stores (DTS and AGs) contain STIM1 in their membranes [43][44][45][46] which is the main regulator of SOCE. 43,44 Hence, we first analysed the content of Orai1 channel (main SOCE channel in platelets); thus, no statistical differences in Orai1 expression were found between neonatal and maternal platelets (Figure 3A; p > 0.05; N = 6).…”

“…Conversely, differences in STIM1 expression, as well as the presence of an alternative STIM1 band were previously reported by our group in neonatal platelets. 45 Additionally, we analysed SARAF, a STIM1 regulator during SOCE. SARAF has been described as the main factor responsible for the mechanism of SCDI.…”

Ginsenoside Re: Are we Close to a Safer Antiplatelet Therapy?

“…5 Plenty of novel insights about platelet physiology, in particular regarding complex molecular dynamics and regulatory networks involving even ion homeostasis are being provided. 6 A physiological pathway that is currently matter of research involves the glycoprotein V, a platelet transmembrane protein which is proteolytically cleaved by thrombin during thrombus formation: it was recently shown that the soluble glycoprotein V released from platelet surface specifically reduces fibrin formation in vitro and in vivo and thereby may prevent occlusive thrombosis without causing hemostatic impairments. 7 Other recent studies found that shear-induced platelet aggregation (SIPA) is primarily involved in platelet activation and aggregation, because local shear stress generated by blood flow in obstructed vessels can be more than 3-fold higher compared with normal vessels.…”