Carbon Nanotubes Activate Store Operated Calcium Entry (SOCE) In Human Platelets Manifested by STIM1 Capping
Authors: Lacerda Silvia H. , Semberova Jana , Holada Karel , Simakova Olga , Simak Jan 
Published: October 2019

Abstract
Abstract 3190
Carbon nanotubes (CNTs) are a promising new class of materials with many potential biomedical applications. CNTs have profound impact on the development of diagnostic biosensors, drug delivery nanosystems, imaging nanoprobes for intravascular use or other devices that come in contact with blood. Therefore, the biocompatibility/toxicity of CNTs in blood is a critical safety issue. We have previously shown that agglomerates of structurally different CNTs but not fullerene (nC60), activate human platelets by inducing extracellular Ca2+ influx (Semberova, J. et al.: Nano Lett., 2009). In addition, we demonstrated that CNT-induced Ca2+ entry in platelets could be inhibited by calcium channel blockers SKF 96365 and 2-APB. Here we investigate a mechanism of CNT-induced platelet activation. Since both unmodified CNTs and nC60 are hydrophobic and insoluble in aqueous solutions, characterization of the dispersed nanomaterial agglomerates in plasma is critical for evaluation of effects on platelets. Flow particle image analysis showed that both multiwalled carbon nanotubes (MWCNTs) M60 (>95% purity; outer diameter 60–100 nm, SES) and nC60 (99.9%; MER) form agglomerates of a similar size range in plasma: median particle size 1.0 μm (10th-90th%: 0.4–2.9) μm for M60, and 0.7 μm (0.4-1.5) μm for nC60. However, roughness and surface area are markedly higher for CNT M60 (128±28 nm; 122 m2/g) compared to fullerene nC60 (4.4±3.9 nm; 0.9 m2/g). Both Field-Emission Scanning Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM) demonstrated that MWCNTs self-assemble to fiber networks which leads in platelet rich plasma to platelet entrapment, formation of pseudopodia, aggregation, platelet plasma membrane budding and release of membrane microparticles. To test whether a surface modification of CNTs leading to increased solubility would affect activity on platelets, we prepared carboxy-MWCNTs (M60(COOH)) by refluxing pristine M60 in a concentrated 3:1 (v/v) H2SO4 and HNO3 mixture at 70°C for 2 hours; followed by washing and dialysis in water. Interestingly, we observed that M60(COOH), which dispersed well in PBS, activate human blood platelets similarly like their pristine counterparts. FESEM showed that both pristine and carboxy-modified M60 self-assemble in plasma forming rope-like structures. Numerous protuberances existed on the top surface of the M60 and M60(COOH) clusters, which were formed by the relatively long, dense net, highly tangled CNTs without specific orientation. In contrast, nC60 particles are largely devoid of these surface features and minimal platelet activation was observed. In addition, TEM analysis with DAB stained platelet dense tubular system (DTS) showed nanopenetration of CNTs through platelet membranes likely causing DTS injury. Changes in calcium concentration in the intracellular compartments were studied in Fura FF loaded platelets in the presence of streptolysin and EGTA (100μM). We demonstrated a marked decrease of calcium in the intracellular stores induced by M60 or M60(COOH), as well as by SERCA inhibitor thapsigargin (TPG). Under the same conditions, nC60 did not induce depletion of calcium from the stores, similarly to the control untreated platelets. Moreover, Laser Scanning Confocal Microscopy (LSCM) using anti STIM1 (clone 44/GOK) monoclonal antibody showed that M60 caused capping of STIM1 molecules in platelets, which indicates activation of the store operated calcium entry (SOCE). Western blotting of platelet lysates showed that the anti STIM1 antibody 44/GOK detected a band at the expected MW of about 85 kDa. This was also confirmed using a polyclonal antibody against carboxy-terminal of STIM1 and corresponding blocking peptide (ProSci, Inc.). LSCM further showed a colocalization of the STIM1 and ORAI1 molecules in platelets. Similar picture of STIM1 capping in platelets was observed after treatment with TPG. In contrast, no STIM1 capping was observed in platelets treated with fullerene nC60. In conclusion, our results showed that the MWCNTs nanopenetrate membranes of DTS and activate SOCE in human platelets. The findings and conclusions in this study have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

Disclosures:
No relevant conflicts of interest to declare.