Carbon Nanotubes Activate Platelets by Facilitating Extracellular Ca²⁺ Influx

Due to a unique combination of their physico-chemical properties, carbon nanotubes (CNTs) are among the principal materials used in biomedical nanotechnologies. Superior mechanical characteristics like elasticity (CNTs 10¹²Pa vs. steel 10⁸Pa) and tensile strength (CNTs 100 GPa vs. steel 2 GPa) make CNTs very attractive candidates as materials for plastic composites for various biomedical applications including devices for collection, processing, and storage of blood transfusion products. Moreover, CNTs have a profound impact on the development of diagnostic biosensors, drug delivery nanosystems, or imaging nanoprobes for intravascular use. Thus the investigation of CNT activities on platelets is a critical safety issue. We studied the effects of PBS suspensions of structurally different purified CNT materials from different manufacturers (Nanolab, NanoAmor, SES, MER) on human platelets (PLTs) and compared their effects to amorphous carbon black nanoparticles (ACB; > 99%, mean size 30 nm, Aldrich), C₆₀ fullerene (nC₆₀; 99.9%), fullerenol C₆₀(OH)₂₄ (both from MER Corp.), and NIST standard polystyrene nanobeads (PNBs; 20nm and 200nm; Duke Scientific Corp.). Using aggregometry of human platelet rich plasma (PRP), we found that CNT materials induced PLT aggregation in a concentration dependent manner. Single wall CNTs (SWCNTs) at 100 μg/mL showed PLT-aggregation 34 ± 10%, similarly to most tested multiwall CNTs (MWCNTs), which was significantly higher compared to ACB 15 ± 4%; p = 0.03. In contrast, fullerene nC₆₀, fullerenol C₆₀OH₂₄, PNBs, or vehicle (PBS) did not cause any significant PLT aggregation (4 ± 0.5%; positive control collagen 100%). Interestingly, the thicker MWCNTs of d = 60nm (MWCNT60) exhibited significantly higher aggregating activity (27 ± 6%) compared to MWCNTs of d = 15nm (13 ± 3%; p = 0.007). Moreover, the bamboo structured MWCNTs showed only very low aggregating activity (7 ± 6%) compared to the corresponding hollow MWCNTs (33 ± 15%; p = 0.001). The PLT aggregation response (MA, AUC) induced by CNTs was proportional to the extracellular Ca²⁺concentration and was completely inhibited by 100 μM EDTA. The activation-dependent Ca²⁺ channel blocker SKF 96365 at 100 μM caused a significant inhibition (64 ± 16%) of MWCNT60-induced PLT-aggregation response. In contrast, no inhibitory effect on CNT-induced PLT-aggregation response was observed with 2-APB (20 μM; IP3 receptor inhibitor), NF 449 (1 μM; P2X1 blocker), MRS 2500 (5 nM; P2Y1 blocker), or TBHQ (20 μM; SERCA3 blocker). The effect of CNTs on intracellular free Ca²⁺ concentration [Ca²⁺]_i was studied using a ratio fluorometry in platelets loaded with a Ca²⁺-sensitive probe FURA-2AM. Changes in fluorescence in individual platelets (n=100) were monitored using an epifluorescence/contrast phase microscope with a low light-level CCD camera/microphotometer assembly. We demonstrated that in contrast to ACB or PNBs, MWCNTs induced in platelets a rapid concentration dependent elevation of [Ca²⁺]_i. For example, MWCNT60 at 100 μg/mL caused a 107 ± 4 nM increase in [Ca²⁺]_i in platelets. No response was observed in Ca²⁺ free buffer. In addition, the flow cytometric analysis demonstrated that CNT-induced PLT activation led to surface exposure of CD62P and CD63 and a release of CD62P⁺ and CD63⁺ platelet membrane microparticles (MPs). In conclusion, we demonstrated that CNTs activate platelets and cause a platelet release reaction, aggregation, and release of MPs. The PLT-activating effect of CNTs is structure dependent: the hollow and thicker (60 nm) MWCNTs were most potent. A likely mechanism is that CNTs induce extracellular Ca²⁺ influx with involvement of activation-dependent Ca²⁺ channels. Our results warrant further studies on blood compatibility of CNT materials for biomedical applications. Caution needs to be applied when considering CNT materials as additives for plastics for transfusion devices and other devices that come in contact with blood. 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.