Due to recent advances in nanotechnology, it is expected that carbon nanostructures will soon be used for different medical applications including devices for transfusion medicine. Fullerenes and carbon nanotubes have a profound impact on the development of diagnostic biosensors, drug delivery nanosystems, or imaging nanoprobes for intravascular use. In addition, fullerenes can also be used as components of plastic and filtration membranes. Furthermore, fullerene C60 has been shown to have antiviral and antibacterial properties and its photodynamic potential for pathogen-reduction treatment of blood products has been suggested. Hydroxylated C60 derivatives have potent antioxidant properties, however, there are concerns about possible cytotoxic effects of fullerenes and/or their oxidative products. We studied the effects of C60 on human umbilical vein endothelial cells (HUVECs) in culture. We used a water suspension of nonsoluble C60 (C60 preparations of 99.5% and 99.9% purity from SES Res., Houston, TX, and MER Corp., Tuscon, AZ, respectively) at 4 μg/mL, particle size < 500 nm, and the water soluble polyhydroxylated fullerene derivative fullerenol C60(OH)24 at 1 – 100 μg/mL (MER Corp., Tuscon, AZ). We found that 24 hr treatment of HUVECs with C60(OH)24 at 100 μg/mL significantly increased cell surface expression of ICAM-1(CD54) (67±4%CD54+ cells vs. 19±2 % CD54+ cells in control; p< 0.001). Moreover, this treatment induced expression of tissue factor (CD142, detected by HTF-1 Mab) on HUVECs (54±20% CD142+ cells vs. 4±2% CD142+ cells in control; p=0.008) and increased exposure of phosphatidylserine (PS, detected by Annexin V) (29±2% PS+ cells vs. 12±5% PS+ cells in control; p<0.001). In addition, using the MTS proliferation assay, we found that C60(OH)24 significantly inhibited HUVEC proliferation (35± 8% inhibition at 10 μg/mL). Analysis of cell cycle and DNA fragmentation (TUNEL) by flow cytometry showed that both C60 and C60(OH)24 caused G1 arrest of HUVECs and C60(OH)24 induced significant apoptosis (21±2% TUNEL+ cells at 100 μg/mL of C60(OH)24 vs. 4±2% TUNEL+ cells in control; p<0.001). The acute effect of fullerenes on intracellular free Ca2+ concentration [Ca2+]i was studied, using a ratio fluorometry in HUVECs, GT1-7 and PC12 cells. Cells were loaded with a Ca2+-sensitive probe FURA-2AM. We demonstrated that both C60 and C60(OH)24 induced a rapid concentration dependent elevation of [Ca2+]i. For example, C60(OH)24 at 100 μg/mL caused 149±30 nM increase in [Ca2+]i in HUVECs. The activity could be inhibited by EGTA, suggesting that the source of [Ca2+]i in fullerene stimulated calcium flux is predominantly from the extracellular environment. In contrast, fullerenes tested at the given concentrations, did neither induce platelet aggregation nor affect TRAP or ADP/epinephrine-induced platelet aggregation, as tested with human PRP. In conclusion, our results indicate possible adverse effects of fullerenes on the endothelium. Hydroxyfullerene C60(OH)24, which may be formed as an oxidative product of C60 fullerenes, inhibited cell proliferation and had both proinflammatory and proapoptotic effects on endothelial cells. These findings warrant further studies on vascular biocompatibility of carbon nanostructures. The views of the authors represent their scientific opinion and should not be construed as FDA policy.

UV light can be used to irradiate platelets to reduce immune responses and activate chemically-mediated pathogen reduction agents. Platelets subjected to UV-based processing methods develop demonstrable losses of in vivo performance. We compared the “lesions” induced by the different wavelengths of UV light on human platelet performance in a SCID mouse animal model of recovery and survival. Previously we demonstrated that severe combined immunodeficient (SCID) mice could be used as an animal model to identify both severely damaged (Blood 106(11), p537a, 2005) and moderately damaged human platelets (Blood 108(11), p175–176a, 2006). Apheresis human platelets, stored for 1 or 7 days, were exposed to UVA(320–400 nm) or UVB(290–320 nm) light for 20 min (4.8 J/mL) or 40 min (9.6 J/mL). Control platelets were processed in the same manner without UV exposure. Effects on platelets were compared on platelet counts, activation measured by p-selectin (anti-human CD62P, clone AK-4) and in vivo recovery. Platelet counts and in vivo recovery are expressed as % of control platelets, p-selectin measurement represents % of cells expressing the antigen. For in vivo recovery, approximately 1×10 9 platelets (UV-treated or control) were injected into the tail vein of SCID mice (n=4 per each condition) and serial blood samples were collected. Human platelets were detected in mouse whole blood by flow cytometry using an anti-human GPIIbIIIa mAb (clone P2). Recovery was defined as percent of human platelets in mouse circulation 30 minutes post infusion. Comparison of recovery between control and UV treatment platelets was done at 2 hours post infusion as shown in Table 1. These results indicate that UVA produces less activation of platelets and less damage to human platelets recognized by the in vivo model than UVB. The UVA lesion detected by increased in vivo clearance is not cumulative in that longer exposures do not cause an increased loss of in vivo recovery. In comparison, UVB mediated damage is associated with lower in vivo recovery and the damage appears to be cumulative with longer exposure. These differences suggest that UVA and UVB exposure may produce platelet lesions through different mechanism(s). Further investigation into the molecular mechanisms of UVA and UVB lesions may lead to methods that could reduce the negative aspects of UV exposure. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Table 1 Treatment Day 1 Day 7 1= percent of control platelets;2=human platelet recovery in SCID mouse model Platelet count1 P-selectin (%) In vivo recovery2 Platelet count1 P-selectin (%) In vivo recovery2 UVA 20 min 100 13.3±2.7 55.2±7.6 100 20.3±3.8 58.2±4.9 40 min 100 9.9±0.9 55.5±2.9 100 17.3±2.9 54.3±6.7 UVB 20 min 82±3.7 34.6±5.0 30.8±8.9 86±5.3 51.1±6.4 18.5±4.6 40 min 51±10.1 29.5±1.5 6.9±2.6 63±10.5 36.2±1.8 2.47±0.9

Beta-thalassemia results from mutations of the β-hemoglobin (Hbb) gene and reduced functional Hbb synthesis. Excess α-Hb causes globin chain aggregation, oxidation, cytoskeletal damage, and increased red blood cell clearance. These events result in anemia, altered iron homeostasis, and expansion of extramedullary erythropoiesis. Serum transferrin (Tf) is suggested to be an important regulator of erythropoiesis in murine models of thalassemia. The present study was conducted to establish a quantitative proteomic and transcriptomic analysis of transferrin-modulated extramedullary erythropoiesis in the spleen of wild type and thalassemic Hbb(th3/+) mice. Our LC-MS/MS protein analysis and mRNA sequencing data provide quantitative expression estimates of 1590 proteins and 24,581 transcripts of the murine spleen and characterize key processes of erythropoiesis and RBC homeostasis such as the whole heme synthesis pathway as well as critical components of the red blood cell antioxidant systems and the proliferative cell cycling pathway. The data confirm that Tf treatment of nontransfused Hbb(th3/+) mice induces a systematic correction of these processes at a molecular level. Tf treatment of Hbb(th3/+) mice for 60 days leads to a complete molecular restoration of the normal murine spleen phenotype. These findings support further investigation of plasma-derived Tf as a treatment for thalassemia.

3358 Pathophysiology of acute lung injury (ALI) includes an inflammatory component with recruitment and activation of neutrophils to the lungs. One proposed mechanism of transfusion related acute lung injury (TRALI) involves two events; the first is a generalized inflammatory response, as would occur in sepsis, which leads to activation of endothelial cells and sequestration of neutrophils to the lungs. The second is an infusion of a transfusion product that contains HLA or HNA antibodies or biologic modifiers such as lipids from stored cells. The second event activates the neutrophils sequestered in the lungs which lead to neutrophil degranulation, superoxide release and localized tissue damage. Growing evidence suggests that platelets exert proinflammatory actions which include supporting tissue infiltration of neutrophils in septic lung injury. In a separate 2010 ASH abstract we show that ultraviolet B light (UVB, 2.4 J/cm2) exposed human platelets (HPs) mediate lung injury in a two-event animal model of ALI. UVB exposure has been reported to activate platelet protein kinase C (PKC). We compared the effects of UVB exposure to PKC activation by a PKC agonist, PMA (30 nM), in aggregation, activation and potential to cause lung injury in the two-event animal model. HPs were collected by apheresis and stored overnight with experiments performed on day 1 post collection. Platelet aggregation induced by increasing concentrations of ADP (5-20 mM) was potentiated by pretreatment with UVB or PMA. TRAP (20 mM) induced aggregation was inhibited by UVB, but unchanged by PMA pretreatment. Both UVB and PMA increased platelet PAC-1 binding and p-selectin expression. Pretreating HPs with a PKC inhibitor prevented all of PMA induced PAC-1 binding and inhibited UVB induced PAC-1 binding by 40%. Furthermore, the PKC inhibitor partially reduced p-selectin expression on PMA and UVB treated HPs, whereas p-selectin expression on control HPs remained unchanged. The UVB HPs or PMA HPs were evaluated in the two-event animal model of ALI. Immunodeficient (SCID) mice were used to minimize the species difference (Piper et al., Transfusion 47:1540-9, 2007). MIP-2 elevation in plasma is a marker of acute inflammation and was increased following LPS administration. Infusion of control HPs as the second event moderately increased MIP-2. When UVB HPs or PMA HPs were infused MIP-2 was significantly elevated compared to control HPs. Pretreatment of UVB HPs with the PKC inhibitor (RO31-8425) reduced MIP-2 elevation to the level of control platelets. In summary, UVB HPs can cause ALI in animals pretreated with LPS (separate 2010 ASH abstract as mentioned above). Changes to the platelets induced by UVB appear to be mediated by PKC since a PKC agonist (PMA) has similar effects on platelets in aggregation and activation as does UVB and PKC inhibitor partially inhibits UVB induced platelet activation. In vivo, both UVB and PMA treated HPs elevated MIP-2 plasma levels when injected after LPS and this response was prevented by treatment of platelets with a PKC inhibitor prior to UVB exposure. The UVB induced activation leads to a conformational change in GpIIb/IIIa which potentiates weak agonist induced aggregation and mediates an acute in vivo inflammatory response that may be responsible for the acute lung injury in the animal model. Understanding the underlying mechanisms of UVB exposure induced changes in platelets would be beneficial in designing methods to reduce the UVB associated ALI in an animal model and potentially in patients susceptible to TRALI by a primary sensitizing event and infused with high dose UVB exposed platelets. The findings and conclusions in this abstract 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.

720 Platelets are currently limited to 5 days of storage at room temperature to prevent growth of bacteria to high levels. Cold storage of platelets could reduce bacterial proliferation but platelets stored in cold for over 48 hours are cleared rapidly from circulation through the hepatocyte Ashwell-Morell (AM) receptor thus limiting the applicability of cold temperatures to platelet storage. We used a temperature cycling method to store human platelets in the cold without decreasing their in vivo recovery in an immunodeficient (SCID) animal model of transfusion. Temperature cycled (TC) apheresis human platelets were stored in the cold (4°C) for 12 hours and then incubated at 37°C for 30 minutes before returning back to cold storage. The TC (37°C pulses for 30 minutes at 12 hour intervals) was continued for 2, 5 and 7 days. Human platelets stored either at room temperature (RT), cold or TC for 2, 5 and 7 days were infused into 6 to 8 SCID mice per group and their in vivo recovery in circulation was determined at 5, 20 and 60 minutes after transfusion by flow cytometry. Carbohydrate exposure on the surface of the platelets was analyzed for galactose by Erythrina cristagalli agglutinin (ECA), and for β-GlnNAc by succinyl wheat germ agglutinin (sWGA) using flow cytometry. Involvement of the AM receptor was examined by monitoring clearance of cold stored platelets in the presence of asialofetuin, a competitive ligand for the receptor. In vivo recovery of human platelets stored for two-days in SCID mice circulation is shown in Figure 1. As expected, cold platelets had significantly decreased recovery compared to RT platelets, from 22.1±2.5% to 11.1±3.3% (P<0.01), 11.5±2.9% to 5.5±3.6% (P<0.01) and 11.2±1.4% to 6.2±1.8% (P<0.01) respectively at 5, 20 and 60 min post platelets injection. Compared to cold platelets, TC platelets recovery increased significantly from 11.1±3.3% to 15.9±4.4% (P<0.01), 5.5±3.6+% to 10.5±4.7% (P<0.01) and 6.2±1.8% to 9.5±2.2% (P<0.05) respectively at 5, 20 and 60 min post platelets injection. At 20 and 60 min post injection, the TC platelets have recovery of 10.5±4.7% and 9.5±2.2% respectively, that are comparable (P>0.05%) to RT platelet recoveries of 11.5±2.9% and 11.2±1.4% for the same time points. Similar increases of in vivo recovery for TC platelets as compared to cold platelets were obtained for at 5 and 7 days.Figure 1Human Platelet Recovery (% of total platelets circulating) * p< 0.05, ** p< 0.01, *** p< 0.001Figure 1. Human Platelet Recovery (% of total platelets circulating) * p< 0.05, ** p< 0.01, *** p< 0.001 Binding of the galactose specific lectin, ECA, was increased by 142±22% from RT to cold platelets (P<0.01) as previously reported. However, binding of ECA was also increased by 134±16% from RT to TC platelets (P<0.01). β-GlnNAc exposure, as measured by sWGA lectin binding, was increased after cold and TC storage by 222±65% (P<0.01) and 197±14% (P<0.01), respectively, when compared to RT platelets. Platelets stored in the cold for >48 hours have been reported to be cleared through the hepatic AM receptor which recognizes asialocarbohydrates. Co-injection of asialofetuin significantly improved the recovery of two-day cold stored platelets from 9.5±5.1% to 18.4±7.3% (P<0.05) and 4.8±3.7% to 12.1±4.9% (P<0.01), at 5 min and 20 min post injection, respectively. Native fetuin did not alter the clearance of cold platelets. However, there was no significant increase in the recovery of TC platelets in the presence of asialofetuin as compared to fetuin injection (P>0.28), even though the TC platelets, like cold platelets, have significantly increased β-galactose exposure. Our results indicate that ‘temperature cycling' during cold storage of platelets may be an effective method to store human platelets up to 7 days without loss of in vivo recovery after transfusion when compared to RT platelets. Temperature cycling does not alter the cold induced increases in β-gal or β-GlcNAc expression which suggests that there are other mechanisms besides binding to the AM receptor that mediate clearance of platelets stored in the cold for >48 hours. The findings and conclusions in this abstract 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.