P38 mitogen activated protein kinase inhibitor improves platelet in vitro parameters and in vivo survival in a SCID mouse model of transfusion for platelets stored at cold or temperature cycled conditions for 14 days.

Platelets for transfusion are stored at room temperature (20-24°C) up to 7 days but decline in biochemical and morphological parameters during storage and can support bacterial proliferation. This decline is reduced with p38MAPK inhibitor, VX-702. Storage of platelets in the cold (4-6°C) can reduce bacterial proliferation but platelets get activated and have reduced circulation when transfused. Thermocycling (cold storage with brief periodic warm ups) reduces some of the effects of cold storage. We evaluated in vitro properties and in vivo circulation in SCID mouse model of human platelet transfusion of platelets stored in cold or thermocycled for 14 days with and without VX-702. Apheresis platelet units (N = 15) were each aliquoted into five storage bags and stored under different conditions: room temperature; cold temperature; thermocycled temperature; cold temperature with VX-702; thermocycled temperature with VX-702. Platelet in vitro parameters were evaluated at 1, 7 and 14 days. On day 14, platelets were infused into SCID mice to assess their retention in circulation by flow cytometry. VX-702 reduced negative platelet parameters associated with cold and thermocycled storage such as an increase in expression of activation markers CD62, CD63 and of phosphatidylserine (marker of apoptosis measured by Annexin binding) and lowered the rise in lactate (marker of increase in anaerobic metabolism). However, VX-702 did not inhibit agonist-induced platelet aggregation indicating that it does not interfere with platelet hemostatic function. In vivo, VX-702 improved initial recovery and area under the curve in circulation of human platelets infused into a mouse model that has been previously validated against a human platelet infusion clinical trial. In conclusion, inhibition of p38MAPK during 14-days platelet storage in cold or thermocycling conditions improved in vitro platelet parameters and platelet circulation in the mouse model indicating that VX-702 may improve cell physiology and clinical performance of human platelets stored in cold conditions.

Validation of a SCID mouse model for transfusion by concurrent comparison of circulation kinetics of human platelets, stored under various temperature conditions, between human volunteers and mice.

BACKGROUND: Initial evaluation of new platelet (PLT) products for transfusion includes a clinical study to determine in vivo recovery and survival of autologous radiolabeled PLTs in healthy volunteers. These studies are expensive and do not always produce the desired results. A validated animal model of human PLTs in vivo survival and recovery used pre-clinically could reduce the risk of failing to advance product development. STUDY DESIGN AND METHODS: An immunodeficient (SCID) mouse model to evaluate recovery of human PLTs was compared to a radiolabeling study in human volunteers. Autologous apheresis PLTs stored for 7 days at room temperature (RT), thermo-cycled (TC), and cold temperature (CT) were radiolabeled and infused into healthy humans (n = 16). The same PLTs, non-radiolabeled, were also infused into mice (n = 160) on the same day. Blood samples from humans and mice were collected to generate clearance curves of PLTs in circulation. Flow cytometry was used to detect human PLTs in mouse blood. RESULTS: Human and mouse PLTs were cleared with one phase exponential clearance. Relative differences for initial recovery and AUC, expressed as ratio of test and control PLTs, were similar in humans and mice. The initial recovery ratio of TC/RT was 0.73 ± 0.07 in humans and 0.67 ± 0.14 in mice. The ratio for CT/TC was 0.53 ± 0.06 in humans and 0.75 ± 0.18 in mice. CONCLUSION: The SCID mouse model can provide information on relative differences of initial in vivo recovery and AUC between control and alternatively stored/processed human PLTs that is predictive of performance in healthy human volunteers.

Non-ionizing 405 nm Light as a Potential Bactericidal Technology for Platelet Safety: Evaluation of in vitro Bacterial Inactivation and in vivo Platelet Recovery in Severe Combined Immunodeficient Mice.

Bacterial contamination of ex vivo stored platelets is a cause of transfusion-transmitted infection. Violet-blue 405 nm light has recently demonstrated efficacy in reducing the bacterial burden in blood plasma, and its operational benefits such as non-ionizing nature, penetrability, and non-requirement for photosensitizing agents, provide a unique opportunity to develop this treatment for in situ treatment of ex vivo stored platelets as a tool for bacterial reduction. Sealed bags of platelet concentrates, seeded with low-level Staphylococcus aureus contamination, were 405 nm light-treated (3-10 mWcm-2) up to 8 h. Antimicrobial efficacy and dose efficiency was evaluated by quantification of the post-treatment surviving bacterial contamination levels. Platelets treated with 10 mWcm-2 for 8 h were further evaluated for survival and recovery in severe combined immunodeficient (SCID) mice. Significant inactivation of bacteria in platelet concentrates was achieved using all irradiance levels, with 99.6-100% inactivation achieved by 8 h (P < 0.05). Analysis of applied dose demonstrated that lower irradiance levels generally resulted in significant decontamination at lower doses: 180 Jcm-2/10 mWcm-2 (P = 0.008) compared to 43.2 Jcm-2/3 mWcm-2 (P = 0.002). Additionally, the recovery of light-treated platelets, compared to non-treated platelets, in the murine model showed no significant differences (P = >0.05). This report paves the way for further comprehensive studies to test 405 nm light treatment as a bactericidal technology for stored platelets.

Proceedings of the Food and Drug Administration's public workshop on new red blood cell product regulatory science 2016.

The US Food and Drug Administration (FDA) held a workshop on red blood cell (RBC) product regulatory science on October 6 and 7, 2016, at the Natcher Conference Center on the National Institutes of Health (NIH) Campus in Bethesda, Maryland. The workshop was supported by the National Heart, Lung, and Blood Institute, NIH; the Department of Defense; the Office of the Assistant Secretary for Health, Department of Health and Human Services; and the Center for Biologics Evaluation and Research, FDA. The workshop reviewed the status and scientific basis of the current regulatory framework and the available scientific tools to expand it to evaluate innovative and future RBC transfusion products. A full record of the proceedings is available on the FDA website (http://www.fda.gov/BiologicsBloodVaccines/NewsEvents/WorkshopsMeetingsConferences/ucm507890.htm). The contents of the summary are the authors' opinions and do not represent agency policy.

Temperature cycling during platelet cold storage improves in vivo recovery and survival in healthy volunteers.

BACKGROUND: Room temperature (RT) storage of platelets (PLTs) can support bacterial proliferation in contaminated units, which can lead to transfusion-transmitted septic reactions. Cold temperature storage of PLTs could reduce bacterial proliferation but cold exposure produces activation-like changes in PLTs and leads to their rapid clearance from circulation. Cold-induced changes are reversible by warming and periodic rewarming during cold storage (temperature cycling [TC]) has been proposed to alleviate cold-induced reduction in PLT circulation. STUDY DESIGN AND METHODS: A clinical trial in healthy human volunteers was designed to compare in vivo recovery, survival, and area under the curve (AUC) of radiolabeled autologous apheresis PLTs stored for 7 days at RT or under TC or cold conditions. Paired comparisons of RT versus TC and TC versus cold PLTs were conducted. RESULTS: Room temperature PLTs had in vivo recovery of 55.7 ± 13.9%, survival of 161.3 ± 28.8 hours, and AUC of 5031.2 ± 1643.3. TC PLTs had recovery of 42.6 ± 16.4%, survival of 48.1 ± 14.4% hours, and AUC of 1331.3 ± 910.2 (n = 12, p < 0.05). In a separate paired comparison, cold PLTs had recovery of 23.1 ± 8.8%, survival of 33.7 ± 14.7 hours, and AUC of 540.2 ± 229.6 while TC PLTs had recovery of 36.5 ± 12.9%, survival of 49.0 ± 17.3 hours, and AUC of 1164.3 ± 622.2 (n = 4, AUC had p < 0.05). CONCLUSION: TC storage for 7 days produced PLTs with better in vivo circulation kinetics than cold storage but is not equivalent to RT storage.

Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

Automated cold temperature cycling improves in vitro platelet properties and in vivo recovery in a mouse model compared to continuous cold storage.

BACKGROUND: Platelets (PLTs) stored at cold temperatures (CTs) for prolonged time have dramatically reduced bacterial growth but poor survival when infused. A previous study demonstrated that human PLTs stored with manual cycling between 4 °C (12 hr) and 37 °C (30 min) and infused into severe combined immunodeficient (SCID) mice had survivals similar to or greater than those stored at room temperature (RT). In this study, the in vitro and in vivo properties of PLTs stored in an automated incubator programmed to cycle between 5 °C (11 hr) and 37 °C (1 hr) were evaluated. STUDY DESIGN AND METHODS: A Trima apheresis unit (n = 12) was aliquoted (60 mL) in CLX bags. One sample was stored with continuous agitation (RT), a second sample was stored at 4-6 °C without agitation (CT), and a third sample was placed in an automated temperature cycler with 5 minutes of agitation during the warm-up period (thermocycling [TC]). PLTs were assayed for several relevant quality variables. On Day 7, PLTs were infused into SCID mice and in vivo recovery was assessed at predetermined time points after transfusion. RESULTS: The glucose consumption rate, morphology score, hypotonic shock recovery level, and aggregation levels were increased and mitochondrial reactive oxygen species accumulations were decreased in TC-PLTs compared to those of CT-PLTs. The pH and Annexin V binding were comparable to those of RT-PLTs. All TC-PLTs had greater recovery than CT-PLTs and were comparable to RT-PLTs. CONCLUSION: PLTs stored under automated TC conditions have improved in vivo recovery and improved results for a number of in vitro measures compared to CT-PLTs.

Expression of the cellular prion protein affects posttransfusion recovery and survival of red blood cells in mice.

BACKGROUND: Cellular prion protein (PrP(C) ) is expressed on various cell types including red blood cells (RBCs). The PrP(C) plays a key role in the pathogenesis of prion diseases, but its physiologic function remains unclear. PrP(C) is expressed on CD34+ hematopoietic stem cells and its expression is regulated during blood cell differentiation including the erythroid line. STUDY DESIGN AND METHODS: We investigated the role of PrP(C) in RBC survival in circulation by transfusing a mix of biotin-labeled RBCs from wild-type (WT) and PrP knockout (KO) mice to groups of recipient mice (WT and KO). The proportion of biotinylated RBCs in peripheral blood was estimated by flow cytometry. RESULTS: KO RBCs displayed a markedly higher first-day posttransfusion recovery but had a decreased survival in circulation when compared to WT RBCs. Similar results were obtained in all groups of transfused mice, irrespective of RBCs biotinylation level. In addition, we confirmed this finding in an analogous study using Tga20 mice overexpressing PrP(C) and KO mice of a different genetic background. CONCLUSION: Our results demonstrate that PrP(C) expression affects RBC recovery and survival in circulation.

Reversal of hemochromatosis by apotransferrin in non-transfused and transfused Hbbth3/+ (heterozygous B1/B2 globin gene deletion) mice.

Intermediate beta-thalassemia has a broad spectrum of sequelae and affected subjects may require occasional blood transfusions over their lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron in plasma. The iron scavenger and transport protein transferrin is a potential treatment being studied for correction of anemia. However, transferrin may also function to prevent or reduce iron loading of tissues when exposure to non-transferrin bound iron increases. Here we evaluate the effects of apotransferrin administration on tissue iron loading and early tissue pathology in non-transfused and transfused Hbb(th3/+) mice. Mice with the Hbb(th3/+) phenotype have mild to moderate anemia and consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Chronic apotransferrin administration resulted in normalization of the anemia. Furthermore, it normalized tissue iron content in the liver, kidney and heart and attenuated early tissue changes in non-transfused Hbb(th3/+) mice. Apotransferrin treatment was also found to attenuate transfusion-mediated increases in plasma non-transferrin bound iron and associated excess tissue iron loading. These therapeutic effects were associated with normalization of transferrin saturation and suppressed plasma non-transferrin bound iron. Apotransferrin treatment modulated a fundamental iron regulatory pathway, as evidenced by decreased erythroid Fam132b gene (erythroferrone) expression, increased liver hepcidin gene expression and plasma hepcidin-25 levels and consequently reduced intestinal ferroportin-1 in apotransferrin-treated thalassemic mice.

Preclinical safety evaluation of human platelets treated with antimicrobial peptides in severe combined immunodeficient mice.

BACKGROUND: Bacterial sepsis is a complication attributed to room temperature (RT)-stored platelets (PLTs) in transfusion medicine. Antimicrobial peptides (AMPs) are emerging as new therapeutic agents against microbes. We had previously demonstrated bactericidal activity of select synthetic AMPs against six types of bacteria in stored PLTs. In this report, we tested these AMPs for their potential antibody response and interference with the recovery and survival of human PLTs in an animal model. STUDY DESIGN AND METHODS: Two separate studies were conducted to evaluate the safety of the synthetic AMPs. 1) Two AMPs (PD3 and PD4), derived from thrombin-induced human PLT microbicidal protein, and four repeats of arginine-tryptophan (RW), containing two to five repeats (RW2-RW5), were tested in rabbits for potential antibody response. 2) RT-stored human PLTs treated for 2 hours with each of the six AMPs individually or with phosphate-buffered saline (PBS) alone were infused into severe combined immunodeficient (SCID) mice to evaluate their in vivo recovery and survival by flow cytometry. RESULTS: Except for PD3, which showed a weak immune response, all other peptides did not induce any detectable antibodies in rabbits. Furthermore, all six AMPs tested did not significantly affect the in vivo recovery and survival of human PLTs in SCID mice compared to PBS alone-treated PLTs. CONCLUSION: Preclinical evaluation studies reported here demonstrate that the selected AMPs used in the study did not adversely affect the human PLT recovery and survival in the SCID mouse model, suggesting further study of AMPs toward addressing the bacterial contamination of PLTs.

Activation of platelet protein kinase C by ultraviolet light B mediates platelet transfusion-related acute lung injury in a two-event animal model.

BACKGROUND: We recently reported that infusion of ultraviolet light B (UVB)-exposed human platelets (HPs) can be the second event that mediates acute lung injury (ALI) in a two-event mouse model of transfusion-related acute lung injury (mTRALI). We have now identified changes in HPs induced by UVB light and responses of the recipient animal that mediate the mTRALI. STUDY DESIGN AND METHODS: Effects of UVB on HPs were monitored by flow cytometry and aggregation. HPs exposed to UVB, with or without inhibitors to specific biochemical pathways, were infused into lipopolysaccharide (LPS)-primed severe combined immunodeficient (SCID) mice. ALI was monitored by protein elevations in bronchoalveolar lavage fluid (BALF). RESULTS: UVB increased fibrinogen binding and potentiated HP aggregation. Infusion of UVB HPs into LPS-primed SCID mice led to macrophage inflammatory protein 2 (MIP-2) elevations in plasma and BALF and resulted in ALI. Protein kinase C (PKC) inhibitors prevented UVB-induced HP changes in vitro and reduced MIP-2 elevation and mTRALI in vivo. Blocking of fibrinogen binding to HP αIIbß3 with c7E3 monoclonal antibody prevented mTRALI. MIP-2 elevation in vivo in response to UVB HPs was essential for ALI since blocking of MIP-2 receptor in vivo prevented mTRALI. CONCLUSION: PKC signaling mediates UVB-induced HP fibrinogen binding and aggregation in vitro. The host animal responds to an infusion of UVB HPs by MIP-2 elevation that mediates downstream mTRALI. Elucidation of molecular mechanisms in UVB HP-mediated mTRALI may provide insight into pulmonary adverse events reported with UV-irradiated pathogen-reduced platelets.

Temperature cycling improves in vivo recovery of cold-stored human platelets in a mouse model of transfusion.

BACKGROUND: Platelet (PLT) storage at room temperature (RT) is limited to 5 days to prevent growth of bacteria, if present, to high levels. Storage in cold temperatures would reduce bacterial proliferation, but cold-exposed PLTs are rapidly cleared from circulation by the hepatic Ashwell-Morell (AM) receptor, which recognizes PLT surface carbohydrates terminated by ß-galactose. We cycled storage temperature between 4 and 37°C to preserve PLT function and reduce bacterial growth. STUDY DESIGN AND METHODS: Temperature-cycled (TC) human PLTs were stored at 4°C for 12 hours and then incubated at 37°C for 30 minutes before returning back to cold storage. PLTs stored at RT or at 4°C (COLD) or TC for 2, 5, and 7 days were infused into SCID mice and the in vivo recovery was determined at 5, 20, and 60 minutes after transfusion. RESULTS: PLTs stored for 2 days in COLD had significantly lower in vivo recoveries than RT PLTs. TC PLTs had improved recoveries over COLD and comparable to RT PLTs. After 5- and 7-day storage, TC PLTs had better recoveries than RT and COLD PLTs. PLT surface ß-galactose was increased significantly for both COLD and TC PLTs compared to RT. Blocking of the AM receptor by asialofetuin increased COLD but not TC PLT recovery. CONCLUSION: TC cold storage may be an effective method to store PLTs without loss of in vivo recovery. The increased ß-galactose exposure in TC PLTs suggests that mechanisms in addition to AM receptors may mediate clearance of cold-stored PLTs.

Host platelets and, in part, neutrophils mediate lung accumulation of transfused UVB-irradiated human platelets in a mouse model of acute lung injury.

We previously reported that ultraviolet light B (UVB)-treated human platelets (hPLTs) can cause acute lung injury (ALI) in a two-event SCID mouse model in which the predisposing event was Lipopolysaccharide (LPS) injection and the second event was infusion of UVB-treated hPLTs. To delineate contributions of host mouse platelets (mPLTs) and neutrophils in the pathogenesis of ALI in this mouse model, we depleted mPLTs or neutrophils and measured hPLT accumulation in the lung. We also assessed lung injury by protein content in bronchoalveolar lavage fluid (BALF). LPS injection followed by infusion of UVB-treated hPLTs resulted in sequestration of both mPLTs and hPLTs in the lungs of SCID mice, although the numbers of neutrophils in the lung were not significantly different from the control group. Depletion of mouse neutrophils caused only a mild reduction in UVB-hPLTs accumulation in the lungs and a mild reduction in protein content in BALF. In comparison, depletion of mPLTs almost completely abolished hPLTs accumulation in the lung and significantly reduced protein content in BALF. UVB-treated hPLTs bound to host mPLTs, but did not bind to neutrophils in the lung. Aspirin treatment of hPLTs in vitro abolished hPLT accumulation in the lung and protected mice from lung injury. Our data indicate that host mPLTs accumulated in the lungs in response to an inflammatory challenge and subsequently mediated the attachment of transfused UVB-hPLTs. Neutrophils also recruited a small percentage of platelets to the lung. These findings may help develop therapeutic strategies for ALI which could potentially result from transfusion of UV illuminated platelets.

Ultraviolet B light-exposed human platelets mediate acute lung injury in a two-event mouse model of transfusion.

BACKGROUND: Ultraviolet B (UVB) light has been used alone on platelet (PLT) transfusion products to prevent alloimmunization or with chemical sensitizers to reduce pathogens. Such processing can damage PLTs and potentiate their storage lesion. Transfusion-related acute lung injury (ALI) has occurred in patients whose underlying condition led to an inflamed endothelium and who were transfused with products that contained either HLA or HNA antibodies or biologic modifiers such as lipids or antigens from stored cells. Clinical trials of UV-treated PLTs in patients with thrombocytopenia generated controversy regarding association of these cells with respiratory distress. We evaluated whether UVB PLTs could mediate ALI in an animal model of ALI. STUDY DESIGN AND METHODS: We used a two-event animal model where the sensitizing event was lipopolysaccharide (LPS) and the second event was infusion of human PLTs or UVB human PLTs (2.4 J/cm(2) ). Infused human PLTs were followed with whole animal imaging, lung histology, confocal microscopy, lung water, and changes in bronchoalveolar lavage fluid (BALF) related to ALI. RESULTS: In LPS-treated mice UVB human PLTs accumulated in the lungs and were associated with ALI manifested by increased protein and white blood cells (WBCs) in BALF. Untreated human PLTs did not accumulate in the lungs or increase BALF protein or WBC counts. CONCLUSIONS: We provide a proof of principle that UVB human PLTs can accumulate in lungs of LPS-primed animals and mediate ALI. PLTs exposed to high doses of UVB could potentially mediate similar effects in patients predisposed with sepsis or other causes of endothelial cell inflammation.

Blood aging, safety, and transfusion: capturing the "radical" menace.

Throughout their life span, circulating red blood cells (RBCs) transport oxygen (O(2)) primarily from the lungs to tissues and return with carbon dioxide (CO(2)) from respiring tissues for final elimination by lungs. This simplistic view of RBCs as O(2) transporter has changed in recent years as other gases, for example, nitric oxide (NO), and small molecules, such as adenosine triphosphate (ATP), have been shown to either be produced and/or carried by RBCs to perform other signaling and O(2) sensing functions. In spite of the numerous biochemical and metabolic changes occurring within RBCs during storage, prior to, and after transfusion, perturbations of RBC membrane are likely to affect blood flow in the microcirculation. Subsequent hemolysis due to storage conditions and/or hemolytic disorders may have some pathophysiological consequences as a result of the release of Hb. In this review, we show that evolution has provided a multitude of protection and intervention strategies against free Hb from "cradle" to "death"; from early biosynthesis to its final degradation and a lot more in between. Furthermore, some of the same naturally occurring protective mechanisms can potentially be employed to oxidatively inactivate this redox active protein and control its damaging side reactions when released outside of the RBC.

Rejuvenation improves roller pump-induced physical stress resistance of fresh and stored red blood cells.

BACKGROUND: Retrospective studies on transfusion recipients suggested that transfusion of older red blood cells (RBCs) was associated with higher morbidity. Similar studies were also done on cardiac surgery patients who were placed on cardiac bypass pumps. It is possible that stored RBCs are more fragile and could be more easily damaged by these pumps, thus leading to additional morbidity. STUDY DESIGN AND METHODS: Fresh and stored (42 days) RBCs, rejuvenated and nonrejuvenated, were compared in resistance to physical stress, induced by a roller pump, and osmotic fragility changes during physical stress to model RBCs going through cardiac bypass instruments. In addition, posttransfusion in vivo recovery was evaluated in an immunodeficient mouse model to minimize species differences between transfusion product and recipient. RESULTS: Fresh RBCs were more resistant to both osmotic and physical stress than stored cells. After 2 hours of physical stress, the osmotic stress resistance of fresh cells declined and was the same as for stored cells. Rejuvenated fresh cells did not demonstrate a decline in osmotic resistance during the stress test and both fresh and stored cells had the same improved resistance to osmotic stress before and after the physical stress. Rejuvenation slightly improved recovery of fresh RBCs but almost doubled the recovery of stored cells in the mouse model. CONCLUSIONS: Our studies suggest that rejuvenation improves roller pump-induced physical and osmotic stress resistance of stored RBCs.

Current and future cellular transfusion products.

Novel red blood cell and platelet transfusion products may be synthetic or may result from modifications to approved collection, processing, and storage procedures for existing cellular products. They must be reviewed and evaluated by the Food and Drug Administration before being legally marketed in the United States to ensure they are safe, pure, and potent. This article reviews the literature and discusses the current and future state of cellular transfusion products.

Carbon nanotubes activate blood platelets by inducing extracellular Ca2+ influx sensitive to calcium entry inhibitors.

To elucidate a mechanism of prothrombotic effects of carbon nanotubes (CNTs), we report here that multiwalled CNTs activate blood platelets by inducing extracellular Ca(2+) influx that could be inhibited by calcium channel blockers SKF 96365 and 2-APB. We also demonstrate platelet aggregating activity of different single-walled and multiwalled CNTs. In addition, we show that CNT-induced platelet activation is associated with a marked release of platelet membrane microparticles positive for the granular secretion markers CD62P and CD63.

Flow cytometric analysis of cell membrane microparticles.

Cell membrane microparticles (MPs) are phospholipid microvesicles shed from the plasma membrane of most eukaryotic cells undergoing activation or apoptosis. The presence of MPs is common in healthy individuals. However, an increase in their release is a controlled event and is considered a hallmark of cellular alteration. Microparticles display cell surface proteins that indicate their cellular origin. In addition, they may also express other markers, e.g., markers of cellular activation. Elevated levels of circulating MPs are associated with various vascular pathologies and their pathogenic potential has been widely documented. MPs have been analyzed in plasma and cell cultures by means of flow cytometry or solid phase assays. Here we present a three-color flow cytometric assay for immunophenotyping of MPs in plasma. This assay has been used to study elevated counts of different phenotypes of circulating endothelial MPs in several hematological and vascular diseases. A modified version of this assay can also be used for MP analysis in blood products and cell cultures.

Adverse effects of fullerenes on endothelial cells: fullerenol C60(OH)24 induced tissue factor and ICAM-I membrane expression and apoptosis in vitro.

We studied the effects of a C60 water suspension at 4 microg/mL (nC60) and the water soluble fullerenol C60(OH)24 at final concentrations of 1-100 microg/mL on human umbilical vein endothelial cells (HUVECs) in culture. We found that a 24 hr treatment of HUVECs with C60(OH)24 at 100 microg/mL significantly increased cell surface expression of ICAM-1(CD54) (67 +/- 4% CD54+ cells vs. 19 +/- 2 % CD540 cells in control; p < 0.001). In addition, this treatment induced the expression of tissue factor (CD142) on HUVECs (54 +/- 20% CD142+ cells vs 4 +/- 2% CD142+ cells in control; p = 0.008) and increased exposure of phosphatidylserine (PS) (29 +/- 2% PS+ cells vs. 12 +/- 5% PS+ cells in control; p < 0.001). Analysis of cell cycle and DNA fragmentation (TUNEL) showed that both nC60 and C60(OH)24 caused G1 arrest of HUVECs and C60(OH)24 induced significant apoptosis (21 +/- 2% TUNEL+ cells at 100 microg/mL of C60(OH)24 vs. 4 +/- 2% TUNEL+ cells in control; p < 0.001). We also demonstrated that both nC60 and C60(OH)24 induced a rapid concentration dependent elevation of intracellular calcium [Ca2+]i. This could be inhibited by EGTA, suggesting that the source of [Ca2+]i in fullerene stimulated calcium flux is predominantly from the extracellular environment. In conclusion, fullerenol C60(OH)24 had both pro-inflammatory and pro-apoptotic effects on HUVECs, indicating possible adverse effects of fullerenes on the endothelium.