Surface Expression Of Phosphatidylserine Research Articles

Platelet and endothelial cell responses under concurrent shear stress and tensile strain

Thrombosis can lead to significant mortality and morbidity. Both platelets and vascular endothelial cells play significant roles in thrombosis. Platelets' response to blood flow-induced shear stress can vary greatly depending on shear stress magnitude, pattern and shear exposure time. Endothelial cells are also sensitive to the biomechanical environment. Endothelial cell activation and dysfunction can occur under low oscillatory shear stress and low tensile strain. Platelet and endothelial cell interaction can also be affected by mechanical conditions. The goal of this study was to investigate how blood flow-induced shear stress, vascular wall tensile strain, platelet-endothelial cell stress history, and platelet-endothelial cell interaction affect platelet thrombogenicity. Platelets and human coronary artery endothelial cells were pretreated with physiological and pathological shear stress and/or tensile strain separately. The pretreated cells were then put together and exposed to pulsatile shear stress and cyclic tensile strain simultaneously in a shearing-stretching device. Following treatment, platelet thrombin generation rate, platelet and endothelial cell activation, and platelet adhesion to endothelial cells was measured. The results demonstrated that shear stress pretreatment of endothelial cells and platelets caused a significant increase in platelet thrombin generation rate, cell surface phosphatidylserine expression, and adhesion to endothelial cells. Shear stress pretreatment of platelets and endothelial cells attenuated endothelial cell ICAM-1 expression under stenosis conditions, as well as vWF expression under recirculation conditions. These results indicate that platelets are sensitized by prior shearing, while in comparison, the interaction with shear stress-pretreated platelets may reduce endothelial cell sensitivity to pathological shear stress and tensile strain.

Chronic Immune Platelet Activation Is Followed by Platelet Refractoriness and Impaired Contractility.

Autoimmune diseases, including systemic lupus erythematosus (SLE), have a high risk of thrombotic and hemorrhagic complications associated with altered platelet functionality. We studied platelets from the blood of SLE patients and their reactivity. The surface expression of phosphatidylserine, P-selectin, and active integrin αIIbβ3 were measured using flow cytometry before and after platelet stimulation. Soluble P-selectin was measured in plasma. The kinetics of platelet-driven clot contraction was studied, as well as scanning and transmission electron microscopy of unstimulated platelets. Elevated levels of membrane-associated phosphatidylserine and platelet-attached and soluble P-selectin correlated directly with the titers of IgG, anti-dsDNA-antibodies, and circulating immune complexes. Morphologically, platelets in SLE lost their resting discoid shape, formed membrane protrusions and aggregates, and had a rough plasma membrane. The signs of platelet activation were associated paradoxically with reduced reactivity to a physiological stimulus and impaired contractility that revealed platelet exhaustion and refractoriness. Platelet activation has multiple pro-coagulant effects, and the inability to fully contract (retract) blood clots can be either a hemorrhagic or pro-thrombotic mechanism related to altered clot permeability, sensitivity of clots to fibrinolysis, obstructiveness, and embologenicity. Therefore, chronic immune platelet activation followed by secondary platelet dysfunction comprise an understudied pathogenic mechanism that supports hemostatic disorders in autoimmune diseases, such as SLE.

Detection of Phosphatidylserine Exposure on Leukocytes Following Treatment with Human Galectins.

Cellular turnover represents a fundamental aspect of immunological homeostasis. While many factors appear to regulate leukocyte removal during inflammatory resolution, recent studies suggest that members of the galectin family play a unique role in orchestrating this process. Unlike cellular removal through apoptotic cell death, several members of the galectin family induce surface expression of phosphatidylserine (PS), a phagocytic marker on cells undergoing apoptosis, in the absence of cell death. However, similar to PS on cells undergoing apoptosis, galectin-induced PS exposure sensitizes cells to phagocytic removal. As galectins appear to prepare cells for phagocytic removal without actually inducing apoptotic cell death, this process has recently been coined preaparesis. Given the unique characteristics of galectin-induced PS exposure in the context of preaparesis, we will examine unique considerations when evaluating the potential impact of different galectin family members on PS exposure and cell viability.

Decreased platelet surface phosphatidylserine predicts increased bleeding in patients with severe factor VIII deficiency

BackgroundBleeding phenotypes among individuals with severe factor VIII (FVIII) deficiency are variable, even with routine prophylactic FVIII administration. Activated platelets, in addition to their role in primary hemostasis, play a major role in coagulation by providing a phospholipid surface to which coagulation factors bind. ObjectivesThe aim of this study was to determine whether platelet function is associated with past and/or future bleeding in patients with severe FVIII deficiency on prophylaxis. Patients/MethodsPlatelet function quantified by platelet surface expression of phosphatidylserine, platelet surface glycoprotein (GP) VI, platelet surface activated GPIIb‐IIIa, platelet surface P‐selectin, the percentage of coated platelets, and the percentage of platelet‐derived microparticles in the presence or absence of in vitro activation by various agonists was assessed in 34 patients. ResultsDecreased platelet surface phosphatidylserine expression (identified by annexin V binding), both in the presence and absence of ADP/thrombin receptor activating peptide, demonstrated a significant association with both prior and subsequent bleeding in any location and specifically with hemarthrosis. No significant difference between patients with and without bleeding was observed in any of the other platelet activity markers. ConclusionsDecreased platelet surface phosphatidylserine expression measured by annexin V binding predicts increased bleeding in severe FVIII deficient patients on prophylaxis.

Maximising platelet availability by delaying cold storage.

Cold-stored platelets may be an alternative to conventional room temperature (RT) storage. However, cold-stored platelets are cleared more rapidly from circulation, reducing their suitability for prophylactic transfusion. To minimise wastage, it may be beneficial to store platelets conventionally until near expiry (4 days) for prophylactic use, transferring them to refrigerated storage to facilitate an extended shelf life, reserving the platelets for the treatment of acute bleeding. Two ABO-matched buffy-coat-derived platelets (30% plasma/70% SSP+) were pooled and split to produce matched pairs (n = 8 pairs). One unit was stored at 2-6°C without agitation (day 1 postcollection; cold); the second unit was stored at 20-24°C with constant agitation until day 4 then stored at 2-6°C thereafter (delayed-cold). All units were tested for in vitro quality periodically over 21 days. During storage, cold and delayed-cold platelets maintained a similar platelet count. While pH and HSR were significantly higher in delayed-cold platelets, other metabolic markers, including lactate production and glucose consumption, did not differ significantly. Furthermore, surface expression of phosphatidylserine and CD62P, release of soluble CD62P and microparticles were not significantly different, suggesting similar activation profiles. Aggregation responses of delayed-cold platelets followed the same trend as cold platelets once transferred to cold storage, gradually declining over the storage period. The metabolic and activation profile of delayed-cold platelets was similar to cold-stored platelets. These data suggest that transferring platelets to refrigerated storage when near expiry may be a viable option for maximising platelet inventories.

Sperm-associated antigen 9 (SPAG9) promotes the survival and tumor growth of triple-negative breast cancer cells.

Recently, we demonstrated the association of sperm-associated antigen 9 (SPAG9) expression with breast cancer. Among breast cancer, 15% of the cancers are diagnosed as triple-negative breast cancers (TNBC) based on hormone receptor status and represent an important clinical challenge because of lack of effective available targeted therapy. Therefore, in the present investigation, plasmid-based small hairpin (small hairpin RNA (shRNA)) approach was used to ablate SPAG9 in aggressive breast cancer cell line model (MDA-MB-231) in order to understand the role of SPAG9 at molecular level in apoptosis, cell cycle, and epithelial-to-mesenchymal transition (EMT) signaling. Our data in MDA-MB-231 cells showed that ablation of SPAG9 resulted in membrane blebbing, increased mitochondrial membrane potential, DNA fragmentation, phosphatidyl serine surface expression, and caspase activation. SPAG9 depletion also resulted in cell cycle arrest in G0-G1 phase and induced cellular senescence. In addition, in in vitro and in vivo xenograft studies, ablation of SPAG9 resulted in upregulation of p21 along with pro-apoptotic molecules such as BAK, BAX, BIM, BID, NOXA, AIF, Cyto-C, PARP1, APAF1, Caspase 3, and Caspase 9 and epithelial marker, E-cadherin. Also, SPAG9-depleted cells showed downregulation of cyclin B1, cyclin D1, cyclin E, CDK1, CDK4, CDK6, BCL2, Bcl-xL, XIAP, cIAP2, MCL1, GRP78, SLUG, SNAIL, TWIST, vimentin, N-cadherin, MMP2, MMP3, MMP9, SMA, and β-catenin. Collectively, our data suggests that SPAG9 promotes tumor growth by inhibiting apoptosis, altering cell cycle, and enhancing EMT signaling in in vitro cells and in vivo mouse model. Hence, SPAG9 may be a potential novel target for therapeutic use in TNBC treatment.

Lung Neutrophilia in Myeloperoxidase Deficient Mice during the Course of Acute Pulmonary Inflammation.

Systemic inflammation accompanying diseases such as sepsis affects primarily lungs and induces their failure. This remains the most common cause of sepsis induced mortality. While neutrophils play a key role in pulmonary failure, the mechanisms remain incompletely characterized. We report that myeloperoxidase (MPO), abundant enzyme in neutrophil granules, modulates the course of acute pulmonary inflammatory responses induced by intranasal application of lipopolysaccharide. MPO deficient mice had significantly increased numbers of airway infiltrated neutrophils compared to wild-type mice during the whole course of lung inflammation. This was accompanied by higher levels of RANTES in bronchoalveolar lavage fluid from the MPO deficient mice. Other markers of lung injury and inflammation, which contribute to recruitment of neutrophils into the inflamed lungs, including total protein and other selected proinflammatory cytokines did not significantly differ in bronchoalveolar lavage fluid from the wild-type and the MPO deficient mice. Interestingly, MPO deficient neutrophils revealed a decreased rate of cell death characterized by phosphatidylserine surface expression. Collectively, the importance of MPO in regulation of pulmonary inflammation, independent of its putative microbicidal functions, can be potentially linked to MPO ability to modulate the life span of neutrophils and to affect accumulation of chemotactic factors at the inflammatory site.

Collagen can selectively trigger a platelet secretory phenotype via glycoprotein VI.

Platelets are not only central actors of hemostasis and thrombosis but also of other processes including inflammation, angiogenesis, and tissue regeneration. Accumulating evidence indicates that these “non classical” functions of platelets do not necessarily rely on their well-known ability to form thrombi upon activation. This suggests the existence of non-thrombotic alternative states of platelets activation. We investigated this possibility through dose-response analysis of thrombin- and collagen-induced changes in platelet phenotype, with regards to morphological and functional markers of platelet activation including shape change, aggregation, P-selectin and phosphatidylserine surface expression, integrin activation, and release of soluble factors. We show that collagen at low dose (0.25 µg/mL) selectively triggers a platelet secretory phenotype characterized by the release of dense- and alpha granule-derived soluble factors without causing any of the other major platelet changes that usually accompany thrombus formation. Using a blocking antibody to glycoprotein VI (GPVI), we further show that this response is mediated by GPVI. Taken together, our results show that platelet activation goes beyond the mechanisms leading to platelet aggregation and also includes alternative platelet phenotypes that might contribute to their thrombus-independent functions.

Erythrocyte-Derived Microvesicles Amplify Systemic Inflammation by Thrombin-Dependent Activation of Complement

Transfusion of aged blood has been associated with increased morbidity and mortality in critically ill patients. During storage, erythrocytes release increasing numbers of microvesicles (red blood cell-derived microvesicles [RBC-MV]). We hypothesized that RBC-MV mediate some of the deleterious effects of aged blood transfusions. We established a murine transfusion model using RBC-MV purified from aged mouse erythrocytes. Injection of RBC-MV into healthy mice had no effect. However, they aggravated pulmonary leukocyte sequestration and peripheral blood leukopenia induced by lipopolysaccharides. Lipopolysaccharide-induced proinflammatory cytokines were significantly increased in plasma after RBC-MV injection. These effects were not seen in C5aR-deficient mice. In vitro, RBC-MV bound C3 fragments after incubation with plasma but failed to bind immunoglobulins, C1q, or mannose-binding lectin. Preventing thrombin generation inhibited complement activation in vitro and in vivo and reversed the proinflammatory effects of RBC-MV in lipopolysaccharide-primed mice. Finally, the RBC-MV-induced phenotype was recapitulated using phosphatidylserine-expressing liposomes, suggesting that surface expression of phosphatidylserine by RBC-MV was mechanistically involved. These results point toward a thrombin-dependent mechanism of complement activation by RBC-MV independent of the classical, lectin, or alternative pathway. Besides identifying RBC-MV as potential mediators of transfusion-related morbidity, our findings may be relevant for other inflammatory disorders involving intravascular microvesicle release, for example, sickle cell disease or thrombotic microangiopathy.

Association Of Platelet Function Markers, Independent Of Platelet Count, With Bleeding Score In Patients With Immune Thrombocytopenia

BackgroundImmune thrombocytopenia (ITP) patients with similarly low platelet counts differ in their tendency to bleed. AimTo determine if differences in platelet function in ITP patients with similarly low platelet counts partly account for the variation in bleeding tendency. MethodsThe relationship between bleeding scores and platelet function markers was investigated in a single center cross-sectional study of pediatric patients with ITP. Following informed consent, blood was collected from ITP patients and bleeding was graded using the Buchanan and Adix Score (J Pediatr 2002) at routine clinic visits or while admitted to the hospital. Bleeding scores were obtained by one of three hematologists blinded to platelet function results, and investigators performing platelet function tests were blinded to clinical results. Platelet function was assessed by whole blood flow cytometric measurement of unstimulated, ADP- or TRAP-stimulated platelet surface activated GPIIb-IIIa (as measured by PAC1 binding), P-selectin, and GPIb and by unstimulated, convulxin-, or ADP plus TRAP-stimulated platelet surface phosphatidylserine expression (as determined by annexin V binding). Platelet count, immature platelet fraction (IPF) and mean platelet volume (MPV) were determined by a Sysmex XE-2100, and platelet forward angle light scatter (FSC) was measured by flow cytometry. ResultsPlatelet function and bleeding scores were evaluated in 34 consecutive consenting pediatric ITP patients (16 female, 18 male, age 9.7 ± 5.7 years [mean ± SD]). ITP was newly diagnosed (< 3 months) in 10 patients, persistent (3 -- 12 months) in 7 patients, and chronic (>12 months) in 17 patients. Platelet count at the time of the blood draw was 47 ± 55 x 109/L. The median bleeding score on day of blood draw was 1 (range 0 to 4). By univariate analysis, higher IPF, and lower platelet count were significantly associated with a higher bleeding score (odds ratio [OR] >1, p<0.05) but MPV was not. Multiple measures of platelet function were associated with bleeding scores by univariate analysis: higher levels of platelet FSC (a measure affected by multiple variables including size) surface GPIb on unstimulated, ADP- or TRAP-stimulated platelets, surface P-selectin on unstimulated platelets, and platelet FSC were associated with increased odds for higher bleeding scores (ORs each >1, p<0.05), while higher ADP- and TRAP-stimulated platelet surface activated GPIIb-IIIa and P-selectin were associated with reduced odds of higher bleeding scores (ORs each <1, p<0.05). After adjustment for platelet count, higher levels of platelet surface P-selectin on unstimulated platelets, GPIb on TRAP-stimulated platelets, and FSC remained significantly associated with increased odds for higher bleeding scores (Figure), but IPF did not. Similarly, after adjustment for platelet count, higher TRAP-stimulated percentage of P-selectin and activated GPIIb-IIIa positive platelets remained significantly associated with reduced odds of higher bleeding scores (Figure). These findings were independent of recent ITP-related treatment. [Display omitted] ConclusionsIn this study of pediatric ITP patients, we identified selected platelet function markers which, independent of platelet count, are associated with increased (platelet FSC, platelet surface P-selectin on unstimulated platelets, and GPIb on TRAP-stimulated platelets) or decreased (TRAP-stimulated percent P-selectin and GPIIb-IIIa positive platelets) odds of high bleeding scores. Possible hypotheses to explain these associations are as follows: 1) Increased P-selectin on unstimulated platelets demonstrates in vivo platelet activation, possibly as a consequence of the recent bleeding. 2) Because platelet activation results in a reduction in platelet surface GPIb and increases in platelet surface activated GPIIb-IIIa and P-selectin, the ORs associated with all of these markers could be explained by reduced ability of platelets in patients with higher bleeding scores to respond to agonists. 3) While platelet FSC is partly related to size, the finding that MPV and IPF, adjusted for platelet count, were not associated with bleeding score suggests that factors other than size account for the association of platelet FSC with higher bleeding scores. Further study is required to validate these findings and determine if differences in platelet function are associated with future risk for bleeding. Disclosures:Off Label Use: Eltrombopag was given to WAS/XLT patients for treatment of thrombocytopenia. Neufeld:Shire: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Apopharma: Consultancy. Michelson:Sysmex: Honoraria.

Dosed Deficiency of Iron Restricts Terminal Maturation and Enucleation of Cultured Human Erythroblasts

Abstract 1041In addition to reduced hemoglobin production, iron deficiency causes anemia though poorly understood defects in erythropoiesis that reduce the number of erythroid cells released from the marrow space. To study the negative effects of iron deficiency upon erythropoiesis, CD34+ cells from three healthy human donors were cultured in serum-free media supplemented with transferrin-bound iron. Dosed titrations of holo-transferrin (1.2, 1.0, 0.8, 0.6, 0.4, 0.2, 0.1 mg/ml) were balanced by apo-transferrin supplementation to maintain total transferrin levels of 1.2 mg/ml in all cultures. Analyses were performed over the 21-day culture period that included assessment of proliferation, differentiation, enucleation and surface phosphatidylserine expression. Diminution of holo-transferrin in the culture medium caused a decrease in total cell counts and reduced cell divisions detected by CFSE analysis. During the first two weeks of culture, the progenitor cells differentiated into a glycophorin A positive population at all holo-transferrin doses. However, several iron-dependent changes were detected in the final stages of differentiation during culture days 14–21. Hemoglobin accumulation was reduced by 72% at the lowest holo-transferrin dose. Flow cytometry demonstrated a decrease in the loss of surface transferrin receptor in iron-deficient cultures. Thiazole orange analysis also demonstrated significantly lower levels of enucleation from 25.9±8.2% (1.2 mg/ml) to 4.4±4.2% (0.1mg/ml). Microscopic examination of the enucleated cells demonstrated a hypochromatic phenotype at lower holo-transferrin concentrations. Phosphatidylserine on the surface membrane (annexin V staining) increased with reduction of holo-transferrin from 6.3±2.6% (1.2 mg/ml) to 78.3±9.4% (0.1 mg/ml, p=0.003). The effects of iron on surface phosphatidylserine expression were limited to holo-transferrin concentrations of <0.6 mg/ml. Isocitrate supplemented at a concentration of 5.0 mM increased the cell counts in all concentrations of holo-transferrin, and partially rescued the iron deficient phenotype. These data suggest that iron deficiency ex vivo causes decreased proliferation, decreased loss of surface transferrin receptor, and decreased enucleation of normoblasts. There was also a significant increase in phosphatidylserine detected on the surface of the iron deficient normoblasts that provides a mechanism whereby macrophages can identify, phagocytose, and efficiently recycle iron from those cells to other normoblasts in the local marrow environment via erythroblastic islands. Disclosures:No relevant conflicts of interest to declare.

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca(2+) entry (SOCE) with Orai1 as principal Ca(2+) entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca(2+) entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1(-/-) and Orai1(-/-) platelets had greatly impaired glycoprotein (GP) VI-dependent Ca(2+) signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2(-/-) platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca(2+) signals of Stim1(-/-) and Orai1(-/-) platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1(-/-) and Orai1(-/-) platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca(2+) entry, inhibited Ca(2+) and procoagulant responses even in Stim1(-/-) and Orai1(-/-) platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca(2+) entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca(2+) entry and PS exposure, only one relying on STIM1-Orai1 interaction.

Cytometry in malaria: From research tool to practical diagnostic approach?

Malaria is a major global health problem, responsible forseveral hundred million debilitating acute infectious episodesand killing an estimated 800,000 people annually, most ofthem children under 5 years of age and many of the rest preg-nant women.Diagnosis and management of malaria and determinationof efficacy of drugs, vaccines, and other interventions alldepend on accurate detection of the Plasmodium parasites thatcause the disease, determination of which species is/areinvolved, and, often, quantification of parasite density,expressed as the number of parasites/lL of blood or as the frac-tion or percentage of parasitized erythrocytes. The mainstay ofdiagnosis since the late 1800’s has been microscopy, usingGiemsa’s and similar stains to detect parasites in blood smears,but results are limited in accuracy and precision by inadequatetraining of observers and by the relatively small amount of sam-ple theycan practically examine.Both automated microscopy and flow cytometry havebeen found effective for parasite detection, but require appara-tus too expensive and complex to be usable fordiagnosis in theresource-poor areas most affected by malaria. Flow cytometryhas, however, been utilized in malaria research since the 1970’s(1), and newer cytometric applications, such as the paper byPattanapanyasat et al. published in this issue reflect an increas-ChimmaP,ChitjamnongchaiS,PolsrilaK,Chotivanich K.Feb-rile temperature but not proinflammatory cytokines promotesphosphatidylserine expression on Plasmodium falciparummalaria-infected red blood cells during parasite maturation.CytometryA 2010;inpress.DOI:10.1002/cyto.a.20879).These authors used fluorescein-labeled annexin to studythe effects of P. falciparum infection on phosphatidylserineexpression of erythrocytes, demonstrating that cell surfacephosphatidylserine expression increases with parasite matura-tion (measured by nucleic acid content detected with ethi-dium produced by intracellular hydroethidine oxidation) andis also influenced by fever, infection rate, and the parasitestrain. This work is particularly important as it provides evi-dence that P. falciparum causes asymmetry of membranephospholipids and is crucial for senescence of uninfected ery-throcytes, whereas proinflammatory cytokine action is not im-portant for erythrocyte senescence.Giemsa’s stain binds to both DNA and RNA, but cannotbe used for quantification of either. Flow cytometric analysesof malaria began in the 1970’s (1). Most early studies utilizeddyes, such as acridine orange, ethidium, and propidium,which are readily excited by 488 nm lasers, the only illumina-tion then available on most instruments. These dyes, and thenewer SYTO and YOYO series, typically stain DNA and RNAindistinguishably, and most require that cells be fixed or per-meabilized. The few investigators who did have early access tothe ultraviolet (UV) sources needed to excite DNA-specific,stoichiometric stains were likely to use Hoechst 33258 orDAPI, which require permeabilization, rather than Hoechst

A role for c‐Jun N‐terminal kinases in apoptosis triggered in human macrophages by carboxydextran‐coated superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide nanoparticles are widely used for cell labeling and as diagnostic contrast media. The aim of this study was to investigate how carboxydextran‐coated superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) nanosized particles used for magnetic resonance imaging might affect signaling and functions of human macrophages. Within 1 h, both SPIO and USPIO were rapidly taken up by macrophages leading to increased intracellular iron contents. One day after treatment, most nanoparticles were localized within the lysosomal compartment. Prolonged exposure of the macrophages to SPIO and USPIO led to a significantly enhanced production of reactive oxygen species associated with a long‐lasting activation of JNK kinases and induction of apoptosis as measured by surface expression of phosphatidylserine and caspase 3 activation. USPIO elicited a more pronounced cytotoxicity than SPIO. Treatment of macrophages with Trolox® or N‐acetyl‐L‐cysteine, two strucurally different scavengers of reactive oxygen species, or with the JNK inhibitor V abolished the JNK activation as well as the cytotoxicity caused by the iron oxide nanoparticles. These data indicate that nanosized contrast media hamper macrophage function, an effect that can be antagonized with suitable oxygen radical scavengers. This work was supported by the DFG.

PO-56 Potential contribution of microparticles to the thrombotic risk in high grade glioma

poorly defined. We hypothesized that treatment of tumor cells with cytotoxic chemotherapeutic drugs increases phosphatidylserine (PS) on the cell surface, cellular tissue factor (TF) activity and release of TF positive microparticles (MPs). Aim: The aim of the study was to determine the effect of anthracyclines on TF activity and MP release from the human monocytic leukemia cell line THP-1. Materials and Methods: THP-1 cells were treated with different concentrations of doxorubicin (0–10mg/ml) and daunorubicin (0–10mg/ml) for 24 hours. These concentrations are similar to those found in plasma of cancer patients. The intact cells were analyzed for TF activity using a two stage clotting assay and PS exposure was analyzed by flow cytometry. In addition, TF activity of MPs released into the culture supernatant was measured using a two stage clotting assay. Results: We found that doxorubicin and daunorubicin both increased cellular TF and MP TF activity in a concentration-dependent manner. This effect was partially abrogated by the caspase inhibitor ZVAD-FMK. Increased PS exposure on the cell surface was also noted in cells treated with anthracyclines. Conclusions: These results support our hypothesis that treatment of THP-1 cells with chemotherapeutic agents induces apoptosis and increases cellular TF activity. This increased activity appears primarily mediated by an increase in cell surface expression of PS. Additionally, the chemotherapeutics increase the release of TF positive MPs from the THP-1 cells. We propose that this increase in cellular TF activity in tumor cells and increased numbers of TF positive MPs leads to an increase in thrombosis in cancer patients treated with chemotherapeutic drugs.

Non-redundant Roles of Phosphoinositide 3-Kinase Isoforms α and β in Glycoprotein VI-induced Platelet Signaling and Thrombus Formation

Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cgamma2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3Kalpha or -beta strongly and selectively suppressed GPVI-induced Ca(2+) mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cgamma2 by PI3Kalpha/beta. That PI3Kalpha and -beta have a non-redundant function in GPVI-induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca(2+) increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3Kalpha/beta was blocked or p85alpha was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kdelta or -gamma. Furthermore, PI3Kalpha/beta, but not PI3Kgamma, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kalpha and -beta isoforms are required for full GPVI-dependent platelet Ca(2+) signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kalpha an interesting new target for anti-platelet therapy.

Impaired Platelet Procoagulant Mechanisms in Patients with Bleeding Disorders

Activated platelets contribute to the arrest of bleeding by forming aggregates at sites of vascular injury and by providing a surface for assembling enzyme complexes involved in fibrin formation (platelet procoagulant activity; PCA). Impairment in the latter property of platelets has been observed in some disorders of hemostasis. In Scott syndrome, there is a defect in membrane vesiculation and in the surface expression of phosphatidylserine (PS), the phospholipid that is necessary for assembling the factor VIIIa/IXa (tenase) and factor Va/Xa (prothrombinase) complexes involved in thrombin formation. A family with an isolated defect in vesiculation, but normal prothrombinase activity, has also been reported. In the Quebec platelet disorder, overexpression of the fibrinolytic enzyme urokinase-type plasminogen activator results in the degradation of alpha-granule proteins, including factor V, and a specific abnormality in platelet factor V is the basis for the prothrombinase defect in platelet factor V-New York. The impaired prothrombinase activity in patients with delta-storage pool deficiency may be due to a failure to provide sufficient amounts of secreted adenine nucleotides which, when bound to P2 purinergic receptors, are necessary to maintain the intracellular Ca (2+) levels that are required for the surface expression of PS. Platelet prothrombinase activity and thrombin potential in patients with Glanzmann thrombasthenia (GPIIb-IIIa deficiency) may be decreased, normal, or increased, depending on the experimental conditions, for reasons that are not currently clear. The most consistent platelet PCA abnormality in the Bernard-Soulier syndrome (GPIb-complex deficiency) is an abnormally short serum prothrombin time, associated with a defect in the process by which an interaction between fibrin, von Willebrand factor, and GPIb promotes PCA.

Phosphatidylserine surface expression and integrin αIIbβ3 activity on thrombin/convulxin stimulated platelets/particles of different sizes

Platelets stimulated by a combination of thrombin/convulxin have been shown to develop two to three populations characterized by different phosphatidylserine (PS) surface expression and integrin alpha IIb beta 3 activity. To determine how these markers are distributed on the surface of platelets/particles, we studied Annexin V and PAC-1 binding to platelets/particles of different sizes by flow cytometry analysis and evaluated influences of calpain and caspase inhibitors on thrombin/convulxin-activated platelets. Analysed platelets/particles were divided by their sizes, according to the standard size beads, into seven populations from 0.37 to 4.8 microm. PAC-1 binding/microm(2) was almost equal in platelets/particles ranging from 1.2 to 4.8 microm and was significantly lower on smaller-sized particles sizes (0.37-0.7 microm). PS surface exposure/microm(2) was high in the particles of 0.37-1.2 microm and very low in platelets (2.6-4.8 microm). Upon thrombin/convulxin stimulation caspase inhibitors prevented microparticle (MP) formation, while a calpain inhibitor stimulated MP formation. It was also shown that stimulated platelets are heterogeneous not only in their ability to activate alpha IIb beta 3 integrin complex and expose PS on their surface, but also in the distribution of activation markers, which strongly depends on platelet/particle size and that platelets/particles of different sizes provide different responses to the same stimulus.

The active metabolite of prasugrel inhibits adenosine diphosphate- and collagen-stimulated platelet procoagulant activities.

Prasugrel is a novel antiplatelet prodrug of the same thienopyridine class as clopidogrel and ticlopidine. Metabolism of prasugrel generates the active metabolite R-138727, an antagonist of the platelet P2Y(12) adenosine diphosphate (ADP) receptor, leading to inhibition of ADP-mediated platelet activation and aggregation. ADP also enhances the platelet response to collagen, and these two agonists contribute to the generation of platelet procoagulant activity. We therefore examined whether R-138727 inhibits ADP- and collagen-triggered platelet procoagulant activities. As shown by whole blood flow cytometry, R-138727 inhibited surface phosphatidylserine expression on ADP plus collagen-stimulated platelets and tissue factor (TF) expression on ADP-, collagen-, and ADP plus collagen-stimulated monocyte-platelet aggregates. R-138727 reduced monocyte-platelet aggregate formation, thereby further inhibiting TF expression. ADP, collagen, and ADP plus collagen accelerated the kinetics of thrombin generation in recalcified whole blood and R-138727 significantly inhibited this acceleration. Clot strength in a modified thromboelastograph system was also inhibited by R-138727 (IC50 0.7 +/- 0.1 microM). In addition to its previously known inhibitory effects on platelet activation and aggregation, the active metabolite of prasugrel, R-138727, inhibits platelet procoagulant activity in whole blood (as determined by phosphatidylserine expression on platelets and TF expression on monocyte-platelet aggregates), resulting in the functional consequences of delayed thrombin generation and impaired clot development.

The active metabolite of prasugrel inhibits adenosine diphosphate- and collagen-stimulated platelet procoagulant activities

Prasugrel is a novel antiplatelet prodrug of the same thienopyridine class as clopidogrel and ticlopidine. Metabolism of prasugrel generates the active metabolite R-138727, an antagonist of the platelet P2Y(12) adenosine diphosphate (ADP) receptor, leading to inhibition of ADP-mediated platelet activation and aggregation. ADP also enhances the platelet response to collagen, and these two agonists contribute to the generation of platelet procoagulant activity. We therefore examined whether R-138727 inhibits ADP- and collagen-triggered platelet procoagulant activities. As shown by whole blood flow cytometry, R-138727 inhibited surface phosphatidylserine expression on ADP plus collagen-stimulated platelets and tissue factor (TF) expression on ADP-, collagen-, and ADP plus collagen-stimulated monocyte-platelet aggregates. R-138727 reduced monocyte-platelet aggregate formation, thereby further inhibiting TF expression. ADP, collagen, and ADP plus collagen accelerated the kinetics of thrombin generation in recalcified whole blood and R-138727 significantly inhibited this acceleration. Clot strength in a modified thromboelastograph system was also inhibited by R-138727 (IC50 0.7 +/- 0.1 microM). In addition to its previously known inhibitory effects on platelet activation and aggregation, the active metabolite of prasugrel, R-138727, inhibits platelet procoagulant activity in whole blood (as determined by phosphatidylserine expression on platelets and TF expression on monocyte-platelet aggregates), resulting in the functional consequences of delayed thrombin generation and impaired clot development.