Platelet-derived extracellular vesicles (PEVs) are a potential alternative to apheresis platelets (PLTs) for hemorrhage control; however, the platelet fraction that yields the most prohemostatic PEVs remains unknown. The objective of this study was to determine whether PEVs derived from platelet additive solution (PAS) supernatant or from washed PLTs exhibit greater hemostatic efficacy. Apheresis PLTs from six healthy donors were processed to generate PAS supernatant and washed PLTs. PEVs were isolated using tangential flow filtration (TFF) and characterized. Procoagulant activity was assessed using calibrated automated thrombography (CAT) and plasma clotting assays. In vivo efficacy was evaluated using a rat liver laceration model of uncontrolled hemorrhage. PAS-derived PEVs supported faster clot initiation (p = 0.0187) and clot formation (p = 0.0019) compared with washed platelet-derived PEVs. Thrombin generation was significantly increased in PAS-derived PEVs, with higher peak thrombin and faster thrombin generation rates (p = 0.0431 and p = 0.0463). In vivo, PAS-derived PEVs reduced cumulative blood loss at 60 minutes following liver laceration (p = 0.049). Post-hemorrhage plasma from PAS-PEV-treated rats demonstrated enhanced thrombin generation kinetics and increased endogenous thrombin potential (ETP; p ≤ 0.040). Procoagulant activity was positively correlated with surface phosphatidylserine expression. PAS-derived PEVs exhibit superior hemostatic activity compared with washed platelet-derived PEVs across ex vivo and in vivo models. These findings support PAS as a preferred source for PEV isolation and inform optimization of PEV production for development as a transfusion alternative for hemorrhage control.

Enhanced procoagulant activity of phosphatidylserine-positive platelets in antiphospholipid syndrome

Mice with reduced protease-activated receptor 4 reactivity show decreased venous thrombosis and platelet procoagulant activity

BackgroundHypercoagulation and thrombin generation are major risk factors for venous thrombosis. Sustained thrombin signaling through PAR4 promotes platelet activation, phosphatidylserine exposure, and subsequent thrombin generation. A single-nucleotide polymorphism in PAR4 (rs2227376) changes proline to leucine extracellular loop 3 (P310L), which decreases PAR4 reactivity and is associated with a lower risk for venous thromboembolism (VTE) in a GWAS meta-analysis.ObjectiveThe goal of this study is to determine the mechanism for the association of rs2227376 with reduced risk for VTE in using mice with a homologous mutation (PAR4-P322L).MethodsVenous thrombosis was examined using our recently generated PAR4-P322L mice in the inferior vena cava stasis and stenosis models. Coagulation and clot stability was measured using rotational thromboelastometry (ROTEM). Thrombin generating potential was measured in platelet-rich plasma. Phosphatidylserine surface expression and platelet-neutrophil aggregates were analyzed using flow cytometry.ResultsPAR4P/L and PAR4L/L had reduced incidence and size of venous clots at 48 hours. PAR4P/L and PAR4L/L platelets had progressively decreased phosphatidylserine in response to thrombin and convulxin, which led to decreased thrombin generation and decreased PAR4-mediated platelet-neutrophil aggregation.ConclusionsThe leucine allele in extracellular loop 3, PAR4–322L leads to fewer procoagulant platelets and decreased endogenous thrombin potential. This decreased ability to generate thrombin offers a mechanism for PAR4’s role in VTE highlighting a key role for PAR4 signaling.

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

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.

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

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.

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.

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.

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.

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

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

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.

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

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

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.