Platelet Viability Research Articles

The study of morphofunctional properties of platelets in the process of storing them in additive solution containing sodium fumarate

Background . Improving the quality of platelet concentrate by choosing an adequate medium for storing isolated platelets is a relevant problem of a blood function. Objective . The study of metabolic activity, platelet viability and their aggregation properties during storage of platelet concentrate in an additive solution containing sodium fumarate. Design and methods . The object of the study was donor platelet concentrates (PC). PC was obtained from donors who passed necessary examinations. Biochemical, hematological and coagulological research methods were utilized in carrying out the study. Results . During the research, reliable evidence was obtained on the best preservation of platelets by 9 days of their storage in a fumarate-containing additive solution (FCAS) as compared to storage in a commercial SSP+ solution (France). It was found that with practically the same glucose content in the compared solutions on the day of PC preparation, the glucose concentration by 9th day of observation was significantly higher in the FCAS than in the SSP+ solution. Furthermore, the FCAS was determined to have a consistently lower lactate content as compared to the SSP+ solution. In regard to their aggregation activity, the platelets prepared in the FCAS were not inferior to the platelets stored in the SSP+. Based on the results of the study, a methodology has been developed to obtain additive solution containing sodium fumarate. Conclusion . The developed composition of the additive solution allows to increase the shelf life of platelets up to 9 days, which is important for a more economical and effective use of this blood component in order to correct thrombocytopenia of various etiologies.

Activated platelets kill Staphylococcus aureus, but not Streptococcus pneumoniae—The role of FcγRIIa and platelet factor 4/heparinantibodies

BackgroundHeparin induced thrombocytopenia (HIT) is likely a misdirected bacterial host defense mechanism. Platelet factor 4 (PF4) binds to polyanions on bacterial surfaces exposing neo‐epitopes to which HIT antibodies bind. Platelets are activated by the resulting immune complexes via FcγRIIA, release bactericidal substances, and kill Gram‐negative Escherichia coli. ObjectivesTo assess the role of PF4, anti‐PF4/H antibodies and FcγRIIa in killing of Gram‐positive bacteria by platelets. MethodsBinding of PF4 to protein‐A deficient Staphylococcus aureus (SA113Δspa) and non‐encapsulated Streptococcus pneumoniae (D39Δcps) and its conformational change were assessed by flow cytometry using monoclonal (KKO,5B9) and patient derived anti‐PF4/H antibodies. Killing of bacteria was quantified by counting colony forming units (cfu) after incubation with platelets or platelet releasate. Using flow cytometry, platelet activation (CD62P‐expression, PAC‐1 binding) and phosphatidylserine (PS)‐exposure were analyzed. ResultsMonoclonal and patient‐derived anti‐PF4/H antibodies bound in the presence of PF4 to both S. aureus and S. pneumoniae (1.6‐fold increased fluorescence signal for human anti‐PF4/H antibodies to 24.0‐fold increase for KKO). Staphylococcus aureus (5.5 × 104cfu/mL) was efficiently killed by platelets (2.7 × 104cfu/mL) or their releasate (2.9 × 104cfu/mL). Killing was not further enhanced by PF4 or anti‐PF4/H antibodies. Blocking FcγRIIa had no impact on killing of S. aureus by platelets. In contrast, S. pneumoniae was not killed by platelets or releasate. Instead, after incubation with pneumococci platelets were unresponsive to TRAP‐6 stimulation and exposed high levels of PS. ConclusionsAnti‐PF4/H antibodies seem to have only a minor role for direct killing of Gram‐positive bacteria by platelets. Staphylococcus aureus is killed by platelets or platelet releasate. In contrast, S. pneumoniae affects platelet viability.

A complement-mediated rat xenotransfusion model of platelet refractoriness

BackgroundPlatelet refractoriness remains a challenging clinical dilemma although significant advancements have been made in identifying human leukocyte antigen (HLA) matched or HLA compatible units. Antiplatelet antibodies are the major risk factor for immune-mediated platelet refractoriness, yet the role of antibody-initiated complement-mediated platelet destruction remains poorly understood. Study Design and MethodsHuman complement-mediated opsonization and killing of platelets was assayed ex vivo using antibody-sensitized human platelets incubated with complement-sufficient human sera. A new animal model of platelet refractoriness utilizing Wistar rats transfused with human platelets is described. ResultsHuman platelets sensitized with anti-platelet antibodies were rapidly opsonized with iC3b upon incubation in human sera. This opsonization could be completely blocked with a classical pathway complement inhibitor, PA-dPEG24. Complement activation decreased platelet viability, which was also reversible with complement inhibitor PA-dPEG24. A new rat model of platelet refractoriness was developed that demonstrated some platelet removal from the blood stream was complement mediated. ConclusionsComplement activation initiated by anti-platelet antibodies leads to complement opsonization and decreased platelet viability. A new rat model of platelet refractoriness was developed that adds a new tool for elucidating the mechanisms of platelet refractoriness.

The Effect of Composol Medium on miR-16 Expression during Platelet Storage up to Day 7 at Room Temperature.

Objective MicroRNAs (miRNAs) are short, noncoding RNAs that play vital roles in gene regulation. It has been shown that storage has an effect on platelet miRNAs. MiR-16 is highly expressed in platelets and it appears to target the genes involved in cell death. It has been shown that platelets could be stored in Composol for a longer period of time. The aim of the present study was to assess and compare the expression pattern of miR-16 in platelet concentrates (PCs) in plasma and Composol media. Materials and Methods In an experimental study, ten PC bags were collected and each bag was divided into two separate bags, one with the 70% Composol and the other with only plasma. Both bags were stored for 7 days at 22˚C and tested on days 1, 3, 5, and 7 of storage. For each sample, we performed quantitative real-time polymerase chain reaction (qRT-PCR). The water-soluble tetrazolium salt-1 (WST-1) test was used to assess platelet viability in all of the samples. Statistical analysis was done by SPSS and REST software. A P<0.05 was considered statistically significant. Results miR-16 was significantly elevated during the storage days, with fold changes of 3.47 (plasma) and 2.77 (Composol). The Composol group had significantly decreased miR-16 expression compared with the plasma group. Results of the WST-1 test showed less decrease in optical density (OD) in the Composol group (0.93 ± 0.4) during the storage days compared with the plasma group (0.75 ± 0.3). ConclusionOur finding supported results from previous studies that reported an increase in miR-16 expression during platelet storage. In addition, miR-16 down-regulation in Composol medium implied that Composol might be a good solution for long-term platelet storage because it has the potential to elevate the shelf-life of platelets stored at 22˚C.

The Marine-Derived Triterpenoid Frondoside A Inhibits Thrombus Formation.

Background: The marine-derived triterpenoid frondoside A inhibits the phosphatidylinositol-3-kinase (PI3K) pathway in cancer cells. Because this pathway is also crucially involved in platelet activation, we studied the effect of frondoside A on thrombus formation. Methods: Frondoside A effects on platelet viability, surface adhesion molecule expression, and intracellular signaling were analyzed by flow cytometry and Western blot. The effect of frondoside A was analyzed by photochemically induced thrombus formation in the mouse dorsal skinfold chamber model and by tail vein bleeding. Results: Concentrations of up to 15 µM frondoside A did not affect the viability of platelets, but reduced their surface expression of P-selectin (CD62P) and the activation of glycoprotein (GP)IIb/IIIa after agonist stimulation. Additional mechanistic analyses revealed that this was mediated by downregulation of PI3K-dependent Akt and extracellular-stimuli-responsive kinase (ERK) phosphorylation. Frondoside A significantly prolonged the complete vessel occlusion time in the mouse dorsal skinfold chamber model of photochemically induced thrombus formation and also the tail vein bleeding time when compared to vehicle-treated controls. Conclusion: Our findings demonstrated that frondoside A inhibits agonist-induced CD62P expression and activation of GPIIb/IIIa. Moreover, frondoside A suppresses thrombus formation. Therefore, this marine-derived triterpenoid may serve as a lead compound for the development of novel antithrombotic drugs.

Defective Platelet Thromboxane A2 Signaling and Serotonin Release in the Pathogenesis of Bleeding during Viral Infection

Viral hemorrhagic fever (VHF) refers to a group of diverse acute human diseases caused by single stranded RNA viruses typically associated with fever, vascular damage and abnormal vascular barrier function. These infections are a continuing threat to public health due to the high case-fatality rate and potential bioterrorism threats. Critically infected VHF patients with unfavorable prognosis often present bleeding symptoms and shock. Thrombocytopenia associated with endothelial, coagulation and platelet dysfunction has been reported during these infections, but their relationship to bleeding is still poorly defined. We have previously established that infection by lymphocytic choriomeningitis virus (LCMV) in extremely thrombocytopenic mice causes severe mucocutaneous bleeding and death mediated by interferon α/β receptor (IFNAR) signaling. We now show in vivo that IFNAR is expressed on megakaryocytes but not on circulating platelets, contrary to previously reported evidence. To explain how increased IFN-I in LCMV-infected mice alters the function of platelets lacking IFNAR, we cross-transplanted WT or IFNAR-/- bone marrow (BM) cells into IFNAR-/- and WT mice. Upon platelet depletion and LCMV infection, only chimeras with WT bone marrow died, suggest that IFN-I-sensitive bone marrow-derived cells are responsible for the mucocutaneous bleeding observed in conditions of extreme thrombocytopenia. Moreover, the results of BM transplantation studies prove that endothelial cell alterations and infection-related inflammatory conditions are not alone sufficient to cause hemorrhage. Circulating platelets during LCMV infection show a generalized aggregation disfunction that persists longer for the arachidonic acid/thromboxane A2 (AA/TBXA2) activation pathway. These aggregation defects are associated, and possibly linked to an impaired secretory ability of both α- and δ-granule. Since circulating platelets are unresponsive to IFN-I, these findings are consistent with the possibility that bone marrow-resident megakaryocytes are targets of IFN-I stimulation during LCMV infection and are the origin of structural and functional platelet abnormalities observed in the circulation. Indeed, megakaryocytes from LCMV-infected mice exhibited fewer granules limited to the perinuclear zone rather than normally diffused throughout the demarcation membrane system; the latter was also underdeveloped. Moreover, platelet transcriptome evaluation of genes involved in platelet production and function showed that IFN-I downregulates bone marrow expression of NF-E2 - a key transcription factor for thrombocytopoiesis - as well as of cyclooxygenase-1 and thromboxane-A synthase. The latter enzymes are required for TBXA2 synthesis from AA amplifying platelet activation. Thus, in addition to being decreased in number, circulating platelets of LCMV-infected animals have impaired activation-induced cargo release from storage granules supporting hemostasis. Experimentally, we observed that bleeding during LCMV infection is increased following aspirin, but not clopidogrel, administration. This suggested that serotonin secretion may be the final effector required to control erythrocyte extravasation. Accordingly, bleeding associated with LCMV infection was markedly enhanced in mice lacking platelet serotonin (TPH1-/-mice), and this was ameliorated by transfusion of normal platelets. In conclusion, we have identified a IFN-I targeting of megakaryocytes leading to reduced production of dysfunctional platelets with abnormal serotonin release as the cause of potentially lethal bleeding in a mouse model of arenavirus infection. Our demonstration that infusion of viable platelets can prevent lethal anemia in LCMV-infected susceptible mouse strains indicates that the same therapeutic intervention monitored by platelet response to AA stimulation may aid in treating severe VHF cases in human patients. Disclosures Aiolfi: MERU-VasImmune, Inc: Other: Stock option. Ruggeri:MERU-VasImmune Inc.: Equity Ownership, Other: CEO and Founder.

Platelets Apoptosis and Clearance in The Presence of Sodium Octanoate during Storage of Platelet Concentrate at 4˚C.

Objective Platelet (PLT) storage at 4˚C has several benefits, however, it is accompanied by increased clearance of PLTs after transfusion. In this study, we evaluated the potential of sodium octanoate (SO) for reducing apoptosis and clearance rate of PLTs after long-term storage in cold. Materials and Methods In this experimental study, PLT concentrates (PCs) were stored for 5 days under the following three conditions: 20-24˚C, 4˚C, and 4˚C in the presence of SO. To measure the viability of PLTs, the water-soluble tetrazolium salt (WST-1) assay was performed. Phosphatidylserine (PS) exposure was determined on PLTs using flow cytometry technique. The amount of human active caspase-3 was determined in PLTs using an enzyme-linked immunosorbent assay. Additionally, the amount of PLT ingestion or clearance was determined by using HepG2 cell line. Results The viability was higher in the SO-treated PLTs compared to the other groups. The level of PS exposure on PLTs was lower in the SO-treated PLTs compared to the other groups. The amount of active caspase-3 increased in all groups during 5-day storage. The highest increase in the amount of caspase-3 levels was observed at cold temperature. However, PLTs kept at 4˚C in the presence of SO had a lower amount of active caspase-3 compared to PLTs kept at 4˚C. The amount of PLTs removal by HepG2 cells was increased for 4˚C-kept PLTs but it was lower for PLTs kept at 4˚C in the presence of SO but, the differences were not significant (P>0.05). ConclusionSO could partially moderate the effects of cold temperature on apoptosis and viability of platelets. It also decreases the ingestion rate of long-time refrigerated PLTs in vitro. Further studies using higher numbers of samples are required to demonstrate the effect of SO on reducing the clearance rate of PLTs.

Antimicrobial blue light for decontamination of platelets during storage.

Platelet (PLT) storage is currently limited to 5 days in clinics in the United States, in part, due to an increasing risk for microbial contamination over time. In light of well-documented antimicrobial activity of blue light (405-470 nm), we investigated potentials to decontaminate microbes during PLT storage by antimicrobial blue light (aBL). We found that PLTs produced no detectable levels of porphyrins or their derivatives, the chromophores that specifically absorb blue light, in marked contrast to microbes that generated porphyrins abundantly. The difference formed a basis with which aBL selectively inactivated contaminated microbes prior to and during the storage, without incurring any harm to PLTs. In accordance with this, when contamination with representative microbes was simulated in PLT concentrates supplemented with 65% of PLT additive solution in a standard storage bag, all "contaminated" microbes tested were completely inactivated after exposure of the bag to 405 nm aBL at 75 J/cm2 only once. While killing microbes efficiently, this dose of aBL irradiation exerted no adverse effects on the viability, activation or aggregation of PLTs ex vivo and could be used repeatedly during PLT storage. PLT survival in vivo was also unaltered by aBL irradiation after infusion of aBL-irradiated mouse PLTs into mice. The study provides proof-of-concept evidence for a potential of aBL to decontaminate PLTs during storage.

Antibacterial Activity and Cytocompatibility of Bone Cement Enriched with Antibiotic, Nanosilver, and Nanocopper for Bone Regeneration.

Bacterial infections due to bone replacement surgeries require modifications of bone cement with antibacterial components. This study aimed to investigate whether the incorporation of gentamicin or nanometals into bone cement may reduce and to what extent bacterial growth without the loss of overall cytocompatibility and adverse effects in vitro. The bone cement Cemex was used as the base material, modified either with gentamicin sulfate or nanometals: Silver or copper. The inhibition of bacterial adhesion and growth was examined against five different bacterial strains along with integrity of erythrocytes, viability of blood platelets, and dental pulp stem cells. Bone cement modified with nanoAg or nanoCu revealed greater bactericidal effects and prevented the biofilm formation better compared to antibiotic-loaded bone cement. The cement containing nanoAg displayed good cytocompatibility without noticeable hemolysis of erythrocytes or blood platelet disfunction and good viability of dental pulp stem cells (DPSC). On the contrary, the nanoCu cement enhanced hemolysis of erythrocytes, reduced the platelets aggregation, and decreased DPSC viability. Based on these studies, we suggest the modification of bone cement with nanoAg may be a good strategy to provide improved implant fixative for bone regeneration purposes.

Reflex arc recovery after spinal cord dorsal root repair with platelet rich plasma (PRP)

Spinal dorsal roots can be affected by a wide range of lesions, leading to a significant loss of proprioceptive information transmission and greatly affecting motor behavior. In this context, the reimplantation of lesioned roots with platelet-rich plasma (PRP) may allow nerve regeneration. Therefore, the present study evaluated sensorimotor improvement following dorsal root rhizotomy and repair with PRP. For this purpose, female Lewis rats were subjected to unilateral rhizotomy (RZ) of the L4–L6 dorsal roots and divided into the following groups: (1) the unlesioned control group; (2) the group that underwent rhizotomy (RZ) without repair; and (3) the group that underwent RZ followed by root repair with PRP. PRP was obtained from human blood and characterized regarding platelet concentration, integrity, and viability. Reflex arc recovery was evaluated weekly for eight weeks by the electronic von Frey method. The spinal cords were processed 1 week postlesion to evaluate the in vivo gene expression of TNFα, TGF-β, BDNF, GDNF, VEGF, NGF, IL-4, IL-6, IL-13 by qRT-PCR and eight weeks postlesion to evaluate changes in the glial response (GFAP and Iba-1) and excitatory synaptic circuits (VGLUT1) by immunofluorescence. The results indicated that PRP therapy partially restores the paw withdrawal reflex over time, indicating the reentry of primary afferents from the dorsal root ganglia into the spinal cord without exacerbating glial reactivity. Additionally, the analysis of mRNA levels showed that PRP therapy has immunomodulatory properties. Overall, the present data suggest that the repair of dorsal roots with PRP may be considered a promising approach to improve sensorimotor recovery following dorsal rhizotomy.

The role of proteasome activity for activating and inhibitory signalling in human platelets

Platelets express key proteins of the proteasome system, but its functional role in the regulation of platelet integrity, however, is not fully understood yet. Therefore, this study evaluated activating and inhibitory platelet signalling pathways using the potent and selective proteasome inhibitor bortezomib.In washed platelets, the effect of bortezomib on viability and on aggregation was assessed. In addition, fibrinogen binding and CD62P expression were determined. The influence on activating and inhibitory signalling was detected by phosphorylation levels of essential messenger molecules.Platelet viability was maintained after incubation with 0.01 μM to 1 μM bortezomib, but tampered with 100 μM bortezomib. Agonist-induced aggregation was only reduced under 100 μM bortezomib and with weak induction by 10 μM adenosine diphosphate. Similarly, phosphorylated kinase levels of the activating signalling pathways were not affected by 0.01 μM to 1 μM bortezomib. In contrast, proteasome inhibition resulted in the reduction of inhibitor-induced vasodilator-stimulated phosphoprotein phosphorylation, accompanied with the partial decrease of induced inhibition of fibrinogen binding and CD62P expression.In conclusion, platelet activation and aggregation are not dependent on proteasome activity. Instead, inhibitory signalling is partially attenuated under proteasome inhibition. Supramaximal inhibitory concentrations of bortezomib (above 1 μM) lead to heterogeneous effects on activating or inhibitory systems, probably caused by decreasing platelet viability.

Acute bilirubin ditaurate exposure attenuates ex vivo platelet reactive oxygen species production, granule exocytosis and activation

BackgroundBilirubin, a by-product of haem catabolism, possesses potent endogenous antioxidant and platelet inhibitory properties. These properties may be useful in inhibiting inappropriate platelet activation and ROS production; for example, during storage for transfusion. Given the hydrophobicity of unconjugated bilirubin (UCB), we investigated the acute platelet inhibitory and ROS scavenging ability of a water-soluble bilirubin analogue, bilirubin ditaurate (BRT) on ex vivo platelet function to ascertain its potential suitability for inclusion during platelet storage. MethodsThe inhibitory potential of BRT (10–100 μM) was assessed using agonist induced platelet aggregation, dense granule exocytosis and flow cytometric analysis of P-selectin and GPIIb/IIIa expression. ROS production was investigated by analysis of H2DCFDA fluorescence following agonist simulation while mitochondrial ROS production investigated using MitoSOX™ Red. Platelet mitochondrial membrane potential and viability was assessed using TMRE and Zombie Green™ respectively. ResultsOur data shows ≤35 μM BRT significantly inhibits both dense and alpha granule exocytosis as measured by ATP release and P-selectin surface expression, respectively. Significant inhibition of GPIIb/IIIa expression was also reported upon ≤35 μM BRT exposure. Furthermore, platelet exposure to ≤10 μM BRT significantly reduces platelet mitochondrial ROS production. Despite the inhibitory effect of BRT, platelet viability, mitochondrial membrane potential and agonist induced aggregation were not perturbed. ConclusionsThese data indicate, for the first time, that BRT, a water-soluble bilirubin analogue, inhibits platelet activation and reduces platelet ROS production ex vivo and may, therefore, may be of use in preserving platelet function during storage.

Platelets stored in whole blood at 4°C: in vivo posttransfusion platelet recoveries and survivals and in vitro hemostatic function.

Ordinarily, whole blood (WB) is separated into components before storage. We assessed the posttransfusion viability and function of platelets (PLTs) if they were stored within WB at 4°C. Whole blood was obtained from 30 normal subjects and stored at 4°C without agitation for 12 days and for 10, 15, or 22 days with agitation. After WB storage, a PLT concentrate was prepared, and a fresh PLT sample was obtained from each donor. The stored PLTs were labeled with 111 In and the fresh with 51 Cr, and both were simultaneously transfused into their donor. Blood samples were obtained after transfusion to determine PLT recoveries and survivals. PLT samples from WB before and after storage were also assayed for PLT function and biochemistry. After storage for 12 days without WB rotation, poststorage PLT counts averaged only 49 ± 12% of baseline values. After storage for 10, 15, or 22 days with end-over-end WB rotation, PLT counts averaged 76 ± 14% of baseline values. Fifteen-day poststorage radiolabeled PLT recoveries averaged 27 ± 11% (49 ± 16% of fresh), and survivals averaged 1.2 ± 0.4 days (16 ± 6% of fresh). in vitro assays demonstrated marked PLT activation after any storage time, and although PLT function decreased over time, stored PLTs were still considered acceptable. These data suggest that, during rotated WB storage at 4°C for up to 15 days, PLT yields, poststorage PLT recoveries and survivals, and PLT function should be sufficient to support the short-term hemostatic needs of traumatized patients.

Enhancing autophagy protects platelets in immune thrombocytopenia patients.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder and involves increased apoptosis of platelets. Autophagy is an essential process for platelets to maintain their life and physiological functions. However, the role of autophagy in ITP platelets was previously unclear. In the present study, the expression of autophagy-related protein and autophagy flux were detected in platelets from ITP patients and healthy controls by immunofluorescence staining and immunoblotting, and the influence of autophagy on the viability and apoptosis of ITP platelets was further explored. We found that platelet autophagy was diminished in ITP patients. Platelet autophagy in ITP was regulated by the PI3K/AKT/mTOR pathway, with mTOR (mammalian target of rapamycin) as a negative regulator and class III PtdIns3K playing a crucial role in the process. Importantly, the small-molecule compound ABO (6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine) enhanced autophagy in ITP platelets. Enhancing platelet autophagy alleviated platelet destruction by inhibiting apoptosis and improving platelet viability. These results suggest a role for autophagy regulation in the pathogenesis of ITP, and offer a novel treatment for these patients.

Edoxaban improves atrial fibrillation and thromboembolism through regulation of the Wnt-β-induced PI3K/ATK-activated protein C system.

Thromboembolism is a commonly observed condition in geriatrics that is caused by vascular endothelial injury, platelet activation, physiological coagulation processes, reduction of anticoagulant activity, decreased fibrinolytic activity and abnormal flow in the heart chamber, artery or vein. The protein C anticoagulant system serves a crucial role in anticoagulant therapy for the treatment of thromboembolism. Previous findings have suggested that edoxaban is an efficient oral anticoagulant in the acute treatment of venous thromboembolism. In the present study, the efficacy of edoxaban on thromboembolism induced by atrial fibrillation was investigated in a mouse model. Inflammatory factors interleukin (IL)-1, −4, −8 and tumor necrosis factor (TNF)-α were analyzed in the sera of mice with fibrillation induced by thromboembolism. Expression and activity of thymic stromal lymphopoietin (TSLP) and activated protein C resistance were investigated in platelets and vascular endothelial cells (VECs). TSLP-induced platelet viability, Wnt-β phosphorylation and integrin expression were analyzed in platelets. Furthermore, Wnt-β expression and the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in VECs were analyzed. Results demonstrated that the expression levels of IL-1, −4, −8 and TNF-α were significantly downregulated in the sera of mice with fibrillation and thromboembolism following treatment with edoxaban (P<0.01). Furthermore, the expression levels of prostacyclin (PGI2), prostaglandin (PG)E2, PGD2 and PGF2α were significantly increased in the sera of experimental mice that received edoxaban therapy (P<0.01). Results also indicated that edoxaban significantly stimulated the protein expression of TSLP and activated Wnt-β phosphorylation and integrin expression in platelets (P<0.01). In addition, edoxaban therapy significantly upregulated the expression levels of PI3K and AKT, and subsequently increased the activity of protein C and S in VECs (P<0.01). Notably, edoxaban treatment improved atrial fibrillation and thromboembolism, as determined by pathological analysis. In conclusion, these results suggested that edoxaban elicited beneficial effects for mice with atrial fibrillation induced by thromboembolism through the regulation of the Wnt-β-induced PI3K/ATK-activated protein C system.

A platelet viability assay (PVA) for the diagnosis of heparin-induced thrombocytopenia

Diagnosing heparin-induced thrombocytopenia (HIT) requires functional assays measuring platelet activation as they are highly specific and sensitive. A useful functional test for diagnosing HIT is the serotonin release assay (SRA), but this assay is technically demanding and requires a radioactive marker. We describe an alternate functional HIT assay, the platelet viability assay (PVA), that overcomes the need for a radioactive marker by using a viability dye endpoint to measure platelet activation. We compared the performance characteristics of the PVA to the SRA. Serum samples from 76 patients with suspected HIT were tested in both the PVA and the SRA. The PVA uses calcein-AM as a marker of platelet viability, with decreases in fluorescence and cell size as surrogate markers for platelet activation. A significant linear correlation (Spearman correlation, r = −0.78, P < 0.0001) was observed between the PVA and SRA. Calcein-AM fluorescence decreased in a negative linear relationship with platelet activation as measured by 14C-serotonin release. The PVA detected all positive SRA samples, with an overall sensitivity of 100% and a specificity of 97% in comparison to the SRA. The measurement of platelet viability using the PVA provided similar results to the SRA when testing suspected HIT patient samples.

ROS scavenger, N-acetyl-l-cysteine and NOX specific inhibitor, VAS2870 reduce platelets apoptosis while enhancing their viability during storage.

Platelet storage is often complicated by deleterious changes that are started by reversible activation of the cells and can lead to procoagulant function and apoptosis during longer periods of storage. Given that increasing levels of reactive oxygen species (ROS) generation are associated with platelet activation and apoptosis, our study investigated whether ROS scavenging or the inhibition of ROS production can enhance the viability of stored platelets. For this study platelet-rich plasma platelet concentrates (PRP-PCs) were either treated with ROS-reducing agents (1 mM N-acetyl-L -cysteine [NAC] or 30 μM NADPH oxidase [NOX] inhibitor, VAS2870) or kept untreated during storage. P-selectin expression, phosphatidylserine (PS) exposure, levels of microparticle (MP) formation, and intraplatelet caspase activity of PCs were analyzed by flow cytometry during 7 days of storage while the platelet viability was also evaluated by MTT assay. Both NAC- and VAS2870-treated platelets had significantly lower caspase activity, MP formation, and PS exposure during storage while they also showed improved viability. The platelet count and mean platelet volume (MPV) were also better preserved in the presence of NAC. Our results confirmed that either the inhibition of ROS generation or the scavenging of these oxidant agents can attenuate platelet apoptosis while improving their viability during storage. In this study, the significant improvement of platelet viability obtained by NAC suggested that its supplementation with a designated safe concentration into PCs may better preserve the quality of these products, especially for longer storage.

Cooling and Cryopreservation of Equine Platelet-Rich Plasma With Dimethyl Sulfoxide and Trehalose

Equine platelet-rich plasma (PRP) has been used in horses to repair bone, articular and tendinous lesions, laminitis, and even endometritis. However, platelets have a very limited lifespan, which makes it difficult to prepare and use PRP, except in loco. With the aim to produce PRP with higher platelet viability for clinical purposes, the effects of the cryoprotectants dimethyl sulfoxide (DMSO) and trehalose were evaluated on cooled (4°C) and cryopreserved (−196°C) equine PRP. The protocols of cooling and cryopreservation were performed independently, comparing the following treatments: fresh PRP, PRP + 6% DMSO, PRP + 300 mM of trehalose, and PRP only. The PRP samples were prepared by double centrifugation of the blood of six ponies, further divided into four aliquots. The cooled or cryopreserved aliquots were stored for 14 days. All samples were evaluated for the platelet count, the mean platelet volume, and the release of transforming growth factor beta 1 (TGF-β1). The number of platelets in the fresh PRP and cooled samples was similar; however, platelet count was higher in the fresh PRP than in cryopreserved samples. The release of TGF-β1 was higher in the fresh PRP (105891 ± 52398 pg/mL), but the stored samples still released significant amounts of this growth factor (27291 ± 9625 pg/mL).

Platelet activity is negatively modulated by tumor necrosis factor alpha through reductions of cytosolic calcium levels and integrin alphaIIbbeta3 phosphorylation

IntroductionTumor necrosis factor-alpha (TNF-α) exerts a critical role in inflammatory events through two distinct receptors, TNFR1 and TNFR2. Platelets have been recognized as important inflammatory cells, but little is known about the effects of TNF-α on the platelet activity. ObjectivesIn the present study we have studied the role of TNF-α on ADP-induced platelet aggregation and its downstream signaling (c-Src and fibrinogen receptor phosphorylation, cytosolic Ca2+ mobilization, cAMP and cGMP levels and cell viability). Methods and resultsWashed rat platelets were incubated with TNF-α (1–3000 pg/ml) for different time-periods (5–60 min) before the addition of ADP (5 μM) to induce platelet aggregation. TNF-α concentration- and time-dependently inhibits ADP-induced aggregation, which was significantly prevented by incubation with the non-selective TNF-α receptor antagonist R7050. TNF-α (300 pg/ml, 30 min) decreases thrombin-induced elevation of cytosolic Ca++ levels by 2.2- fold compared to untreated platelets. TNF-α decreases the cAMP levels, while significantly increases the intracellular cyclic cGMP levels. However, the pre-incubation of platelets with the guanylyl cyclase inhibitor ODQ, despite decreasing the cGMP levels, does not modify the inhibitory effect of TNF-α on ADP-induced platelet aggregation. Additionally, western blotting analysis showed that TNF-α significantly reduced (Tyr 416)-c-Src and (Tyr773)-β3 subunit of αIIbβ3 integrin phosphorylation. TNF-α does not affect the platelet viability in any condition tested. ConclusionTherefore, our results show that TNF-α negatively modulates ADP-induced aggregation via TNFR1/TNFR2 receptors by reducing cytosolic Ca++ levels and by inhibiting c-Src and fibrinogen receptor activation, which take place through cAMP- and cGMP-independent mechanisms.

Multifaceted effects of arachidonic acid and interaction with cyclic nucleotides in human platelets

IntroductionArachidonic acid induced aggregation is a generally accepted test for aspirin resistance. However, doubts have been raised that arachidonic acid stimulated aggregation can be regarded as reliable testing for aspirin resistance. Arachidonic acid, in addition to platelet activation, can induce phosphatidylserine translocation on the outer surface of platelet membrane which could be mediated by apoptosis pathways or transformation of platelets to the procoagulant state. Materials and methodsWe explored effects of arachidonic acid over a vast range of concentrations and a wide range of read-outs for human platelet activation, procoagulant activity, and platelet viability. Additionally we tested whether cAMP- or cGMP-dependent protein kinase activation can inhibit procoagulant activity or platelet viability. ResultsArachidonic acid-induced washed platelet activation was detected at low micromolar concentrations during the first 2 min of stimulation. After longer incubation and/or at higher concentrations arachidonic acid triggered platelet procoagulant activity and reduced platelet viability. At the same time, arachidonic acid stimulated adenylate cyclase mediated protein phosphorylation which correlated with reduced platelet activation. Moreover, additional stimulation of cAMP- or cGMP-dependent protein kinase inhibited only platelet activation, but did not prevent pro-coagulant activity and platelet death. ConclusionsWhile arachidonic acid induces platelet activation at low concentrations and during short incubation time, higher concentrations and lasting incubation evokes adenylate cyclase activation and subsequent protein phosphorylation corresponding to reduced platelet activation, but also enhanced pro-coagulant activity and reduced viability. Our observations provide further proof for the complex fine tuning of platelet responses in a time and agonist concentration dependent manner.