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Sample records for platelet lysate preparation

  1. Preparation, quality criteria, and properties of human blood platelet lysate supplements for ex vivo stem cell expansion.

    Science.gov (United States)

    Shih, Daniel Tzu-Bi; Burnouf, Thierry

    2015-01-25

    Most clinical applications of human multipotent mesenchymal stromal cells (MSCs) for cell therapy, tissue engineering, regenerative medicine, and treatment of immune and inflammatory diseases require a phase of isolation and ex vivo expansion allowing a clinically meaningful cell number to be reached. Conditions used for cell isolation and expansion should meet strict quality and safety requirements. This is particularly true for the growth medium used for MSC isolation and expansion. Basal growth media used for MSC expansion are supplemented with multiple nutrients and growth factors. Fetal bovine serum (FBS) has long been the gold standard medium supplement for laboratory-scale MSC culture. However, FBS has a poorly characterized composition and poses risk factors, as it may be a source of xenogenic antigens and zoonotic infections. FBS has therefore become undesirable as a growth medium supplement for isolating and expanding MSCs for human therapy protocols. In recent years, human blood materials, and most particularly lysates and releasates of platelet concentrates have emerged as efficient medium supplements for isolating and expanding MSCs from various origins. This review analyzes the advantages and limits of using human platelet materials as medium supplements for MSC isolation and expansion. We present the modes of production of allogeneic and autologous platelet concentrates, measures taken to ensure optimal pathogen safety profiles, and methods of preparing PLs for MSC expansion. We also discuss the supply of such blood preparations. Produced under optimal conditions of standardization and safety, human platelet materials can become the future 'gold standard' supplement for ex vivo production of MSCs for translational medicine and cell therapy applications.

  2. Platelet lysates produced from expired platelet concentrates support growth and osteogenic differentiation of mesenchymal stem cells.

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    Sandra Mjoll Jonsdottir-Buch

    Full Text Available BACKGROUND: Mesenchymal stem cells are promising candidates in regenerative cell therapy. Conventional culture methods involve the use of animal substances, specifically fetal bovine serum as growth supplement. Since the use of animal-derived products is undesirable for human applications, platelet lysates produced from human platelets are an attractive alternative. This is especially true if platelet lysates from already approved transfusion units at blood banks can be utilized. The purpose of this study was to produce human platelet lysates from expired, blood bank-approved platelet concentrates and evaluate their use as growth supplement in the culture of mesenchymal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study, bone marrow-derived mesenchymal stem cells were cultured with one of three culture supplements; fetal bovine serum, lysates from freshly prepared human platelet concentrates, or lysates from expired human platelet concentrates. The effects of these platelet-derived culture supplements on basic mesenchymal stem cell characteristics were evaluated. All cultures maintained the typical mesenchymal stem cell surface marker expression, trilineage differentiation potential, and the ability to suppress in vitro immune responses. However, mesenchymal stem cells supplemented with platelet lysates proliferated faster than traditionally cultured cells and increased the expression of the osteogenic marker gene RUNX-2; yet no difference between the use of fresh and expired platelet concentrates was observed. CONCLUSION/SIGNIFICANCE: Our findings suggest that human platelet lysates produced from expired platelet concentrates can be used as an alternative to fetal bovine serum for mesenchymal stem cell culture to the same extent as lysates from fresh platelets.

  3. Analysis of Reparative Activity of Platelet Lysate: Effect on Cell Monolayer Recovery In Vitro and Skin Wound Healing In Vivo.

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    Sergeeva, N S; Shanskii, Ya D; Sviridova, I K; Karalkin, P A; Kirsanova, V A; Akhmedova, S A; Kaprin, A D

    2016-11-01

    Platelet lysate prepared from donor platelet concentrate and pooled according to a developed technique stimulates migration of multipotent mesenchymal stromal cells of the human adipose tissue and promotes healing of the monolayer defect in cultures of human fibroblasts and multipotent mesenchymal stromal cells in vitro in concentrations close those of fetal calf serum (5-10%). Lysate of platelets from platelet-rich rat blood plasma stimulated healing of the skin defect by promoting epithelialization and granulation tissue formation. The regenerative properties of platelet lysate in vivo increased with increasing its concentration.

  4. Platelet Lysates Produced from Expired Platelet Concentrates Support Growth and Osteogenic Differentiation of Mesenchymal Stem Cells

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    Sandra Mjoll Jonsdottir-Buch; Ramona Lieder; Olafur Eysteinn Sigurjonsson

    2013-01-01

    BACKGROUND: Mesenchymal stem cells are promising candidates in regenerative cell therapy. Conventional culture methods involve the use of animal substances, specifically fetal bovine serum as growth supplement. Since the use of animal-derived products is undesirable for human applications, platelet lysates produced from human platelets are an attractive alternative. This is especially true if platelet lysates from already approved transfusion units at blood banks can be utilized. The purpose ...

  5. Exosomes: novel effectors of human platelet lysate activity

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    E Torreggiani

    2014-09-01

    Full Text Available Despite the popularity of platelet-rich plasma (PRP and platelet lysate (PL in orthopaedic practice, the mechanism of action and the effectiveness of these therapeutic tools are still controversial. So far, the activity of PRP and PL has been associated with different growth factors (GF released during platelet degranulation. This study, for the first time, identifies exosomes, nanosized vesicles released in the extracellular compartment by a number of elements, including platelets, as one of the effectors of PL activity. Exosomes were isolated from human PL by differential ultracentrifugation, and analysed by electron microscopy and Western blotting. Bone marrow stromal cells (MSC treated with three different exosome concentrations (0.6 μg, 5 μg and 50 μg showed a significant, dose-dependent increase in cell proliferation and migration compared to the control. In addition, osteogenic differentiation assays demonstrated that exosome concentration differently affected the ability of MSC to deposit mineralised matrix. Finally, the analysis of exosome protein content revealed a higher amount of basic fibroblast growth factor (bFGF, vascular endothelial growth factor (VEGF, platelet-derived growth factor (PDGF-BB and transforming growth factor beta 1 (TGF-β1 as compared to PL. In regards to RNA content, an enrichment of small RNAs in exosomes as compared to donor platelets has been found. These results suggest that exosomes consistently contribute to PL activity and could represent an advantageous nanodelivery system for cell-free regeneration therapies.

  6. Effect of platelet lysate on human cells involved in different phases of wound healing.

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    Barsotti, Maria Chiara; Chiara Barsotti, Maria; Losi, Paola; Briganti, Enrica; Sanguinetti, Elena; Magera, Angela; Al Kayal, Tamer; Feriani, Roberto; Di Stefano, Rossella; Soldani, Giorgio

    2013-01-01

    Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization). Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v). Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (pwound healing.

  7. Effect of platelet lysate on human cells involved in different phases of wound healing.

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    Maria Chiara Barsotti

    Full Text Available BACKGROUND: Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization. METHODOLOGY/PRINCIPAL FINDINGS: Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2 and inflammatory response evaluation (NFκB. Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v. Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (p<0.05 vs. control, comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. CONCLUSION/SIGNIFICANCE: These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

  8. Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications.

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    Fortunato, Tiago M; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A; Pula, Giordano

    2016-05-04

    Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs.

  9. Overcoming the bottleneck of platelet lysate supply in large-scale clinical expansion of adipose-derived stem cells

    DEFF Research Database (Denmark)

    Glovinski, Peter V; Herly, Mikkel; Mathiasen, Anders B

    2017-01-01

    BACKGROUND: Platelet lysates (PL) represent a promising replacement for xenogenic growth supplement for adipose-derived stem cell (ASC) expansions. However, fresh platelets from human blood donors are not clinically feasible for large-scale cell expansion based on their limited supply. Therefore...... may be an efficient and reliable source of human growth supplement allowing for large-scale ASC expansion for clinical use....... stored in the platelet additive solution, InterSol. Three types of PLs were prepared from outdated PCs with platelets suspended in either (1) InterSol (not manipulated), (2) InterSol + supplemented with plasma or (3) plasma alone (InterSol removed). Using these PLs, we compared ASC population doubling...

  10. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

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    Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

    2014-02-01

    Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  11. Use of platelet lysate for bone regeneration-are we ready for clinical translation?

    Institute of Scientific and Technical Information of China (English)

    Ala; Altaie; Heather; Owston; Elena; Jones

    2016-01-01

    Current techniques to improve bone regeneration following trauma or tumour resection involve the use of autograft bone or its substitutes supplemented with osteoinductive growth factors and/or osteogenic cells such as mesenchymal stem cells(MSCs).Although MSCs are most commonly grown in media containing fetal calf serum,human platelet lysate(PL) offers an effective alternative.Bone marrow- derived MSCs grown in PLcontaining media display faster proliferation whilst maintaining good osteogenic differentiation capacity.Limited pre-clinical investigations using PL-expanded MSCs seeded onto osteoconductive scaffolds indicate good potential of such constructs to repair bone in vivo.In an alternative approach,nude PL-coated scaffolds without seeded MSCs have been proposed as novel regenerative medicine devices.Even though methods to coat scaffolds with PL vary,in vitro studies suggest that PL allows for MSC adhesion,migration and differentiation inside these scaffolds.Increased new bone formation and vascularisation in comparison to uncoated scaffolds have also been observed in vivo.This review outlines the state-of-the-art research in the field of PL for ex vivo MSC expansion and in vivo bone regeneration.To minimise inconsistency between the studies,further work is required towards standardisation of PL preparation in terms of the starting material,platelet concentration,leukocyte depletion,and the method of platelet lysis.PL quality control procedures and its "potency" assessment are urgently needed,which could include measurements of key growth and attachment factors important for MSC maintenance and differentiation.Furthermore,different PL formulations could be tailor-made for specific bone repair indications.Such measures would undoubtedly speed up clinical translation of PL-based treatments for bone regeneration.

  12. Comparison of corneal epitheliotrophic capacities among human platelet lysates and other blood derivatives

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    Huang, Chien-Jung; Sun, Yi-Chen; Christopher, Karen; Pai, Amy Shih-I; Lu, Chia-Ju; Hu, Fung-Rong; Lin, Szu-Yuan; Chen, Wei-Li

    2017-01-01

    Purpose To evaluate the corneal epitheliotropic abilities of two commercialized human platelet lysates (HPLs) and to compare the results with other blood derivatives, including human peripheral serum (HPS) and bovine fetal serum (FBS). Methods In vitro, human corneal epithelial cells were incubated in various concentrations (0%, 3%, 5% and 10%) of blood derivatives. Two commercialized HPLs, including UltraGRO TM (Helios, Atlanta, GA) and PLTMax (Mill Creek, Rochester, MI), were tested and compared with HPS and FBS. Scratch-induced directional wounding assay was performed to evaluate cellular migration. MTS assay was used to evaluate cellular proliferation. Cellular differentiation was examined by scanning electron microscopy, inverted microscopy and transepithelial electrical resistance. Sprague-Dawley rats were used to evaluate the effects of the blood derivatives on corneal epithelial wound healing in vivo. Different blood derivatives were applied topically every 2 hours for 2 days after corneal epithelial debridement. The concentrations of epidermal growth factor (EGF), transforming growth factor -β1 (TGF-β1), fibronectin, platelet-derived growth factor-AB (PDGF-AB), PDGF-BB, and hyaluronic acid in different blood derivatives were evaluated by enzyme-linked immunosorbent assay (ELISA). Results In vitro experiments demonstrated statistically comparable epitheliotropic characteristics in cellular proliferation, migration, and differentiation for the two commercialized HPLs compared to FBS and HPS. Cells cultured without any serum were used as control group. The epitheliotropic capacities were statistically higher in the two commercialized HPLs compared to the control group (p<0.05). Among the different concentrations of blood derivatives, the preparations with 3% yielded better outcomes compared to 5% and 10%. In rats, HPLs also caused improved but not statistically significant wound healing compared to HPS. All the blood derivatives had better wound healing

  13. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study

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    Antonio M. Fea

    2016-01-01

    Full Text Available Purpose. To determine the effectiveness of autologous platelet lysate (APL eye drops in patients with primary Sjögren syndrome (SS dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM. Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI, best corrected visual acuity (BCVA, Schirmer test, fluorescein score, and breakup time (BUT. A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n=20 patients mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n=10 patients. The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p<0.05. Conclusion. APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications.

  14. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study

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    Fea, Antonio M.; Testa, Valeria; Machetta, Federica; Parisi, Simone; D'Antico, Sergio; Spinetta, Roberta; Fusaro, Enrico; Grignolo, Federico M.

    2016-01-01

    Purpose. To determine the effectiveness of autologous platelet lysate (APL) eye drops in patients with primary Sjögren syndrome (SS) dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM). Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI), best corrected visual acuity (BCVA), Schirmer test, fluorescein score, and breakup time (BUT). A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n = 20 patients) mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n = 10 patients). The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p < 0.05). Conclusion. APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications. PMID:27200376

  15. Composition of growth factors and cytokines in lysates obtained from fresh versus stored pathogen-inactivated platelet units.

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    Sellberg, Felix; Berglund, Erik; Ronaghi, Martin; Strandberg, Gabriel; Löf, Helena; Sommar, Pehr; Lubenow, Norbert; Knutson, Folke; Berglund, David

    2016-12-01

    Platelet lysate is a readily available source of growth factors, and other mediators, which has been used in a variety of clinical applications. However, the product remains poorly standardized and the present investigation evaluates the composition of platelet lysate obtained from either fresh or stored pathogen-inactivated platelet units. Platelet pooled units (n = 10) were obtained from healthy blood donors and tested according to standard procedures. All units were pathogen inactivated using amotosalen hydrochloride and UVA exposure. Platelet lysate was subsequently produced at two separate time-points, either from fresh platelet units or after 5 days of storage, by repeated freeze-thaw cycles. The following mediators were determined at each time-point: EGF, FGF-2, VEGF, IGF-1, PDGF-AB/BB, BMP-2, PF4, TGF-β isoform 1, IL-1β, IL-2, IL-6, IL-10, IL-12p70, 1L-17A, TNF-α, and IFN-γ. The concentration of growth factors and cytokines was affected by time in storage. Notably, TGF-β, PDGF-AB/BB, and PF4 showed an increase of 27.2% (p concentrations and all other growth factors remained stable with time in storage. The composition of mediators in platelet lysate obtained from pathogen-inactivated platelet units differs when produced from fresh and stored platelet units, respectively. This underscores the need for further standardization and optimization of this important product, which potentially may influence the clinical effects. Copyright © 2016. Published by Elsevier Ltd.

  16. Topical gel formulation and stability assessment of platelet lysate based on turbidimetric method

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    Soliman Mohammadi Samani

    2015-06-01

    Full Text Available Platelet-rich growth factors have attracted attentions of scientists and clinical practitioners who are involved in wound healing and regenerative medicine extensively, according to their unprecedented potential of promoting and catalyzing healing process. Platelet-rich growth factors are cost-benefit, available and more stable than recombinant human growth factors. These appealing characteristics have converted PRGF to one of the popular candidates for treatment of variety of wounds. According to these valuable properties, we decided to formulate and assess the effect of different excipients on the stability of such valuable protein based formulations. Different excipients have been chosen according to their effective ness on the stability of proteins and their application in other similar formulations. The stabilizing effect of excipients was evaluated by measuring heat-induced aggregation of growth factors by turbidimetric assay. Glycerol, glycine and dextrose were chosen as stabilizing excipients for these formulations. The results show that dextrose has more stabilizing effect on prevention of heat induced aggregation of the platelet lysate growth factors than glycerol and glycine. All of the formulations also contained antioxidant, chelating agents, preservative and carbopol934 in order to form appropriate gel.

  17. Umbilical Cord Blood Platelet Lysate as Serum Substitute in Expansion of Human Mesenchymal Stem Cells.

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    Shirzad, Negin; Bordbar, Sima; Goodarzi, Alireza; Mohammad, Monire; Khosravani, Pardis; Sayahpour, Froughazam; Baghaban Eslaminejad, Mohamadreza; Ebrahimi, Marzieh

    2017-10-01

    The diverse clinical applications for human mesenchymal stem cells (hMSCs) in cellular therapy and regenerative medicine warrant increased focus on developing adequate culture supplements devoid of animal-derived products. In the present study, we have investigated the feasibility of umbilical cord blood-platelet lysate (UCB-PL) as a standard substitute for fetal bovine serum (FBS) and human peripheral blood-PL (PB-PL). In this experimental study, platelet concentrates (PC) from UCB and human PB donors were frozen, melted, and sterilized to obtain PL. Quality control included platelet cell counts, sterility testing (viral and microbial), total protein concentrations, growth factor levels, and PL stability. The effects of UCB-PL and PB-PL on hMSCs proliferation and differentiation into osteocytes, chondrocytes, and adipocytes were studied and the results compared with FBS. UCB-PL contained high levels of protein content, platelet-derived growth factor- AB (PDGF-AB), and transforming growth factor (TGF) compared to PB-PL. All growth factors were stable for at least nine months post-storage at -70˚C. hMSCs proliferation enhanced following treatment with UCB-PL. With all three supplements, hMSCs could differentiate into all three lineages. PB-PL and UCB-PL both were potent in hMSCs proliferation. However, PB promoted osteoblastic differentiation and UCB-PL induced chondrogenic differentiation. Because of availability, ease of use and feasible standardization of UCB-PL, we have suggested that UCB-PL be used as an alternative to FBS and PB-PL for the cultivation and expansion of hMSCs in cellular therapy.

  18. Platelet-Rich Fibrin Lysate Can Ameliorate Dysfunction of Chronically UVA-Irradiated Human Dermal Fibroblasts.

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    Wirohadidjojo, Yohanes Widodo; Budiyanto, Arief; Soebono, Hardyanto

    2016-09-01

    To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm⁻²) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined.

  19. Human platelet lysate versus minoxidil stimulates hair growth by activating anagen promoting signaling pathways.

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    Dastan, Maryam; Najafzadeh, Nowruz; Abedelahi, Ali; Sarvi, Mohammadreza; Niapour, Ali

    2016-12-01

    Minoxidil and human platelet lysate (HPL) are commonly used to treat patients with hair loss. However, the roles of HPL versus minoxidil in hair follicle biology largely remain unknown. Here, we hypothesized that bulge and dermal papilla (DP) cells may express specific genes, including Kras, Erk, Akt, Shh and β-catenin after exposure to minoxidil or HPL. The mouse hair follicles were isolated on day 10 after depilation and bulge or DP regions were dissected. The bulge and DP cells were cultured for 14days in DMEM/F12 medium. Then, the cells were treated with 100μM minoxidil and 10% HPL for 10 days. Nuclear morphology was identified using DAPi staining. Reverse transcriptase and real-time polymerase chain reaction (PCR) analysis were also performed to examine the expression of Kras, Erk, Akt, Shh and β-catenin mRNA levels in the treated bulge and DP regions after organ culture. Here, we found that minoxidil influences bulge and DP cell survival (Pminoxidil treatment in both bulge and DP cells. HPL mediated Erk upregulation in both bulge and DP cells (Pminoxidil-treated bulge cells. In contrast, the expression of β-cateinin and Shh in the DP cells was not meaningfully increased after treatment with HPL. Our results suggest that minoxidil and HPL can promote hair growth by activating the main anagen inducing signaling pathways. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  20. Platelet-Rich Fibrin Lysate Can Ameliorate Dysfunction of Chronically UVA-Irradiated Human Dermal Fibroblasts

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    Budiyanto, Arief; Soebono, Hardyanto

    2016-01-01

    To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm-2) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined. PMID:27401663

  1. Highly Efficient In Vitro Reparative Behaviour of Dental Pulp Stem Cells Cultured with Standardised Platelet Lysate Supplementation

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    Palmieri, Francesca; Marrelli, Massimo

    2016-01-01

    Dental pulp is an accessible source of multipotent mesenchymal stromal cells (MSCs). The perspective role of dental pulp stem cells (DPSCs) in regenerative medicine demands an in vitro expansion and in vivo delivery which must deal with the safety issues about animal serum, usually required in cell culture practice. Human platelet lysate (PL) contains autologous growth factors and has been considered as valuable alternative to fetal bovine serum (FBS) in cell cultures. The optimum concentration to be added of such supplement is highly dependent on its preparation whose variability limits comparability of results. By in vitro experiments, we aimed to evaluate a standardised formulation of pooled PL. A low selected concentration of PL (1%) was able to support the growth and maintain the viability of the DPSCs. The use of PL in cell cultures did not impair cell surface signature typically expressed by MSCs and even upregulated the transcription of Sox2. Interestingly, DPSCs cultured in presence of PL exhibited a higher healing rate after injury and are less susceptible to toxicity mediated by exogenous H2O2 than those cultured with FBS. Moreover, PL addition was shown as a suitable option for protocols promoting osteogenic and chondrogenic differentiation of DPSCs. Taken together, our results indicated that PL is a valid substitute of FBS to culture and differentiate DPSCs for clinical-grade use. PMID:27774106

  2. Human platelet lysate permits scale-up of dental pulp stromal cells for clinical applications.

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    Govindasamy, Vijayendran; Ronald, Veronica Sainik; Abdullah, Aimi Naim Binti; Ganesan Nathan, Kavitha R; Aziz, Zeti Adura Che Abdul; Abdullah, Mariam; Zain, Rosnah Binti; Kasim, Noor Hayaty Abu; Musa, Sabri; Bhonde, Ramesh R

    2011-11-01

    BACKGROUND AIMS. Dental pulp stromal cells (DPSC) are considered to be a promising source of stem cells in the field of regenerative therapy. However, the usage of DPSC in transplantation requires large-scale expansion to cater for the need for clinical quantity without compromising current good manufacturing practice (cGMP). Existing protocols for cell culturing make use of fetal bovine serum (FBS) as a nutritional supplement. Unfortunately, FBS is an undesirable additive to cells because it carries the risk of transmitting viral and prion diseases. Therefore, the present study was undertaken to examine the efficacy of human platelet lysate (HPL) as a substitute for FBS in a large-scale set-up. METHODS. We expanded the DPSC in Dulbecco's modified Eagle's medium-knock-out (DMEM-KO) with either 10% FBS or 10% HPL, and studied the characteristics of DPSC at pre- (T25 culture flask) and post- (5-STACK chamber) large-scale expansion in terms of their identity, quality, functionality, molecular signatures and cytogenetic stability. RESULTS. In both pre- and post-large-scale expansion, DPSC expanded in HPL showed extensive proliferation of cells (c. 2-fold) compared with FBS; the purity, immune phenotype, colony-forming unit potential and differentiation were comparable. Furthermore, to understand the gene expression profiling, the transcriptomes and cytogenetics of DPSC expanded under HPL and FBS were compared, revealing similar expression profiles. CONCLUSIONS. We present a highly economized expansion of DPSC in HPL, yielding double the amount of cells while retaining their basic characteristics during a shorter time period under cGMP conditions, making it suitable for therapeutic applications.

  3. The role of a platelet Lysate-Based compartmentalized system as a carrier of cells and platelet-origin cytokines for periodontal tissue regeneration

    OpenAIRE

    Babo, Pedro Miguel Sousa; Xinjie Cai; Plachokova, A.; Reis, R. L.; Jansen, John A.; Gomes, Manuela E.; Walboomers, X. Frank

    2016-01-01

    Currently available clinical therapies are not capable to regenerate tissues that are lost by periodontitis. Tissue engineering can be applied as a strategy to regenerate reliably the tissues and function of damaged periodontium. A prerequisite for this regeneration is the colonization of the defect with the adequate cell populations. In this study, we proposed a bilayered system composed of (1) a platelet lysate (PL)-based construct produced by crosslinking of PL proteins with genipin (gP...

  4. Platelet lysate as a substitute for animal serum for the ex-vivo expansion of mesenchymal stem/stromal cells: present and future.

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    Astori, Giuseppe; Amati, Eliana; Bambi, Franco; Bernardi, Martina; Chieregato, Katia; Schäfer, Richard; Sella, Sabrina; Rodeghiero, Francesco

    2016-07-13

    The use of fetal bovine serum (FBS) as a cell culture supplement is discouraged by regulatory authorities to limit the risk of zoonoses and xenogeneic immune reactions in the transplanted host. Additionally, FBS production came under scrutiny due to animal welfare concerns. Platelet derivatives have been proposed as FBS substitutes for the ex-vivo expansion of mesenchymal stem/stromal cells (MSCs) since platelet-derived growth factors can promote MSC ex-vivo expansion. Platelet-derived growth factors are present in platelet lysate (PL) obtained after repeated freezing-thawing cycles of the platelet-rich plasma or by applying physiological stimuli such as thrombin or CaCl2.PL-expanded MSCs have been used already in the clinic, taking advantage of their faster proliferation compared with FBS-expanded preparations. Should PL be applied to other biopharmaceutical products, its demand is likely to increase dramatically. The use of fresh platelet units for the production of PL raises concerns due to limited availability of platelet donors. Expired units might represent an alternative, but further data are needed to define safety, including pathogen reduction, and functionality of the obtained PL. In addition, relevant questions concerning the definition of PL release criteria, including concentration ranges of specific growth factors in PL batches for various clinical indications, also need to be addressed. We are still far from a common definition of PL and standardized PL manufacture due to our limited knowledge of the mechanisms that mediate PL-promoting cell growth. Here, we concisely discuss aspects of PL as MSC culture supplement as a preliminary step towards an agreed definition of the required characteristics of PL for the requirements of manufacturers and users.

  5. Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against Stroke

    Directory of Open Access Journals (Sweden)

    Chengbo Tan

    2016-01-01

    Full Text Available Currently, there is increasing interest in human bone marrow stromal cells (hBMSCs as regeneration therapy against cerebral stroke. The aim of the present study was to evaluate the feasibility and validity of hBMSC cultures with allogeneic platelet lysates (PLs. Platelet concentrates (PC were harvested from healthy volunteers and made into single donor-derived PL (sPL. The PL mixtures (mPL were made from three different sPL. Some growth factors and platelet cell surface antigens were detected by enzyme-linked immunosorbent assay (ELISA. The hBMSCs cultured with 10% PL were analyzed for their proliferative potential, surface markers, and karyotypes. The cells were incubated with superparamagnetic iron oxide (SPIO agents and injected into a pig brain. MRI and histological analysis were performed. Consequently, nine lots of sPL and three mPL were prepared. ELISA analysis showed that PL contained adequate growth factors and a particle of platelet surface antigens. Cell proliferation capacity of PLs was equivalent to or higher than that of fetal calf serum (FCS. No contradiction in cell surface markers and no chromosomal aberrations were found. The MRI detected the distribution of SPIO-labeled hBMSCs in the pig brain. In summary, the hBMSCs cultured with allogeneic PL are suitable for cell therapy against stroke.

  6. Morphological Study of Isolated Ovarian Preantral Follicles Using Fibrin Gel Plus Platelet Lysate after Subcutaneous Transplantation

    Directory of Open Access Journals (Sweden)

    Ali Reza Rajabzadeh

    2015-04-01

    Full Text Available Objective: Ovarian and follicle transplantation may preserve fertility in young cancer survivors. In this study, we have transplanted preantral follicles using fibrin gel as a carrier and fibrin gel supplemented with platelet lysate (PL as a rich source of angiogenic and growth factors. The purpose of this study was to evaluate the role of fibrin gel and PL in follicle transplantation. Materials and Methods: In this experimental study, ovaries were taken from 14-dayold Naval Medical Research Institute (NMRI mice. Preantral follicles were dissected from the ovaries and encapsulated into fibrin gel supplemented with 5, 10, 15 or 20% PL, then transplanted back into the same donor mice. Fibrin gels supplemented with PL that contained preantral follicles were placed in a subcutaneous pocket in the back of the neck of the recipient, donor mouse (the same mouse that follicles were collected. After 14 days the grafts were processed and embedded in paraffin blocks, then serially sectioned for histological evaluation. We counted the follicles and classified them according to stage (preantral or antral. Data were presented as mean ± standard error of mean (SEM and analysed by analysis of variance (ANOVA and the Kruskal-Wallistest. Results: The mean percentage of recovered follicles encapsulated and transplanted in each group were 33.30 ± 2.47 (fibrin gel, 31.96 ± 1.90 (fibrin gel+5% PL, 34.02 ± 2.44 (fibrin gel+10% PL, 48.31 ± 2.06 (fibrin gel+15% PL and 17.60 ± 2.79 (fibrin gel+20% PL. There was a significant increase in the recovery rate of grafted follicles with fibrin gel+15% PL (48.31%; p<0.001. The percentage of preantral follicles showed no significant difference in all groups (p<0.05. The percentage of antral follicles showed a significant decrease in follicles grafted with fibrin gel+20% PL when compared to the other groups (11.77%; p<0.005 but no significant difference was observed in the other groups. Conclusion: The use of PL in follicle

  7. Bone Formation with Deproteinized Bovine Bone Mineral or Biphasic Calcium Phosphate in the Presence of Autologous Platelet Lysate: Comparative Investigation in Rabbit

    Directory of Open Access Journals (Sweden)

    Carole Chakar

    2014-01-01

    Full Text Available Bone substitutes alone or supplemented with platelet-derived concentrates are widely used to promote bone regeneration but their potency remains controversial. The aim of this study was, therefore, to compare the regenerative potential of preparations containing autologous platelet lysate (APL and particles of either deproteinized bovine bone mineral (DBBM or biphasic calcium phosphate (BCP, two bone substitutes with different resorption patterns. Rabbit APL was prepared by freeze-thawing a platelet suspension. Critical-size defects in rabbit femoral condyle were filled with DBBM or DBBM+APL and BCP or BCP+APL. Rabbits were sacrificed after six weeks and newly formed bone and residual implanted material were evaluated using nondemineralized histology and histomorphometry. New bone was observed around particles of all fillers tested. In the defects filled with BCP, the newly formed bone area was greater (70%; P<0.001 while the residual material area was lower (60%; P<0.001 than that observed in those filled with DBBM. New bone and residual material area of defects filled with either APL+DBBM or APL+BCP were similar to those observed in those filled with the material alone. In summary, osteoconductivity and resorption of BCP were greater than those of DBBM, while APL associated with either DBBM or BCP did not have an additional benefit.

  8. Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem cells.

    Science.gov (United States)

    Wang, Chuan; Xiao, Ran; Cao, Yi-Lin; Yin, Hong-Yu

    2017-09-09

    Cryopreservation provides an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs). Dimethylsulfoxide (DMSO) and fetal bovine serum (FBS) are frequently used as cryoprotectants for this purpose. However, the use of DMSO can result in adverse effects and toxic reactions and FBS can introduce risks of viral, prion, zoonose contaminations and evoke immune responses after injection. It is therefore crucial to reduce DMSO concentrations and use serum-free solution in the cryopreservation process. Human platelet lysate (PL) is a promising candidate for use as an alternative to DMSO and FBS. Therefore, in this study, with an aim to identify a cryoprotective agent for ASC cryopreservation, we determined the viability, proliferation potential, phenotype, and differentiation potential of fresh ASCs and ASCs cryopreserved using different combinations of three cryoprotective agents: fetal bovine serum (FBS), dimethylsulfoxide (DMSO), and human platelet lysate (PL). The viability of the ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO was >80%, and the proliferation potentials, cell phenotypes, and differentiation potentials of these groups were similar to those of fresh ASCs. Together, our findings suggest that a combination of 97% PL and 3% DMSO is an ideal cryoprotective agent for the efficient cryopreservation of human ASCs. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Comparison of clinical grade human platelet lysates for cultivation of mesenchymal stromal cells from bone marrow and adipose tissue

    DEFF Research Database (Denmark)

    Juhl, Morten; Tratwal, Josefine; Follin, Bjarke

    2016-01-01

    BACKGROUND: The utility of mesenchymal stromal cells (MSCs) in therapeutic applications for regenerative medicine has gained much attention. Clinical translation of MSC-based approaches requires in vitro culture-expansion to achieve a sufficient number of cells. The ideal cell culture medium should...... be devoid of any animal derived components. We have evaluated whether human Platelet Lysate (hPL) could be an attractive alternative to animal supplements. METHODS: MSCs from bone marrow (BMSCs) and adipose tissue-derived stromal cells (ASCs) obtained from three donors were culture expanded in three...... culture conditions with 10% fetal bovine serum (FBS). Cell morphology, proliferation, phenotype, genomic stability, and differentiation potential were analyzed. RESULTS: Regardless of manufacturer, BMSCs and ASCs cultured in hPL media showed a significant increase in proliferation capacity compared to FBS...

  10. The effects of human platelet lysate on dental pulp stem cells derived from impacted human third molars.

    Science.gov (United States)

    Chen, Bo; Sun, Hai-Hua; Wang, Han-Guo; Kong, Hui; Chen, Fa-Ming; Yu, Qing

    2012-07-01

    Human platelet lysate (PL) has been suggested as a substitute for fetal bovine serum (FBS) in the large-scale expansion of dental pulp stem cells (DPSCs). However, the biological effects and the optimal concentrations of PL for the proliferation and differentiation of human DPSCs remain unexplored. We isolated and expanded stem cells from the dental pulp of extracted third molars and evaluated the effects of PL on the cells' proliferative capacity and differentiation potential in vitro and in vivo. Before testing, immunocytochemical staining and flow cytometry-based cell sorting showed that the cells derived from human dental pulp contained mesenchymal stem cell populations. Cells were grown on tissue culture plastic or on hydroxyapatite-tricalcium phosphate (HA/TCP) biomaterials and were incubated with either normal or odontogenic/osteogenic media in the presence or absence of various concentrations of human PL for further investigation. The proliferation of DPSCs was significantly increased when the cells were cultured in 5% PL under all testing conditions (P biomaterials and had fully covered the surface of the scaffold with an extensive sheet-like structure 14 d after seeding. In addition, 5% PL showed significantly positive effects on tissue regeneration in two in vivo transplantation models. We conclude that the appropriate concentration of PL enhances the proliferation and mineralized differentiation of human DPSCs both in vitro and in vivo, which supports the use of PL as an alternative to FBS or a nonzoonotic adjuvant for cell culture in future clinical trials. However, the elucidation of the molecular complexity of PL products and the identification of both the essential growth factors that determine the fate of a specific stem cell and the criteria to establish dosing require further investigation.

  11. Efficacy of the controlled release of concentrated platelet lysate from a collagen/gelatin scaffold for dermis-like tissue regeneration.

    OpenAIRE

    Ito, Ran; Morimoto, Naoki; Pham, Liem Hieu; Taira, Tsuguyoshi; Kawai, Katsuya; Suzuki, Shigehiko

    2013-01-01

    Introduction: A collagen/gelatin scaffold (CGS) can provide a sustained release of basic fibroblast growth factor (bFGF), which promotes wound healing. However, bFGF is approved for clinical use in Japan and China only. One potential alternative to bFGF is platelet lysate (PL), a safe and easily attainable source of a wide range of growth factors necessary for tissue repair. In the present study, we investigated the use of PL with CGS to repair wounds and identified the optimal concentration ...

  12. Comparison of osteo/odontogenic differentiation of human adult dental pulp stem cells and stem cells from apical papilla in the presence of platelet lysate.

    Science.gov (United States)

    Abuarqoub, Duaa; Awidi, Abdalla; Abuharfeil, Nizar

    2015-10-01

    Human dental pulp cells (DPSCs) and stem cells from apical papilla have been used for the repair of damaged tooth tissues. Human platelet lysate (PL) has been suggested as a substitute for fetal bovine serum (FBS) for large scale expansion of dental stem cells. However, biological effects and optimal concentrations of PL for proliferation and differentiation of human dental stem cells remain to be elucidated. DPSCs and SCAP cells were isolated from impacted third molars of young healthy donors, at the stage of root development and identified by markers using flow cytometry. For comparison the cells were cultured in media containing PL (1%, 5% and 10%) and FBS, with subsequent induction for osteogenic/odontogenic differentiation. The cultures were analyzed for; morphology, growth characteristics, mineralization potential (Alizarin Red method) and differentiation markers using ELISA and real time -polymerase chain reaction (qPCR). The proliferation rates of DPSCs and SCAP significantly increased when cells were treated with 5% PL (7X doubling time) as compared to FBS. 5% PL also enhanced mineralized differentiation of DPSCs and SCAP, as indicated by the measurement of alkaline phosphatase activity, osteocalcin and osteopontin, calcium deposition and q-PCR. Our findings suggest that using 5% platelet lysate, proliferation and osteo/odontogenesis of DPSCs and SCAP for a short period of time (15 days), was significantly improved. This may imply its use as an optimum concentration for expansion of dental stem cells in bone regeneration. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Human platelet lysate is an alternative to fetal bovine serum for large-scale expansion of bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Gottipamula, Sanjay; Sharma, Archana; Krishnamurthy, Sagar; Majumdar, Anish Sen; Seetharam, Raviraja N

    2012-07-01

    Human platelet lysate (HPL) was evaluated as an alternative to fetal bovine serum (FBS) in large-scale culturing of bone marrow-derived mesenchymal stromal cells (BM-MSCs) for therapeutic applications. Dulbecco's modified Eagle medium (DMEM)of low glucose (LG) and Knock Out (KO) were used with human platelet lysate (HPL) as LG-HPL and KO-HPL, and with FBS as LG-FBS and KO-FBS to culture the BM-MSCs. HPL at 10 % (v/v) supported BM-MSCs growth and subsequent isolation efficiency generated >90 × 10(6) MSCs in LG-HPL. Population doublings (PDs) and population doubling times of LG-HPL and KO-HPL (PDT) were not significantly different but LG-HPL showed a significant clonogenic potential and HPL cultures had an average PDT of 36.5 ± 6.5 h and an average PDs of 5 ± 0.7/passage. BM-MSCs cultured with LG-HPL had significantly higher immunosuppression compared to LG-FBS, but KO-HPL and KO-FBS-grown cultures were not significantly different. HPL is therefore alternative to FBS for large-scale production of BM-MSCs for therapeutic applications.

  14. Human Platelet Lysate as a Xeno Free Alternative of Fetal Bovine Serum for the In Vitro Expansion of Human Mesenchymal Stromal Cells

    Science.gov (United States)

    Mohammadi, Saeed; Nikbakht, Mohsen; Malek Mohammadi, Ashraf; Zahed Panah, Mahdi; Ostadali, Mohammad Reza; Nasiri, Hajar; Ghavamzadeh, Ardeshir

    2016-01-01

    Background: Mesenchymal stromal cells (MSCs) are employed in various different clinical settings in order to modulate immune response. Human autologous and allogeneic supplements including platelet derivatives such as platelet lysate (PL), platelet-released factors (PRF) and serum are assessed in clinical studies to replace fetal bovine serum (FBS). The immunosuppressive activity and multi-potential characteristic of MSCs appear to be maintained when the cells are expanded in platelet derivatives. Materials and Methods: Platelet-rich plasma was collected from umbrical cord blood (UCB). Platelet-derived growth factors obtained by freeze and thaw methods. CD62P expression was determined by flow cytometry. The concentration of PDGF-BB and PDGF-AB was detemined by ELISA. We tested the ability of a different concentration of PL-supplemented medium to support the ex vivo expansion of Wharton's jelly derived MSCs. We also investigated the biological/functional properties of expanded MSCs in presence of different concentration of PL. The conventional karyotyping was performed in order to study the chromosomal stability. The gene expression of Collagen I and II aggrecan and SOX-9 in the presence of different concentrations of PL was evaluated by Real-time PCR. Results: We observed 5% and 10% PL, causing greater effects on proliferation of MSCs .These cells exhibited typical morphology, immunophenotype and differentiation capacity. The genetic stability of these derivative cells from Wharton's jelly was demonstrated by a normal karyotype. Furthermore, the results of Real-time PCR analysis showed that the expression of chondrocyte specific genes was higher in MSCs in the presence of 5% and 10% PL, compared with FBS supplement. Conclusions: We demonstrated that PL could be used as an alternative safe source of growth factors for expansion of MSCs and also maintained similar growing potential and phenotype without any effect on chromosomal stability. PMID:27489592

  15. 血小板裂解液对人间充质干细胞生物学特性的影响%Effect of platelet lysate on the biological characteristics of human mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    吴迪; 武晓云; 吴岩

    2015-01-01

    BACKGROUND:Platelet lysate has been known as a kind of lysate of autologous or alogeneic platelet-rich products. It not only removes the residual cel structure, reduces immunogenicity, but also retains many growth factors. Platelet lysate has been suggested as a substitute for fetal bovine serum to expand mesenchymal stem cels in vitro. OBJECTIVE:To observe the effects of platelet lysate on biological characteristics of human bone marrow mesenchymal stem cels and adipose mesenchymal stem cels, and provide some experimental data for clinical cel therapy and regenerative medicine. METHODS:Platelet lysate was prepared by repeated freezing and thawing from fresh blood. Healthy adult bone marrow and adipose tissue were colected. Human bone marrow mesenchymal stem cels and adipose mesenchymal stem cels were obtained by density gradient centrifugation and type I colagenase digestion. We tested the morphology, cel phenotype, differentiation characteristics, proliferation capacity, colony forming ability and the level of cytokine secretion of bone marrow mesenchymal stem cels and adipose mesenchymal stem cels after cultured with platelet lysis. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cels and adipose mesenchymal stem cels were successfuly cultured in vitro using platelet lysate. There were no significant differences in morphology, cel phenotype, colony forming ability and the level of cytokine secretion, and chondrogenic, osteogenic and adipogenic capacities between bone marrow mesenchymal stem cels and adipose mesenchymal stem cels. Adipose mesenchymal stem cels had a high cumulative population doublings than bone marrow mesenchymal stem cels (P < 0.05). These findings suggest adipose mesenchymal stem cels had a stronger proliferative ability, and are more suitable for large-scale expansionin vitro cultivation system of platelet lysate compared with bone marrow mesenchymal stem cels.%背景:血小板裂解液是自体或异体血小板富集物经裂解后

  16. THE USE OF PREPARATIONS BASED ON BACTERIAL LYSATES IN THE TREATMENT OF CHRONIC TONSILLITIS IN PATIENTS WITH RHEUMATOID ARTHRITIS

    OpenAIRE

    Kolyada T.І.; Tupotіlov A.V.; Vdovіchenko N.І; Litvinenko O.Y.

    2014-01-01

    In the therapy of decompensated form of chronic tonsillitis (CT) were used as immunomodulatory agents IRS and Ismigen. These bacterial lysates differ in the bacterial setting, the method of preparation (chemical, mechanical) and the method of application.Rheumatoid arthritis (RA) is one of the important factors that could significantly complicate the therapy of chronic tonsillitis. RA is a chronic immune inflammatory disease that progressively affects connective tissue...

  17. Increase in phosphotidylinositide-3 kinase activity by nitrotyrosylation of lysates of platelets from patients with systemic sclerosis.

    Science.gov (United States)

    Chiang, Thomas M; Postlethwaite, Arnold E

    2006-01-01

    We have observed that the platelet non-integrin collagen receptor (65 kDa) and another protein (M(r) 185 kDa) are altered in the posttranslational modification by nitrotyrosylation in platelets from patients with systemic sclerosis (SSc). We reported the identification of nitrotyrosylated 65-kDa proteins in a previous study. In the present investigation, using Western blots, one- and two-dimensional gel electrophoreses and matrix assisted ionization/desorption-time of flight (MALDI-TOF) we have identified the 185-kDa protein as phosphoinositide kinase C2beta (PI 3-K). There is a positive correlation between the nitrotyrosylation of PI 3-K and activity of the enzyme, i.e., the nitrotyrosylation of PI 3-K increases its enzymatic activity. In addition, the activity of PI 3-K increases in nitrotyrosylated platelet lystaes from patients with SSc compared to normal volunteer controls, suggesting that this is an alteration in the posttranslational modification of PI 3-K in platelets from patients with SSc. The increased nitrotyrosylation of PI 3-K may contribute to the impairment of platelet function in patients with SSc by increasing platelet reactivity to matrix components within the vascular walls of patients with this disease.

  18. Optimized human platelet lysate as novel basis for a serum-, xeno- and additive-free corneal endothelial cell and tissue culture.

    Science.gov (United States)

    Thieme, Daniel; Reuland, Lynn; Lindl, Toni; Kruse, Friedrich; Fuchsluger, Thomas

    2017-09-21

    The expansion of donor derived corneal endothelial cells is a promising approach for regenerative therapies in corneal diseases. To achieve the best GMP standard the entire cultivation process should be devoid of non-human components. However, so far there is no suitable xeno-free protocol for clinical applications. We therefore introduce a processed variant of a platelet lysate for the use in corneal cell and tissue culture based on a GMP-grade thrombocyte concentrate. This processed human platelet lysate (phPL), free of any animal components and of anti-coagulants like heparin with a physiological ionic composition, was used to cultivate corneal endothelial cells (EC) in vitro and ex vivo in comparison to standard cultivation with FCS. Human donor corneas were cut in quarters while two quarters of each cornea were incubated with the respective medium supplement. Three fields of view per quarter were taken into account for the analysis. Evaluation of phPL as a medium supplement in cell culture of immortalized EC showed a superior viability compared to fetal calf serum (FCS) control with reduced cell proliferation. Furthermore, the viability during the expansion of primary cells is significantly (3fold+-0.5) increased with phPL compared to FCS standard medium. Quartering donor corneas was traumatic for the endothelium and therefore resulted in increased EC loss. Interestingly, however, cultivation of the quartered pieces for two weeks in 0.1mg/mL pHPL in Biochrome I showed a 21 (+-10) % EC loss compared to 67 (+-12) % EC loss when cultivated in 2% FCS in Biochrome I. The cell culture protocol with pHPL as FCS replacement seems to be superior to the standard FCS protocols with respect to EC survival. It offers a xeno-free and physiological environment for corneal endothelial cells. This alternative cultivation protocol could facilitate the use of EC for human corneal cell therapy. This article is protected by copyright. All rights reserved.

  19. Platelet-rich plasma preparation using three devices : Implications for platelet activation and platelet growth factor release

    NARCIS (Netherlands)

    Everts, Peter A. M.; Mahoney, Christine Brown; Hoffmann, Johannes J. M. L.; Schonberger, Jacques P. A. M.; Box, Henk A. M.; Van Zundert, Andre; Knape, Johannes T. A.

    2006-01-01

    Background: In this study, three commercial systems for the preparation of platelet-rich plasma (PRP) were compared and platelet growth factors release was measured. Methods: Ten healthy volunteers donated whole blood that was fractionated by a blood cell separator, and a table-top centrifuge to pre

  20. The Potential of GMP-Compliant Platelet Lysate to Induce a Permissive State for Cardiovascular Transdifferentiation in Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Camilla Siciliano

    2015-01-01

    Full Text Available Human adipose tissue-derived mesenchymal stem cells (ADMSCs are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL, a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs.

  1. The Potential of GMP-Compliant Platelet Lysate to Induce a Permissive State for Cardiovascular Transdifferentiation in Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

    Science.gov (United States)

    Siciliano, Camilla; Chimenti, Isotta; Bordin, Antonella; Ponti, Donatella; Iudicone, Paola; Peruzzi, Mariangela; Rendina, Erino Angelo; Calogero, Antonella; Pierelli, Luca; Ibrahim, Mohsen; De Falco, Elena

    2015-01-01

    Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs. PMID:26495284

  2. Removal process of prion and parvovirus from human platelet lysates used as clinical-grade supplement for ex vivo cell expansion.

    Science.gov (United States)

    Kao, Yu-Chun; Bailey, Andy; Samminger, Bernhard; Tanimoto, Junji; Burnouf, Thierry

    2016-07-01

    Pooled human platelet lysate (HPL) is becoming the new gold standard as supplement for ex vivo cell culture for clinical protocols. However, the risk of pathogen contamination of HPL increases with the platelet pool size. We hypothesized that hollow fiber anion exchange membrane chromatography using QyuSpeed D (QSD) could remove resistant and untested bloodborne pathogens, such as parvoviruses and prions, from HPL-supplemented growth media without substantially affecting their capacity to support ex vivo cell expansion. Frozen or thawed platelet concentrates were serum-converted and centrifuged for obtaining HPL that was added to various growth media (ca. 100 mL), filtered through a 0.6-mL QSD membrane and characterized for proteins, growth factors and chemical composition. Capacity to expand Chinese hamster ovary, periodontal ligament, gingival fibroblast cells and Wharton's jelly mesenchymal stromal cells was studied. Removal of porcine parvovirus (PPV) and of the 263K prion strain of hamster-adapted scrapie was studied by spiking experiments following international guidelines. QSD had minimal impact on HPL-supplemented medium composition in proteins, growth factors and chemical content, nor capacity to expand and differentiate cells. In addition, QSD could remove ≥5.58 log10 [TCID50/mL] and ≥3.72 log10 of PPV and the 263K prion, respectively. QSD hollow fiber chromatography can be used to improve the virus and prion safety of HPL-supplemented media to safely expand cells for clinical protocols. These data bring new perspectives for increasingly safer use of pooled HPL in cell therapy and regenerative medicine applications. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  3. Enhancement of osteogenic differentiation of human adipose derived stem cells by the controlled release of platelet lysates from hybrid scaffolds produced by supercritical fluid foaming.

    Science.gov (United States)

    Santo, Vítor E; Duarte, Ana Rita C; Popa, Elena G; Gomes, Manuela E; Mano, João F; Reis, Rui L

    2012-08-20

    A new generation of scaffolds capable of acting not only as support for cells but also as a source of biological cues to promote tissue regeneration is currently a hot topic of in bone Tissue Engineering (TE) research. The inclusion of growth factor (GF) controlled release functionalities in the scaffolds is a possible strategy to achieve such goal. Platelet Lysate (PL) is an autologous source of GFs, providing several bioactive agents known to act on bone regeneration. In this study, chitosan-chondroitin sulfate nanoparticles loaded with PL were included in a poly(D,L-lactic acid) foam produced by supercritical fluid foaming. The tridimensional (3D) structures were then seeded with human adipose-derived stem cells (hASCs) and cultured in vitro under osteogenic stimulus. The osteogenic differentiation of the seeded hASCs was observed earlier for the PL-loaded constructs, as shown by the earlier alkaline phosphatase peak and calcium detection and stronger Runx2 expression at day 7 of culture, in comparison with the control scaffolds. Osteocalcin gene expression was upregulated in presence of PL during all culture period, which indicates an enhanced osteogenic induction. These results suggest the synergistic effect of PL and hASCs in combinatory TE strategies and support the potential of PL to increase the multifunctionality of the 3D hybrid construct for bone TE applications.

  4. Pooled human platelet lysate versus fetal bovine serum—investigating the proliferation rate, chromosome stability and angiogenic potential of human adipose tissue-derived stem cells intended for clinical use

    DEFF Research Database (Denmark)

    Trojahn Kølle, Stig-Frederik; Oliveri, Roberto S; Glovinski, Peter V

    2013-01-01

    Because of an increasing focus on the use of adipose-derived stem cells (ASCs) in clinical trials, the culture conditions for these cells are being optimized. We compared the proliferation rates and chromosomal stability of ASCs that had been cultured in Dulbecco's modified Eagle's Medium (DMEM......) supplemented with either pooled human platelet lysate (pHPL) or clinical-grade fetal bovine serum (FBS) (DMEM(pHPL) versus DMEM(FBS))....

  5. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

    Science.gov (United States)

    Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

    2016-01-01

    Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins. PMID:27768741

  6. Quality assessment of platelet concentrates prepared by platelet rich plasma-platelet concentrate, buffy coat poor-platelet concentrate (BC-PC and apheresis-PC methods

    Directory of Open Access Journals (Sweden)

    Singh Ravindra

    2009-01-01

    Full Text Available Background: Platelet rich plasma-platelet concentrate (PRP-PC, buffy coat poor-platelet concentrate (BC-PC, and apheresis-PC were prepared and their quality parameters were assessed. Study Design: In this study, the following platelet products were prepared: from random donor platelets (i platelet rich plasma - platelet concentrate (PRP-PC, and (ii buffy coat poor- platelet concentrate (BC-PC and (iii single donor platelets (apheresis-PC by different methods. Their quality was assessed using the following parameters: swirling, volume of the platelet concentrate, platelet count, WBC count and pH. Results: A total of 146 platelet concentrates (64 of PRP-PC, 62 of BC-PC and 20 of apheresis-PC were enrolled in this study. The mean volume of PRP-PC, BC-PC and apheresis-PC was 62.30±22.68 ml, 68.81±22.95 ml and 214.05±9.91 ml and ranged from 22-135 ml, 32-133 ml and 200-251 ml respectively. The mean platelet count of PRP-PC, BC-PC and apheresis-PC was 7.6±2.97 x 1010/unit, 7.3±2.98 x 1010/unit and 4.13±1.32 x 1011/unit and ranged from 3.2-16.2 x 1010/unit, 0.6-16.4 x 1010/unit and 1.22-8.9 x 1011/unit respectively. The mean WBC count in PRP-PC (n = 10, BC-PC (n = 10 and apheresis-PC (n = 6 units was 4.05±0.48 x 107/unit, 2.08±0.39 x 107/unit and 4.8±0.8 x 106/unit and ranged from 3.4 -4.77 x 107/unit, 1.6-2.7 x 107/unit and 3.2 - 5.2 x 106/unit respectively. A total of 26 units were analyzed for pH changes. Out of these units, 10 each were PRP-PC and BC-PC and 6 units were apheresis-PC. Their mean pH was 6.7±0.26 (mean±SD and ranged from 6.5 - 7.0 and no difference was observed among all three types of platelet concentrate. Conclusion: PRP-PC and BC-PC units were comparable in terms of swirling, platelet count per unit and pH. As expected, we found WBC contamination to be less in BC-PC than PRP-PC units. Variation in volume was more in BC-PC than PRP-PC units and this suggests that further standardization is required for preparation of BC

  7. Safety and complications reporting update on the re-implantation of culture-expanded mesenchymal stem cells using autologous platelet lysate technique.

    Science.gov (United States)

    Centeno, Christopher J; Schultz, John R; Cheever, Michelle; Freeman, Michael; Faulkner, Stephen; Robinson, Brent; Hanson, Ronald

    2011-12-01

    Mesenchymal stem cells (MSCs) hold great promise as therapeutic agents in regenerative medicine. Numerous animal studies have documented the multipotency of MSCs, showing their capabilities for differentiating into orthopedic tissues such as muscle, bone, cartilage, and tendon. However, the safety of culture expanded MSC's for human use has only just begun to be reported. Between 2006 and 2010, two groups of patients were treated for various orthopedic conditions with culture-expanded, autologous, bone marrow-derived MSCs (group 1: n=50; group 2: n=290-one patient in both groups). Cells were cultured in monolayer culture flasks using an autologous platelet lysate technique and re-injected into peripheral joints or into intervertebral discs with use of c-arm fluoroscopy. While both groups had prospective surveillance for complications, Group 1 additionally underwent 3.0T MRI tracking of the re-implant sites. The mean age of patients treated was 53 +/- 13.85 years; 214 were males and 125 females with mean follow-up time from any procedure being 435 days +/- 261 days. Number of contacts initiated based on time from first procedure was 482 at 3 months, 433 at 6 months, 316 contacts at 12 months, 110 contacts at 24 months, and 22 contacts at 36 months. For Group 1, 50 patients underwent 210 MRI surveillance procedures at 3 months, 6 months, 1, 2, and 3 years which failed to demonstrate any tumor formation at the re-implant sites. Formal disease surveillance for adverse events based on HHS criteria documented significantly less morbidity than is commonly reported for more invasive surgical procedures, all of which were either self-limited or were remedied with therapeutic measures. Two patients were diagnosed with cancer out of 339 patients treated since study inception; however, this was almost certainly unrelated to the MSC therapy and the neoplasm rate in similar to that seen in the U.S. Caucasian population. Knee outcome data was collected on a subset of patients

  8. A Novel Platelet Concentrate: Titanium-Prepared Platelet-Rich Fibrin

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    Mustafa Tunalı

    2014-01-01

    Full Text Available We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF. The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun’s leukocyte- and platelet-rich fibrin (L-PRF method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn using a syringe. Nine milliliters was transferred to a dry glass tube, and 9 mL was transferred to a titanium tube. Half of each clot (i.e., the blood that was clotted using T-PRF or L-PRF was processed with a scanning electron microscope (SEM. The other half of each clot was processed for fluorescence microscopy analysis and light microscopy analysis. The T-PRF samples seemed to have a highly organized network with continuous integrity compared to the other L-PRF samples. Histomorphometric analysis showed that T-PRF fibrin network covers larger area than L-PRF fibrin network; also fibrin seemed thicker in the T-PRF samples. This is the first human study to define T-PRF as an autogenous leukocyte- and platelet-rich fibrin product. The platelet activation by titanium seems to offer some high characteristics to T-PRF.

  9. A novel platelet concentrate: titanium-prepared platelet-rich fibrin.

    Science.gov (United States)

    Tunalı, Mustafa; Özdemir, Hakan; Küçükodacı, Zafer; Akman, Serhan; Yaprak, Emre; Toker, Hülya; Fıratlı, Erhan

    2014-01-01

    We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF). The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun's leukocyte- and platelet-rich fibrin (L-PRF) method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn using a syringe. Nine milliliters was transferred to a dry glass tube, and 9 mL was transferred to a titanium tube. Half of each clot (i.e., the blood that was clotted using T-PRF or L-PRF) was processed with a scanning electron microscope (SEM). The other half of each clot was processed for fluorescence microscopy analysis and light microscopy analysis. The T-PRF samples seemed to have a highly organized network with continuous integrity compared to the other L-PRF samples. Histomorphometric analysis showed that T-PRF fibrin network covers larger area than L-PRF fibrin network; also fibrin seemed thicker in the T-PRF samples. This is the first human study to define T-PRF as an autogenous leukocyte- and platelet-rich fibrin product. The platelet activation by titanium seems to offer some high characteristics to T-PRF.

  10. THE USE OF PREPARATIONS BASED ON BACTERIAL LYSATES IN THE TREATMENT OF CHRONIC TONSILLITIS IN PATIENTS WITH RHEUMATOID ARTHRITIS

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    Kolyada T.І.

    2014-12-01

    Full Text Available In the therapy of decompensated form of chronic tonsillitis (CT were used as immunomodulatory agents IRS and Ismigen. These bacterial lysates differ in the bacterial setting, the method of preparation (chemical, mechanical and the method of application.Rheumatoid arthritis (RA is one of the important factors that could significantly complicate the therapy of chronic tonsillitis. RA is a chronic immune inflammatory disease that progressively affects connective tissue mostly of the peripheral joints and it has a wide range of extra-articular manifestations. The aim of our study was to explore the dynamics of immunologic indicators during the active disease and treatments in patients with decompensate form of chronic tonsillitis, including tonsillitis complicated with RA. Materials and methods. 33 patients with decompensate form of chronic tonsillitis in active period of disease observed during the study. Patients were divided into the following groups: 24 persons with the decompensate form of CT, 9 persons with the rheumatoid arthritis and 9 persons with the decompensate form of CT complicated with RA in remission stage. The control group consisted of 15 apparently healthy persons. Concentrations of serum IgA, IgM, IgG were determined by the method of radial immune diffusion by Manchini. Levels of sIgA, IFN – γ and rheumatoid factor in the blood serum of patients were evaluated using ELISA test systems of "Vector-best". Patients of group CTD (with decompensate form of chronic tonsillitis were divided into subgroups CTD1 and CTD2, depending on the applied treatment. Both subgroups treated with standard therapy for two weeks and received Derynat during 1 month by 2 drops in each nostril twice a day. After 30 days of the standard therapy beginning the subgroup CTD1 patients received IRS 19 during two weeks, one intranasal inhalation in each nostril 3 times a day. Patients subgroup CTD2 and CTD+RA instead IRS 19 received Ismigen after 30 days of

  11. Cultivation and irradiation of human fibroblasts in a medium enriched with platelet lysate for obtaining feeder layer in epidermal cell culture; Cultivo e irradiacao de fibroblastos humanos em meio enriquecido com lisado de plaquetas para obtencao de camada de sustentacao em culturas de celulas da epiderme

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele

    2011-07-01

    For over 30 years, the use of culture medium, enriched with bovine serum, and murines fibroblasts, with the rate of proliferation controlled by irradiation or by share anticarcinogenic drugs, has been playing successfully its role in assisting in the development of keratinocytes in culture, for clinical purposes. However, currently there is a growing concern about the possibility of transmitting prions and animals viruses to transplanted patients. Taking into account this concern, the present work aims to cultivate human fibroblasts in a medium enriched with human platelets lysate and determine the irradiation dose of these cells, for obtaining feeder layer in epidermal cell culture. For carrying out the proposed objective, platelets lysis has standardized, this lysate was used for human fibroblasts cultivation and the irradiation dose enough to inhibit its duplication was evaluated. Human keratinocytes were cultivated in these feeder layers, in culture medium enriched with the lysate. With these results we conclude that the 10% platelets lysate promoted a better adhesion and proliferation of human fibroblasts and in all dose levels tested (60 to 300 Gy), these had their mitotic activity inactivated by ionizing irradiation, being that the feeder layers obtained with doses from 70 to 150 Gy were those that provided the best development of keratinocytes in medium containing 2.5% of human platelet lysate. Therefore, it was possible to standardize both the cultivation of human fibroblasts as its inactivation for use as feeder layer in culture of keratinocytes, so as to eliminate xenobiotics components. (author)

  12. Differential modulation of transforming growth factor-betas and cyclooxygenases in the platelet lysates of male F344 rats by dietary lipids and piroxicam.

    Science.gov (United States)

    Raju, Jayadev; Bird, Ranjana P

    2002-02-01

    Platelets are implicated in the pathogenesis of various chronic diseases including cancer. The main objective of the present study was to determine if dietary fish oil and piroxicam, known modulators of colon tumorigenesis, effect transforming growth factor (TGF)-betas and cyclooxygenase (COX) isozymes in the platelets of colon tumor-bearing male F344 rats. TGF-betas and COXs are important in the development of chronic illnesses including colon cancer. Animals harboring preneoplastic colonic lesions were randomly allocated to a low fat diet (5% by weight--low corn oil, LFC) and three high fat diets (23% by weight--high corn oil, HFC; high corn oil containing 150-ppm piroxicam, HFC+P; and high fish oil, HFF) for 16 weeks. TGF-beta1, TGF-beta2, COX-1 and COX-2 protein levels were assessed in the platelets by Western blot analysis. Active TGF-beta1 (12.5 kDa) level was significantly lower in the platelets of the HFC+P group (p level was significantly lower in the platelets of the HFF group (p protein in the platelets. However a 29-kDa protein, potentially a precursor of TGF-beta2, was detected in the platelets of all the groups and was significantly lower in the HFC+P and HFF groups than in LFC and HFC (p level was significantly lower in the HFF group than the other three groups (p protein was detected in the platelets of all diet groups. Piroxicam in the presence of high corn oil (HFC+P) significantly lowered the level of COX-2 (p level. These findings conclusively show that LFC and HFC differ from HFF and HFC+P, and piroxicam differs from fish oil, in regulating the levels of TGF-betas and COX in the platelets. This supports the conjecture that the levels of bioactive constituents of the platelets are profoundly modulated by dietary lipids, which in turn could influence the pathogenesis of chronic illnesses.

  13. Platelets

    Science.gov (United States)

    ... tiny fraction of the blood volume. The principal function of platelets is to prevent bleeding. Red blood cells are ... forming a long string. This illustrates the basic function of platelets, to stick to any foreign surface and then ...

  14. Platelet-rich preparations to improve healing. Part I: workable options for every size practice.

    Science.gov (United States)

    Davis, Vicki L; Abukabda, Alaeddin B; Radio, Nicholas M; Witt-Enderby, Paula A; Clafshenkel, William P; Cairone, J Vito; Rutkowski, James L

    2014-08-01

    Numerous studies have demonstrated that platelet-rich preparations applied to surgical sites, injuries, or wounds are a safe and effective way to promote soft tissue healing and bone growth. Various protocols have been developed for preparing platelet-rich preparations, with subtle but important differences between them. Unfortunately, only a minority of clinicians use platelet-rich preparations, such as platelet-rich plasma and platelet-rich fibrin, in their practice, possibly due to confusion about the different methods and their advantages and disadvantages. Therefore, the different types of preparations are described to help guide the selection of the best method for any size practice. Classic methods generally require large volumes of blood and can be expensive, complicated, and time-intensive. Simpler protocols have been developed recently, which require relatively inexpensive equipment and small blood volumes and, thus, may be more applicable for small clinical practices. Platelet-rich preparations accelerate healing at earlier time points to reduce discomfort and the potential for adverse outcomes, including infection, poor wound closure, and delays in forming strong bone for subsequent procedures (such as implants). However, platelet-rich preparations may also improve long-term outcomes in patients expected to have impaired healing, such as with lifestyle choices (eg, smoking), medications (eg, steroids), diseases (eg, diabetes, osteoporosis, atherosclerosis), and aging, by supplementing the deficient wound environment to restore proper healing. Therefore, both large and small clinical practices would benefit from utilizing platelet-rich preparations to enhance healing in their patients.

  15. Vascular endothelium and platelet preparations for the prediction of xenobiotic effects on the vascular system.

    Science.gov (United States)

    Togna, G; Togna, A R; Caprino, L

    1985-01-01

    Platelets and vascular cells play a fundamental role in the pathogenesis of cardiovascular diseases including thrombus formation and atherosclerotic phenomena. Preparations of platelets and aortic rings have been developed to study the potential of xenobiotics to produce evidence of vascular toxicity in vitro. The xenobiotics cadmium and mercury which exert vascular toxicity in vivo, modify platelet and endothelial-cell reactivity in these in vitro systems.

  16. An Evaluation of the Accuracy of the Subtraction Method Used for Determining Platelet Counts in Advanced Platelet-Rich Fibrin and Concentrated Growth Factor Preparations

    Directory of Open Access Journals (Sweden)

    Taisuke Watanabe

    2017-01-01

    Full Text Available Platelet concentrates should be quality-assured of purity and identity prior to clinical use. Unlike for the liquid form of platelet-rich plasma, platelet counts cannot be directly determined in solid fibrin clots and are instead calculated by subtracting the counts in other liquid or semi-clotted fractions from those in whole blood samples. Having long suspected the validity of this method, we herein examined the possible loss of platelets in the preparation process. Blood samples collected from healthy male donors were immediately centrifuged for advanced platelet-rich fibrin (A-PRF and concentrated growth factors (CGF according to recommended centrifugal protocols. Blood cells in liquid and semi-clotted fractions were directly counted. Platelets aggregated on clot surfaces were observed by scanning electron microscopy. A higher centrifugal force increased the numbers of platelets and platelet aggregates in the liquid red blood cell fraction and the semi-clotted red thrombus in the presence and absence of the anticoagulant, respectively. Nevertheless, the calculated platelet counts in A-PRF/CGF preparations were much higher than expected, rendering the currently accepted subtraction method inaccurate for determining platelet counts in fibrin clots. To ensure the quality of solid types of platelet concentrates chairside in a timely manner, a simple and accurate platelet-counting method should be developed immediately.

  17. In vivo evaluation of titanium-prepared platelet-rich fibrin (T-PRF): a new platelet concentrate.

    Science.gov (United States)

    Tunalı, Mustafa; Özdemir, Hakan; Küçükodacı, Zafer; Akman, Serhan; Fıratlı, Erhan

    2013-07-01

    We have developed a new, titanium-prepared, platelet-rich fibrin (T-PRF) together with the protocol for forming it, which is based on the hypothesis that titanium tubes may be more effective at activating platelets than the glass tubes used by Chouckroun in his platelet-rich fibrin (PRF) method. The aim of this study was to find a suitable animal model in which to evaluate the method and to investigate the efficacy of T-PRF for wound healing. Blood samples from 6 rabbits were used to confirm the protocol for formation of T-PRF. We evaluated T-PRF or T-PRF-like clots morphologically using scanning electron microscopy (EM). Blood samples from 5 rabbits were used to develop an experiment in which to evaluate the effects of T-PRF on wound healing. The mucoperiosteal flaps were filled with autologous T-PRF membranes from the vestibule in the anterior mandibular regions. Samples collected from the surgical sites were stained with haematoxylin and eosin. We found a mature fibrin network in T-PRF clots that had been centrifuged for 15 min at 3500 rpm and, 15 days after placement of the membrane, we found newly-forming connective tissue and islets of bony tissue in the T-PRF membrane. These results show that T-PRF could induce the formation of new bone with new connective tissue in a rabbit model of wound healing within 30 days of treatment. Published by Elsevier Ltd.

  18. A standardized research protocol for platelet- rich plasma (PRP preparation in rats

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    Michel Reis Messora

    2011-07-01

    Full Text Available Introduction: The urgent need for studies using standardized protocols to evaluate the real biological effects of PRP has been emphasized by several authors. Objective: The purpose of this study was to standardize a methodology for autologous Platelet-Rich Plasma (PRP preparation in rats. Material and methods: Twenty-four, 5 to 6-month-old, male rats, weighing 450 to 500 g were used. After general anesthesia, 3.15 ml of blood was collected from each animal, via cannulation of the jugular vein. A standardized technique of double centrifugation was used to prepare PRP. PRP samples and peripheral blood platelets were then manually counted using a Neubauer chamber. Student’s t-test was used to compare the differences between the number of platelets in peripheral blood and PRP samples (p < 0.05. In addition, PRP and peripheral blood smears were stained to see platelets’ morphology. Results: All surgical procedures were well tolerated by the animals and they were healthy during the entire experimental period. PRP samples showed higher significantly platelet concentrations than peripheral blood samples (2,677,583 and 683,680 respectively. Conclusion: Within the limits of this study, it can be concluded that the method used produced autologous PRP with appropriated platelet quantity and quality, in rats.

  19. Platelet-rich plasma (PRP) and Platelet-Rich Fibrin (PRF): surgical adjuvants, preparations for in situ regenerative medicine and tools for tissue engineering.

    Science.gov (United States)

    Bielecki, Tomasz; Dohan Ehrenfest, David M

    2012-06-01

    The recent developement of platelet concentrate for surgical use is an evolution of the fibrin glue technologies used since many years. The initial concept of these autologous preparations was to concentrate platelets and their growth factors in a plasma solution, and to activate it into a fibrin gel on a surgical site, in order to improve local healing. These platelet suspensions were often called Platelet-Rich Plasma (PRP) like the platelet concentrate used in transfusion medicine, but many different technologies have in fact been developed; some of them are even no more platelet suspensions, but solid fibrin-based biomaterials called Platelet-Rich Fibrin (PRF). These various technologies were tested in many different clinical fields, particularly oral and maxillofacial surgery, Ear-Nose-Throat surgery, plastic surgery, orthopaedic surgery, sports medicine, gynecologic and cardiovascular surgery and ophthalmology. This field of research unfortunately suffers from the lack of a proper accurate terminology and the associated misunderstandings, and the literature on the topic is quite contradictory. Indeed, the effects of these preparations cannot be limited to their growth factor content: these products associate many actors of healing in synergy, such as leukocytes, fibrin matrix, and circulating progenitor cells, and are in fact as complex as blood itself. If platelet concentrates were first used as surgical adjuvants for the stimulation of healing (as fibrin glues enriched with growth factors), many applications for in situ regenerative medicine and tissue engineering were developed and offer a great potential. However, the future of this field is first dependent on his coherence and scientific clarity. The objectives of this article is to introduce the main definitions, problematics and perspectives that are described in this special issue of Current Pharmaceutical Biotechnology about platelet concentrates.

  20. Human platelet lysates as an alternative to fetal bovine serum for the culture of mesenchymal stem cells%血小板裂解液替代胎牛血清培养间充质干细胞

    Institute of Scientific and Technical Information of China (English)

    华杰; 龚健; 何志刚; 徐斌; 杨庭松; 宋振顺

    2014-01-01

    目的 观察人血小板裂解液(HPL)对脐带间充质干细胞(UC-MSCs)的形态、增殖、表面标志、诱导分化能力的影响.方法 取正常足月妊娠产妇脐带血,离心后收集血沉棕黄层及血清按4∶1混合后-20℃冻存,反复冻融2次后制成HPL,按10%比例加入培养基.分离获得UC-MSCs,在含10% HPL或10%胎牛血清(FBS)的培养基中培养,比较培养30 d所获得的累积倍增级、最大传代代数所获得的细胞数及第3代细胞6孔板中培养6d所获得的细胞量;通过流式细胞仪检测HPL对培养细胞的表面标志的影响;观察HPL对培养细胞的成骨、成脂及成软骨诱导分化能力的影响.结果 HPL或FBS培养的UC-MSCs形态相同,均为长梭形,传至第3代形态无明显改变.在HPL或FBS中培养30 d所获得的累积倍增级比较差异无统计学意义(P>0.05);HPL培养的细胞在第10代的细胞量[(7.5±0.4)×106个]明显多于10% FBS组[(4.3±0.2)×106个];第3代UC-MSCs在6孔板培养6d后,HPL组所得细胞数明显多于FBS组(P<0.05).流式结果显示,UC-MSCs表面高表达CD44、CD73、CD90、CD105,不表达或低表达CD11b、CD19、CD31、CD34、CD45及人类白细胞抗原DR基因(HLA-DR).与10% FBS培养的UC-MSCs比较,10% HPL培养并未影响其表面标记的表达.HPL或FBS培养的UC-MSCs均能进行三系诱导分化,其分化能力无明显差异.结论 HPL可以代替FBS来进行培养和扩增UC-MSCs.在保持其形态、表面标志、分化能力不变的情况下,UC-MSCs在HPL环境下增殖更快.%Objective To investigate the impact of human platelet lysates (HPL) on the morphology,proliferation,cell surface markers,and differentiation capacity of umbilical cord-derived mesenchymal stem cells (UC-MSCs).Methods Cord blood was collected from umbilical cord after the delivery of placenta.Four buffy coat units and one serum unit were pooled and frozen at-20 ℃.After two freeze-thaw cycles,at least 10 units of freeze-thaw lysed

  1. Platelet matching for alloimmunized patients

    Institute of Scientific and Technical Information of China (English)

    S H.Hsu

    2010-01-01

    @@ Platelets play an essential role in blood coagulation,hemostasis and maintenance of vascular integrity.Platelets are utilized primarily to prevent or treat bleeding in thrombocytopenic patients and patients with impaired platelet production in the bone marrow and/or with dysfunctional platelets.In current practice,platelet transfusion begins with randomly selected platelet products:either pooled platelets prepared from whole blood derived platelets; or single donor platelets prepared by apheresis procedures.

  2. Preparation and Characterization of BAS Reinforced with SiC Platelets

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    @@BAS glass-ceramic powders were prepared by sol-gel processing. SiC platelets reinforced BAS glass-ceramic matrix composites with high density and uniform microstructure could be made using hot-pressing method. The related processing parameters were studied in this work, including effects of additional seed on transformation from H to M of barium aluminosilicate. DTA method was used to determine exact time to press mixture powders during hot-pressing for the purpose of effective densification. Microstructure and mechanical properties of the composites were investigated by mean of SEM, XRD and TEM techniques as well as three points bending test and single edge notched beam measurement. The results showed that the flexural strength and fracture toughness value of the BAS glass-ceramic matrix composites could be effectively improved by the addition of the SiC platelets. But the relative densities of composites were slightly decreased. The main toughening mechanism is crack deflection, platelets' pull-out and bridging. The increased value of flexural strength is contributed to the load transition effect from matrix to SiC platelets.

  3. Defining an appropriate leucoreduction strategy by serial assessment of cytokine levels in platelet concentrates prepared by different methods

    Directory of Open Access Journals (Sweden)

    Daljit Kaur

    2015-01-01

    Full Text Available Background and Objectives: Different methods of platelet concentrate preparations leave behind certain number of residual leukocytes, accounting for most of the febrile nonhemolytic transfusion reactions, especially in multitransfused patients. Various inflammatory cytokines, such as tumor necrosis factor-α (TNF-α, interleukin-1β (IL-1β, and IL-6 are generated during storage and have been implicated for these adverse effects. We have studied the levels of these cytokines and their correlation with leucocyte contents in platelet concentrates prepared by three different methods. Study Design and Methods: Five pools of platelet rich plasma platelet concentrates (PRP-PC and buffy-coat platelet concentrates (BC-PC each were prepared and divided into two halves. One half of the pool was leucofiltered (LF, whereas the other half was stored as such. Ten apheresis units were also included in the study. All the platelet concentrates were assessed for leucocyte load and cytokine content (IL-1β, IL-6, and TNF-α on different days of storage (0, 3, and 5 using Nageotte chamber and commercially available immunoassays respectively. Results: There was a statistically significant rise in cytokine levels (IL-1β, IL-6, and TNF-α in nonleucofiltered (NLF random donor platelet concentrates (RDPs (PRP-PC and BC-PC during storage (day 3 and 5 whereas LF RDP concentrates (PRP-PC and BC-PC and apheresis platelet concentrates (AP-PC did not show any significant rise in cytokine levels (on day 3 and 5 over the baseline values at day 0. Conclusion: This data suggests that although AP-PCs are superior to PRP-PC (NLF and BC-PC (NLF in terms of in vitro quality control parameters and cytokine generation during storage, BC-PC mode of platelet preparation followed by leucofiltration is the best method to store platelets and minimise the cytokine accumulation. This strategy is best suited for transfusion in multitransfused hematooncologic patients, who cannot afford

  4. Platelet-rich Preparation may serve as a Powerful Tool for Therapeutic Dental Pulp Regeneration

    Directory of Open Access Journals (Sweden)

    Hai-Hua Sun

    2011-09-01

    Full Text Available Introduction: Regeneration of dental pulp tissues presents one of the most challenging issues in regenerative dentistry due to their extremely poor intrinsic ability for self-healing and re-growth.The hypothesis: We hypothes-ize that patient-derived platelet-rich preparation can be used in clinical endodontic regenerative procedure, serving as a powerful tool for therapeutic dental pulp regeneration.Evaluation of the hypothesis: The cell transplantation does not always obtain the good result because of the low survival rate of transplanted cells. In addition, the use of ex vivo manipulated cell products faces many translational hurdles in treating non-vital disease. Recently, the body cells are focused as a potential source for therapeutics. Some researchers have demonstrated that endogenous stem cells may be recruited to a desired anatomic site pharma-cologically. This is spurring interest in developing new generation of biomaterials that incorporate and release selected powerful extracellular influences in a near-physiological fashion, and subsequently capture endogenous stem cells and influence their fates for regene-ration. The use of patient-derived products such as platelet-rich preparations that contain a multitude of endogenous growth factors and proteins is a clinically translatable biotechnology for this proposes. These simple and cost efficient procedures may have a potential impact in reducing the economic costs for standard medical treatments in regenerative endodontics.

  5. Evaluation of the Correct Use of Virkon Disinfectant in Reducing Bacterial Contamination of Platelets Components Prepared

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    M Sadeh

    2007-06-01

    Full Text Available Background: The purpose of the present study was to test the bactericidal effect of virkon on native species of bacteria seen in Iran by selecting a suitable concentration of virkon which would effectively reduce the level of environmental bacterial contamination including contamination of platelets components while following the exact instruction of the use of the disinfectant. Methods: This was an interferal -applied study. 160 samples were taken from laboratory benches, instruments and outer surface of blood component bags. The growing organisms were identified by using the McFarland constant standard protocols and the CFU/ml of bacteria was determined. Later all the laboratory benches and instruments involved in the preparation of platelet components were disinfected using (1% virkon solution. 101 samples were taken from disinfected areas and swabs were plated on to standard bacteriological media and plates were read. In addition, 1100 segments from platelet bags were separated and the platelet contents were plated and any bacterial growths were assessed using quality control department guidelines. Results: Out of 169 samples which were plated before disinfection by virkon following organisms were separated 56/8% gram positive b. (spore forming and without spores 96 samples, 59/8% gram positive cocci (staph. &strept. 101samples, 94/8% gram negative b. 159 samples, 82/2% gram negative cocci 139 samples. Post disinfection by virkon solution, out of 101 samples following organisms was separated: 24/8% gram positive b. 25 samples, 16/8% gram positive cocci 17 samples, 0% gram negative b. 5/9% gram negativ 6 samples, out of 1100 segment separated from platelets bags 4 samples (segments had bacterial growth (0036% 2 samples had gram positive b. growth and 2 samples had staph. Conclusion: By using correct concentration of virkon solution and following the exact manufactures instruction for use we were able to observe log reduction in bacterial

  6. Analysis of efficiency of the double- centrifugation protocol to prepare platelet rich plasma (PRP – an experimental study in rabbits

    Directory of Open Access Journals (Sweden)

    Michel Reis MESSORA

    2009-09-01

    Full Text Available Introduction and objective:The purpose of this study was to evaluatethe concentrations of platelets obtained from platelet rich plasma (PRP prepared according to the double-centrifugation protocol. Material and methods: Eight adult male rabbits (White New Zealand weighing 2.8 to 4 kg were used.After general anesthesia, 10 ml of blood were drawn from each animal via cardiac puncture. Each blood sample was centrifuged according to the protocol of Sonnleitner et al.(2000.The peripheral blood (total from each animal and the PRP samples platelets were counted manually. Data were submitted to statistical analysis.The normality of the data was confirmed and the Student’s t test was applied (p < 0.05.Results: PRP samples presented an average platele count significantly higher than that of peripheral blood.Conclusion Within the limits of this study, it was concluded that the double centrifugation protocol was adequate to prepare PRP .

  7. Influence of abciximab on the adhesion of platelets on a shielded plasma gradient prepared on polyethylene

    NARCIS (Netherlands)

    Spijker, HT; Busscher, HJ; van Oeveren, W

    2002-01-01

    Introduction: Thrombotic effects of biomaterial implants are mediated merely through activation of the platelet glycoprotein IIb-Illa (GpIIb-IIIa) receptor. Consequently, platelet GpIIb-IIIa receptor inhibitors are successfully used during stent implantation procedures to prevent thrombosis. However

  8. PELA microspheres loaded H.pylori lysates and their mucosal immune response

    Institute of Scientific and Technical Information of China (English)

    Jian-MinRen; Quan-MingZou; Fu-KunWang; QingHe; WeiChen; Wen-KunZen

    2002-01-01

    AIM:To prepare poly(D,L-lactiede)-polyethylene glycol copolymer(PELA)micrspheres loaded H.pylori lysates or cystografin and observe their targeting in gastrointestinal mucous membrane or analyze the mucosal immune responses by oral administration.

  9. Preparation of small volume, leuko and erythrocyte very poor platelet concentrates.

    Science.gov (United States)

    Valbonesi, M; Angelini, G; Malfanti, L; Lercari, G; Fella, M; Calderisi, S; Anselmo, A; Balistreri, M

    1986-05-01

    Recently developed automated discontinuous flow centrifuge (DFC) separators can produce leuko- and erythrocyte-poor platelet concentrates (PC). According to general experience with these machines it is difficult to obtain more than 4 X 10(11) platelets, though a second program set up by Coffe et al. appears to produce PC containing approximately 5 X 10(11) platelets suspended in a plasma volume of 390 ml. At our center we employed a new Dideco cell separator equipped with the surge pump and a technique developed for the production of small volume, RBC and WBC-very poor PC. In 60 routine procedures we obtained the following results: mean processing time 87 +/- 11 minutes; final volume of PC 136 +/- 19 ml, with a mean platelet yield of 5.21 X 10(11) platelets. WBC contamination was 1.8 X 10(8) (93% lymphocytes) and RBC were 3.1 X 10(8). Plasma volume as well as WBC and RBC contamination were reduced by recirculating PC after the 6th pass. The demand for single donor platelet concentrates (PC) is increasing progressively. Recently developed automated cell separators can produce leukocyte (WBC) and erythrocyte (RBC) poor PC. With these machines it may be difficult to obtain PC containing at least 4 X 10(11) platelets and less than 1 X 10(9) leukocytes (1, 2, 3) since donor variables such as hematocrit, precounts, buffy coat formation and initial plasma light transmission are of paramount importance for the efficiency of the program. At our center a prototype discontinuous flow centrifuge (DFC) cell separator equipped with the surge pump was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Longitudinal effects of menopausal hormone treatments on platelet characteristics and cell-derived microvesicles.

    Science.gov (United States)

    Miller, Virginia M; Lahr, Brian D; Bailey, Kent R; Heit, John A; Harman, S Mitchell; Jayachandran, Muthuvel

    2016-01-01

    Activated platelets serve as a catalyst for thrombin generation and a source of vasoactive and mitogenic factors affecting vascular remodeling. Oral menopausal hormone treatments (MHT) may carry greater thrombotic risk than transdermal products. This study compared effects of oral and transdermal MHT on platelet characteristics, platelet proteins, and platelet-derived microvesicles (MV) in recently menopausal women. Platelets and MV were prepared from blood of a subset of women (n = 117) enrolled in the Kronos Early Estrogen Prevention Study prior to and after 48 months of treatment with either oral conjugated equine estrogen (0.45 mg/day), transdermal 17β-estradiol (50 µg/day), each with intermittent progesterone (200 mg/day for 12 days a month), or placebo pills and patch. Platelet count and expression of platelet P-selectin and fibrinogen receptors were similar across groups. An aggregate measure of 4-year change in vasoactive and mitogenic factors in platelet lysate, by principle component analysis, indicated significantly lower values in both MHT groups compared to placebo. Increases in numbers of tissue factor positive and platelet-derived MV were significantly greater in the transdermal compared to placebo group. MHT was associated with significantly reduced platelet content of vasoactive and mitogenic factors representing a potential mechanism by which MHT may affect vascular remodeling. Various hormonal compositions and doses of MHT could differentially regulate nuclear transcription in bone marrow megakaryocytes and non-genomic pathways in circulating platelets thus determining numbers and characteristics of circulating MV. Thrombotic risk associated with oral MHT most likely involves liver-derived inflammatory/coagulation proteins rather than circulating platelets per se.

  11. EFFECT OF PLATELET LYSATE ON CHONDROGENIC DIFFERENTIATION OF HUMAN UMBILICAL CORD DERIVED MESENCHYMAL STEM CELLS IN VITRO%血小板裂解液对人脐带间充质干细胞体外成软骨分化的影响

    Institute of Scientific and Technical Information of China (English)

    冯学涛; 田少奇; 孙康; 张积华; 张才龙; 刘世海; 周明; 吕江涛

    2011-01-01

    目的 探讨血小板裂解液(platelet lysate,PL)在体外定向诱导人脐带间充质干细胞(human umbilicalcord derived mesenchymal stem cells,hUCMSCs)分化成软骨细胞中的作用.方法 取健康产妇自愿捐赠脐带,采用胶原酶消化法分离hUCMSCs,体外培养扩增,流式细胞仪进行细胞表型鉴定.根据加入诱导培养基成分不同将实验分为以下3组:A组为H-DMEM培养基、10%FBS及10%PL,B组为H-DMEM培养基、10%FBS、10 ng/mL TGF-β1、1×10-7 mol/L地塞米松、50 μg/mL维生素C及1%胰岛素铁硒传递蛋白(insulin-transferrin-selenium,ITS),C组为H-DMEM培养基、10%FBS、10 ng/mL TGF-β1、1×10 7 mo1/L地塞米松、50 μg/mL维生素C、1%ITS及10%PL.诱导培养2周,甲苯胺蓝染色检测各组软骨细胞基质的分泌,免疫荧光检测软骨特异性Ⅱ型胶原表达,半定量RT-PCR检测蛋白聚糖(Aggrecan)和Ⅱ型胶原表达.结果 分离得到的hUCMSCs不表达造血细胞的表面标记CD45、CD34和HLA-DR,而表达黏附分子和MSCs表面标记CD44、CD105和CD146.甲苯胺蓝染色和Ⅱ型胶原免疫荧光染色示C组呈阳性,B组呈弱阳性,而A组均呈阴性.半定量RT-PCR检测示Aggrecan和Ⅱ型胶原在B、C组中均有表达,A组中未见表达;C组Aggrecan mRNA和Ⅱ型胶原mRNA表达明显高于B组,差异均有统计学意义(P<0.05).结论单纯10%PL不能诱导hUCMSCs成软骨分化,但它可当作成软骨诱导培养基的辅助添加剂,对hUCMSCs成软骨分化有明显促进作用,为构建组织工程软骨提供了新的可利用条件.%Objective To study the effect of platelet lysate (PL) on chondrogenic differentiation of human umbilical cord derived mesenchymal stem cells (hUCMSCs) in vitro. Methods Umbilical cords were voluntarily donated by healthy mothers. The hUCMSCs were isolated by collagenase digestion and cultured in vitro. The surface markers of the cells were detected by flow cytometer. According to different components of inductive medium, the

  12. Preparation and activity of rabbit platelet-rich plasma%兔富血小板血浆制备及其活性分析*★

    Institute of Scientific and Technical Information of China (English)

    王鹏飞; 时长江; 王玉亮; 姜文学

    2013-01-01

      背景:富血小板血浆是目前已知富含多种生长因子并能将其释放的自体提取物,并已应用于骨、软骨组织工程再生的研究。目的:通过比较不同方法制备兔富血小板血浆中血小板浓度,并测定其血小板源性生长因子、转化生因子β1水平,探讨富血小板血浆制备方法及影响因素。方法:采用 Petrungaro 法、Landesberg 法、Aghaloo 法制备新西兰大耳白兔富血小板血浆。检测3组富血小板血浆中血小板计数,以及3组富血小板血浆活化前后及正常血浆、贫血小板血浆中血小板源性生长因子、转化生因子β1水平。结果与结论:3种方法制备的富血小板血浆中血小板计数、血小板回收率、血小板富集系数差异有非常显著性意义(P <0.001),Landesberg 法和 Aghaloo 法均可制备有效浓度的富血小板血浆,且 Aghaloo法制备血小板浓度及活性高于 Landesberg 法(P <0.05)。活化前3组富血小板血浆中血小板源性生长因子、转化生因子β1水平与正常血浆组、贫血小板血浆组比较差异无显著性意义。活化后,Landesberg法和 Aghaloo 法制备的血小板血浆中血小板源性生长因子、转化生因子β1水平明显高于活化前(P <0.001),且 Aghaloo 法最高(P <0.05)。富血小板血浆中血小板计数与血小板源性生长因子水平(r=0.872, P <0.001),转化生因子β1水平(r=0.917,P <0.001)呈正相关。%BACKGROUND: Platelet-rich plasma is an autogeneic extract containing and releasing a variety of growth factors, which has been used in the research of bone and cartilage tissue engineering and regeneration. OBJECTIVE: To investigate the preparation methods of platelet-rich plasma and its influencing factors by comparing the platelet concentration in the platelet-rich plasma prepared by different methods in rabbits and detecting the levels of platelet-derived growth factor and transforming growth factor β1

  13. 血小板凝胶的制备方法及其影响因素★%Platelet gel preparation methods and relevant parameters

    Institute of Scientific and Technical Information of China (English)

    温天杨; 王爱红; 许樟荣

    2013-01-01

    BACKGROUND: The preparation methods of platelet gel are various, but there is no uniform standard. OBJECTIVE: To conclude the methods of platelet gel preparation and to explore the relevant parameters. METHODS: The first author searched the PubMed and Wanfang databases for relevant articles published from 1990 to 2011 using the keywords of “platelet gel, classification, parameters” in English and Chinese, respectively. RESULTS AND CONCLUSION: There are two main parameters to classify the different preparation methods, which are the yield and composition of gel and the fibrin network of gel. According to the two main parameters, the preparation methods of platelet gel can be classified into four categories, namely, pure platelet-rich plasma, leukocyte- and platelet-rich plasma, pure platelet-rich fibrin, and leukocyte- and platelet-rich fibrin. According to the preparation process, the preparation methods of PRP gel also can be divided into manual protocol and automatic protocol. There are inadequacies in al the preparation methods.%  背景:血小板凝胶制备方法繁多,分类标准不统一。目的:总结血小板凝胶制备方法,并讨论影响因素。方法:由第一作者检索1990至2011年 PubMed 数据库及万方数据库。英文检索词为“Platelet gel, Classification,Parameters”,中文检索词为“血小板凝胶,分类,影响因素”。结果与结论:依据凝胶产量与成分、凝胶中纤维蛋白结构两个主要影响因素可将血小板凝胶制备方法分为4大类,即纯富血小板血浆凝胶、富白细胞-血小板血浆凝胶、纯富血小板纤维蛋白凝胶和富白细胞-血小板纤维蛋白凝胶;根据制备流程不同,血小板凝胶的每一种制备方法还可以再分为手工制备方法和全自动制备方法,但各种分类方法均存在不足之处。

  14. Evaluation of the correlation between pH and MPV platelet concentrates prepared in Tirana Blood Transfusion Center.

    Directory of Open Access Journals (Sweden)

    MERITA XHETANI

    2014-06-01

    Full Text Available The quality of platelet concentrates is an important option in transfusion therapy. pH and platelet indices have been found to be valuable parameters for monitoring the in vitro quality of platelet concentrates. Platelet activation which leads to loss of its functionality has been demonstrated by changes in those two parameters. The aim of the study was to evaluate the correlation between pH and mean platelet volume (MPV in platelet concentrates in order to examine the quality of platelet concentrate. 150 units of platelet concentrates were produced by platelet reach plasma (PRP, and stored for 5 days. Then MPV and pH were analyzed by automated hematological cell counter and Ph meter respectively. Regression analysis showed that there was a significant influence of pH changes on the changes in MPV. On the other hand, increase in pH lead to decrease in MPV. Storing platelet concentrates up to 5 days may stimulate platelet activity, enhancing its size and resulted in its destruction, so the remaining platelet are those with significantly lower MPV. Also platelet activation was those with an increase in pH. As a result measurements of MPV and pH have a great potential as quality markers of platelet concentrates.

  15. 改良白膜法制备浓缩血小板及回收率的影响因素%Improved preparation of buffy-coat-derived platelet concentrates and factors affecting platelet recovery

    Institute of Scientific and Technical Information of China (English)

    单泓; 王姣杰; 别立莉; 刘敏; 戚正; 段艳丽

    2014-01-01

    背景:白膜法和富含血浆法制备的浓缩血小板有无效输注发生率高和不良反应发生率高的缺点。  目的:观察改良白膜法制备浓缩血小板的实验研究,分析制备浓缩血小板回收率的影响因素。  方法:随机抽取126例站内采集后4-6 h的400 mL血液,随机分成改良白膜法组、白膜法组和富含血浆法组。改良白膜法采用3步离心,第1次采用次重离心,离心转速2300 r/min,离心时间12 min,降速5,离心温度(22±2)℃;第2次采用轻离心,离心转速910 r/min,离心时间10 min,离心温度(22±2)℃;第3次离心转速2800 r/min,离心时间12 min,离心温度(22±2)℃,离心后,挤去上层含血小板较少的血浆,袋中留30 mL血浆悬浮血小板,即为浓缩血小板。通过数据库文献检索的方法分析制备浓缩血小板回收率的影响因素。  结果与结论:改良白膜法、白膜法以及富含血浆法制备的手工浓缩血小板中,制备前各组血小板总数差别无统计学意义(P >0.05);富含血浆法组和改良白膜法组较白膜法组血小板回收率高,差异有显著性意义(P 0.05);白膜法组和改良白膜法组较富含血浆法组残留红细胞和残留白细胞的量少,差异有显著性意义(P0.05)。制备浓缩血小板的回收率受到全血量、离心转速、离心时间、离心方法等因素的影响。改良白膜法制备浓缩血小板减少红细胞和白细胞的残留量,提高了血小板的回收率,可在血液中心或中心血站推广应用。%BACKGROUND:Buffy-coat-derived platelet concentrates and plasma-rich platelet concentrates have a high incidence of invalid infusion and adverse reactions. OBJECTIVE:To observe the improved preparation of buffy-coat-derived platelet concentrates and to analyze the influential factors relevant to platelet recovery. METHODS:400 mL of blood sample extracted from 126 cases were randomly

  16. Surface characterization and platelet adhesion studies on fluorocarbons prepared by plasma-induced graft polymerization.

    Science.gov (United States)

    Lin, J C; Tiong, S L; Chen, C Y

    2000-01-01

    It is believed that the interactions between the biological environment and biomaterial surface are the key factors influencing its biocompatibility. Therefore, plasma processing, which can vary the surface properties without altering the bulk properties, has been considered as one of the important techniques for improving a materials' biocompatibility. In this investigation, plasma-induced grafting polymerization of vinylidene fluoride (VDF) and chlorotrifluoroethylene (CTFE), instead of direct plasma polymerization, was attempted with an aim to improve the substrate blood compatibility. Contact angle measurement indicated both fluorocarbon-grafted Pdyethylenes (PEs) are hydrophobic. Due to the additional fluorine and chlorine atoms on the CTFE chain, the PCTFE-grafted PE exhibited a higher hydrophobicity than the PVDF-grafted one. ESCA analysis has revealed that these two plasma-induced fluorocarbon deposits contain almost no CFx (x > 2) binding on the surface layer, indicating the grafting polymerization mainly follows the free radical mechanism instead of the molecule-highly-fragmented reaction steps commonly seen in the direct plasma polymerization treatment. In addition, ATR-FTIR has shown the surface chemical configuration of these PVDF- and PCTFE-grafted PEs to be very similar to those of the bulk samples of PVDF and PCTFE. The surface roughness decreased after oxygen plasma treatment and was further reduced by VDF and CTFE grafting polymerization. In vitro platelet adhesion testing indicated these two fluorocarbon grafted PEs are less platelet-activating than the nontreated PE control and oxygen plasma activated one.

  17. Anti-Human Platelet Antigen-1a Immunoglobulin G Preparation Intended to Prevent Fetal and Neonatal Alloimmune Thrombocytopenia

    Science.gov (United States)

    Weng, Ying-Jan; Husebekk, Anne; Skogen, Björn; Kjaer, Mette; Lin, Liang-Tzung; Burnouf, Thierry

    2016-01-01

    Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe disease that is caused by maternal alloantibodies generated during pregnancy or at delivery as a result of incompatibility between maternal and fetal human platelet antigens (HPAs) inherited from the father. Antibody-mediated immune suppression using anti-HPA-1a immunoglobulins is thought to be able to prevent FNAIT caused by HPA-1a. A fractionation process to prepare anti-HPA-1a immunoglobulin (Ig) G (IgG) from human plasma was therefore developed. Anti-HPA-1a plasma was obtained from volunteer mothers who underwent alloimmunization against HPA-1a during a previous pregnancy. Plasma was cryoprecipitated and the supernatant treated with caprylic acid and solvent/detergent (S/D), purified by chromatography, nanofiltered, concentrated, and sterile-filtered. The anti-HPA-1a immunoglobulin fraction was characterized for purity and safety. PAK12 and quantitative monoclonal antibody immobilization of platelet antigen (MAIPA) assays were used to detect anti-HPA-1a IgG. Hepatitis C virus (HCV) removal during nanofiltration was assessed by spiking experiments, using cell culture-derived reporter HCV and luciferase analysis. The caprylic acid treatment precipitated non-Ig proteins yielding a 90% pure Ig supernatant. S-HyperCel chromatography of the S/D-treated supernatant followed by HyperCel STAR AX provided high IgG recovery (>80%) and purity (>99.5%), and efficient IgA and IgM removal. Concentrations of complement factors C3 and C4 were HPA-1a throughout the process. Clinical-grade HPA-1a IgG can be prepared using a process compliant with current quality requirements opening perspectives for the prevention of FNAIT. PMID:27627660

  18. The Clinical Effect of Platelet Rich Plasma Prepared Through Different Activation Methods on Patients with Knee Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Nevres Aydogan

    2016-11-01

    Full Text Available Aim: Intra-articular application of platelet rich plasma (PRP can be an alternative treatment method for knee osteoarthritis. The objective of this study was to compare the activation methods of platelet rich plasma before intra-articular application. Material and Method: A total 51 patients (76 knees was randomly selected into two groups. In group 1, activation of PRP was managed by adding calcium chloride (CaCl. In group 2, activation of PRP was managed by keeping the solution at -70° degrees for 24 hours after preparation and immersed in water at 37oC for a period of 5 minutes for complete dissolution. Then PRP was applied. The patients were assessed with VAS and WOMAC pain scores both baseline and after 2nd, 6th and 12th months of the treatment. Results: VAS and WOMAC pain scores were significantly higher at baseline compared to the results obtained at the 2nd, 6th and 12th months (p=0.06. Following 2nd, 6th and 12th months a gradual downward tendency was seen in both scores, even though no significant difference was found between the groups after 2nd, 6th and 12th months. Discussion: Patients received some clinical benefits from both activation methods. There is no significant difference between activating PRP by CaCl or -70°C which compared in terms of clinical benefits. Therefore, blood storage at -70ºC may be preferred primary due to no need for additional material such as CaCl.

  19. Evaluation of platelets prepared by apheresis and stored for 5 days. In vitro and in vivo studies

    Energy Technology Data Exchange (ETDEWEB)

    Shanwell, A.; Gulliksson, H.; Berg, B.K.; Jansson, B.A.; Svensson, L.A.

    1989-11-01

    To evaluate the effect of storage on apheresis platelets collected with a closed-system blood cell separator, an in vitro investigation was performed, with measurements of pH, lactate, ATP, the ratio of ATP to the total adenine nucleotide content, and adenylate kinase. Unmodified apheresis platelets and apheresis platelets with plasma added were compared with conventional platelets stored in PL-1240 or PL-732 plastic containers. During 6 days of storage, there were similar changes in all variables with one exception: the extracellular activity of adenylate kinase was lower in apheresis platelets with plasma than in the other three groups (p less than 0.01). In vivo studies were carried out with 111Indium-labeled autologous platelets in eight volunteers. Apheresis platelets with 100 mL of plasma added were stored in two 1000-mL containers (PL-732) at 22 degrees C during agitation. Platelets from one of the containers were labeled with 111Indium and transfused into the volunteer within 24 hours. Platelets from the other container were labeled after 5 days of storage and transfused into the same donor. There were no significant differences between apheresis platelets stored for 1 day and those stored for 5 days: the mean percentage of recovery was 58.4 and 57.6 percent, t1/2 was 69 and 67 hours, and the survival time was 5.5 and 5.6 days, respectively.

  20. Preparation and catalytic performance of ZrO2 - nanographene platelets composites

    Science.gov (United States)

    Kristianto, Y.; Taufik, A.; Saleh, R.

    2016-11-01

    The ZrO2/Nanographene platelets (NGP) composites with different NGP weight percents were successfully synthesized using two-step methods (sol-gel followed by coprecipitation methods). The structural properties of the composites were analyzed using X-Ray Diffraction (XRD), Energy Dispersive X-Ray (EDX), Transmission Electron Microscope (TEM), and Thermalgravimetric Analysis (TGA). The presence of NGP in the composites confirms by the graphitic-like structure in the XRD and layer-like structure in the TEM. Thermal stability of the composites tends to reduce with the incorporation of NGP. The catalytic performances of the composites were evaluated by three catalytic processes. The degradation of methylene blue as a model pollutant by the composites are monitored using UV-Vis Spectroscopy. The result shows that ZrO2 with 10 wt% of NGP exhibits the highest performance for all of three processes. The scavenger experiment shows that hole acts as an active species that play a role in the catalytic process of the composites.

  1. Heparin-induced platelet aggregation (H-IPA): dose/response relationship for two low molecular weight (LMW) heparin preparations (CY 216 and CY 222)

    Energy Technology Data Exchange (ETDEWEB)

    Brace, L.D.; Fareed, J.

    1986-03-01

    The authors have previously demonstrated that heparin and a LMW heparin derivative (PK 10169) causes platelet aggregation in a dose-dependent manner that can be inhibited by antagonists of the thromboxane pathway. Using fractions of these agents separated on the basis of molecular weight (MW) by gel permeation chromatography, the authors showed that H-IPA was directly dependent upon the MW of the agents tested. In order to further examine this MW dependence, the authors tested two other LMW heparin preparations, CY 216 and CY 222 and subfractions of these agents separated on the basis of MW. Citrate anticoagulated whole blood was drawn from drug-free normal healthy donors whose platelets aggregated when heparin was added to their platelet-rich plasma (PRP). PRP was prepared, various concentrations of the agents or their subfractions were added and aggregation was monitored for 40 minutes at 37/sup 0/C. The results demonstrate that like heparin and PK 10169, CY 216 and CY 222 caused platelet aggregation in a dose and MW dependent manner. Fractions with MW less than 2500 daltons caused aggregation only at concentrations exceeding the therapeutic range of the agents. The authors conclude that the ability to cause H-IPA is an inherent property of heparin and its fractions.

  2. Platelets Inhibit Migration of Canine Osteosarcoma Cells.

    Science.gov (United States)

    Bulla, S C; Badial, P R; Silva, R C; Lunsford, K; Bulla, C

    2017-01-01

    The interaction between platelets and tumour cells is important for tumour growth and metastasis. Thrombocytopenia or antiplatelet treatment negatively impact on cancer metastasis, demonstrating potentially important roles for platelets in tumour progression. To our knowledge, there is no information regarding the role of platelets in cancer progression in dogs. This study was designed to test whether canine platelets affected the migratory behaviour of three canine osteosarcoma cell lines and to give insights of molecular mechanisms. Intact platelets, platelet lysate and platelet releasate inhibited the migration of canine osteosarcoma cell lines. Addition of blood leucocytes to the platelet samples did not alter the inhibitory effect on migration. Platelet treatment also significantly downregulated the transcriptional levels of SNAI2 and TWIST1 genes. The interaction between canine platelets or molecules released during platelet activation and these tumour cell lines inhibits their migration, which suggests that canine platelets might antagonize metastasis of canine osteosarcoma. This effect is probably due to, at least in part, downregulation of genes related to epithelial-mesenchymal transition. Copyright © 2016. Published by Elsevier Ltd.

  3. Anti-Human Platelet Antigen-1a Immunoglobulin G Preparation Intended to Prevent Fetal and Neonatal Alloimmune Thrombocytopenia.

    Science.gov (United States)

    Weng, Ying-Jan; Husebekk, Anne; Skogen, Björn; Kjaer, Mette; Lin, Liang-Tzung; Burnouf, Thierry

    2016-01-01

    Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe disease that is caused by maternal alloantibodies generated during pregnancy or at delivery as a result of incompatibility between maternal and fetal human platelet antigens (HPAs) inherited from the father. Antibody-mediated immune suppression using anti-HPA-1a immunoglobulins is thought to be able to prevent FNAIT caused by HPA-1a. A fractionation process to prepare anti-HPA-1a immunoglobulin (Ig) G (IgG) from human plasma was therefore developed. Anti-HPA-1a plasma was obtained from volunteer mothers who underwent alloimmunization against HPA-1a during a previous pregnancy. Plasma was cryoprecipitated and the supernatant treated with caprylic acid and solvent/detergent (S/D), purified by chromatography, nanofiltered, concentrated, and sterile-filtered. The anti-HPA-1a immunoglobulin fraction was characterized for purity and safety. PAK12 and quantitative monoclonal antibody immobilization of platelet antigen (MAIPA) assays were used to detect anti-HPA-1a IgG. Hepatitis C virus (HCV) removal during nanofiltration was assessed by spiking experiments, using cell culture-derived reporter HCV and luciferase analysis. The caprylic acid treatment precipitated non-Ig proteins yielding a 90% pure Ig supernatant. S-HyperCel chromatography of the S/D-treated supernatant followed by HyperCel STAR AX provided high IgG recovery (>80%) and purity (>99.5%), and efficient IgA and IgM removal. Concentrations of complement factors C3 and C4 were < 0.5 and < 0.4 mg/dL, respectively. The final IgG could be nanofiltered on Planova 20N under conditions removing more than 3 log HCV infectivity to baseline mock infection level, and concentrated to ca. 30 g/L. Proteolytic activity and thrombin generation were low in the final fraction. The Pak12 and MAIPA assays showed good recovery of anti-HPA-1a throughout the process. Clinical-grade HPA-1a IgG can be prepared using a process compliant with current quality requirements

  4. Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544.

    Science.gov (United States)

    Jain, Lata; Rawat, Mayank; Prajapati, Awadhesh; Tiwari, Ashok Kumar; Kumar, Bablu; Chaturvedi, V K; Saxena, H M; Ramakrishnan, Sarvanan; Kumar, Jatin; Kerketta, Priscilla

    2015-09-01

    The prophylactic efficacies of plain and alum adsorbed lysate were evaluated by direct virulent challenge in mice model. A recently isolated brucellaphage 'ϕLd' was used for generation of lysates. Twenty four h incubated Brucella abortus S19 broth cultures standardized to contain approximately 10(8) CFU/ml were found suitable for generation of lysates. Three lysate batches produced through separate cycles did not show any significant variation with respect to protein and polysaccharide contents, endotoxin level and phage counts, indicating that compositionally stable lysate preparations can be generated through an optimized production process. Three polypeptides of ∼16, 19 and 23 kDa could be identified as immuno-dominant antigens of the lysate which induced both humoral and cell-mediated immune responses in a dose dependent manner. Results of efficacy evaluation trial confirmed dose-dependent protective potencies of lysate preparation. The lysate with an antigenic dose of 0.52 μg protein and 60 μg CHO adsorbed on aluminium gel (0.1 percent aluminium concentration) exhibited the highest protective potency which was greater than that induced by standard S19 vaccine. Phage lysate methodology provides a very viable option through which an improved immunizing preparation with all desirable traits can be developed against brucellosis, and integrated with immunization programmes in a more efficient manner.

  5. Cost-effectiveness of transfusion of platelet components prepared with pathogen inactivation treatment in the United States

    NARCIS (Netherlands)

    Bell, C.F.; Botteman, M.F.; Gao, X.; Weissfeld, J.L.; Postma, Maarten; Pashos, C.L.; Triulzi, D.; Staginnus, U.; Gao, [No Value

    2003-01-01

    Background: The intercept Blood System (IBS) for platelets has been developed to reduce pathogen transmission risks during transfusions. Objective: This study was a comprehensive economic analysis of the cost-effectiveness of using the IBS for single-donor apheresis platelets (AP) and random-donor p

  6. In vitro amplification of misfolded prion protein using lysate of cultured cells.

    Directory of Open Access Journals (Sweden)

    Charles E Mays

    Full Text Available Protein misfolding cyclic amplification (PMCA recapitulates the prion protein (PrP conversion process under cell-free conditions. PMCA was initially established with brain material and then with further simplified constituents such as partially purified and recombinant PrP. However, availability of brain material from some species or brain material from animals with certain mutations or polymorphisms within the PrP gene is often limited. Moreover, preparation of native PrP from mammalian cells and tissues, as well as recombinant PrP from bacterial cells, involves time-consuming purification steps. To establish a convenient and versatile PMCA procedure unrestricted to the availability of substrate sources, we attempted to conduct PMCA with the lysate of cells that express cellular PrP (PrP(C. PrP(Sc was efficiently amplified with lysate of rabbit kidney epithelial RK13 cells stably transfected with the mouse or Syrian hamster PrP gene. Furthermore, PMCA was also successful with lysate of other established cell lines of neuronal or non-neuronal origins. Together with the data showing that the abundance of PrP(C in cell lysate was a critical factor to drive efficient PrP(Sc amplification, our results demonstrate that cell lysate in which PrP(C is present abundantly serves as an excellent substrate source for PMCA.

  7. 白膜法制备手工血小板方法的探讨%Exploration on Preparation Method of Buffy Coat-platelet Concentrates

    Institute of Scientific and Technical Information of China (English)

    刘广亚; 单桂秋; 张雅妮; 耿文艳; 谭美芳; 丘勇新

    2012-01-01

    目的 通过研究采集与制备时间对手工血小板质量的影响,探讨白膜法制备手工血小板的方法.方法 采集28例健康献血者400ml全血,采用(两步法)白膜法分离制备手工血小板,按照采集与制备时间的不同均分为4组,每组7例.对照组:新鲜全血采集6h之内立即进行手工血小板的分离制备(对照组);B组:新鲜全血采集6h之内进行第一步分离,白膜放置6h进行第二步分离;C组:新鲜全血采集6h之内进行第一步分离,白膜放置24 h进行第二步分离;D组:新鲜全血采集6~8 h之后进行手工血小板的分离制备.检测血小板计数(platelet count,PLT)、血小板平均体积(mean platelet volum,MPV)、白细胞残余率、pH值、低渗休克反应(hypotonic shock response,HSR)、血小板聚集率以及血小板膜P-选择素(CD62p)的变化.结果 白膜放置6h之后(B组),制备手工血小板其血小板计数(779.9±83.8)×109/L和HSR(62.61±5.24)%高于其他3组;MPV(6.44±0.17)fl和CD62p(47.67±7.40)%低于其他3组.B、C、D3组血小板计数、pH值、HSR和血小板聚集的比较均优于对照组,且差异有统计学意义(P<0.05),4组CD62p流式细胞术检测结果无统计学差异.结论 手工制备血小板可以打破传统6~8 h之内必须制备的概念,尤以新鲜全血立即制备第一步,白膜放置6h之后制备手工血小板为最优.%Objective To study the effect of collection time and preparation time on the quality of buffy coat-platelet concentrates (BC-PC) and explore the preparation method of BC-PC. Methods 400 ml of whole blood were collected from 28 healthy donors, and then were separated and prepared to obtain the platelet though two-step buffy coat method. The samples were divided into four groups (7 samples in each group) according to the collection time and preparation time. In group A (control), platelet concentrates (PCs) were prepared from fresh whole blood within 6 hours on the day of collection

  8. Effect of holding buffy coats before platelet preparation on the quality of platelet concentrates%白膜放置时间对制备手工浓缩血小板质量的影响

    Institute of Scientific and Technical Information of China (English)

    刘凤君; 蒋秋容; 王泽蓉; 王世春; 张红; 陈敏; 贾睿平; 赵树铭

    2013-01-01

    Objective To evaluate the quality of platelet concentrates (PCs) prepared from buffy coats held for different time periods. Methods Buffy coats (BCs) ( n = 80) were prepared from 400 mL whole blood ( WB) units within 6 h of collection. Every 5 BCs were pooled and then each pool were divided into three equal bags. One bag was taken to prepare PCs immediately (0 h group) ,and the other two bags of BCs were agitated in platelet incubator, and agitated for 4 h (4 h group) ,and 16 h (16 h group) at 22℃ before PC preparation. The in vitro quality of platelets was investigated. Results Platelet, RBC counts showed little difference between these groups. In comparison to 0 h and 4 h groups, the decrease of WBC counts in the 16 h group was significant. Free hemoglobin increased as the agitation time extended. Conclusion The agitation time of BCs may affect the quality of platelet concentrates.%目的 评价金血当天分离的白膜(BC)在不同时间分离制备的浓缩血小板(PC)的质量,为手工制备PC 提供参考.方法 将80袋400 mL全血于采集6h内分离出BC,将5袋同血型BC由无菌接驳机对接合并成1袋(1个治疗量)后,再均分在3个血小板保存袋内,1袋即刻(Oh组)轻离心分离制备PC,另2袋在22℃血小板保存箱分别振摇4 h(4 h组)和16 h(16 h组)后再分离PC.对所有标本留样进行血小板质量检测,包括Plt、血小板回收率、CD62P、聚集率、RBC混入量、WBC混入量、FHb含量.结果 3组PC制剂RBC混入量、Plt、血小板回收率、CD62P、聚集率差异无统计学意义;WBC混入量:0 h(6.76±1.29)和4h组变化不明显,16 h(3.78±0.45)组降低明显(P <0.05);FHb含量:随BC处理时间延长有增高趋势,16 h(65.62土11.11)与0 h(33.45±6.95)比差异具统计学意义(P<0.05).结论 随BC放置时间延长对制备PC制剂的质量有一定影响.

  9. Quality analysis of new buffy coat method preparation platelet concentrate%新白膜法制备浓缩血小板的质量分析

    Institute of Scientific and Technical Information of China (English)

    朱仕清; 危燕芬

    2015-01-01

    Objective:To improve the quality of the platelets concentrate and ensure the clinic therapy by exploring the best way to prepare platelets concentrate as well as analyzing the factors on affecting its quality. Methods:Select 50 bags of whole blood with 400 ml within 6h at random which are respectively used to prepare the platelets concentrate by PRP and new BC. The platelets concentrate prepared by PRP comes from the control as Group A. The Buffy coat prepared and centrifuged by new BC for the first time are respectively placed and kept in the Platelet concussion instrument for 2h as Group B, 6h as Group C, 12h as Group D, and 26h as Group E. After that, they are centrifuged for the second time to prepare the platelets concentrate which came from experimental group. Testing the PLT, count the Residual Volume of white blood cells and red blood cells, pH and bacterial culture, observing different preparation or observing the effect of different periods of keeping Buffy coat to platelets concentrate.Results:The amount of platelets concentrate by new BC(except Group B) is more than the PRP, including Group D with the most platelets concentrate, Group C and Group E more than Group A. The difference is statistically significant. No bacteria grow up on every bacterial culture. Conclusions:The platelets concentrate prepared by new BC is superior to the one by PRP. In addition, the platelets concentrate by new BC can be done over 6-8h, even can reach certain quality over 24h.%目的:探讨浓缩血小板的最佳制备方法,同时,对影响浓缩血小板质量的因素进行分析,以提高浓缩血小板的质量,确保临床应用疗效。方法:随机抽取6h内采集的400ml全血200袋,分别采用富浆法(PRP)及新白膜法(BC)制备浓缩血小板,以PRP制备的浓缩血小板为对照组(A组),用新BC法第一次离心后制备的白膜层,分别放置在血小板震荡仪中保存2h(B组)、6h(C组)、12h(D组)、26h

  10. Transfusion of pooled buffy coat platelet components prepared with photochemical pathogen inactivation treatment: the euroSPRITE trial

    NARCIS (Netherlands)

    D.J. van Rhenen (Dirk Jan); S. Marblie (Stephane); M. Laforet (Michel); K. Davis (Kathryn); M. Conlan (Maureen); B. Lioure (Bruno); H. Gulliksson (Hans); J.P. Cazenave; P. Metzel (Peyton); D. Pamphilon (Derwood); L. Corash (Laurence); J. Flament (Jocelyne); P. Ljungman (Per); H. Kluter; H. Vermeij (Hans); V. Mayaudon (Veronique); L. Lin (Lily); M.C. Kappers-Klunne (Mies); D. Buchholz (Don); G.E. de Greef (Georgine)

    2003-01-01

    textabstractA nucleic acid-targeted photochemical treatment (PCT) using amotosalen HCl (S-59) and ultraviolet A (UVA) light was developed to inactivate viruses, bacteria, protozoa, and leukocytes in platelet components. We conducted a controlled, randomized, double-blinded trial in thrombocytopenic

  11. Transfusion of pooled buffy coat platelet components prepared with photochemical pathogen inactivation treatment: the euroSPRITE trial

    NARCIS (Netherlands)

    D.J. van Rhenen (Dirk Jan); S. Marblie (Stephane); M. Laforet (Michel); K. Davis (Kathryn); M. Conlan (Maureen); B. Lioure (Bruno); H. Gulliksson (Hans); J.P. Cazenave; P. Metzel (Peyton); D. Pamphilon (Derwood); L. Corash (Laurence); J. Flament (Jocelyne); P. Ljungman (Per); H. Kluter; H. Vermeij (Hans); V. Mayaudon (Veronique); L. Lin (Lily); M.C. Kappers-Klunne (Mies); D. Buchholz (Don); G.E. de Greef (Georgine)

    2003-01-01

    textabstractA nucleic acid-targeted photochemical treatment (PCT) using amotosalen HCl (S-59) and ultraviolet A (UVA) light was developed to inactivate viruses, bacteria, protozoa, and leukocytes in platelet components. We conducted a controlled, randomized, double-blinded trial in thrombocytopenic

  12. Platelet mimicry

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Hunter, Alan Christy; Peer, Dan

    2016-01-01

    Here we critically examine whether coating of nanoparticles with platelet membranes can truly disguise them against recognition by elements of the innate immune system. We further assess whether the "cloaking technology" can sufficiently equip nanoparticles with platelet-mimicking functionalities...

  13. Platelet Count

    Science.gov (United States)

    ... their spleen removed surgically Use of birth control pills (oral contraceptives) Some conditions may cause a temporary (transitory) increased ... increased platelet counts include estrogen and birth control pills (oral contraceptives). Mildly decreased platelet counts may be seen in ...

  14. Effect of platelet age on adhesiveness to collagen and platelet surface charge

    Energy Technology Data Exchange (ETDEWEB)

    Castellan, R.M.; Steiner, M.

    1976-11-30

    Adhesion to collagen was investigated as a function of platelet age in rat platelets. Platelet adherence was measured using EDTA-containing platelet- rich plasma which was added to preparations of collagen fibers clamped between magnetic stirrers by recording changes in light transmission. The plot of light transmission versus logarithm of time was linear and allowed calculation of a slope factor which related to the rate of adherence. Neither the amount of collagen nor the platelet count were limiting in the test. Young platelet populations (less than or equal to 1 day old) were obtained during the recovery phase from immune induced thrombocytopenia. Old platelet populations were prepared by blocking thrombopoiesis with cyclophosphamide. Young platelets did not differ significantly from randomly aged platelets in this function. The electrophoretic mobility of platelets was not affected by their age.

  15. In vitro function of random donor platelets stored for 7 days in composol platelet additive solution

    Directory of Open Access Journals (Sweden)

    Gupta Ashish

    2011-01-01

    Full Text Available Background and Aim: Platelets are routinely isolated from whole blood and stored in plasma for 5 days. The present study was done to assess the in vitro function of random donor platelets stored for 7 days in composol platelet additive solution at 22°C. Materials and Methods: The study sample included 30 blood donors of both sex in State Blood Bank, CSM Medical University, Lucknow. Random donor platelets were prepared by platelet rich plasma method. Whole blood (350 ml was collected in anticoagulant Citrate Phosphate Dextrose Adenine triple blood bags. Random donor platelets were stored for 7 days at 22°C in platelet incubators and agitators, with and without additive solution. Results: Platelet swirling was present in all the units at 22°C on day 7, with no evidence of bacterial contamination. Comparison of the mean values of platelet count, platelet factor 3, lactate dehydrogenase, pH, glucose and platelet aggregation showed no significant difference in additive solution, whereas platelet factor 3, glucose and platelet aggregation showed significant difference (P < 0.001 on day 7 without additive solution at 22°C. Conclusion: Our study infers that platelet viability and aggregation were best maintained within normal levels on day 7 of storage in platelet additive solution at 22°C. Thus, we may conclude that in vitro storage of random donor platelets with an extended shelf life of 7 days using platelet additive solution may be advocated to improve the inventory of platelets.

  16. 混合浓缩血小板的制备及取消白细胞过滤的评估分析%Preparation of Pooled Platelets and Assessment Analysis of Leukocytes Unfiltered

    Institute of Scientific and Technical Information of China (English)

    叶柱江; 刘赴平; 邓穗英

    2013-01-01

    目的 制备混合浓缩血小板并过滤,评价3种血小板过滤器的过滤效果;评估分析混合12U浓缩血小板及取消过滤后混合浓缩血小板的计数情况.方法 以血小板回收率为70%以上设定离心条件,使用富血小板血浆法(PRP法)制备浓缩血小板并混合过滤,计算过滤后的白细胞清除率、红细胞清除率、血小板回收率并进行比较分析.另外,对混合12U浓缩血小板且不过滤的混合浓缩血小板的血小板含量、红细胞混入量及白细胞混入量进行计数并得出均值及标准差.结果 3种过滤器的白细胞清除率、红细胞清除率、血小板回收率差异均有统计学意义(P<0.01).259例改由12U浓缩血小板制备的混合浓缩血小板的计数结果与GB18469-2012《全血及成分血质量要求》中混合浓缩血小板质量要求的血小板含量符合率为95.4%,红细胞混入量符合率为71.8%,总体符合率为67.2%.结论 可不必对混合浓缩血小板进行白细胞滤除,每例混合浓缩血小板宜采用12U浓缩血小板制备以达到质量要求.%Objective To prepare pooled platelets and filtration, to evaluate the efficiency of three kinds of platelet filter, and assess the count situation of the pooled platelets that mixed 12U platelets without filtration. Methods Platelets were prepared by platelet rich plasma (PRP) method and the centrifugation condition was set based on the recovery rate of platelets more than 70%, missed and filtered. The removal rates of leukocytes, red cells and the recovery rate of platelets were estimated after filtering. 12U platelets was mixed with the pooled platelets without filtering and determined for the platelets count, red cells and leukocytes contamination. Results The were significant differences showed in the leukocytes removal rate,red cells removal rate and platelets recovery rate of using three different kinds of filters.Based on the "GB18469-2012 quality requirements for

  17. Platelets stimulate fibroblast-mediated contraction of collagen gels

    Directory of Open Access Journals (Sweden)

    Lundahl Joachim

    2003-10-01

    Full Text Available Abstract Background Platelets are thought to play a role in a variety of inflammatory conditions in the lung, some of which may lead to fibrosis. In the current study we tested the hypothesis that whole platelets and platelet lysate can mediate remodelling of extracellular matrix in vitro by affecting fibroblast-mediated contraction of a collagen gel. We also sought to determine to what extent platelet-derived growth factor (PDGF and transforming growth factor-β (TGF-β contribute to this effect. Methods Washed platelets, isolated from healthy blood donors, and platelet lysate (freezing and thawing, were cast together with human lung fibroblasts in three-dimensional collagen gels. The gels were then released and cultured for four days. PDGF and TGF-β1 concentrations were measured in culture supernatants by ELISA. Results Both platelets and platelet lysate augmented fibroblast-mediated gel contraction in a time and concentration dependent manner (19.9% ± 0.1 (mean ± SEM of initial area vs. 48.0% ± 0.4 at 48 hours; P 1 and PDGF-AA/AB were released in co-culture. PDGF-AA/AB had a maximum release at 24 hours whereas TGF-β1 release increased with longer culture periods. Neutralising antibodies to these mediators partially inhibited platelet-induced gel contraction. Conclusion We conclude that platelets may promote remodelling of extracellular matrix in vitro and that PDGF and TGF-β partially mediate this effect, also indicating a role for other mediators. The findings may be an important mechanism in regulating repair processes after injury.

  18. ASSESSMENT STUDY ON A SET OF PLATELET-RICH PLASMA PREPARATION%富血小板血浆制备套装的评估研究

    Institute of Scientific and Technical Information of China (English)

    李明; 张长青; 袁霆; 陈圣宝; 吕汝举

    2011-01-01

    目的 通过分析富血小板血浆(platelet-rich plasma,PRP)中血小板、白细胞和生长因子的浓度,计算回收率和富集系数,并做相关性分析,探讨PRP制备套装的实用性和稳定性.方法 取30例符合纳入标准的自愿者自愿捐赠的外周血各40 mL,应用山东威高集团医用高分子制品股份有限公司的PRP制备套装制备PRP各4 mL.全自动血液分析仪计数全血和PRP中血小板和白细胞浓度,并计算血小板或白细胞同收率及富集系数;并分别测定男、女自愿者血小板及白细胞浓度.ELISA法定量分析激活后全血及PRP中PDGF、TGF-β、VEGF的浓度.结果 全血和PRP中血小板浓度分别为(131.40 ±29.44)×109/L和(819.47 ±136.32)×109/L,比较差异有统计学意义(t=-27.020,P=0.000);PRP中血小板回收率为60.85%±8.97%,富集系数为6.40 ±1.06.全血和PRP中自细胞浓度分别为(5.57 ±1.91)×1012/L和(32.20±10.42)× 1012/L,比较差异有统计学意义(t=-13.780,P=0.000);PRP中自细胞回收率为58.30%±19.24%,富集系数为6.10±1.93.PRP中血小板浓度和白细胞浓度分别与全血中血小板浓度(r=0.652,P=0.000)和白细胞浓度(r=0.460,P=0.011)成正相关.男性组和女性组PRP中血小板浓度和白细胞浓度比较差异均无统计学意义(P>0.05).PRP中PDGF、TGF-β、VEGF浓度分别为(698.15 ±64.48)、(681.36±65.90)、(1 071.55 ±106.04)ng/mL,是全血的(5.67 ±1.18)、(6.99 ±0.61)、(5.74 ±0.83)倍.PRP中PDGF浓度(r=0.832,P=0.020)、TGF-β浓度(r=0.835,P=0.019)、VEGF浓度(r=0.824,P=0.023)均与PRP中血小板浓度成正相关.结论 PRP制备套装可以稳定地制备出富含高浓度血小板、白细胞和牛长因子的PRP.%Objective To calculate the recovery rate and enrichment factor and to analyse the correlation by measuring the concentrations of platelets, leukocyte, and growth factors in platelet-rich plasma (PRP) so as to evaluate the feasibility and stability of a set of PRP preparation. Methods

  19. Actualización bibliográfica sobre el uso de preparaciones ricas en plaquetas en la cicatrización de heridas Updated review of the use of platelet rich preparations in wound healing

    Directory of Open Access Journals (Sweden)

    D.A. Mateo de Acosta Andino

    2010-09-01

    Full Text Available Las plaquetas poseen más de 20 factores de crecimiento en sus gránulos. Su secreción promueve la reparación de heridas. La cicatrización es un intrincado proceso en el cual intervienen no solo los factores plaquetarios proangiogénicos sino también factores antiangiogénicos. La preservación por congelación de las plaquetas empleada para su preservación, desestabiliza y activa la membrana plaquetaria alterando sus propiedades. En el campo del tratamiento de heridas recalcitrantes en pacientes debilitados o con enfermedades crónicas, el uso de preparaciones plaquetarias en forma de plasma fresco congelado, las preparaciones plaquetarias sonicadas, las preparaciones congeladas y deshidratadas y el uso de fragmentos de dermis alogénica, confiere efectos beneficiosos, logrando mejores tiempos de cierre en las heridas y menor alteración de la morfología de la cicatriz final. En el presente artículo, describiremos los estudios que han demostrado estos efectos, así como las encrucijadas actuales y los estudios que debemos esperar en un futuro no lejano.Platelets store more than 20 growth factors in their granules. Their secretion promotes wound healing and repair. The intricate wound healing process involves not only the angiogenic platelet factors but also de anti-angiogenic ones. The process of platelet preservation, such as freezing, disrupt the platelet membrane structure and their angiogenic properties causing platelet activation. In the field of wound healing and the management of recalcitrant wounds in debilitated and chronically ill patients, there have been several studies. These studies have focused on the use of the different platelet preparations such as fresh frozen plasma, frozen-dried platelet rich plasma, sonicated platelet rich plasma and the use of alogenic acelular dermis. Platelet preparations posses positive effects on wound healing with little alterations of the final scar tissue. In this article we will describe

  20. Platelet-rich fibrin: Evolution of a second-generation platelet concentrate

    Directory of Open Access Journals (Sweden)

    Sunitha Raja V

    2008-01-01

    Full Text Available Platelet-rich plasma (PRP is a platelet concentrate that has been used widely to accelerate soft-tissue and hard-tissue healing. The preparation of PRP has been described by several authors. Platelet-rich fibrin (PRF was first described by Choukroun et al. in France. It has been referred to as a second-generation platelet concentrate, which has been shown to have several advantages over traditionally prepared PRP. Its chief advantages include ease of preparation and lack of biochemical handling of blood, which makes this preparation strictly autologous. This article describes the evolution of this novel platelet concentrate, referred to as PRF.

  1. STABILIZATION OF STANDARD PLATELET CONCENTRATES AND MINIMIZATION OF THE PLATELET STORAGE LESION BY A PROSTACYCLIN ANALOG

    NARCIS (Netherlands)

    ELIAS, M; HEETHUIS, A; BOM, [No Value; BLOM, N; MCSHINE, RL; HALIE, MR; SIBINGA, CTS

    Platelet concentrates were pretreated with a stable synthetic prostacyclin analogue (Iloprost) at two different concentrations before the second centrifugation step (pelleting step) of preparation. This resulted in loss. of platelet sensitivity to aggregating agents. To mimic the situation after

  2. An ideal preparation for dermal regeneration: skin renewal growth factors, the growth factor composites from porcine platelets.

    Science.gov (United States)

    Wang, Kuo-Hsien; Wu, Yo-Ping Greg; Lo, Wen-Cheng

    2012-12-01

    The use of growth factor composites from platelets has been introduced to many areas of clinical applications and studies. With the richest source of growth factors (GFs), beneficial effects have been shown on tissue regeneration and wound healing. However, animal and clinical studies have revealed inconsistent outcomes with the use of platelet-derived growth factors (PDGFs), which were likely due to variations in the presence and concentrations of GFs between various sources. Autologous PDGFs are considered to be safer, but they are limited by the feasibility of large-scale production to be used extensively in the acute phase, greater surface area, or general cosmetic applications. This study employed a simple process to obtain growth factor composites from activated platelets of porcine origin, namely skin renewal growth factors (SRGF). The functions of SRGF were subsequently evaluated on cultured human fibroblasts, keratinocytes, and melanocytes. Our data revealed that SRGF significantly promoted the proliferation of fibroblasts, accompanied by increased expression of collagens (types I, III, IV, and VIII) and proteoglycans. Diminished proliferation and arrested differentiation of keratinocytes were evidenced by the attenuated expression of laminin V and keratin 10. In addition, SRGF also suppressed the growth of melanocytes and reduced the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and paired box 3 (PAX3), which mediates melanogensis. Our results suggest that SRGF possesses beneficial properties and is a promising and cost-effective composition for the development of a safe cosmetic agent or topical products for skin regeneration. The development of SRGF may also provide an alternative strategy for tissue engineering.

  3. Platelet proteomics.

    Science.gov (United States)

    Zufferey, Anne; Fontana, Pierre; Reny, Jean-Luc; Nolli, Severine; Sanchez, Jean-Charles

    2012-01-01

    Platelets are small cell fragments, produced by megakaryocytes, in the bone marrow. They play an important role in hemostasis and diverse thrombotic disorders. They are therefore primary targets of antithrombotic therapies. They are implicated in several pathophysiological pathways, such as inflammation or wound repair. In blood circulation, platelets are activated by several pathways including subendothelial matrix and thrombin, triggering the formation of the platelet plug. Studying their proteome is a powerful approach to understand their biology and function. However, particular attention must be paid to different experimental parameters, such as platelet quality and purity. Several technologies are involved during the platelet proteome processing, yielding information on protein identification, characterization, localization, and quantification. Recent technical improvements in proteomics combined with inter-disciplinary strategies, such as metabolomic, transcriptomics, and bioinformatics, will help to understand platelets biological mechanisms. Therefore, a comprehensive analysis of the platelet proteome under different environmental conditions may contribute to elucidate complex processes relevant to platelet function regarding bleeding disorders or platelet hyperreactivity and identify new targets for antiplatelet therapy.

  4. A novel collagen/platelet-rich plasma (COL/PRP) scaffold: preparation and growth factor release analysis.

    Science.gov (United States)

    Zhang, Xiujie; Wang, Jingwei; Ren, Mingguang; Li, Lifeng; Wang, Qingwen; Hou, Xiaohua

    2016-06-01

    Platelet-rich plasma (PRP) has been widely used in clinical practice for more than 20 years because it causes the release of many growth factors. However, the burst release pattern and short release period of PRP have become obstacles to its application. An optimal controllable release system is an urgent need for researchers. This study investigated whether collagen/PRP (COL/PRP) scaffolds can serve as a vehicle for the controllable release of growth factors. We fabricated a novel scaffold that integrates PRP activated by thrombin or collagen into type I collagen. The mechanical properties, cytotoxicity, and transforming growth factor β1 (TGF-β1), platelet derived growth factor (PDGF), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) content were evaluated. Our results demonstrate that the COL/PRP scaffolds were not cytotoxic to L-929 fibroblasts. The PDGF and FGF content in the thrombin group was at a higher level and lasted for a long period of time. Collagen and thrombin played the same role in the release of TGF-β1 and VEGF. These data suggest that the novel COL/PRP scaffolds provide a carrier for the controllable release of growth factors and may be used in tissue- regenerative therapies.

  5. TOPICAL BACTERIAL LYSATES IN PEDIATRIC CHRONIC TONSILLITIS TREATMENT

    Directory of Open Access Journals (Sweden)

    М.М. Polunin

    2011-01-01

    Full Text Available Chronic tonsillitis remains a current problem in pediatric health due to the risk of nonspecific systemic heart, renal and joint diseases. High social significance and prevalence among children call for effective preventive and treatment measures against this disease. Topical bacterial lysates recently have become popular among physicians as safe and effective component of tonsillitis treatment.Key words: chronical tonsillitis, childhood, bacterial lysates. (Voprosy sovremennoi pediatrii — Current Pediatrics. — 2011; 10 (6: 176–178

  6. Platelet lipidomic.

    Science.gov (United States)

    Dolegowska, B; Lubkowska, A; De Girolamo, L

    2012-01-01

    Lipids account for 16-19 percent dry platelet matter and includes 65 percent phospholipids, 25 percent neutral lipids and about 8 percent glycosphingolipids. The cell membrane that surrounds platelets is a bilayer that contains different types phospholipids symmetrically distributed in resting platelets, such as phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine, and sphingomyelin. The collapse of lipid asymmetry is exposure of phosphatidylserine in the external leaflet of the plasma bilayer, where it is known to serve at least two major functions: providing a platform for development of the blood coagulation cascade and presenting the signal that induces phagocytosis of apoptotic cells. During activation, this asymmetrical distribution becomes disrupted, and PS and PE become exposed on the cell surface. The transbilayer movement of phosphatidylserine is responsible for the platelet procoagulant activity. Exposure of phosphatidylserine is a flag for macrophage recognition and clearance from the circulation. Platelets, stored at room temperature for transfusion for more than 5 days, undergo changes collectively known as platelet storage lesions. Thus, the platelet lipid composition and its possible modifications over time are crucial for efficacy of platelet rich plasma therapy. Moreover, a number of substances derived from lipids are contained into platelets. Eicosanoids are lipid signaling mediators generated by the action of lipoxygenase and include prostaglandins, thromboxane A2, 12-hydroxyeicosatetraenoic acid. Isoprostanes have a chemical structure similar to this of prostanoids, but are differently produced into the particle, and are ligands for prostaglandins receptors, exhibiting biological activity like thromboxane A2. Endocannabinoids are derivatives from arachidonic acid which could reduce local pain. Phospholipids growth factors (sphingolipids, lysophosphatidic acid, platelet-activating factor) are involved in tissue

  7. The heat-compression technique for the conversion of platelet-rich fibrin preparation to a barrier membrane with a reduced rate of biodegradation.

    Science.gov (United States)

    Kawase, Tomoyuki; Kamiya, Mana; Kobayashi, Mito; Tanaka, Takaaki; Okuda, Kazuhiro; Wolff, Larry F; Yoshie, Hiromasa

    2015-05-01

    Platelet-rich fibrin (PRF) was developed as an advanced form of platelet-rich plasma to eliminate xenofactors, such as bovine thrombin, and it is mainly used as a source of growth factor for tissue regeneration. Furthermore, although a minor application, PRF in a compressed membrane-like form has also been used as a substitute for commercially available barrier membranes in guided-tissue regeneration (GTR) treatment. However, the PRF membrane is resorbed within 2 weeks or less at implantation sites; therefore, it can barely maintain sufficient space for bone regeneration. In this study, we developed and optimized a heat-compression technique and tested the feasibility of the resulting PRF membrane. Freshly prepared human PRF was first compressed with dry gauze and subsequently with a hot iron. Biodegradability was microscopically examined in vitro by treatment with plasmin at 37°C or in vivo by subcutaneous implantation in nude mice. Compared with the control gauze-compressed PRF, the heat-compressed PRF appeared plasmin-resistant and remained stable for longer than 10 days in vitro. Additionally, in animal implantation studies, the heat-compressed PRF was observed at least for 3 weeks postimplantation in vivo whereas the control PRF was completely resorbed within 2 weeks. Therefore, these findings suggest that the heat-compression technique reduces the rate of biodegradation of the PRF membrane without sacrificing its biocompatibility and that the heat-compressed PRF membrane easily could be prepared at chair-side and applied as a barrier membrane in the GTR treatment.

  8. Effect of photodynamic therapy on mouse platelets

    Science.gov (United States)

    Zhou, Chuannong; Chi, Shunji; Deng, Jinsheng; Zhang, Hua; Liang, Junlin; Ha, Xian-wen

    1993-06-01

    Normal mice received hematoporphyrin derivative (HpD) i.v. prior to red light irradiation and the platelet-rich plasma was prepared and irradiated by red light. The platelets were processed for EM examination and stereological analysis. It was shown the 16 hrs after irradiation almost all platelets were necrotized; 8 hours after irradiation about one fourth of the platelets were necrotized and the remaining were considerably damaged. Immediately after irradiation a small number of platelets became necrotic and most other platelets were swollen and deformated, showing significantly increased mean area, perimeter and short axis, and mean cell volume and cell surface area. The findings indicate that platelets are highly sensitive to PDT action and can be directly and rapidly damaged by PDT even in the absence of vascular endothelial cells. The early platelet photoactivation may play an important role in the initiation of early vascular damage and microcirculatory alterations induced by PDT in vivo.

  9. Antimicrobial/anti-biofilm activity of expired blood platelets and their released products 

    Directory of Open Access Journals (Sweden)

    Marcin I. Różalski

    2013-04-01

    Full Text Available Introduction. Although platelets are not part of the classical immune system, they have many features that indicate their role in the anti-infective host defense. They come into interactions with microorganisms, which results in co-aggregation and co-adhesion or destruction of themicrobes due to the action of antimicrobial peptides released from platelets.The aim of this study was to evaluate the killing effect of platelets against planktonic and biofilm cultures of Staphylococcus aureus and to test their synergy with antibiotics. Materials and Methods. S. aureus ATCC 29213; platelet rich plasma (1-3 days post shelf life. Evaluation of bactericidal activity of platelets or their lysates against planktonic cultures of S. aureus – CFU calculation after 4- and 24-hour co-incubation. Assessment of S. aureus biofilm viability under the influence of platelets – Live/Dead® BacLightTM Bacterial Viability Kit. Determination of minimum inhibitory concentrations (MICs (oxacillin, vancomycin, linezolid and estimation of the synergistic action of antibiotics and platelet lysates – a gradient-diffusion test strip. Results. Microbicidal activity of “expired” platelets and their lysates has been shown as a significant reduction in the population of staphylococci in their planktonic cultures by 56-87�0and a decrease in metabolic activity of biofilm formation by 7-38�20These activities were enhanced after activation with ADP. Platelet lysates showed a synergistic effect with β-lactam antibiotic (oxacillin and glycopeptide (vancomycin but not with oxazolidinone (linezolid. Conclusions and Discussion. In summary, platelets even after the medical expiry date are still a good source of antimicrobial low molecular weight proteins (PMPs. Testing of bacterialresistance to PMPs may be advisable as a predictive indicator of susceptibility to treatment of infections such as infective endocarditis and other local infections of biofilm nature.

  10. Trehalose lyophilized platelets for wound healing.

    Science.gov (United States)

    Pietramaggiori, Giorgio; Kaipainen, Arja; Ho, David; Orser, Cindy; Pebley, Walter; Rudolph, Alan; Orgill, Dennis P

    2007-01-01

    Fresh platelet preparations are utilized to treat a wide variety of wounds, although storage limitations and mixed results have hampered their clinical use. We hypothesized that concentrated lyophilized and reconstituted platelet preparations, preserved with trehalose, maintain and possibly enhance fresh platelets' ability to improve wound healing. We studied the ability of a single dose of trehalose lyophilized and reconstituted platelets to enhance wound healing when topically applied on full-thickness wounds in the genetically diabetic mouse. We compared these results with the application of multiple doses of fresh platelet preparations and trehalose lyophilized and reconstituted platelets as well as multiple doses of vascular endothelial growth factor (VEGF) and wounds left untreated. Trehalose lyophilized and reconstituted platelets, in single and multiple applications, multiple applications of fresh platelets and multiple applications of VEGF increased granulation tissue deposition, vascularity, and proliferation when compared with untreated wounds, as assessed by histology and immunohistochemistry. Wounds treated with multiple doses of VEGF and a single dose of freeze-dried platelets reached 90% closure faster than wounds left untreated. A single administration of trehalose lyophilized and reconstituted platelet preparations enhanced diabetic wound healing, therefore representing a promising strategy for the treatment of nonhealing wounds.

  11. Acquired platelet function defect

    Science.gov (United States)

    Acquired qualitative platelet disorders; Acquired disorders of platelet function ... blood clotting. Disorders that can cause problems in platelet function include: Idiopathic thrombocytopenic purpura Chronic myelogenous leukemia Multiple ...

  12. Platelet Donation

    Science.gov (United States)

    ... of gratitude that washed over me when I saw those platelets going into my husband’s body. I ... Needles LGBTQ+ Donors Blood Donor Community SleevesUp Games Facebook Avatars and Badges Banners eCards Red Cross Information ...

  13. Platelet-rich fibrin matrix for facial plastic surgery.

    Science.gov (United States)

    Sclafani, Anthony P; Saman, Masoud

    2012-05-01

    Platelets are known primarily for their role in hemostasis, but there is increasing interest in the effect of platelets on wound healing. Platelet isolates such as platelet-rich plasma have been advocated to enhance and accelerate wound healing. This article describes the use of a novel preparation, platelet-rich fibrin matrix (PRFM), for facial plastic surgery applications such as volume augmentation, fat transfer supplementation, and as an adjunct to open surgical procedures.

  14. ROLE OF THE TOPIC BACTERIAL LYSATES IN THE PEDIATRIC PRACTICES

    Directory of Open Access Journals (Sweden)

    L.R. Selimzyanova

    2009-01-01

    Full Text Available The recurrent and chronic infections are an actual problem in pediatrics. The regular use of antibiotics leads to the resistance of the oral cavity pathogens and disturbances in the normal microflora balance. Bacterial lysates are considered to be the efficient means to prevent and treat the acute and chronic respiratory diseases. The article is an executive summary of literature on the bacterial immune response modulating agents, including the description of the mechanism of action of these medications. The article provides for the information on the peculiarities of the topic bacterial lysates.Key words: children, acute respiratory infections, chronic pharyngitis, chronic tonsillitis, stomatitis, bacterial immune response modulating agent.

  15. L-amino acid oxidase from Naja atra venom activates and binds to human platelets

    Institute of Scientific and Technical Information of China (English)

    Rui Li; Shaowen Zhu; Jianbo Wu; Wanyu Wang; Qiumin Lu; Kenneth J.Clemetson

    2008-01-01

    An L-amino acid oxidase (LAAO),NA-LAAO,was purified from the venom of Naja atra.Its N-terminal sequence shows great similarity with LAAOs from other snake venoms.NALAAO dose-dependently induced aggregation of washed human platelets.However,it had no activity on platelets in platelet-rich plasma.A low concentration of NA-LAAO greatly promoted the effect of hydrogen peroxide,whereas hydrogen peroxide itself had little activation effect on platelets.NA-LAAO induced tyrosine phosphorylation of a number of platelet proteins including Src kinase,spleen tyrosine kinase,and phospholipase C γ2.Unlike convulxin,Fc receptor γ chain and T lymphocyte adapter protein are not phosphorylated in NA-LAAO activated platelets,suggesting an activation mechanism different from the glycoprotein VI pathway.Catalase inhibited the platelet aggregation and platelet protein phosphorylation induced by NA-LAAO.NA-LAAO bound to fixed platelets as well as to platelet lysates of Western blots.Furthermore,affinity chromatography of platelet proteins on an NA-LAAO Sepharose 4B column isolated a few platelet membrane proteins,suggesting that binding of NA-LAAO to the platelet membrane might play a role in its action on platelets.

  16. 白膜法少白细胞浓缩血小板的制备及质量分析%Preparation and Quality Analysis of Leucoreduced Platelet Concentrates by Buffy Coats Method

    Institute of Scientific and Technical Information of China (English)

    党娥; 陈晨; 张蕾; 刘晶; 贾登; 穆士杰; 张献清

    2011-01-01

    目的 改进手工制备浓缩血小板方法,提高血小板制备质量.方法 ①全血经特定程序进行离心,利用全自动血液成分分离机分离白膜,白膜解聚后进行二次轻离心.②经白细胞滤器滤除血小板中的白细胞,然后进行细胞计数.结果 10份按该法制备的浓缩血小板(10U/袋),其血小板计数、残余红细胞数、白细胞数分别为(3.8±0.3)×1011/袋,(4.0±0.4)×108/袋,(0.5±0.3)×106/袋,容量250 ml~350 ml.结论 该法制备的手工浓缩血小板含量高,残余红细胞、白细胞数量低.质量指标达到国家质量要求,适宜血站推广应用.%Objective To improve the hand-made method of platelet concentrates and enhance the quality of platelet. Methods ①The whole blood were centrifuged according to definite programme,then the butty coat were separated with automatic blood cell separator. After the disaggregation of butty coats,the second mild centrifugation was done. ②The white cell in the platelet conccentrates were filtrated by white cell filter,then the cellcounts were tested. Results In 10 platelet concentrates prepared according to this methods ,the platelets count,residual red blood cell count and white cell were (3. 8 ± 0. 3) × 1011/bag, (4. 0± 0. 4) × 108/bag, (0. 5±0. 3) × 106/bag, respectively. The volume of platelet concentrates were about 250~350 ml. Conclusion The platelet concentrates prepared by this methods had high content platelet and low residual red blood cells, as well as white blood cells. The quality indexes could meet the national quality reguirement and could be used widely.

  17. Image analysis of blood platelets adhesion.

    Science.gov (United States)

    Krízová, P; Rysavá, J; Vanícková, M; Cieslar, P; Dyr, J E

    2003-01-01

    Adhesion of blood platelets is one of the major events in haemostatic and thrombotic processes. We studied adhesion of blood platelets on fibrinogen and fibrin dimer sorbed on solid support material (glass, polystyrene). Adhesion was carried on under static and dynamic conditions and measured as percentage of the surface covered with platelets. Within a range of platelet counts in normal and in thrombocytopenic blood we observed a very significant decrease in platelet adhesion on fibrin dimer with bounded active thrombin with decreasing platelet count. Our results show the imperative use of platelet poor blood preparations as control samples in experiments with thrombocytopenic blood. Experiments carried on adhesive surfaces sorbed on polystyrene showed lower relative inaccuracy than on glass. Markedly different behaviour of platelets adhered on the same adhesive surface, which differed only in support material (glass or polystyrene) suggest that adhesion and mainly spreading of platelets depends on physical quality of the surface. While on polystyrene there were no significant differences between fibrin dimer and fibrinogen, adhesion measured on glass support material markedly differed between fibrin dimer and fibrinogen. We compared two methods of thresholding in image analysis of adhered platelets. Results obtained by image analysis of spreaded platelets showed higher relative inaccuracy than results obtained by image analysis of platelets centres and aggregates.

  18. In vitro function of random donor platelets stored for 7 days in composol platelet additive solution

    Directory of Open Access Journals (Sweden)

    Gupta Ashish

    2011-01-01

    Full Text Available Background and Aim: Platelets are routinely isolated from whole blood and stored in plasma for 5 days. This study was done to assess the in vitro function of random donor platelets stored for 7 days in composol platelet additive solution at 22°C. Materials and Methods: The study sample included 30 blood donors of both sex in State Blood Bank, C S M Medical University, Lucknow. Random donor platelets were prepared by the platelet-rich plasma method. Whole blood (350 ml was collected in anticoagulant Citrate Phosphate Dextrose Adenine triple blood bags. Random donor platelets were stored for 7 days at 22°C in platelet incubators and agitators with and without additive solution. Results: Platelet swirling was present in all the units at 22°C on day 7 with no evidence of bacterial contamination. Comparison of the mean values of platelet count, platelet factor 3, lactate dehydrogenase, pH, glucose and platelet aggregation showed no significant difference in additive solution while platelet factor 3, glucose and platelet aggregation showed significant difference (P < 0.001 on day 7 without additive solution at 22°C. Conclusion: Our study infers that the platelet viability and aggregation were the best maintained within normal levels on day 7 of storage in platelet additive solution at 22°C. Thus, we may conclude that in vitro storage of random donor platelets with an extended shelf life of 7 days using platelet additive solution may be advocated to improve the inventory of platelets.

  19. Comparison of the methods for the preparation of platelet- rich plasma%富血小板血浆提取方法的探讨

    Institute of Scientific and Technical Information of China (English)

    王悦; 朱喆; 刘昕鸣; 马英智; 周延民; 杨旭芳

    2011-01-01

    Objective; To compare the effects of different methods for the preparation of platelet-rich plasma ( PRP). Methods; Venous blood was obtained from a health volunteer and 30 patients of the department of ophthalmology. PRP was prepared by PCCS system, Curasan system and Trindade method respectively. The levels of PDGF, VEGF and TGF-p in the PRP samples were measured using ELJSA. A phase contrast microscope was used to observe the morphology of MG63 cells cultured wilh PRP. Results: PDGF level in the PRP prepared by Trindade method and treated with CaCI, was higher, TGF-p level in PRP prepared by PCCS and treated with CaCl2 and thrombase was higher, the concentration of VEGF in PRP prepared by Curasan and treated by CaCl2 was higher. CaCl2 increased PDGF level in the PRP prepared by Trindade melhod, TGF-p by PCCS and Curasan, and VEGF by the three methods(P 0. 05). Cenlrifuged PRP did not affect the observation of the morphology of cultured MG63 cells. Conclusion; Three methods are effective in the preparation of PRP. Preparation at low temperature and activation of PRP by CaCl2 or CaCK plus thrombin may increase the cytoktn content. Cenlrifuged PRP is suitable for cell culture.%目的:比较不同离心和激活方法处理富血小板血浆(PRP)的效果.方法:1名健康志愿者、30名眼科住院患者静脉采血,分别用PCCSkit法,Curasan法及Trindade法制备PRP.ELISA检测各组血小板衍化生长因子(PDGF)、转化生长因子(TGF-β)、血管内皮细胞生长因子(VEGF),倒置相差显微镜观察PRP添加方式对人成骨样细胞MG63观察效果的影响.结果:Trindade离心法提取的PRP加CaCl2后,PDGF含量高于其他2种方法.PCCSkit法提取的PRP加CaCl2和凝血酶后,TGF-β含量高于其他2种方法,Curasan法提取的PRP加CaCl2后,VEGF浓度高于其他2种方法.3种离心方法中未激活和激活的PRP中生长因子浓度差异有显著性(P<0.05),激活组中CaCl2组和CaCl2加凝血酶组PRP中生长因

  20. The effects of selective serotonin reuptake inhibitors on platelet function in whole blood and platelet concentrates.

    Science.gov (United States)

    Reikvam, Anne-Grete; Hustad, Steinar; Reikvam, Håkon; Apelseth, Torunn Oveland; Nepstad, Ina; Hervig, Tor Audun

    2012-01-01

    Several studies report that patients who are treated with selective serotonin reuptake inhibitors (SSRIs) for depression may have increased risk of bleeding, particularly from the gastrointestinal tract. This may be related to low intraplatelet serotonin concentrations. Several blood banks do not store platelets from donors using SSRIs for transfusion, although the possible effects of SSRIs on platelet storage are not documented. We conducted a case-control pilot study of apheresis platelet concentrates prepared from donors using SSRIs (n=8) and from donors without medication (n=10). The platelet concentrates were stored for 5 days. Light transmission aggregometry (LTA), thrombelastography (TEG), and flow cytometric analyses were preformed for in vitro measurements of platelet function. Platelet function and platelet serotonin content were investigated in whole blood and in platelet concentrates stored for up to 5 days. LTA, TEG, and flow cytometric analysis of glycoprotein expression did not reveal any significant differences between the two groups. All 18 platelet concentrates performed well according to the standards set for platelet quality in relation to transfusion. Blood donors using SSRIs had significantly lower platelet serotonin compared to blood donors without medication. The results from our pilot study indicate that platelets from donors using SSRIs may be suitable for transfusion after storage for 5 days, but further laboratory and clinical studies are necessary to confirm this.

  1. Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

    Science.gov (United States)

    Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna Freitas Oliveira; Krieger, Marco A; Foti, Leonardo

    2016-08-01

    Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Single-step separation of platelets from whole blood coupled with digital quantification by interfacial platelet cytometry (iPC).

    Science.gov (United States)

    Basabe-Desmonts, L; Ramstrom, S; Meade, G; O'Neill, S; Riaz, A; Lee, L P; Ricco, A J; Kenny, D

    2010-09-21

    We report the efficient single-step separation of individual platelets from unprocessed whole blood, enabling digital quantification of platelet function using interfacial platelet cytometry (iPC) on a chip. iPC is accomplished by the precision micropatterning of platelet-specific protein surfaces on solid substrates. By separating platelets from whole blood using specific binding to protein spots of a defined size, iPC implements a simple incubate-and-rinse approach, without sample preparation, that enables (1) the study of platelets in the physiological situation of interaction with a protein surface, (2) the choice of the number of platelets bound on each protein spot, from one to many, (3) control of the platelet-platelet distance, including the possibility to study noninteracting single platelets, (4) digital quantification (counting) of platelet adhesion to selected protein matrices, enabling statistical characterization of platelet subpopulations from meaningfully large numbers of single platelets, (5) the study of platelet receptor expression and spatial distribution, and (6) a detailed study of the morphology of isolated single platelets at activation levels that can be manipulated. To date, we have demonstrated 1-4 of the above list. Platelets were separated from whole blood using iPC with fibrinogen, von Willebrand factor (VWF), and anti-CD42b antibody printed "spots" ranging from a fraction of one to several platelet diameters (2-24 μm). The number of platelets captured per spot depends strongly on the protein matrix and the surface area of the spot, together with the platelet volume, morphology, and activation state. Blood samples from healthy donors, a May-Hegglin-anomaly patient, and a Glanzmann's Thrombasthenia patient were analyzed via iPC to confirm the specificity of the interaction between protein matrices and platelets. For example, the results indicate that platelets interact with fibrinogen spots only through the fibrinogen receptor (

  3. LPS-activated monocytes are unresponsive to T4 phage and T4-generated Escherichia coli lysate

    Directory of Open Access Journals (Sweden)

    Katarzyna Bocian

    2016-08-01

    Full Text Available A growing body of data shows that bacteriophages can interact with different kinds of immune cells. The objective of this study was to investigate whether T4 bacteriophage and T4-generated Escherichia coli lysate affect functions of monocytes, the key population of immune cells involved in antibacterial immunity. To that end we evaluated how T4 and E. coli lysate influence the expression of main costimulatory molecules including CD40, CD80 and CD86, TLR2, TLR4 on monocytes, as well as the production of IL-6 and IL-12 in cultures of peripheral blood mononuclear cells (PBMCs. Separate experiments were performed on unactivated and LPS-activated PBMCs cultures. Both studied preparations significantly increased the percentage of CD14+CD16-CD40+ and CD14+CD16-CD80+ monocytes in unactivated PBMCs cultures, as well as the concentration of IL-6 and IL-12 in culture supernates. However, neither purified T4 nor E. coli lysate had any significant effect on monocytes in LPS-activated PBMCs cultures. We conclude that LPS-activated monocytes are unresponsive to phages and products of phage-induced lysis of bacteria. This study is highly relevant to phage therapy because it suggests that in patients with infections caused by Gram-negative bacteria the administration of phage preparations to patients and lysis of bacteria by phages are not likely to overly stimulate monocytes.

  4. A3R Phage and Staphylococcus aureus Lysate Do Not Induce Neutrophil Degranulation

    Science.gov (United States)

    Borysowski, Jan; Międzybrodzki, Ryszard; Wierzbicki, Piotr; Kłosowska, Danuta; Korczak-Kowalska, Grażyna; Weber-Dąbrowska, Beata; Górski, Andrzej

    2017-01-01

    The objective of this study was to evaluate the effects of A3R phage and Staphylococcus aureus lysate obtained after phage infection on neutrophil degranulation. The exocytosis of primary and secondary granules from neutrophils was investigated in vitro in whole blood specimens by flow cytometry based on the expression of specific markers of exocytosis (CD63 for primary granules and CD66b for secondary granules). We found that both A3R and S. aureus lysate had no significant effect on the exocytosis of primary and secondary granules. These data suggest that neither A3R virions nor any products of phage-induced lysis of S. aureus are likely to induce neutrophil degranulation in patients who are treated with phage preparations. Since neutrophil granules contain some potentially toxic proteins, our results provide an important argument for the safety of phage therapy. Moreover, these data indicate that the induction of neutrophil degranulation is not likely to contribute to antibacterial effects of phages. PMID:28230780

  5. A3R Phage and Staphylococcus aureus Lysate Do Not Induce Neutrophil Degranulation

    Directory of Open Access Journals (Sweden)

    Jan Borysowski

    2017-02-01

    Full Text Available The objective of this study was to evaluate the effects of A3R phage and Staphylococcus aureus lysate obtained after phage infection on neutrophil degranulation. The exocytosis of primary and secondary granules from neutrophils was investigated in vitro in whole blood specimens by flow cytometry based on the expression of specific markers of exocytosis (CD63 for primary granules and CD66b for secondary granules. We found that both A3R and S. aureus lysate had no significant effect on the exocytosis of primary and secondary granules. These data suggest that neither A3R virions nor any products of phage-induced lysis of S. aureus are likely to induce neutrophil degranulation in patients who are treated with phage preparations. Since neutrophil granules contain some potentially toxic proteins, our results provide an important argument for the safety of phage therapy. Moreover, these data indicate that the induction of neutrophil degranulation is not likely to contribute to antibacterial effects of phages.

  6. Platelet Function Tests

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Platelet Function Tests Share this page: Was this page helpful? ... their patients by ordering one or more platelet function tests. Platelet function testing may include one or more of ...

  7. Congenital platelet function defects

    Science.gov (United States)

    ... storage pool disorder; Glanzmann's thrombasthenia; Bernard-Soulier syndrome; Platelet function defects - congenital ... Congenital platelet function defects are bleeding disorders that ... function, even though there are normal platelet numbers. Most ...

  8. The effect of subarachnoid erythrocyte lysate on brain injury: a preliminary study.

    Science.gov (United States)

    Zhang, Zi-Huan; Han, Yan-Ling; Wang, Chun-Xi; Zhou, Chen-Hui; Wu, Ling-Yun; Zhang, Hua-Sheng; Chen, Qiang; Fan, Jie-Mei; Zhou, Meng-Liang; Hang, Chun-Hua

    2016-08-01

    Abundant erythrocytes remain and lyse partially in the subarachnoid space after severe subarachnoid haemorrhage (SAH). But the effect of subarachnoid erythrocyte lysate on brain injury is still not completely clear. In this study, autologous erythrocytes (the non-lysate group) and their lysate (the lysate group) were injected separately into the cistern magna of rabbits to induce a model of experimental SAH, although the control group received isotonic sodium chloride solution instead of erythrocyte solution. Results showed that vasospasm of the basilar artery was observed at 72 h after experimental SAH, but there was no significant difference between the non-lysate group and the lysate group. Brain injury was more severe in the lysate group than in the non-lysate group. Meanwhile, the levels of peroxiredoxin 2 (Prx2), IL-6 and TNF-α in brain cortex and in CSF were significantly higher in the lysate group than those in the non-lysate group. These results demonstrated that brain injury was more likely to be caused by erythrocyte lysate than by intact erythrocytes in subarachnoid space, and inflammation response positively correlated with Prx2 expression might be involved in mechanism of brain injury after SAH.

  9. The effect of centrifugation speed and time on pre-analytical platelet activation

    DEFF Research Database (Denmark)

    Söderström, Anna Cecilia; Nybo, Mads; Nielsen, Christian

    2016-01-01

    . METHODS: Citrate- and EDTA-anticoagulated blood from healthy volunteers were centrifuged at 80-10,000 g for 5-15 min to prepare plasma and platelet-rich plasma. Pre-analytical platelet activation was assessed by flow cytometric measurement of platelet P-selectin (CD62p) expression. Blood cell counts, mean...... platelet volume (MPV), immature platelet fraction (IPF), and platelet distribution width (PDW) were measured. Platelet aggregation in platelet-rich plasma induced by arachidonic acid (AA), ADP or thrombin receptor activator peptide-6 (TRAP) was tested by 96-well aggregometry. RESULTS: The median percentage...... of platelets expressing P-selectin in citrate- and EDTA-plasma centrifuged at 2000 g for 10 min were 43% [interquartile range (IQR), 38%-53%] and 56% (IQR, 31%-78%), respectively (p=0.82). Platelet-rich plasma prepared at 100-250 g for 10 min had significantly lower platelet P-selectin expression (11%-15%), p...

  10. Modification of solid phase red cell adherence assay for the detection of platelet antibodies in patients with thrombocytopenia.

    Science.gov (United States)

    Vongchan, Preeyanat; Nawarawong, Weerasak; Linhardt, Robert J

    2008-09-01

    Platelet refractoriness is caused by HLA antibodies and platelet-specific antibodies. Current methods used to detect antiplatelet antibodies have limitations. Solid phase red cell adherence (SPRCA) lacks sensitivity and requires a second assay using chloroquine-treated intact platelets to specify the response due to anti-HLA. We modified SPRCA by using 2 types of antihuman platelet antibodies with different specificities toward platelet lysate and tested samples from 361 patients (69 with unexplained thrombocytopenia and 292 with poor response to platelet transfusions not explicable by alloimmunization or the clinical situation) and 50 from healthy volunteers. Our method compared favorably with platelet suspension direct immunofluorescence. All samples from healthy volunteers were negative; of the samples from the patient population, 240 were positive (147 samples had only antiplatelet and 3 samples had only anti-HLA antibodies). This modified technique had a sensitivity of 98% and a specificity of 91%.

  11. 骨髓来源富血小板血浆制备的初步研究%Preparation of bone marrow-derived platelet-rich plasma: a preliminary study

    Institute of Scientific and Technical Information of China (English)

    张永涛; 金丹

    2013-01-01

    目的 探讨BM-PRP制备的可行性并比较BM-PRP与PB-PRP中PLT数量的差异.方法 成年新西兰白兔8只,BM组和PB组分别采用骨髓穿刺和耳中央动脉穿刺抽取标本各10ml/只(含1.5ml4.7%的枸橼酸钠作为抗凝剂),采用二次离心的方法制备新西兰白兔的BM-PRP和PB-PRP,手工计数其中的血小板数量,采用t检验进行统计学分析.结果 BM-PRP中的的PLT数量(2296.76±148.80)×103/μl是PB-PRP中PLT浓度(1633.26±89.64)×103/μl的1.41倍(P <0.001).结论 骨髓穿刺是获取PRP的一种有效方法,并可获得较PB-PRP更高浓度的PRP.%Objective To discuss the preparation of bone marrow-derived platelet-rich plasma ( BM-PRP) and to compare the differences in the number of platelets between BM-PRP and peripheral blood derived platelet-rich plasma (PB-PRP). Methods 10 ml (containing 1. 5 ml 4. 7% sodium citrate as an anticoagulant ) of BM were obtained using bone marrow aspirate techniques and peripheral blood (PB) was drawn from ear central artery of eight adult New Zealand white rabbits. BM-PRP and PB-PRP were obtained using two cen-trifugation techniques. The platelets in samples were counted manually and the data were statistically analyzed by t-test. Results The number of platelets increased 1. 41-fold in samples of BM-PRP (2296. 76 ± 148.80) ×l03/ml compared to PB-PRP (1633.26 ±89.64) ×10-3/ml ( P<0.001). Conclusion Bone marrow aspirate is one of an effective method to obtain BM-PRP ,the number of platelets in BM-RPR samples was significantly higher than PB-PRP samples.

  12. 富血小板凝胶制备方法的比较与优选%Comparison and selective preference of preparation methods of platelet-rich gel

    Institute of Scientific and Technical Information of China (English)

    康健; 袁文

    2014-01-01

    背景:富血小板凝胶含有多种细胞生长因子,对于局部创伤及骨缺损的修复具有非常重要的作用。富血小板凝胶作为组织工程的支架的研究越来越多,但是对于其最优化的制备方法目前尚无统一标准。  目的:简要综述近年来富血小板凝胶所含生长因子及制备方法的相关研究,总结多种富血小板凝胶的制备方法并进行比较,以此找出较为优化的制备方法。  方法:以“富血小板血浆,富血小板凝胶,组织工程”为检索词检索中国期刊全文数据库(1980年1月至2013年1月),以“Platelet-rich plasma,Platelet-rich plasma gel,Tissue Engineering”为检索词检索PubMed数据库(1980年1月至2013年1月),纳入与富血小板凝胶检测及制备方法相关的研究,排除不相关学科及内容的研究,检索得到322篇相关文献,筛选后最终纳入67篇。  结果与结论:目前富血小板凝胶的多种制备方法都存在各自的优势,不能单纯凭借某一项数据断定何种制备方法最好,需根据不同需要确定使用不同的方法。随着富血小板凝胶研究的不断深入,将会对创伤及骨缺损的临床修复以及组织工程研究起到至关重要的作用,也是未来组织工程支架研究的重要方向。%BACKGROUND:Platelet-rich plasma gel contains some kinds of growth factors, which play important roles in repair of local trauma and bone defects. Platelet-rich plasma gel is now more and more studied as tissue engineering scaffolds, but there are no unified preparation methods. OBJECTIVE:To summarize the studies about the preparation methods of platelet-rich plasma gel and the growth factors in platelet-rich plasma gel in recent years, and to compare different methods in order to find which one is better. METHODS:A online search of PubMed database from January 1980 to January 2013 and China National Knowledge Infrastructure from January 1980

  13. Platelet derivatives in regenerative medicine: an update.

    Science.gov (United States)

    De Pascale, Maria Rosaria; Sommese, Linda; Casamassimi, Amelia; Napoli, Claudio

    2015-01-01

    Prior preclinical and clinical studies support the use of platelet-derived products for the treatment of soft and hard tissue lesions. These regenerative effects are controlled by autocrine and paracrine biomolecules including growth factors and cytokines contained in platelet alpha granules. Each growth factor is involved in a phase of the healing process, such as inflammation, collagen synthesis, tissue granulation, and angiogenesis collectively promoting tissue restitution. Platelet derivatives have been prepared as platelet-rich plasma, platelet gel, platelet-rich fibrin, and platelet eye drops. These products vary in their structure, growth factors, composition, and cytokine concentrations. Here, we review the current use of platelet-derived biological products focusing on the rationale for their use and the main requirements for their preparation. Variation in the apparent therapeutic efficacy may have resulted from a lack of reproducible, standardized protocols for preparation. Despite several individual studies showing favorable treatment effects, some randomized controlled trials as well as meta-analyses have found no constant clinical benefit from the application of platelet-derived products for prevention of tissue lesions. Recently, 3 published studies in dentistry showed an improvement in bone density. Seven published studies showed positive results in joint regeneration. Five published studies demonstrated an improvement in the wound healing, and an improvement of eye epithelial healing was observed in 2 reports. Currently, at least 14 ongoing clinical trials in phase 3 or 4 have been designed with large groups of treated patients (n > 100). Because the rationale of the therapy with platelet-derived compounds is still debated, a definitive insight can be acquired only when these large randomized trials will be completed.

  14. A Multicenter study on preparation of Leukodepleted Platelet Concentrates from Pooled whole blood-derived platelets%全血制备浓缩血小板的汇集及滤除白细胞的多中心研究

    Institute of Scientific and Technical Information of China (English)

    王红; 吴瑕; 钟锐; 贺曾; 曹晔; 何语良; 陈洁; 刘嘉馨

    2012-01-01

    Objective To cooperate with a number of blood centers and evaluate the quality of pooled platelet and leukocyte depletion, which aims to supply basis for establishing operating procedures and quality standard of pooled platelet and leukocyte depletion. Methods PCs was prepared from 400 ml fresh whole blood by platelet-rich plasma (PRP) or buffy-coat (BC) method. 10 to 16 units of ABO-matched PCs were pooled, and then filtered with two types of domestic filters (namely group A and B) randomly. The conventional and functional indicators of platelet were evaluated before and after filtering, and the total samples were 202. Results The total of cases that number of platelet was more than 2. 4 × 1011 was 147, including 77 cases in group A and 70 cases in group B. In group A the number of platelet and leukocyte before and after filtering were (2. 90 ±0.45) xlO" 75(2.60±0.43) × 1011 ,(176.45 ±135.67) × 106 VS( 1.00 ±2.29) ×106 respectively ,and in group B the number of platelet and leukocyte before and after filtering were(2. 80 ±0. 36) × 1011 VS(2. 40 ± 0.37) × 1011, (175. 76 ±147. 84) × 106 VS(0. 30 ±0. 72) × 106 respectively. While the total of cases that number of platelet was less than 2. 4 × 1011 was 55, including 29 cases in group A and 26 cases in group B. In group A the number of platelet and leukocyte before and after filtering were (1. 71 ±0.39)×1011 VS( 1.43 ±0.42) × 1011 ,(65. 85 ±110. 97) ×106 VS (3. 7 ±3. 98) × 106 respectively,and in group B the number of platelet and leukocyte before and after filtering were(l. 79 ±0.48) ×1011 VS(1.42±0.46) × 1011 ,(70. 63 ±145. 55) × 106 VS(1. 45 ±2. 66) × 106 respectively. There were no significant difference(P>0. 05) of these indicators such as pH value,hypotonic shock response (HSR) ,CD62p expression ( % ) and platelet aggregation after filtering in group A and B. Conclusion Pooling and filtering platelet concentrates prepared by PRP and BC method can remove leukocyte effectively and

  15. Radiation-induced volatile hydrocarbon production in platelets

    Energy Technology Data Exchange (ETDEWEB)

    Radha, E.; Vaishnav, Y.N.; Kumar, K.S.; Weiss, J.F.

    1989-01-01

    Generation of volatile hydrocarbons (ethane, pentane) as a measure of lipid peroxidation was followed in preparations from platelet-rich plasma irradiated in vitro. The hydrocarbons in the headspace of sealed vials containing irradiated and nonirradiated washed platelets, platelet-rich plasma, or platelet-poor plasma increased with time. The major hydrocarbon, pentane, increased linearly and significantly with increasing log radiation dose, suggesting that reactive oxygen species induced by ionizing radiation result in lipid peroxidation. Measurements of lipid peroxidation products may give an indication of suboptimal quality of stored and/or irradiated platelets.

  16. Platelet-functionalized three-dimensional poly-ε-caprolactone fibrous scaffold prepared using centrifugal spinning for delivery of growth factors.

    Science.gov (United States)

    Rampichová, Michala; Buzgo, Matej; Míčková, Andrea; Vocetková, Karolína; Sovková, Věra; Lukášová, Věra; Filová, Eva; Rustichelli, Franco; Amler, Evžen

    2017-01-01

    Bone and cartilage are tissues of a three-dimensional (3D) nature. Therefore, scaffolds for their regeneration should support cell infiltration and growth in all 3 dimensions. To fulfill such a requirement, the materials should possess large, open pores. Centrifugal spinning is a simple method for producing 3D fibrous scaffolds with large and interconnected pores. However, the process of bone regeneration is rather complex and requires additional stimulation by active molecules. In the current study, we introduced a simple composite scaffold based on platelet adhesion to poly-ε-caprolactone 3D fibers. Platelets were used as a natural source of growth factors and cytokines active in the tissue repair process. By immobilization in the fibrous scaffolds, their bioavailability was prolonged. The biological evaluation of the proposed system in the MG-63 model showed improved metabolic activity, proliferation and alkaline phosphatase activity in comparison to nonfunctionalized fibrous scaffold. In addition, the response of cells was dose dependent with improved biocompatibility with increasing platelet concentration. The results demonstrated the suitability of the system for bone tissue.

  17. Multiwavelength UV/visible spectroscopy for the quantitative investigation of platelet quality

    Science.gov (United States)

    Mattley, Yvette D.; Leparc, German F.; Potter, Robert L.; Garcia-Rubio, Luis H.

    1998-04-01

    The quality of platelets transfused is vital to the effectiveness of the transfusion. Freshly prepared, discoid platelets are the most effective treatment for preventing spontaneous hemorrhage or for stopping an abnormal bleeding event. Current methodology for the routine testing of platelet quality involves random pH testing of platelet rich plasma and visual inspection of platelet rich plasma for a swirling pattern indicative of the discoid shape of the cells. The drawback to these methods is that they do not provide a quantitative and objective assay for platelet functionality that can be used on each platelet unit prior to transfusion. As part of a larger project aimed at characterizing whole blood and blood components with multiwavelength UV/vis spectroscopy, isolated platelets and platelet in platelet rich plasma have been investigated. Models based on Mie theory have been developed which allow for the extraction of quantitative information on platelet size, number and quality from multi-wavelength UV/vis spectra. These models have been used to quantify changes in platelet rich plasma during storage. The overall goal of this work is to develop a simple, rapid quantitative assay for platelet quality that can be used prior to platelet transfusion to ensure the effectiveness of the treatment. As a result of this work, the optical properties for isolated platelets, platelet rich plasma and leukodepleted platelet rich plasma have been determined.

  18. Evaluation of platelet aggregation in platelet concentrates: storage implications

    Directory of Open Access Journals (Sweden)

    Neiva Teresinha J.C.

    2003-01-01

    Full Text Available The use of hemo-derivatives is nowadays a fundamentally important therapeutic modality in the exercise of medicine. Among the various hemo-components employed, we have the platelet concentrate (PC, indicated in cases of hemorrhagic disturbances. We previously showed that platelet function in blood donors is reduced in their screening phase and after the separation process of PCs. Currently, we are providing evidence for the existence of biochemical and functional changes in PC preparations stored for three days at temperatures of 20 ± 2 ºC. Platelet concentrates from 40 healthy donors, collected in CPD anticoagulant and PL-146 polyvinylchloride containers, were examined in order to determine the pH value, pCO2 ,pO2 and lactate concentrations. In addition, the aggregation of platelets with thrombin and collagen were examined to evaluate platelet function. A pH increase from 7.07 ± 0.04 to 7.36 ± 0.07 (p < 0.01 was observed. The pCO2 concentration decreased progressively from 69.2 ± 7.7 mmHg to 28.8 ± 6.2 mmHg (p < 0.001 during the storage period. In contrast, pO2 value increase from 103.4 ± 30.6 to 152.3 ± 24.6 mmHg (p < 0.001 was evidenced during the 48 hours of storage. The lactate concentration increased from 17.97 ± 5.2 to 57.21 ± 5.7 mg/dl (p < 0.001. Platelet aggregation using 0.25 U/ml-thrombin and 2.0 µg/ml-collagen showed significant hypofunction from 61.8 ± 2.7% to 24.8 ± 9.8% and 62.7±5.0 to 33.4± 6.2 (p < 0.001, respectively. We concluded that the evaluated biochemical parameters and the platelet function changed significantly when the platelets were kept under routine storage conditions.

  19. Detection of circulating tumor lysate-reactive CD4+ T cells in melanoma patients

    DEFF Research Database (Denmark)

    Ladekarl, Morten; Agger, Ralf; Fleischer, Charlotte C

    2004-01-01

    PURPOSE: We wanted to study whether an allogeneic melanoma lysate would be a feasible stimulatory antigen source for detection of a peripheral CD4+ T-cell immune response in patients with medically untreated malignant melanoma. The lysate was produced from a melanoma cell line (FM3.29) which expr...

  20. Clinical application of radiolabelled platelets

    Energy Technology Data Exchange (ETDEWEB)

    Kessler, C. (Medical Univ. Lubeck, Lubeck (DE))

    1990-01-01

    This book presents papers on the clinical applications of radiolabelled platelets. The papers are grouped into six sections on platelet labelling techniques, radiolabelled platelets in cardiology, monitoring of antiplatelet therapy, platelet scintigraphy in stroke patients, platelet scintigraphy in angiology, and platelet scintigraphy in hematology and other clinical applications, including renal transplant rejection.

  1. 抗凝剂对富含血小板血浆质量的影响%The study of anticoagulants selection in platelet-rich plasma preparation

    Institute of Scientific and Technical Information of China (English)

    雷华; 归来; 刘珍君; 马桂娥

    2015-01-01

    目的 探讨抗凝剂对富含血小板血浆中血小板活性的影响,以明确抗凝剂的选择是否影响富含血小板血浆的质量.方法 分别用肝素钠、枸橼酸钠、抗凝枸橼酸葡萄糖、枸橼酸-茶碱-腺苷-潘生丁作为抗凝剂,抽取志愿者全血,分离血小板、制备富含血小板血浆.电镜观察血小板的微观结构;检测血浆中可溶性P-选择素浓度,以观察血小板的自发激活情况;测定富含血小板血浆激活后转移生长因子释放量.结果 枸橼酸-茶碱-腺苷-潘生丁抗凝剂和抗凝枸橼酸葡萄糖与肝素钠和枸橼酸钠相比,能长时间保持血小板结构完整,减少血小板在富含血小板血浆制备过程中的自发激活.抗凝枸橼酸葡萄糖对血小板的保护作用较枸橼酸-茶碱-腺苷-潘生丁弱,但二者与肝素钠和枸橼酸钠相比,均能使富含血小板血浆释放较多的生长因子.结论 不同抗凝剂对富含血小板血浆的质量和生物活性有显著影响.复合试剂抗凝枸橼酸葡萄糖、枸橼酸-茶碱-腺苷-潘生丁能更长时间地保持富含血小板血浆的生物活性,提高其质量.%Objective To investigate the effect of the anticoagulants on PRP quality,so as to clarify the appropriate anticoagulant used in PRP production.Methods The microstructure change of platelets collected via heparin,citrate,acid citrate dextrose (ACD) and citrate-theophylline-adenosinedipyridamole (CTAD) was observed by TEM following time course.The extent of spontaneous activation of platelets in four groups was detected by measuring sP-selectin in plasma.The TGF-β1 release amount of activated PRP of four groups was measured.Results CTAD is superior to other anticoagulants in maintaining the integrity of platelet structures for a long time and preventing platelet spontaneous activation.ACD slightly surpassed heparin and citrate in above two aspects.ACD-PRP and CTAD-PRP released significantly more TGF-β1 compared with heparin

  2. Protein microarrays using liquid phase fractionation of cell lysates.

    Science.gov (United States)

    Yan, Fang; Sreekumar, Arun; Laxman, Bharathi; Chinnaiyan, Arul M; Lubman, David M; Barder, Timothy J

    2003-07-01

    We describe an approach in which protein microarrays are produced using a two-dimensional (2-D) liquid phase fractionation of cell lysates. The method involves a pI-based fractionation using chromatofocusing in the first dimension followed by nonporous reversed-phase high-performance liquid chromatography (HPLC) of each pI fraction in the second dimension. This allows fractionation of cellular proteins in the liquid phase that could then be arrayed on nitrocellulose slides and used to study humoral response in cancer. Protein microarrays have been used to identify potential serum biomarkers for prostate cancer. It is shown that specific fractions are immunoreactive against prostate cancer serum but not against serum from healthy individuals. These proteins could serve as sero-diagnostic markers for prostate cancer. Importantly, this method allows for use of post-translationally modified proteins as baits for detection of humoral response. Proteins eliciting an immune response are identified using the molecular mass and peptide sequence data obtained using mass spectrometric analysis of the liquid fractions. The fractionation of proteins in the liquid phase make this method amenable to automation.

  3. Preparation of calcium sulfate and platelet-rich plasma bioactive scaffold%硫酸钙/富血小板血浆活性支架材料的制备

    Institute of Scientific and Technical Information of China (English)

    张杨; 尚万兵

    2012-01-01

    目的 探讨硫酸钙/富血小板血浆人工骨活性支架材料的制备.方法 将富血小板血浆和硫酸钙混合后迅速搅拌至糊状,常温常压下压制成型,并用扫描电镜和X线衍射对复合物进行分析.结果 硫酸钙晶体基本保持了原有的晶体形貌.复合支架材料以5~10μm硫酸钙柱状晶体为骨架,其间布满直径约1μm的片状晶体,分布均匀,使晶体间的结合更加紧密.硫酸钙支架材料产物为二水硫酸钙,主要出峰位置与二水硫酸钙基本一致,但强度有所变化,主峰附近小的衍射峰为血浆中的蛋白质成分形成.结论 以硫酸钙/富血小板血浆制备的人工骨活性支架材料具骨诱导性,支架材料结构致密.%Objective To explore the preparation of calcium sulfate and platelet-rich plasma bioactive scaffold. Methods The calcium sulfate and platelet-rich plasma were mixed and then agitated quickly to pasty,and were molded. The compounds was analysed by scanning electron micrograph and X-ray crystal diffraction. Results Calcium sulfate crystals remained of the original crystal morphology. The columnar crystals of calcium sulfate (5 - 10 μm) was the skeleton of composite scaffold. The composite scaffold was covered with flaky crystals which diameter of about 1 μm. The flaky crystals were uniform distribution, which made the crystals were more closely. Calcium sulfate scaffold was composed of calcium sulfate dehydrate,the main peak position was same with the calcium sulfate dehydrate, but the intensity changed. The small diffraction peaks near the main peak were formed by protein of the plasma. Conclusion The artificial bioactive bone scaffolds which were prepared by calcium sulfate/platelet-rich plasma have the bone induction, and the scaffold structure is dense.

  4. Detection of dengue virus in platelets isolated from dengue patients.

    Science.gov (United States)

    Noisakran, Sansanee; Gibbons, Robert V; Songprakhon, Pucharee; Jairungsri, Aroonroong; Ajariyakhajorn, Chuanpis; Nisalak, Ananda; Jarman, Richard G; Malasit, Prida; Chokephaibulkit, Kulkanya; Perng, Guey Chuen

    2009-03-01

    Though thrombocytopenia or dysfunction of platelets is common in dengue virus infection, the role of platelets has not been established. We enrolled 33 hospitalized children with serologically confirmed dengue virus infection. Blood specimens were collected during hospitalization. Platelets and plasma were isolated from the whole blood. Detection of dengue virus in plasma and platelets was carried out by RT-PCR with primers that can differentiate different dengue serotypes simultaneously, and by electron transmission microscopy (EM). Dengue viral RNA was detected in the platelets and plasma by conventional RT-PCR. A significantly higher percentage of dengue viral RNA was detected in platelets than in plasma (p = 0.03). Platelets isolated 5 days after onset of fever were most likely positive for viral RNA. Concurrent infection or co-circulation with multiple dengue serotypes was observed in 12% of patients. Infrequently, negative-stranded dengue viral RNA was detected in platelets and in plasma. Importantly, EM confirmed the presence of dengue viral-like particles inside platelets prepared from dengue patients. Our findings suggest the presence of dengue virus in platelets may be associated with the dysfunction of platelets observed in dengue patients.

  5. Pooled platelet concentrates: an alternative to single donor apheresis platelets?

    Science.gov (United States)

    Pietersz, R N I

    2009-10-01

    Three types of platelet concentrates (PC) are compared: PC either processed with the platelet-rich plasma (PRP) or the Buffy coat (BC) method from whole blood units and PC obtained by apheresis. Leuko-reduction (LR) pre-storage is advocated to improve quality of the PC during storage and reduce adverse reactions in recipients. Standardization of methods allow preparation of PC with comparable yields of approximately 400 x 10(9) platelets in pooled non-LR-PRP, approximately 370 x 10(9) in pooled LR-BC-PC and in LR apheresis PC the number of platelets can be targeted on 350 x 10(9) or more with devices of various manufacturers. While viral transmission can be prevented by outstanding laboratory tests, the risk of bacterial contamination should be reduced by improved arm disinfection, deviation of the first 20-30 ml of blood and culture or rapid detection assays of the PC pre-issue. In a large prospective multicenter trial no significant difference was observed between cultures of apheresis PC (n = 15,198): 0.09% confirmed positive units versus 0.06% in pooled BC-PC (n = 37,045), respectively. Though platelet activation as measured by CD62 expression may differ in vitro in PC obtained with various apheresis equipment, and also between PC processed with the two whole blood methods there is scarce literature about the clinical impact of these findings. In conclusion the final products of LR-PC derived from whole blood or obtained by apheresis can be comparable, provided the critical steps of the processing method are identified and covered and the process is in control.

  6. In vitro model of platelet aggregation in stenotic arteries

    Energy Technology Data Exchange (ETDEWEB)

    Morley, D.; Santamore, W.P.

    1988-07-01

    Clinical and experimental evidence suggest a strong relationship between arterial stenosis, platelet aggregation, and subsequent thrombus formation. To facilitate the study of platelet accumulation in stenotic arteries, we developed an in vitro preparation. Arterial segments were perfused with whole citrated blood. A stenosis was created by applying an external plastic constrictor to the artery. Platelet accumulation within the stenosis was assessed by scanning electron microscopy and by radioactive counts from Indium-111 labeled platelets. Utilizing this preparation, 30 carotid arterial segments from 10 mongrel dogs were perfused at 100 mmHg for 15 min. In 10 arteries without a stenosis, scanning electron microscopy and radioactive counts demonstrated little platelet accumulation. In contrast, extensive platelet aggregation was observed in 10 arteries with stenoses. Moreover, in 10 stenotic arteries exposed to the thromboxane mimetic, U46619 (Upjohn Diagnostic Group), scanning electron microscopy and radioactive counts demonstrated a significant increase in platelet deposition. Conversely, we demonstrated a dimunition of platelet accumulation in stenosed arterial segments exposed to the prostacyclin analogue platelet inhibitor, Iloprost. The in vitro preparation allows precise control of hemodynamic variables and makes it possible to perform multiple tests on segments of the same vessel from the same animal.

  7. Platelets and hemostasis

    Directory of Open Access Journals (Sweden)

    M. A. Panteleev

    2014-09-01

    Full Text Available Platelets are anuclear cell fragments playing important role in hemostasis, termination of bleeding after damage, as well as in pathological thrombus formation. The main action of platelets is the formation of aggregates, overlapping the injury. They obtained the ability to aggregate by the transition process called activation. Despite the relatively simple and definite function platelet structure is very difficult: they have almost a full set of organelles, including the endoplasmic reticulum, mitochondria and other entities. When activated platelets secrete various granules interact with plasma proteins and red blood cells and other tissues. Their activation is controlled by multiple receptors and complex signaling cascades. In this review platelet structure, mechanisms of its functioning in health and disease, diagnostic methods of platelet function and approaches to their correction were considered. Particular attention will be given to those areas of the science of platelets, which still lay hidden mysteries.

  8. The influence of environmental variables on platelet concentration in horse platelet-rich plasma

    OpenAIRE

    Rinnovati, Riccardo; Romagnoli, Noemi; Gentilini, Fabio; Lambertini, Carlotta; Spadari, Alessandro

    2016-01-01

    Platelet-rich plasma (PRP) commonly refers to blood products which contain a higher platelet (PLT) concentration as compared to normal plasma. Autologous PRP has been shown to be safe and effective in promoting the natural processes of soft tissue healing or reconstruction in humans and horses. Variability in PLT concentration has been observed in practice between PRP preparations from different patients or from the same individual under different conditions. A change in PLT concentration cou...

  9. Inactivation of human immunodeficiency virus type 1 in blood samples stored as high-salt lysates.

    Science.gov (United States)

    Zolg, J W; Lanciotti, R S; Wendlinger, M; Meyer, W A

    1990-09-01

    Blood samples to be tested for the presence of parasite DNA by using specific DNA probes are routinely stored in our laboratory as high-salt lysates (HSL). To safeguard against the risk of accidental infection with etiological agents such as the human immunodeficiency virus type 1 (HIV-1) while manipulating large numbers of blood samples in preparation for DNA probing, we determined the residual infectivity of HIV-1 after exposure to HSL components. Both high-titer virus stocks or provirus-carrying cells, suspended either in tissue culture medium or freshly drawn blood, were completely inactivated upon contact with the HSL components. This was verified by the absence of any detectable HIV-1-specific antigen in the supernatants of long-term cultures and the absence of virus-specific DNA fragments after amplification by polymerase chain reaction with DNA from such cultures as target DNA. These results support the conclusion that the virus is in fact completely inactivated by contact with the HSL components, rendering blood specimens stored as HSL noninfectious in regard to HIV-1.

  10. Purification of supercoiled plasmid DNA from clarified bacterial lysate by arginine-affinity chromatography: effects of spacer arms and ligand density.

    Science.gov (United States)

    Bai, Jin-Shan; Bai, Shu; Shi, Qing-Hong; Sun, Yan

    2014-06-01

    Efficient loading on a chromatographic column is the dilemma of the process development faced by engineers in plasmid DNA purification. In this research, novel arginine-affinity chromatographic beads were prepared to investigate the effect of spacer arm and ligand density to their chromatographic performance for the purification of plasmid. The result indicated that dynamic binding capacity for plasmid increased with an increasing ligand density and carbon number of spacer arm, and the highest binding capacity for plasmid of 6.32 mg/mL bead was observed in the column of arginine bead with a ligand density of 47 mmol/L and 10-atom carbon spacer. Furthermore, this arginine bead exhibited better selectivity to supercoiled (sc) plasmid. The evidence of a linear gradient elution suggested further that the binding of plasmid on arginine beads was driven by electrostatic interaction and hydrogen bonding. Hence, sc plasmid could successfully be purified from clarified lysate by two-stepwise elution of salt concentration. By the refinement of the elution scheme and loading volume of clarified lysate, the column of arginine bead with a ligand density of 47 mmol/L exhibited the highest recovery yield and a much higher productivity among arginine-affinity columns. Therefore, reshaped arginine beads provided more feasible and practical application in the preparation of sc plasmid from clarified lysate. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Selective Capture of Histidine-tagged Proteins from Cell Lysates Using TEM grids Modified with NTA-Graphene Oxide

    Science.gov (United States)

    Benjamin, Christopher J.; Wright, Kyle J.; Bolton, Scott C.; Hyun, Seok-Hee; Krynski, Kyle; Grover, Mahima; Yu, Guimei; Guo, Fei; Kinzer-Ursem, Tamara L.; Jiang, Wen; Thompson, David H.

    2016-10-01

    We report the fabrication of transmission electron microscopy (TEM) grids bearing graphene oxide (GO) sheets that have been modified with Nα, Nα-dicarboxymethyllysine (NTA) and deactivating agents to block non-selective binding between GO-NTA sheets and non-target proteins. The resulting GO-NTA-coated grids with these improved antifouling properties were then used to isolate His6-T7 bacteriophage and His6-GroEL directly from cell lysates. To demonstrate the utility and simplified workflow enabled by these grids, we performed cryo-electron microscopy (cryo-EM) of His6-GroEL obtained from clarified E. coli lysates. Single particle analysis produced a 3D map with a gold standard resolution of 8.1 Å. We infer from these findings that TEM grids modified with GO-NTA are a useful tool that reduces background and improves both the speed and simplicity of biological sample preparation for high-resolution structure elucidation by cryo-EM.

  12. Transcellular lipoxygenase metabolism between monocytes and platelets

    Energy Technology Data Exchange (ETDEWEB)

    Bigby, T.D.; Meslier, N. (Univ. of California, San Francisco (USA))

    1989-09-15

    We have examined the effects of co-culture and in vitro co-stimulation on lipoxygenase metabolism in monocytes and platelets. Monocytes were obtained from the peripheral blood of normal volunteers by discontinuous gradient centrifugation and adherence to tissue culture plastic. Platelets were obtained from the platelet-rich plasma of the same donor. When 10(9) platelets and 2.5 x 10(6) monocytes were co-stimulated with 1 microM A23187, these preparations released greater quantities of 12(S)-hydroxy-10-trans-5,8,14-cis-eicosatetraenoic acid, 5(S),12-(S)dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, and leukotriene C4, 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic (LTC4) when compared with monocytes alone. Release of arachidonic acid, 5-HETE, delta 6-trans-LTB4, and delta 6-trans-12-epi-LTB4 from monocytes was decreased in the presence of platelets. A dose-response curve was constructed and revealed that the above changes became evident when the platelet number exceeded 10(7). Dual radiolabeling experiments with 3H- and 14C-arachidonic acid revealed that monocytes provided arachidonic acid, 5-HETE, and LTA4 for further metabolism by the platelet. Monocytes did not metabolize platelet intermediates detectably. In addition, as much as 1.2 microM 12(S)-hydroxy-10-trans-5,8,14-cis-eicosatetraenoic acid and 12(S)-hydroperoxy-10-trans-5,8,14-cis-eicosatetraenoic acid had no effect on monocyte lipoxygenase metabolism. Platelets were capable of converting LTA4 to LTC4, but conversion of LTA4 to LTB4 was not detected. We conclude that the monocyte and platelet lipoxygenase pathways undergo a transcellular lipoxygenase interaction that differs from the interaction of the neutrophil and platelet lipoxygenase pathways. In this interaction monocytes provide intermediate substrates for further metabolic conversion by platelets in an unidirectional manner.

  13. Preparation

    Directory of Open Access Journals (Sweden)

    M.M. Dardir

    2014-03-01

    Full Text Available Some hexanamide-mono and di-linoleniate esters were prepared by the reaction of linolenic acid and hexanamide (derived from the reaction of hexanoic acid and diethanolamine. The chemical structure for the newly prepared hexanamide-mono and di-linoleniate esters were elucidated using elemental analysis, (FTIR, H 1NMR and chemical ionization mass spectra (CI/Ms spectroscopic techniques. The results of the spectroscopic analysis indicated that they were prepared through the right method and they have high purity. The new prepared esters have high biodegradability and lower toxicity (environmentally friendly so they were evaluated as a synthetic-based mud (ester-based mud for oil-well drilling fluids. The evaluation included study of the rheological properties, filtration and thermal properties of the ester based-muds formulated with the newly prepared esters compared to the reference commercial synthetic-based mud.

  14. Morphometric analysis of density subpopulations of normal human platelets.

    Science.gov (United States)

    Chamberlain, K G; Froebel, M; Macpherson, J; Penington, D G

    1988-08-30

    Platelets from seven normal subjects were fractionated on continuous Percoll density gradients and low density (LD), intermediate, and high density (HD) platelets were prepared for transmission electron microscopy followed by computerised morphometric analysis. Normal ultrastructural appearance and discoid shape were preserved by incubation of the platelets in nutrient medium at 37 degrees C immediately before fixation. HD platelet sections had a larger mean cross-sectional area but a lower ratio of the major to the minor axis compared to LD platelet sections. HD platelets contained more alpha granules, dense granules and mitochondria per square micron of section area than LD platelets. The percentage of section area occupied by open canalicular system was greater in the LD platelets while the percentage area occupied by glycogen fields was over ten-fold higher in the HD platelets. The mean cross-sectional areas of individual alpha granules and dense granules increased with density while the opposite trend was found for mitochondria. It is suggested that these ultrastructural differences mainly arise during thrombopoiesis and may indicate some functional specialization among platelets.

  15. Feasibility of Using Multilayer Platelets as Toughening Agents

    Directory of Open Access Journals (Sweden)

    Yuan-Liang Chin

    2009-12-01

    Full Text Available It is known that the toughness of brittle ceramics can be improved significantly with the addition of hard platelets. In the present study, platelet-shape multilayer ceramic laminates are utilized as a toughening agent for alumina ceramics. They are prepared by laminating the BaTiO3-based ceramic tapes. Although the elastic modulus of the BaTiO3-based platelets is lower than that of the alumina matrix, and the platelets are also reactive to alumina at elevated temperatures, the weak platelets are found to exhibit the ability to deflect major matrix cracks by forming a large number of microcrack branches within the platelets, thus achieving the desired toughening effect.

  16. Organic extraction of bone lysates improves DNA purification with silica beads.

    Science.gov (United States)

    Desmyter, Stijn; De Cock, Greet; Moulin, Sabine; Noël, Fabrice

    2017-04-01

    In our standard protocol for DNA extraction from skeletal remains of unidentified bodies, bone lysates resulting from decalcification and Proteinase K treatment were purified with the DNA IQ™ Casework Pro Kit for Maxwell(®)16 automate (Promega, WI). Despite its success in the majority of cases, the DNA purification with paramagnetic silica beads failed in some challenging samples. This failure in DNA recovery was often associated with filter clogging during the required volume reduction of the lysate to enable loading on the automate. Two modifications to the standard method were tested for a more efficient filtering and purification. Adding collagenase to the lysate reduced the filter lead time but did not enhance DNA yield, while organic extraction of the crude lysate solved the filter clogging and resulted in successful DNA purification. The modified method in which a phenol treated lysate was loaded on the automate resulted in successful STR-profiling of the skeletal remains of all 13 unidentified bodies tested, which showed a wide variety in post mortem interval and preservation conditions. The variation in DNA yield between the 28 samples tested showed the importance of bone type selection and multiple sampling in successful STR-profiling of skeletal remains. Despite the disadvantages inherent to phenol, the organic extraction of crude bone lysates enhanced the efficiency of DNA purification with paramagnetic silica beads. The combined method of organic extraction and purification with silica beads resulted in STR-profiling of challenging bone samples.

  17. Biochemical characterization of PECAM-1 (CD31 antigen) on human platelets.

    Science.gov (United States)

    Metzelaar, M J; Korteweg, J; Sixma, J J; Nieuwenhuis, H K

    1991-12-02

    The platelet plasma membrane expresses several membrane glycoproteins with a high molecular weight. In this study we have investigated the properties of the CD31 antigen on platelets and endothelial cells using the monoclonal antibody (MoAb) RUU-PL 7E8. Comparative studies revealed that the CD31 antigen, PECAM-1 and endoCAM are the same protein. The CD31 antigen was immunoprecipitated with a molecular mass of 125 kDa nonreduced and 135 kDa reduced from Nonidet-P40 lysates of surface labeled human platelets. The relative position in two-dimensional nonreduced/reduced SDS-PAGE and IEF-PAGE, compared to other glycoproteins of similar molecular weight, was elucidated. The position of the CD31 antigen was clearly distinct from the position of the platelet membrane glycoproteins Ia, Ib, IIa, IIb, IIIa and the granule membrane protein GMP-140. Native resting platelets bound 7,760 +/- 1,670 molecules/platelet, whereas thrombin-stimulated platelets bound 14,500 +/- 3,790 molecules/platelet. Immunoelectron microscopy revealed the presence of the CD31 antigen on the membrane of both resting and thrombin-activated platelets. Immunofluorescence studies showed the presence of the CD31 antigen in the membrane of endothelial cells on sites of cell-cell contact, suggesting that the CD31 antigen might be involved in cell-cell interaction. In functional studies, MoAb RUU-PL 7E8 did not inhibit platelet aggregation, platelet adherence to the extracellular matrix of endothelial cells and purified collagen fibrils under flow conditions, nor was any influence found on endothelial cell detachment and growth.

  18. Effects of argon laser on in vitro aggregation of platelets in platelet rich plasma and whole blood

    Energy Technology Data Exchange (ETDEWEB)

    Doerger, P.T.; Glueck, H.I.; McGill, M.

    1988-06-01

    The effects of an Argon laser on platelet aggregation were studied, since platelets may be exposed to laser energy when used intravascularly. Various preparations of platelets in platelet rich plasma (PRP) and whole blood, with or without aspirin, were tested with the aggregating agents ADP, collagen, thrombin, and epinephrine. Simultaneous release of ATP was also measured in PRP. At relatively low levels of irradiation, platelet aggregation was potentiated. Enhancement was evidenced by an increase in percent aggregation, earlier onset of the reaction, and reduction in the amount of aggregating agent required. In PRP, the mechanism of laser potentiation appeared to be the release of endogenous ATP from platelets. At relatively high levels of irradiation, platelets were destroyed and aggregation abolished. In whole blood, the mechanism was somewhat more complicated since release of ATP occurred from RBCs as well as platelets. Spontaneous aggregation following laser treatment occurred in isolated instances in PRP and in every trial in whole blood preparations. Aspirin ingestion inhibited the laser's effects in PRP but not in whole blood. These results may have important clinical implications for laser angioplasty, and the potentiated aggregation response may prove useful in laboratory studies of platelet function.

  19. Era of blood component therapy: time for mandatory pre-donation platelet count for maximizing donor safety and optimizing quality of platelets.

    Science.gov (United States)

    Das, Sudipta Sekhar; Zaman, R U; Biswas, Dipak

    2013-12-01

    Blood bank regulatory agencies including the Drug and Cosmetics Act (DCA) of India do not mandate a predonation platelet count in whole blood donation. Mandating such practice will definitely optimize the quality of random donor platelets (RDP) in terms of platelet yield and patient therapeutic benefit. We observed poor platelet yield in RDP concentrates prepared at our center with a significant number not meeting the DCA guideline of ≥ 4.5 × 10(10) per bag processed from 450 ml of whole blood. Therefore we planned this study to evaluate the pre-donation hematological values in our blood donor population and effect of these values on the quality of platelet concentrates. The prospective study included 221 blood donors eligible for donating 450 ml of whole blood (WB). Following the departmental standard operating procedure (SOP) RDPs were prepared using the 'Top & Bottom' quadruple bag system and automated component extractor. Quality of RDP was assessed as per departmental protocol. All results were recorded and subsequently transcribed to SPSS working sheet. A significant (pblood counts has been observed after WB donation. Mean donor Hb and platelets reduced by 0.72 g/dl and 22.1 × 10(6)/ml respectively. Quality of RDPs in terms of platelet yield was significantly better (pcount was >200 × 10(6)/ml. Although platelet yield significantly correlated with the donor platelet count however quality of RDPs in terms of red cell contamination showed no correlation with the donor hematocrit. Platelet yield in random donor platelets is a concern in Eastern India. A platelet yield of 4.5 × 10(10) per bag as mandated by the DCA of India was only achieved when the donor platelet count was >200 × 10(6)/ml. Posttransfusion platelet recovery (PPR) was unsatisfactory in the transfused patient. Introduction of pre-donation platelet count in whole blood donation will maximize donor safety and optimize patient platelet transfusion management.

  20. Catabolism of exogenously supplied thymidine to thymine and dihydrothymine by platelets in human peripheral blood

    Energy Technology Data Exchange (ETDEWEB)

    Pero, R.W.; Johnson, D.; Olsson, A.

    1984-11-01

    The interference of platelets with the estimation of unscheduled DNA synthesis in human peripheral mononuclear leukocytes following genotoxic exposure was studied. A 96% reduction in the unscheduled DNA synthesis value was achieved by incubating (/sup 3/H)thymidine with platelet-rich plasma for 5 hr at 37 degrees. Using radioactive thymine-containing compounds, together with quantitative analyses based on thin-layer and ion-exchange chromatographies, we have shown that thymidine was converted to thymine which, in turn, was converted to dihydrothymine in platelet-rich plasma. The enzymes responsible were separated from platelet lysates by gel filtration and were identified as thymidine phosphorylase and dihydrothymine dehydrogenase. The phosphorylase reversibly catalyzed the formation of thymine from thymidine and converted bromodeoxyuridine to bromouracil. The dehydrogenase reversibly catalyzed the interconversion of thymine and dihydrothymine in a reaction dependent on NADP(H), and it was inhibited by diazouracil and by thymine. Nearly all the thymidine-catabolizing activity found in whole blood samples supplied exogenously with thymidine was accounted for by the platelets. Since most genetic toxicological tests that use blood samples do not involve removing platelets from the blood cell cultures, then it is concluded that precautions should be taken in the future to determine the influence of platelets on these test systems. This is particularly true for methods dependent on thymidine pulses such as unscheduled DNA synthesis, or those dependent on bromodeoxyuridine, such as sister chromatid exchanges, since this nucleoside is also a substrate for thymidine phosphorylase.

  1. Platelet alloimmunization after transfusion

    DEFF Research Database (Denmark)

    Taaning, E; Simonsen, A C; Hjelms, E;

    1997-01-01

    BACKGROUND AND OBJECTIVES: The frequency of platelet-specific antibodies after one series of blood transfusions has not been reported, and in multiply transfused patients is controversial. MATERIALS AND METHODS: We studied the frequency of alloimmunization against platelet antigens in 117 patient...

  2. Flavanols and Platelet Reactivity

    Directory of Open Access Journals (Sweden)

    Debra A. Pearson

    2005-01-01

    Full Text Available Platelet activity and platelet-endothelial cell interactions are important in the acute development of thrombosis, as well as in the pathogenesis of cardiovascular disease. An increasing number of foods have been reported to have platelet-inhibitory actions, and research with a number of flavanol-rich foods, including, grape juice, cocoa and chocolate, suggests that these foods may provide some protection against thrombosis. In the present report, we review a series of in vivo studies on the effects of flavanol-rich cocoa and chocolate on platelet activation and platelet-dependent primary hemostasis. Consumption of flavanol-rich cocoa inhibited several measures of platelet activity including, epinephrine- and ADP-induced glycoprotein (GP IIb/IIIa and P-Selectin expression, platelet microparticle formation, and epinephrine-collagen and ADP-collagen induced primary hemostasis. The epinephrine-induced inhibitory effects on GP IIb/IIIa and primary hemostasis were similar to, though less robust than those associated with the use of low dose (81 mg aspirin. These data, coupled with information from other studies, support the concept that flavanols present in cocoa and chocolate can modulate platelet function through a multitude of pathways.

  3. Platelet activation and aggregation

    DEFF Research Database (Denmark)

    Jensen, Maria Sander; Larsen, O H; Christiansen, Kirsten

    2013-01-01

    This study introduces a new laboratory model of whole blood platelet aggregation stimulated by endogenously generated thrombin, and explores this aspect in haemophilia A in which impaired thrombin generation is a major hallmark. The method was established to measure platelet aggregation initiated...

  4. Gasotransmitters and platelets.

    Science.gov (United States)

    Truss, Nicola J; Warner, Timothy D

    2011-11-01

    Platelets are essential to prevent blood loss and promote wound healing. Their activation comprises of several complex steps which are regulated by a range of mediators. Over the last few decades there has been intense interest in a group of gaseous mediators known as gasotransmitters; currently comprising nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H(2)S). Here we consider the action of gasotransmitters on platelet activity. NO is a well established platelet inhibitor which mediates its effects predominantly through activation of soluble guanylyl cyclase leading to a decrease in intraplatelet calcium. More recently CO has been identified as a gasotransmitter with inhibitory actions on platelets; CO acts through the same mechanism as NO but is less potent. The in vivo and platelet functions of the most recently identified gasotransmitter, H(2)S, are still the subject of investigations, but they appear generally inhibitory. Whilst there is evidence for the individual action of these mediators, it is also likely that combinations of these mediators are more relevant regulators of platelets. Furthermore, current evidence suggests that these mediators in combination alter the production of each other, and so modify the circulating levels of gasotransmitters. The use of gasotransmitters as therapeutic agents is also being explored for a range of indications. In conclusion, the importance of NO in the regulation of vascular tone and platelet activity has long been understood. Other gasotransmitters are now establishing themselves as mediators of vascular tone, and recent evidence suggests that these other gasotransmitters may also modulate platelet function.

  5. Fetal stem cells obtained from amniotic fluid and wharton's jelly expanded using platelet lysate for tissue engineering applications

    OpenAIRE

    Pinto, A. R.; Aleixo, I; Frias, A.M.; Fernandes, S.; Rocha, L; Reis, R. L.; Neves, N.M

    2012-01-01

    Extra-embryonic tissues, such as amniotic fluid (AF) and Wharton´s Jelly (WJ) of umbilical cord, offer many advantages over both embryonic and adult stem cell sources. These tissues are routinely discarded at parturition and the extracorporeal nature of these cell sources facilitates isolation, as well as the comparatively large volume and ease of physical manipulation theoretically increases the number of stem cells that can be isolated. Autologous approaches to use MSCs, n...

  6. Alloimmune refractoriness to platelet transfusions.

    Science.gov (United States)

    Sandler, S G

    1997-11-01

    Patients who are transfused on multiple occasions with red cells or platelets may develop platelet-reactive alloantibodies and experience decreased clinical responsiveness to platelet transfusion. This situation, conventionally described as "refractoriness to platelet transfusions," is defined by an unsatisfactory low post-transfusion platelet count increment. If antibodies to HLAs are detected, improved clinical outcomes may result from transfusions of HLA-matched or donor-recipient cross-matched platelets. Because refractoriness is an expected, frequently occurring phenomenon, prevention of HLA alloimmunization is an important management strategy. Prevention strategies include efforts to decrease the number of transfusions, filtration of cellular components to reduce the number of HLA-bearing leukocytes, or pretransfusion ultraviolet B irradiation of cellular components to decrease their immunogenicity. Other investigational approaches include reducing the expression of HLAs on transfused platelets, inducing a transient reticuloendothelial system blockade by infusions of specialized immunoglobulin products, or transfusing semisynthetic platelet substitutes (thromboerythrocytes, thrombospheres) or modified platelets (infusible platelet membranes, lyophilized platelets).

  7. Platelet function in dogs

    DEFF Research Database (Denmark)

    Nielsen, Line A.; Zois, Nora Elisabeth; Pedersen, Henrik D.

    2007-01-01

    Cairn Terriers, 10 Boxers, and 11 Labrador Retrievers) were included in the study. Platelet function was assessed by whole-blood aggregation with ADP (1, 5, 10, and 20 µM) as agonist and by PFA-100 using collagen and epinephrine (Col + Epi) and Cpæ + ADP as agonists. Plasma thromboxane B2 concentration......Background: Clinical studies investigating platelet function in dogs have had conflicting results that may be caused by normal physiologic variation in platelet response to agonists. Objectives: The objective of this study was to investigate platelet function in clinically healthy dogs of 4...... different breeds by whole-blood aggregometry and with a point-of-care platelet function analyzer (PFA-100), and to evaluate the effect of acetylsalicylic acid (ASA) administration on the results from both methods. Methods: Forty-five clinically healthy dogs (12 Cavalier King Charles Spaniels [CKCS], 12...

  8. Cisplatin triggers platelet activation.

    Science.gov (United States)

    Togna, G I; Togna, A R; Franconi, M; Caprino, L

    2000-09-01

    Clinical observations suggest that anticancer drugs could contribute to the thrombotic complications of malignancy in treated patients. Thrombotic microangiopathy, myocardial infarction, and cerebrovascular thrombotic events have been reported for cisplatin, a drug widely used in the treatment of many solid tumours. The aim of this study is to explore in vitro cisplatin effect on human platelet reactivity in order to define the potentially active role of platelets in the pathogenesis of cisplatin-induced thrombotic complications. Our results demonstrate that cisplatin increases human platelet reactivity (onset of platelet aggregation wave and thromboxane production) to non-aggregating concentrations of the agonists involving arachidonic acid metabolism. Direct or indirect activation of platelet phospholipase A(2) appears to be implicated. This finding contributes to a better understanding of the pathogenesis of thrombotic complications occurring during cisplatin-based chemotherapy.

  9. Platelet function in dogs

    DEFF Research Database (Denmark)

    Nielsen, Line A.; Zois, Nora Elisabeth; Pedersen, Henrik D.

    2007-01-01

    Background: Clinical studies investigating platelet function in dogs have had conflicting results that may be caused by normal physiologic variation in platelet response to agonists. Objectives: The objective of this study was to investigate platelet function in clinically healthy dogs of 4...... different breeds by whole-blood aggregometry and with a point-of-care platelet function analyzer (PFA-100), and to evaluate the effect of acetylsalicylic acid (ASA) administration on the results from both methods. Methods: Forty-five clinically healthy dogs (12 Cavalier King Charles Spaniels [CKCS], 12...... applied. However, the importance of these breed differences remains to be investigated. The PFA-100 method with Col + Epi as agonists, and ADP-induced platelet aggregation appear to be sensitive to ASA in dogs....

  10. Radiation-induced volatile hydrocarbon production in platelets. Scientific report

    Energy Technology Data Exchange (ETDEWEB)

    Radha, E.; Vaishnav, Y.N.; Kumar, K.S.; Weiss, J.F.

    1989-01-01

    Thrombocytopenia plays an important role in the development of the post-irradiation hemorrhagic syndrome. Although destruction of platelet precursors in bone marrow is a major effect of high-dose radiation exposure, the effects of radiation on preformed platelets are unclear. The latter is also of concern with respect to blood-banking practices since platelets are often irradiated at doses in the range of 20-50 Gy before transfusions to prevent graft-versus-host disease. With increasing emphasis on allogenic and autologous bone-marrow transplantation, transfusions of irradiated platelets are likely to rise. Generation of volatile hydrocarbons (ethane, pentane) as a measure of lipid peroxidation was followed in preparations from platelet-rich plasma irradiated in vitro. The hydrocarbons in the headspace of sealed vials containing irradiated and nonirradiated washed platelets, platelet-rich plasma, or platelet-poor plasma increased with time. The major hydrocarbon, pentane, increased linearly and significantly with increasing log radiation dose, suggesting that reactive oxygen species induced by ionizing radiation result in lipid peroxidation. Measurements of lipid peroxidation products may give an indication of suboptimal quality of stored and/or irradiated platelets.

  11. Metabolomic analysis of platelets during storage

    DEFF Research Database (Denmark)

    Paglia, Giuseppe; Sigurjónsson, Ólafur E; Rolfsson, Óttar;

    2015-01-01

    BACKGROUND: Platelet concentrates (PCs) can be prepared using three methods: platelet (PLT)-rich plasma, apheresis, and buffy coat. The aim of this study was to obtain a comprehensive data set that describes metabolism of buffy coat-derived PLTs during storage and to compare it with a previously...... measurements. This data set was obtained by combining a series of standard quality control assays to monitor the quality of stored PLTs and a deep coverage metabolomics study using liquid chromatography coupled with mass spectrometry. RESULTS: Stored PLTs showed a distinct metabolic transition occurring 4 days...

  12. Biochemical lesions of platelets stored as concentrates in PVC bags.

    Science.gov (United States)

    Krishnan, L K; Mathai, J; Sulochana, P V; Jacob, J; Sivakumar, R

    1997-02-01

    Acid-base status of platelet suspension during storage is a measure of the gas permeability of the bag material. To assess the efficacy of the bags available in our market to store platelets, we compared biochemical lesions of platelets stored in an Indian polyvinyl chloride (PVC) triple bag against a Japanese PVC bag standardized for 5 days platelet storage. Platelet concentrates prepared in both control and test PVC bags were stored for 72 h. Two ml samples were drawn 1 h after preparation, and then at 24 h intervals, for analysis. Our data show that the mean pH value in the test bags was maintained above 6.5. However, the CO2 tension was high and O2 tension was low. We also analyzed malondialdehyde (MDA) formation which is a measure of arachidonic acid metabolism, and seemed to be unaffected in stored platelets. Lactate dehydrogenase (LDH) was not released into the plasma excessively and hence significant platelet lysis was absent during storage. Hypotonic shock response (HSR) of platelets stored in both test and control bags was comparable, indicating the possibility of satisfactory post-transfusion recovery.

  13. MORPHOGENESIS OF KNEE HYALINE CARTILAGE DURING INTRAARTICULAR INJECTION OF PLATELET-RICH AUTOLOGOUS PLASMA AND/OR HYALURONIC ACID PREPARATION IN RATS WITH EXPERIMENTAL OSTEOARTHRITIS

    Directory of Open Access Journals (Sweden)

    S. A. Demkin

    2016-01-01

    Full Text Available According to current concepts, the influence of autologous platelet-rich plasma (PRP and high molecular hyaluronates (HA on the repair of hyaline cartilage during its inflammatory and degenerative changes has been insufficiently studied yet. The objective of the work was to evaluate the morphological changes in the structure of hyaline cartilage in experimental osteoarthritis after intra-articular injection of PRP and/or HA. Material and methods. The authors used 50 adult rats of Wistar line, weighing 250±2,2 g., distributed into five groups of 10 animals (two control and three experimental groups. An experimental gonarthosis was simulated on four groups of animals. Animals of the first experimental group received intra-articular injection of PRP, the second group – HA, the third – both PRP and HA. Results. No morphological signs of degenerative and inflammatory changes in the first control group were identified. Following osteoarthritis simulation the articular cartilage thinned to 121±20,4 microns (p<0,05 and the volume fraction of chondrocyte decreased to 1,2±0,6% (p<0,05. The authors observed an uneven coloration of collagen fibers with severe tinctorial properties disorder of the articular cartilage matrix. After the RPR introduction the authors observed tickening of the articular cartilage up to 275±18,9 micron (p<0,05 and the volume fraction of chondrocytes up to 18,4±2,0% (p<0,05. The contour of the cartilage surface became smoother with the formation of a cell-free zone. Collagen fibers demonstrated a uniform distribution, tinctorial properties of cartilage matrix in all areas were preserved, no signs of inflammation were noted. After HA introduction the authors observed thickening of the cartilage plate up to 264±21,3 microns (p<0,05 and the volume fraction of chondrocytes up to 11,6±1,2% (p<0,05. The surface of the cartilage featured uneven contours due to multiple areas of pulping. Uneven tinctorial properties of cartilage

  14. Measurement of platelet aggregation, independently of patient platelet count

    DEFF Research Database (Denmark)

    Vinholt, P. J.; Frederiksen, H.; Hvas, A.M.

    2017-01-01

    platelet aggregation ruled out bleeding tendency in thrombocytopenic patients. Summary: Background: Methods for testing platelet aggregation in thrombocytopenia are lacking. Objective: To establish a flow-cytometric test of in vitro platelet aggregation independently of the patient's platelet count......, and examine the association of aggregation with a bleeding history in thrombocytopenic patients. Patients/methods: We established a flow-cytometric assay of platelet aggregation, and measured samples from healthy individuals preincubated with antiplatelet drugs, and samples from two patients with inherited...... platelets at platelet counts of > 10 × 109 L-1; otherwise, platelet isolation was required. The platelet aggregation percentage decreased with increasing antiplatelet drug concentration. Platelet aggregation in patients was reduced as compared with healthy individuals: 42% (interquartile range [IQR] 27...

  15. Comparison of platelets characteristics according to various processing methods

    Directory of Open Access Journals (Sweden)

    O. V. Karpova

    2014-01-01

    Full Text Available To date different techniques of platelet concentrates (PC preparation and processing are proposed to achieve the best efficiency of transfusions and to minimize the risks post-transfusion reactions. However, data on the impact of different approaches to PC preparation and processing on morphological and functional characteristics of platelets and, as a consequence, the clinical efficacy of transfusions is controversial. In this paper we analyzed the impact of the platelet storage solution and different methods of pathogen inactivation (X-rays, UV-irradiation after photosensitization with riboflavin on morphological and functional parameters of platelets. Our findings allow optimizing the technology of preparation and processing of PC to achieve greater effectiveness of transfusion therapy.

  16. Platelet-collagen adhesion enhances platelet aggregation induced by binding of VWF to platelets

    Energy Technology Data Exchange (ETDEWEB)

    Laduca, F.M.; Bell, W.R.; Bettigole, R.E. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA) State Univ. of New York, Buffalo (USA))

    1987-11-01

    Ristocetin-induced platelet aggregation (RIPA) was evaluated in the presence of platelet-collagen adhesion. RIPA of normal donor platelet-rich plasma (PRP) demonstrated a primary wave of aggregation mediated by the binding of von Willebrand factor (VWF) to platelets and a secondary aggregation wave, due to a platelet-release reaction, initiated by VWF-platelet binding and inhibitable by acetylsalicylic acid (ASA). An enhanced RIPA was observed in PRP samples to which collagen had been previously added. These subthreshold concentrations of collagen, which by themselves were insufficient to induce aggregation, caused measurable platelet-collagen adhesion. Subthreshold collagen did not cause microplatelet aggregation, platelet release of ({sup 3}H)serotonin, or alter the dose-responsive binding of {sup 125}I-labeled VWF to platelets, which occurred with increasing ristocetin concentrations. However, ASA inhibition of the platelet release reaction prevented collagen-enhanced RIPA. These results demonstrate that platelet-collagen adhesion altered the platelet-release reaction induced by the binding of VWF to platelets causing a platelet-release reaction at a level of VWF-platelet binding not normally initiating a secondary aggregation. These findings suggest that platelet-collagen adhesion enhances platelet function mediated by VWF.

  17. Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate

    Energy Technology Data Exchange (ETDEWEB)

    Percin, Is Latin-Small-Letter-Dotless-I k [Department of Biology, Hacettepe University, Ankara (Turkey); Karakoc, Veyis [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey); Akgoel, Sinan [Department of Biochemistry, Ege University, Izmir (Turkey); Aksoez, Erol [Department of Biology, Hacettepe University, Ankara (Turkey); Denizli, Adil, E-mail: denizli@hacettepe.edu.tr [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey)

    2012-07-01

    The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m{sup 2}/g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 {mu}g/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 Degree-Sign C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA. - Highlights: Black-Right-Pointing-Pointer Magnetic nanoparticles have several advantages over conventional adsorbents. Black-Right-Pointing-Pointer MAH acted as the pseudospecific ligand, ligand immobilization step was eliminated. Black-Right-Pointing-Pointer pDNA adsorption amount was 154 mg/g. Black-Right-Pointing-Pointer Fifty-fold capacity increase was obtained when compared to conventional matrices.

  18. Fusaric acid, a mycotoxin, and its influence on blood coagulation and platelet function.

    Science.gov (United States)

    Devaraja, Sannaningaiah; Girish, Kesturu S; Santhosh, Martin S; Hemshekhar, Mahadevappa; Nayaka, Siddaiah C; Kemparaju, Kempaiah

    2013-06-01

    The current study intended to explore the effect of fusaric acid on blood coagulation including plasma coagulation and platelet aggregation. Fusaric acid exhibited biphasic effects on citrated human plasma recalcification time. At concentrations below 50 ng, fusaric acid decreased the clotting time of plasma dose-dependently from 130 ± 3s control value to 32 ± 3s; however, above 50 ng, fusaric acid increased the clotting time from 32 ± 3s and reached a maximum of 152 s at 100 ng and remained unaltered thereafter for the increased dose of fusaric acid. Fusaric acid without damaging red blood cells and platelets, inhibited agonists such as collagen, ADP, thrombin, and epinephrine-induced aggregation of both platelet-rich plasma (PRP) and washed platelets preparations of human. Interestingly, fusaric acid showed biphasic effects only in thrombin-induced platelet aggregation of washed platelets, and at lower concentration (below 900 ng) it activated platelet aggregation; however, in increased concentration (above 900 ng) it inhibited the platelet aggregation of washed platelets. In addition, fusaric acid also inhibited the agonist ADP-induced platelet aggregation of washed platelet suspension but did not show biphasic effect. Further, fusaric acid did not induce the platelets to generate reactive oxygen species (ROS) that clearly suggests that the induction of platelet function could be the result of the fusaric acid-mediated receptor interaction but not through the morphological shape change.

  19. Campylobacter jejuni cell lysates differently target mitochondria and lysosomes on HeLa cells.

    Science.gov (United States)

    Canonico, B; Campana, R; Luchetti, F; Arcangeletti, M; Betti, M; Cesarini, E; Ciacci, C; Vittoria, E; Galli, L; Papa, S; Baffone, W

    2014-08-01

    Campylobacter jejuni is the most common cause of bacterial gastroenteritis in humans. The synthesis of cytolethal distending toxin appears essential in the infection process. In this work we evaluated the sequence of lethal events in HeLa cells exposed to cell lysates of two distinct strains, C. jejuni ATCC 33291 and C. jejuni ISS3. C. jejuni cell lysates (CCLys) were added to HeLa cell monolayers which were analysed to detect DNA content, death features, bcl-2 and p53 status, mitochondria/lysosomes network and finally, CD54 and CD59 alterations, compared to cell lysates of C. jejuni 11168H cdtA mutant. We found mitochondria and lysosomes differently targeted by these bacterial lysates. Death, consistent with apoptosis for C. jejuni ATCC 33291 lysate, occurred in a slow way (>48 h); concomitantly HeLa cells increase their endolysosomal compartment, as a consequence of toxin internalization besides a simultaneous and partial lysosomal destabilization. C. jejuni CCLys induces death in HeLa cells mainly via a caspase-dependent mechanism although a p53 lysosomal pathway (also caspase-independent) seems to appear in addition. In C. jejuni ISS3-treated cells, the p53-mediated oxidative degradation of mitochondrial components seems to be lost, inducing the deepest lysosomal alterations. Furthermore, CD59 considerably decreases, suggesting both a degradation or internalisation pathway. CCLys-treated HeLa cells increase CD54 expression on their surface, because of the action of lysate as its double feature of toxin and bacterial peptide. In conclusion, we revealed that C. jejuni CCLys-treated HeLa cells displayed different features, depending on the particular strain.

  20. Defining Platelet Function During Polytrauma

    Science.gov (United States)

    2013-02-01

    using calibrated automated thrombography ( CAT ). 3. Platelet-induced clot contraction and using viscoelastic measures such as TEG with Platelet Mapping...using calibrated automated thrombography ( CAT ) in platelet-rich plasma. 3. Platelet-induced clot contraction and effect on clot structure by platelet...if injury with stable vital signs on initial evaluation.  Pregnancy (confirmed with urine pregnancy testing)  Documented do not resuscitate order

  1. Quantitative assessment of the p53-Mdm2 feedback loop using protein lysate microarrays.

    Science.gov (United States)

    Ramalingam, Sundhar; Honkanen, Peter; Young, Lynn; Shimura, Tsutomu; Austin, John; Steeg, Patricia S; Nishizuka, Satoshi

    2007-07-01

    Mathematical simulations of the p53-Mdm2 feedback loop suggest that both proteins will exhibit impulsive expression characteristics in response to high cellular stress levels. However, little quantitative experimental evaluation has been done, particularly of the phosphorylated forms. To evaluate the mathematical models experimentally, we used lysate microarrays from an isogenic pair of gamma-ray-irradiated cell lysates from HCT116 (p53(+/+) and p53(-/-)). Both p53 and Mdm2 proteins showed expected pulses in the wild type, whereas no pulses were seen in the knockout. Based on experimental observations, we determined model parameters and generated an in silico "knockout," reflecting the experimental data, including phosphorylated proteins.

  2. 采集后6小时与72小时制备的冰冻单采血小板质量研究%A comparative study on qualities of frozen platelets prepared 6 h and 72 h after collection

    Institute of Scientific and Technical Information of China (English)

    刘利明; 陈蓉; 詹鹏飞; 邹志强; 杨湘辉; 江顺琴; 陈雪丽

    2008-01-01

    Objective To offer evidence for preparation criteria of frozen platelet preparation by means of comparison of a number of quality parameters between the frozen platelets prepared at 6 hour and 72 hour time point after collection.Methods Frozen platelet preparation was made 6 h and 72 h after collection of blood platelets gathered in bags by Trima Blood Corpuscle Machine.A week later, the frozen platelet preparation was dissolved and sampled for detection of PLT,MPV,PCT,PDW, platelet adhesiveness,P-selectin,PF3A,PF4,pH value,blood clot contractibility(clot retraction test).Results There were no significant differences in a series of quality parameters between the frozen platelets prepared at 6 hours and 72 hours after collection.Under the different conservation conditions.PF3A kept available activity.When the blood platelets were conserved under the conditions at22℃or at-80℃,the expressions of PF3A and p-Selection were increased,there was no difference of PF3A and p-Selection expression between the frozen platelets prepared at 6 hours and 72 hours after collection.During three days of routine conservation,no change appeared in the conglutinate function and clot contractibility;however,the above parameters were significantly decreased,and the activity of platelets was markedly datined under congealing.Conclusion There are no significant differences in a series of quality parameters between the frozen platelet preparation prepared with gathered blood by Trima Blood Corpuscle Machine at 6 hours and 72 hours after collection,and both of the frozen platelet preparation can effectively improve or control the hemorrhagic tendency of patients suffered urgent massive hemorrhage.%目的 通过比较采集后6 h和72 h制备的冰冻单采血小板制品的多项质量参数,为冰冻单采血小板的制备标准提供依据.方法 以美国产Trims血细胞分离机配套全密闭7 d保存袋采集的单采血小板,分别于采集后6 h和72 h

  3. PLATELET RICH FIBRIN: A PROMISING INNOVATION IN REGENERATIVE THERAPY

    Directory of Open Access Journals (Sweden)

    Arun

    2015-04-01

    Full Text Available Platelets can play a crucial role in regenerative therapy as they are reservoirs of growth factors and cytokines which are the key factors for regeneration of the bone and maturation of the soft tissue. Platelet - rich fibr in (PRF was first described by Choukroun et al. in France. It has been referred to as a second - generation platelet concentrate, which has been shown to have several advantages over traditionally prepared PRP. Platelet - rich fibrin (PRF is autologous plate let concentrates prepared from patient’s own blood. It is a natural fibrin - based biomaterial prepared from an anticoagulant - free blood harvest without any artificial biochemical modification that allows obtaining fibrin membranes enriched with platelets a nd growth factors. Evidence from the literature suggests the potential role of PRF in regeneration and tissue engineering. The slow polymerisation during centrifugation and fibrin - based structure makes PRF a better healing biomaterial than PRP and other fi brin adhesives. The purpose of this review article is to describe the novel second - generation platelet concentrate PRF, which is an improvement over the traditionally prepared PRP for use in regenerative dentistry.

  4. Optimizing parameters for clinical-scale production of high IL-12 secreting dendritic cells pulsed with oxidized whole tumor cell lysate

    Directory of Open Access Journals (Sweden)

    Chiang Cheryl L-L

    2011-11-01

    Full Text Available Abstract Background Dendritic cells (DCs are the most potent antigen-presenting cell population for activating tumor-specific T cells. Due to the wide range of methods for generating DCs, there is no common protocol or defined set of criteria to validate the immunogenicity and function of DC vaccines. Methods Monocyte-derived DCs were generated during 4 days of culture with recombinant granulocyte-macrophage colony stimulating factor and interleukin-4, and pulsed with tumor lysate produced by hypochlorous acid oxidation of tumor cells. Different culture parameters for clinical-scale DC preparation were investigated, including: 1 culture media; 2 culture surface; 3 duration of activating DCs with lipopolysaccharide (LPS and interferon (IFN-gamma; 4 method of DC harvest; and 5 cryomedia and final DC product formulation. Results DCs cultured in CellGenix DC media containing 2% human AB serum expressed higher levels of maturation markers following lysate-loading and maturation compared to culturing with serum-free CellGenix DC media or AIM-V media, or 2% AB serum supplemented AIM-V media. Nunclon™Δ surface, but not Corning® tissue-culture treated surface and Corning® ultra-low attachment surface, were suitable for generating an optimal DC phenotype. Recombinant trypsin resulted in reduced major histocompatibility complex (MHC Class I and II expression on mature lysate-loaded DCs, however presentation of MHC Class I peptides by DCs was not impaired and cell viability was higher compared to cell scraping. Preservation of DCs with an infusible cryomedia containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO yielded higher cell viability compared to using human AB serum containing 10% DMSO. Finally, activating DCs for 16 hours with LPS and IFN-γ stimulated robust mixed leukocyte reactions (MLRs, and high IL-12p70 production in vitro that continued for 24 hours after the cryopreserved DCs were thawed and

  5. Clinical application of radiolabelled platelets

    Energy Technology Data Exchange (ETDEWEB)

    Kessler, C. (Medical University Luebeck (Federal Republic of Germany). Department of Neurology); Hardeman, M.R. (Amsterdam Univ. (Netherlands). Academisch Ziekenhuis); Henningsen, H. (Heidelberg Univ. (Germany, F.R.). Neurologische Klinik); Petrovici, J.-N. (Cologne-Merheim Hospital (Federal Republic of Germany). Department of Neurology) (eds.)

    1990-01-01

    The increasing number of therapeutic modalities available for the management of patients with thromboembolic complications, such as fibrinolytic treatment or vascular surgery, require the development of new imaging techniques to provide more information on the xtent, age and activity of the thromboembolic material causing clinical symptoms. Since the introduction of radiolabelling of platelets with indium-111, platelet scintigraphy (PSC) has been used as a tool in the diagnosis of various thromboembolic diseases. During the International Symposium on Radiolabelled Platelets scientists from a variety of medical backgrounds presented their results on the clinical applictions of radiolabelled platelets. The papers presented there have been updated to take account of the latest results before publication in this volume. The papers are grouped into six sections on platelet labelling techniques, radiolabelled platelets in cardiology, monitoring of antiplatelet therapy, platelet scintigraphy in stroke patients, platelet scintigraphy in angiology, and platelet scintigraphy in hematology and other clinical applications, including renal transplant rejection. refs.; figs.; tabs.

  6. Platelet preservation: agitation and containers.

    Science.gov (United States)

    van der Meer, Pieter F; de Korte, Dirk

    2011-06-01

    For platelets to maintain their in vitro quality and in vivo effectiveness, they need to be stored at room temperature with gentle agitation in gas-permeable containers. The mode of agitation affects the quality of the platelets, and a gentle method of agitation, either a circular or a flat bed movement, provides the best results. Tumblers or elliptical agitators induce platelet activation and subsequent damage. As long as the platelets remain in suspension, the agitation speed is not important. Agitation of the platelet concentrates ensures that the platelets are continuously oxygenated, that sufficient oxygen can enter the storage container and that excess carbon dioxide can be expelled. During transportation of platelet concentrates, nowadays over long distances where they are held without controlled agitation, platelets may tolerate a certain period without agitation. However, evidence is accumulating that during the time without agitation, local hypoxia surrounding the platelets may induce irreversible harm to the platelets. Over the decades, more gas-permeable plastics have been used to manufacture platelet containers. The use of different plastics and their influence on the platelet quality both in vitro and in vivo is discussed. The improved gas-permeability has allowed the extension of platelet storage from 3 days in the early 1980s, to currently at least 7 days. In the light of new developments, particularly the introduction of pathogen reduction techniques, the use of platelet additive solutions and the availability of improved automated separators, further (renewed) research in this area is warranted.

  7. INTERFERON-GAMMA STIMULATING ACTIVITIES OF THE FRACTIONATED NEOSPORA CANINUM TACHYZOITE LYSATE

    Science.gov (United States)

    Neospora caninum is an obligate intracellular protozoan parasite, causing bovine abortion worldwide. Our recent research showed that N. caninum tachyzoite lysate elicits production of the T cell cytokine interferon-gamma (IFN-g) by both bovine and murine T cells, which may be critical to host protec...

  8. Quantitative analysis of HIV-1 protease inhibitors in cell lysates using MALDI-FTICR mass spectrometry.

    NARCIS (Netherlands)

    Kampen, JJ van; Burgers, P.C.; Groot, R. de; Osterhaus, A.D.; Reedijk, M.L.; Verschuren, E.J.; Gruters, R.A.; Luider, T.M.

    2008-01-01

    In this report we explore the use of MALDI-FTICR mass spectrometry for the quantitative analysis of five HIV-1 protease inhibitors in cell lysates. 2,5-Dihydroxybenzoic acid (DHB) was used as the matrix. From a quantitative perspective, DHB is usually a poor matrix due to its poor shot-to-shot and p

  9. Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration.

    Science.gov (United States)

    Mohammedsaeed, Walaa; Cruickshank, Sheena; McBain, Andrew J; O'Neill, Catherine A

    2015-11-05

    A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration.

  10. Red blood cell lysate modulates the expression of extracellular matrix proteins in dermal fibroblasts.

    Science.gov (United States)

    Akbari, Amir; Li, Yunyuan; Kilani, Ruhangiz T; Ghahary, Aziz

    2012-11-01

    During the early stage of wound healing process, blood clots can be served as a temporary extracellular matrix (ECM) to let skin cell migration and proliferation. The red blood cells are generally thought as inert bystanders in the early and inflammatory phase of wound healing. Here, we provide evidence that red blood cells (RBC) also play an important role in modulation of key ECM components such as type-I collagen, α-smooth muscle actin, fibronectin, and matrix metalloproteinases (MMPs). In this study, we used western blot analysis and showed a significant increase in the level of MMP-1, 2, 3. Furthermore, we found that RBC lysate significantly down-regulates type-I collagen and α-smooth muscle actin while up-regulates fibronectin expression in dermal fibroblasts. To further explore the mechanism by which RBC lysate modulates MMP-1 expression, the effect of inhibitors for three MAPK signaling pathways on RBC inducing MMP-1 expression by dermal fibroblasts were tested. The result showed that the inhibitor of ERK1/2 could abrogate the stimulatory effect of RBC lysate on MMP-1 expression in dermal fibroblasts. Consistently, RBC treatment results in an increase of ERK1/2 phosphorylation in dermal fibroblast. In conclusion, these findings suggest that RBC lysate can modulate the expression of MMPs and key ECM components which are important in healing process.

  11. An ultrasensitive LC-MS/MS method with liquid phase extraction to determine paclitaxel in both cell culture medium and lysate promising quantification of drug nanocarriers release in vitro.

    Science.gov (United States)

    Baati, Tarek; Schembri, Thérèse; Villard, Claude; Correard, Florian; Braguer, Diane; Estève, Marie-Anne

    2015-11-10

    The quantification of paclitaxel, a chemotherapy drug used to treat different types of cancers, has been performed from complete cell culture medium and cell lysate samples using a simple liquid-liquid extraction procedure in conjunction with liquid chromatography tandem mass spectrometry (LC-MS/MS). A simple sample preparation using methanol and acetic acid as a weaker acid was applied to avoid paclitaxel destruction and to achieve recovery exceeding 80 % from both matrices spiked with paclitaxel and docetaxel used as internal standard. This rapid, simple, selective and sensitive method enabled the quantification of paclitaxel within the linear range of 1-250nM in culture medium and 5-250nM in cell lysate. The lower limit of quantification was achieved in cell culture medium and cell lysates at 0.2 and 1pmol, respectively. This method was successfully applied to human non-small cell lung carcinoma cells (A549 cells) in order to quantify the amount of paclitaxel in both cell culture medium and lysate after incubation with 5, 50 and 100nM of paclitaxel. This ultra-sensitive method promises the quantification of ultra-low concentrations of paclitaxel released from any nanocarriers, allowing the determination of the kinetic profile of drug release, which is an essential parameter to validate the use of nanocarriers for drug delivery in cancer therapy.

  12. Platelets and cardiac arrhythmia

    Directory of Open Access Journals (Sweden)

    Jonas S De Jong

    2010-12-01

    Full Text Available Sudden cardiac death remains one of the most prevalent modes of death in industrialized countries, and myocardial ischemia due to thrombotic coronary occlusion is its primary cause. The role of platelets in the occurrence of SCD extends beyond coronary flow impairment by clot formation. Here we review the substances released by platelets during clot formation and their arrhythmic properties. Platelet products are released from three types of platelet granules: dense core granules, alpha-granules, and platelet lysosomes. The physiologic properties of dense granule products are of special interest as a potential source of arrhythmic substances. They are released readily upon activation and contain high concentrations of serotonin, histamine, purines, pyrimidines, and ions such as calcium and magnesium. Potential arrhythmic mechanisms of these substances, e.g. serotonin and high energy phosphates, include induction of coronary constriction, calcium overloading, and induction of delayed after-depolarizations. Alpha-granules produce thromboxanes and other arachidonic acid products with many potential arrhythmic effects mediated by interference with cardiac sodium, calcium and potassium channels. Alpha-granules also contain hundreds of proteins that could potentially serve as ligands to receptors on cardiomyocytes. Lysosomal products probably do not have an important arrhythmic effect. Platelet products and ischemia can induce coronary permeability, thereby enhancing interaction with surrounding cardiomyocytes. Antiplatelet therapy is known to improve survival after myocardial infarction. Although an important part of this effect results from prevention of coronary clot formation, there is evidence to suggest that antiplatelet therapy also induces anti-arrhythmic effects during ischemia by preventing the release of platelet activation products.

  13. PLATELET RICH PLASMA (PRP) APPLICATION IN TOTAL KNEE ARTHROPLASTY (TKA)

    OpenAIRE

    2015-01-01

    PURPOSE: To find out the PRP application effects in TKA on pain syndrome, wound healing, postoperative blood loss, range of motion and the knee circumference (centimetry). MATERIAL AND METHOD: The preparation of the platelet-rich plasma is based on our treatment algorithm. 20 patients have been subject to TKA within the period from October 2012 to November 2014 and underwent TKA, as platelet rich plasma was used intraoperatively. The average patient age is 72,3; 9 male and 11 female patien...

  14. Purification of human platelet-derived growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Raines, E.W.; Ross, R.

    1985-01-01

    The paper describes a method for purification of human platelet-derived growth factor (PDGF) from outdated platelet-rich plasma (PRP) using commonly available laboratory reagents and yielding a mitogen purified 800,000-fold over the starting material. (/sup 3/H)thymidine incorporation into DNA of cultured cells responsive to PDGF represents the most readily available method to follow its purification and define the biological activity of a purified preparation. Other assays to quantitate PDGF include radioreceptor assay and radioimmunoassay.

  15. The Platelet and Platelet Function Testing in Liver Disease

    NARCIS (Netherlands)

    Hugenholtz, Greg G. C.; Porte, Robert J.; Lisman, Ton

    2009-01-01

    Patients who have liver disease commonly present with alterations in platelet number and function. Recent data have questioned the contribution of these changes to bleeding complications in these patients. Modern tests of platelet function revealed compensatory mechanisms for the decreased platelet

  16. Investigation of platelet function and platelet disorders using flow cytometry.

    Science.gov (United States)

    Rubak, Peter; Nissen, Peter H; Kristensen, Steen D; Hvas, Anne-Mette

    2016-01-01

    Patients with thrombocytopenia or platelet disorders are at risk of severe bleeding. We report the development and validation of flow cytometry assays to diagnose platelet disorders and to assess platelet function independently of platelet count. The assays were developed to measure glycoprotein levels (panel 1) and platelet function (panel 2) in sodium citrated blood. Twenty healthy volunteers and five patients diagnosed with different platelet disorders were included. Glycoprotein expression levels of the receptors Ia, Ib, IIb, IIIa and IX were measured and normalised with forward scatter (FS) as a measurement of platelet size. Platelet function was assessed by CD63, P-selectin and bound fibrinogen in response to arachidonic acid, adenosine diphosphate (ADP), collagen-related peptide, ristocetin and thrombin receptor-activation peptide-6. All patients except one with suspected δ-granule defect showed aberrant levels of glycoproteins in panel 1. Glanzmann's thrombasthenia and genetically verified Bernard-Soulier syndrome could be diagnosed using panel 1. All patients showed reduced platelet function according to at least one agonist. Using panel 2 it was possible to diagnose Bernard-Soulier syndrome, δ-granule defect and GPVI disorder. By combining the two assays, we were able to diagnose different platelet disorders and investigate platelet function independent of platelet count.

  17. Reproducibility of Manual Platelet Estimation Following Automated Low Platelet Counts

    Directory of Open Access Journals (Sweden)

    Zainab S Al-Hosni

    2016-11-01

    Full Text Available Objectives: Manual platelet estimation is one of the methods used when automated platelet estimates are very low. However, the reproducibility of manual platelet estimation has not been adequately studied. We sought to assess the reproducibility of manual platelet estimation following automated low platelet counts and to evaluate the impact of the level of experience of the person counting on the reproducibility of manual platelet estimates. Methods: In this cross-sectional study, peripheral blood films of patients with platelet counts less than 100 × 109/L were retrieved and given to four raters to perform manual platelet estimation independently using a predefined method (average of platelet counts in 10 fields using 100× objective multiplied by 20. Data were analyzed using intraclass correlation coefficient (ICC as a method of reproducibility assessment. Results: The ICC across the four raters was 0.840, indicating excellent agreement. The median difference of the two most experienced raters was 0 (range: -64 to 78. The level of platelet estimate by the least-experienced rater predicted the disagreement (p = 0.037. When assessing the difference between pairs of raters, there was no significant difference in the ICC (p = 0.420. Conclusions: The agreement between different raters using manual platelet estimation was excellent. Further confirmation is necessary, with a prospective study using a gold standard method of platelet counts.

  18. [Use of reactions with Limulus amoebocyte lysate (LAL) to determine biological activity of lipopolysaccharides from reference and clinical strains of the Bacteroides fragilis group].

    Science.gov (United States)

    Rokosz, Alicja; Fiejka, Maria; Górska, Paulina; Aleksandrowicz, Janina; Meisel-Mikołajczyk, Felicja; Łuczak, MirosŁaw

    2002-01-01

    The aim of this study was to determine and compare a biological activity of lipopolysaccharides (LPS) from reference and clinical strains of strictly anaerobic bacteria belonging to the Bacteroides fragilis group (BFG) by means of quantitative, photometric BET (LAL) method with Limulus polyphemus amoebocyte lysate and chromogenic substrate S-2423. Lipopolysaccharides of five BFG species were extracted by Westphal and Jann method (1965) from eight reference and two clinical strains of B. fragilis group. Crude LPS preparations were purified according to the procedure described by Gmeiner (1975) with ultracentrifugation and nuclease treatment. Biological activities of bacterial endotoxins were determined by quantitative BET method with chromogenic substrate S-2423 (ENDOCHROME kit, Charles River Endosafe Ltd., USA). Tests were performed according to the producer's recommendations. E. coli O55:B5 LPS was applied to compare its activity in reaction with LAL reagent with activities of LPS preparations from rods of the Bacteroides genus. Among examined bacterial compounds the most active in BET method was E. coli O55:B5 LPS. Activities of lipopolysaccharides from five species of BFG rods in reaction with Limulus amoebocyte lysate were differentiated. Greater ability to activate LAL proenzyme revealed lipopolysaccharides of these species of the Bacteroides genus, which are important from the clinical point of view--B. fragilis and B. thetaiotaomicron.

  19. Quality of cryoprecipitate prepared from the plasma after separation of platelets by buffy coat method%全血手工制备浓缩血小板后的血浆再制备冷沉淀的质量评价

    Institute of Scientific and Technical Information of China (English)

    易中梅; 王红苹; 李晨晨; 王世春; 王泽蓉; 黎儒青; 郭辉; 赵树铭

    2013-01-01

    目的 评价室温新鲜全血白膜法制备浓缩血小板后的血浆再制备冷沉淀的质量.方法 实验组为24例,新鲜全血(400 mL)置室温于<8h用白膜法制备浓缩血小板后所得的血浆,冰冻保存.对照组1为12例,常规制备新鲜冰冻血浆,冰冻保存.对照组2为12例,新鲜冰冻单采血浆,血浆单采完毕分装为200 mL/袋并立即冰冻保存.3组血浆按常规制备冷沉淀,评价其质量:外观、凝血因子FⅧ及Fib的含量;血细胞残留量.结果 3组冷沉淀外观均正常;WBC含量在3组间无统计学意义.与对照组1比较:实验组凝血因子FⅧ(81.76±34.07)IU较低,Fib(202.63±48.58)mg及Plt (7.81±5.81)×109均较高.与对照组2比较:实验组凝血因子FⅧ含量相当,Fib(202.63 ±48.58)mg较高、Plt(7.81±5.81)×109较低.结论 全血来源的制备浓缩血小板后的冰冻血浆还可以用于冷沉淀的制备,其质量符合国家标准.%Objective To investigate the quality of cryoprecipitate prepared from plasma after preparation of buffy coat derived platelet concentrate. Methods The experimental group consisted of 24 cryoprecipitates prepared from plasma after platelet concentrates were prepared by buffy coat method, using whole blood stored at room temperature for less than 8 h. Control group 1 consisted of cryoprecipitates prepared from regular fresh frozen plasma,prepared from whole blood stored at 4℃ within 8 h of whole blood collection. Control group 2 consisted of cryoprecipitates prepared from apheresis plasma frozen within 2 h of collection. All the plasma units were frozen for 3 d before preparation of cryoprecipitate. The quality of cryoprecipitate was evaluated by appearance,FⅧ and fibrinogen (Fib) content,and residual blood cells. Results The cryoprecipitates of all 3 groups showed normal appearance. There was no difference in residual WBC counts between the three groups. Compared to control group 1 ,the clotting factor FⅧ in the experimental group was

  20. Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes

    Directory of Open Access Journals (Sweden)

    Andreas Bayer

    2017-01-01

    Full Text Available Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs or their clinically related formulations (e.g., Vivostat PRF® came recently into the physicians’ focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiation status of skin wounds in vivo. Therefore, we investigated the expression of early (keratin 1 and keratin 10 and late (transglutaminase-1 and involucrin differentiation markers. PRGF treatment of primary human keratinocytes decreased keratin 1 and keratin 10 gene expression but induced involucrin and transglutaminase-1 gene expression in an epidermal growth factor receptor- (EGFR- dependent manner. In concordance with these results, microscopic analyses revealed that PRGF-treated human keratinocytes displayed morphological features typical of keratinocytes undergoing terminal differentiation. In vivo treatment of artificial human wounds with Vivostat PRF revealed a significant induction of involucrin and transglutaminase-1 gene expression. Together, our results indicate that PRGF and Vivostat PRF induce terminal differentiation of primary human keratinocytes. This potential mechanism may contribute to the observed beneficial effects in the treatment of hard-to-heal wounds with autologous thrombocyte concentrate lysates in vivo.

  1. Analysis of Platelet-Rich Plasma Extraction

    Science.gov (United States)

    Fitzpatrick, Jane; Bulsara, Max K.; McCrory, Paul Robert; Richardson, Martin D.; Zheng, Ming Hao

    2017-01-01

    Background: Platelet-rich plasma (PRP) has been extensively used as a treatment in tissue healing in tendinopathy, muscle injury, and osteoarthritis. However, there is variation in methods of extraction, and this produces different types of PRP. Purpose: To determine the composition of PRP obtained from 4 commercial separation kits, which would allow assessment of current classification systems used in cross-study comparisons. Study Design: Controlled laboratory study. Methods: Three normal adults each donated 181 mL of whole blood, some of which served as a control and the remainder of which was processed through 4 PRP separation kits: GPS III (Biomet Biologics), Smart-Prep2 (Harvest Terumo), Magellan (Arteriocyte Medical Systems), and ACP (Device Technologies). The resultant PRP was tested for platelet count, red blood cell count, and white blood cell count, including differential in a commercial pathology laboratory. Glucose and pH measurements were obtained from a blood gas autoanalyzer machine. Results: Three kits taking samples from the “buffy coat layer” were found to have greater concentrations of platelets (3-6 times baseline), while 1 kit taking samples from plasma was found to have platelet concentrations of only 1.5 times baseline. The same 3 kits produced an increased concentration of white blood cells (3-6 times baseline); these consisted of neutrophils, leukocytes, and monocytes. This represents high concentrations of platelets and white blood cells. A small drop in pH was thought to relate to the citrate used in the sample preparation. Interestingly, an unexpected increase in glucose concentrations, with 3 to 6 times greater than baseline levels, was found in all samples. Conclusion: This study reveals the variation of blood components, including platelets, red blood cells, leukocytes, pH, and glucose in PRP extractions. The high concentrations of cells are important, as the white blood cell count in PRP samples has frequently been ignored

  2. Mean platelet volume and mean platelet volume/platelet count ratio ...

    African Journals Online (AJOL)

    Amira M. Elsayed

    2016-03-30

    Mar 30, 2016 ... Abstract The mean platelet volume (MPV) is a laboratory marker associated with platelet func- tion and activity. .... the first 24 h of presentation to the emergency department. Severity of ..... J Neurol Neurosurg Psychiatry.

  3. L-carnitine effectively improves the metabolism and quality of platelet concentrates during storage.

    Science.gov (United States)

    Deyhim, Mohammad Reza; Mesbah-Namin, Seyed Alireza; Yari, Fatemeh; Taghikhani, Mohammad; Amirizadeh, Naser

    2015-04-01

    Human platelets undergo structural and biochemical alternations during storage which are collectively called platelet storage lesion (PSL). PSL is characterized as metabolic and functionally changes. It causes decrease in platelet recovery and survival. Here, we evaluated the effect of L-carnitine (LC) on the metabolism, function, and mitochondrial metabolic activity of platelet during storage. Platelet-rich plasma was used to prepare platelet concentrate (PC) in Iranian Blood Transfusion Organization. For this purpose, ten PC bags from healthy donors were stored at 22 °C with gentle agitation in the presence or absence of LC. The effects of LC (15 mM) on the platelet quality were assessed by analyzing the levels of glucose, lactate, ATP, and lactate dehydrogenase (LDH) activity. Platelet aggregations induced by arachidonate and ristocetin were analyzed by aggregometer. Platelet mitochondrial melablolic activity was measured by tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay; platelet count and mean platelet volume were also determined by a hematology analyzer during 5 days of PC storage. The results indicated that LC could significantly decrease lactate concentration and glucose consumption accompanied with the increased oxygen consumption in stored PC. LDH activity also less significantly increased in LC-treated PC on days 2 and 5 of storage. Platelet aggregation in response to the ristocetin and arachidonate was significantly higher in LC-treated PC than that in untreated PC on day 5 of storage. Finally, platelet mitochondrial metabolic activity less significantly decreased in LC-treated PC compared to the control group on days 2 and 5 of storage. It seems that LC would be a good additive to reduce PSL and improve the platelet metabolism and quality of the stored PC for platelet transfusion therapy.

  4. Extracellular Fibrinogen-binding Protein (Efb) from Staphylococcus aureus Inhibits the Formation of Platelet-Leukocyte Complexes.

    Science.gov (United States)

    Posner, Mareike G; Upadhyay, Abhishek; Abubaker, Aisha Alsheikh; Fortunato, Tiago M; Vara, Dina; Canobbio, Ilaria; Bagby, Stefan; Pula, Giordano

    2016-02-05

    Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to platelets proceeds via two independent mechanisms: fibrinogen-mediated and fibrinogen-independent. By proteomic analysis of Efb-interacting proteins within platelets and confirmation by pulldown assays followed by immunoblotting, we identified P-selectin and multimerin-1 as novel Efb interaction partners. The interaction of both P-selectin and multimerin-1 with Efb is independent of fibrinogen. We focused on Efb interaction with P-selectin. Excess of P-selectin extracellular domain significantly impaired Efb binding by activated platelets, suggesting that P-selectin is the main receptor for Efb on the surface of activated platelets. Efb-N interaction with P-selectin inhibited P-selectin binding to its physiological ligand, P-selectin glycoprotein ligand-1 (PSGL-1), both in cell lysates and in cell-free assays. Because of the importance of P-selectin-PSGL-1 binding in the interaction between platelets and leukocytes, we tested human whole blood and found that Efb abolishes the formation of platelet-monocyte and platelet-granulocyte complexes. In summary, we present evidence that in addition to its documented antithrombotic activity, Efb can play an immunoregulatory role via inhibition of P-selectin-PSGL-1-dependent formation of platelet-leukocyte complexes.

  5. 鼠抗人血小板CD36分子单克隆抗体的制备及活性分析%Preparation and Activity Analysis of Mouse Anti Human Platelet CD36 Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    陈麟凤; 张杰; 杨嘉慧; 罗圆圆; 庄远; 李卉; 冯倩; 汪德清

    2013-01-01

    本研究旨在制备具有功能活性的重组人血小板表面CD36抗原的鼠抗人单克隆抗体.提取人肝细胞总RNA,经RT-PCR扩增编码人血小板CD36抗原胞外区(Gly30-Asn439)氨基酸残基cDNA,构建于原核表达载体pMD18并转化大肠杆菌DH5α,筛选获得阳性重组子pMD18-CD36,提取质粒.经序列测定后,将该基因插入到真核细胞瞬时表达载体pTE2上,构建成为pTE2-s-CD36-10 His真核瞬时表达载体.采用lipofectamine 2000转染法,将重组质粒转染至HEK293细胞,表达产物经Ni2+2NTA柱层析纯化.以制备的重组CD36蛋白免疫BALB/c小鼠后,取脾与小鼠骨髓瘤细胞融合,筛选出阳性克隆,行Western blot检测抗体结合活性.结果显示:RT-PCR扩增获得了1.4 kb的片段.经测序,该序列分析结果与GenBank中的NM_001001547.2完全一致.SDS-PAGE证实转染的HEK293细胞表达了人CD36抗原胞外区蛋白片段.鼠单克隆抗体在Western blot中可以识别重组CD36蛋白,灵敏度达到8 ng.结论:成功制备了抗人血小板CD36单克隆抗体,为临床筛选CD36阴性患者及献血员、深入研究人血小板CD36表面抗原对血小板输注无效的影响提供了实验基础.%This study was purposed to prepare eukaryotic expression vector of recombinant human platelet CD36 gene. The total RNA was extracted from human liver tissue and the cDNA encoding human platelet CD36 antigen extracellular region ( Gly30 - Asn439) was amplified by RT-PCR. The cDNA was cloned into the prokaryotic expression vector pMD18 and the recombinant vector was transformed into E. coli DH5a. The positive recombinant pMD18-CD36 plasmid was screened. After sequencing, this combinant vector was inserted into the transient eukaryotic expression vector pTE2, the pTE2-s-CD36-10 His transient eukaryotic expression vector was constructed. The recombinant CD36 Gly30 - Asn439 expressed by HEK-293 cells was purifid with Ni2 + 2NTA chromatography. The results showed that 1.4 kb cDNA was

  6. [Effect of magnesium, acetylsalicilic acid and emoxypine on aggregation of platelets].

    Science.gov (United States)

    Loznikova, S Zh; Sukhodola, A A; Shcharbina, N Iu; Shcharbin, D G

    2014-01-01

    The thrombin-induced platelet aggregation was studied in the absence and presence of magnesium sulfate, acetylsalicylic acid and emoxypine. It was found that all the preparations studied were able separately to decrease platelet aggregation. In contrast, their joint action was not able to affect the aggregation of platelets. The data obtained can be used to choose the treatment strategy for patients with ischemic stroke.

  7. Antitumor immunity by a dendritic cell vaccine encoding secondary lymphoid chemokine and tumor lysate on murine prostate cancer

    Institute of Scientific and Technical Information of China (English)

    Jun Lu; Qi Zhang; Chun-Min Liang; Shu-Jie Xia; Cui-Ping Zhong; Da-Wei Wang

    2008-01-01

    Aim: To investigate the antitumor immunity by a dendritic cell (DC) vaccine encoding secondary lymphoid chemokine gene and tumor lysate on murine prostate cancer. Methods: DC from bone marrow of C57BL/6 were transfected with a plasmid vector expressing secondary lymphoid chemokine (SLC) cDNA by Lipofectamine2000 liposome and tumor lysate. Total RNA extracted from SLC+lysate-DC was used to verify the expression of SLC by reverse transcriptase-polymerase chain reaction (RT-PCR). The immunotherapeutic effect of DC vaccine on murine prostate cancer was assessed. Results: We found that in the prostate tumor model of C57BL/6 mice, the adminstration of SLC+lysate-DC inhibited tumor growth most significantly when compared with SLC-DC, lysate-DC, DC or phos-phate buffer solution (PBS) counterparts (P<0.01). Immunohistochemical fluorescent staining analysis showed the infiltration of more CD4+, CD8+ T cell and CD11c+ DC within established tumor treated by SLC+lysate-DC vaccine than other DC vaccines (P<0.01). Conclusion: DC vaccine encoding secondary lymphoid chemokine and tumor lysate can elicit significant antitumor immunity by infiltration of CD4+, CD8+ T cell and DC, which might provide a potential immunotherapy method for prostate cancer.

  8. Investigating GABA and its function in platelets as compared to neurons.

    Science.gov (United States)

    Kaneez, Fatima Shad; Saeed, Sheikh Arshad

    2009-08-01

    We have recently suggested that platelets could be used as a model for neuronal receptors. In this paper we have investigated gamma-aminobutyric acid (GABA) metabolism and GABA receptors in platelets and in cultured neurons to see whether platelets' GABA mimics neuronal GABA receptor activities. We used the ELISA technique for detecting the GABA concentration in platelet rich plasma and cultured neurons. The functional effects of GABA and its receptor ligands on platelets were determined using an aggregometer. We found that the GABA concentration is 30% lower in platelets than in neurons and in both preparations GABA was metabolized by GABA transaminase (GABA-T). GABA potentiated calcium dependent platelet aggregation with a higher value in washed platelets suspension (WPS) then in platelet rich plasma (PRP). This effect was inhibited by benzodiazepines, calcium channel blockers and the selective phosphoinositide 3-kinase antagonist Wortmannin. GABA neurotransmission is involved in most aspects of normal brain function and can be perturbed in many neuropathologic conditions. We concluded that platelets could be further developed to be used as a peripheral model to study neuronal GABAergic function and its abnormality in diseases such as epilepsy and schizophrenia. Furthermore our results indicated that PI3-kinase is involved in calcium dependent GABA induced platelet aggregation as this synergistic effect is inhibited by Wortmannin in dose dependent manner.

  9. A similar in vitro and in cell lysate folding intermediate for the FF domain.

    Science.gov (United States)

    Latham, Michael P; Kay, Lewis E

    2014-09-23

    Understanding the mechanisms by which proteins fold into their three-dimensional structures, including a description of the intermediates that are formed during the folding process, remains a goal of protein science. Most studies are performed under carefully controlled conditions in which the folding reaction is monitored in a buffer solution that is far from the natural milieu of the cell. Here, we have used (13)C and (1)H relaxation dispersion NMR spectroscopy to study folding of the FF domain in both Escherichia coli and Saccharomyces cerevisiae cellular lysates. We find that a conformationally excited state is populated in both lysates, which is very similar in structure to a folding intermediate observed in previous studies in buffer, with the kinetics and thermodynamics of the interconversion between native and intermediate conformers somewhat changed. The results point to the importance of extending folding studies beyond the test tube yet emphasize that insights can be obtained through careful experiments recorded in controlled buffer solutions.

  10. Prophylactic platelets in dengue

    DEFF Research Database (Denmark)

    Whitehorn, James; Rodriguez Roche, Rosmari; Guzman, Maria G

    2012-01-01

    of platelets in dengue. Respondents were all physicians involved with the treatment of patients with dengue. Respondents were asked that their answers reflected what they would do if they were the treating physician. We received responses from 306 physicians from 20 different countries. The heterogeneity...

  11. IT-24DEVELOPMENT OF A NOVEL AUTOLOGOUS DENDRITIC CELL / ALLOGENEIC GLIOBLASTOMA LYSATE VACCINE PROTOCOL

    OpenAIRE

    Parney, Ian; Peterson, Timothy; Gustafson, Michael; Dietz, Allan

    2014-01-01

    BACKGROUND: Dendritic cell (DC) vaccines for glioblastoma (GBM) are promising but significant conceptual shortcomings may have limited their clinical efficacy. First, most trials have not employed optimal DC culture techniques resulting in large numbers of immature (immunosuppressive) DC's. Second, most have used autologous tumor lysate. While highly personalized, this limits vaccine availability and precludes antigen-specific response testing. Finally, GBM-mediated immunosuppression has been...

  12. Rho GTPases in platelet function.

    Science.gov (United States)

    Aslan, J E; McCarty, O J T

    2013-01-01

    The Rho family of GTP binding proteins, also commonly referred to as the Rho GTPases, are master regulators of the platelet cytoskeleton and platelet function. These low-molecular-weight or 'small' GTPases act as signaling switches in the spatial and temporal transduction, and amplification of signals from platelet cell surface receptors to the intracellular signaling pathways that drive platelet function. The Rho GTPase family members RhoA, Cdc42 and Rac1 have emerged as key regulators in the dynamics of the actin cytoskeleton in platelets and play key roles in platelet aggregation, secretion, spreading and thrombus formation. Rho GTPase regulators, including GEFs and GAPs and downstream effectors, such as the WASPs, formins and PAKs, may also regulate platelet activation and function. In this review, we provide an overview of Rho GTPase signaling in platelet physiology. Previous studies of Rho GTPases and platelets have had a shared history, as platelets have served as an ideal, non-transformed cellular model to characterize Rho function. Likewise, recent studies of the cell biology of Rho GTPase family members have helped to build an understanding of the molecular regulation of platelet function and will continue to do so through the further characterization of Rho GTPases as well as Rho GAPs, GEFs, RhoGDIs and Rho effectors in actin reorganization and other Rho-driven cellular processes. © 2012 International Society on Thrombosis and Haemostasis.

  13. The influence of environmental variables on platelet concentration in horse platelet-rich plasma.

    Science.gov (United States)

    Rinnovati, Riccardo; Romagnoli, Noemi; Gentilini, Fabio; Lambertini, Carlotta; Spadari, Alessandro

    2016-07-04

    Platelet-rich plasma (PRP) commonly refers to blood products which contain a higher platelet (PLT) concentration as compared to normal plasma. Autologous PRP has been shown to be safe and effective in promoting the natural processes of soft tissue healing or reconstruction in humans and horses. Variability in PLT concentration has been observed in practice between PRP preparations from different patients or from the same individual under different conditions. A change in PLT concentration could modify PRP efficacy in routine applications. The aim of this study was to test the influence of environmental, individual and agonistic variables on the PLT concentration of PRP in horses. Six healthy Standardbred mares were exposed to six different variables with a one-week washout period between variables, and PRP was subsequently obtained from each horse. The variables were time of withdrawal during the day (morning/evening), hydration status (overhydration/dehydration) treatment with anti-inflammatory drugs and training periods on a treadmill. The platelet concentration was significantly higher in horses treated with a non-steroidal anti-inflammatory drug (P = 0.03). The leukocyte concentration increased 2-9 fold with respect to whole blood in the PRP which was obtained after exposure to all the variable considered. Environmental variation in platelet concentration should be taken into consideration during PRP preparation.

  14. Probing non-specific interactions of Ca{sup 2+}-calmodulin in E. coli lysate

    Energy Technology Data Exchange (ETDEWEB)

    Latham, Michael P.; Kay, Lewis E., E-mail: kay@pound.med.utoronto.ca [University of Toronto, Departments of Molecular Genetics, Biochemistry and Chemistry (Canada)

    2013-03-15

    The biological environment in which a protein performs its function is a crowded milieu containing millions of molecules that can potentially lead to a great many transient, non-specific interactions. NMR spectroscopy is especially well suited to study these weak molecular contacts. Here, non-specific interactions between the Ca{sup 2+}-bound form of calmodulin (CaM) and non-cognate proteins in Escherichia coli lysate are explored using Ile, Leu, Val and Met methyl probes. Changes in CaM methyl chemical shifts as a function of added E. coli lysate are measured to determine a minimum 'average' dissociation constant for interactions between Ca{sup 2+}-CaM and E. coli lysate proteins. {sup 2}H R{sub 2} and {sup 13}C R{sub 1} spin relaxation rates report on the binding reaction as well. Our results further highlight the power of methyl containing side-chains for characterizing biomolecular interactions, even in complex in-cell like environments.

  15. Influência da coleta, da produção e da estocagem na qualidade dos concentrados de plaquetas Influence of collection, preparation and storage on the quality of platelet concentrates

    Directory of Open Access Journals (Sweden)

    Maria Aparecida V. Tostes

    2008-10-01

    Full Text Available Como o controle de qualidade dos concentrados de plaquetas (CP, feito na data de seu vencimento, não possibilita distinguir o momento e o procedimento que determina a eventual redução de sua qualidade, decidimos investigar, separadamente, a influência da coleta, da produção e da estocagem sobre a qualidade deste hemocomponente. Foram avaliados, em 33 CP randômicos, diariamente, durante cinco dias, os seguintes parâmetros: a agregação, o número de plaquetas e leucócitos, a pO2 e pCO2, o pH, sódio e potássio, a presença de swirling, grumos, hemácias e lipemia e cultura para bactérias. Observamos maior queda da agregação plaquetária com pares de agonistas durante a produção dos CP (de 99,4% para 59,8%, pAs quality control of platelet concentrates obtained at the expiration date does not distinguish between the different stages that may cause reductions in quality, such as collection and storage, we decided to separately investigate the influence of collection, processing and storage on the quality of these blood components. This study evaluated 33 random platelet concentrates daily for five days for the following parameters: aggregation, number of platelets and leukocytes, PO2 and PCO2, pH, sodium and potassium, the presence of swirling, platelet clots, red blood cells and lipemia, and bacteria culture. We observed a greater decrease in platelet aggregation with pairs of agonists during the production of platelet concentrates (from 99.4% to 59.8%, p<0.05, followed by a gradual drop during storage reaching 40.4% on day five. During storage the following were also observed: 1. a gradual drop of platelet concentration (p<0.05 although the values always remained higher than 5x1010/70 mL; 2. a decrease in leukocyte concentration (p<0.05; 3. increases in pO2 and decreases in pCO2 (p<0.05; 4. increase in the pH (p<0.05 from day 4 and in concentrations of sodium and potassium (in general p<0.05; 5. swirling in all platelet

  16. [Risk Assessment of Single-Donor (Apheresis) Platelet Concentrates and Pooled Whole-Blood-Derived Platelet Concentrates].

    Science.gov (United States)

    Hitzler, Walter; Hutschenreuter, Gabriele; Wartensleben, Herbert

    2015-01-01

    According to the risk estimates of the Robert-Koch-Institute (RKI) and the Paul Ehrlich-Institute (PEI) an equivalence cannot be assumed to exist between the two different platelet preparations. Differences between single-donor (apheresis) platelet concentrates (ATK) and pooled whole-blood-derived platelet concentrates (PTK) result from donor populations, donation intervals, and preparation techniques. There are no prospective randomized studies with regard to the clinical efficacy, which would unambiguously demonstrate equivalence of the therapeutic efficacy of PTK (buffy coat method) in comparison to ATK. The German Association of Blood Transfusion Services (StKB) points out that, due to the non-equivalence of PTK and ATK, it is incumbent on the transfusion physician to select the platelet concentrate, make the appropriate disclosures, and assume treatment responsibility. Proper compensation for ATK and PTK must be ensured by the health insurance companies, whereby a special indication for the selection of either PTK or ATK is not given. Exceptions are patients with known HLA antibodies in which only selected platelet concentrates may be administered. Otherwise, no indication exists in the selection of the different platelet concentrates (Article is in German).

  17. A practical synthesis of sarpogrelate hydrochloride and in vitro platelet aggregation inhibitory activities of its analogues

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A convenient approach for the preparation of sarpogrelate hydrochloride was developed.Two series of sarpogrelate hydrochloride analogues were designed and synthesized in order to improve their platelet aggregation inhibitory activities, biological tests suggested that these compounds have platelet aggregation inhibitory activities to some extent.

  18. Formation of nematic liquid crystals of sterically stabilized layered double hydroxide platelets

    NARCIS (Netherlands)

    Mourad, M.C.D.; Devid, E.J.; van Schooneveld, M.M.; Vonk, Ch.; Lekkerkerker, H.N.W.

    2008-01-01

    Colloidal platelets of hydrotalcite, a layered double hydroxide, have been prepared by coprecipitation at pH 11−12 of magnesium nitrate and aluminum nitrate at two different magnesium to aluminum ratios. Changing the temperature and ionic strength during hydrothermal treatment, the platelets were ta

  19. Actively induced platelet-bound IgG associated with thrombocytopenia in the marmoset

    Energy Technology Data Exchange (ETDEWEB)

    Gengozian, N.; McLaughlin, C.L.

    1978-06-01

    Interspecies platelet immunizations among marmosets lead to antibody formation to the donor platelets and a profound thrombocytopenia, which when associated with anemia may result in death of the animal. This actively induced immonologic thrombocytopenia closely resembles two clinical disease entities manifesting autoimmune thrombocytopenia, posttransfusion purpura and idiopathic thrombocytopenic purpura. Although antibody to donor-type platelets could be demonstrated readily, antihost activity was most often nondetectable or, when present, was in very low titer. A consistent finding was the appearance of IgG on the host's platelets shortly after immunization and concomitant with the appearance of antidonor platelet antibody. In 3 of 13 immunized animals thromoocytopenia did not occur even though antibody was formed and the host's platelets became IgG positive. In those animals that recovered from the induced thrombocytopenia IgG-positive platelets were found for periods ranging from 30 to greater than 100 days. Splenectomy before or after immunization did not alter the sequential development of antibody formation, appearance of IgG-positive platelets, and thrombocytopenia. Eluates prepared from IgG-positive platelets contained IgG and platelet antigens; the eluted IgG could attach nonspecifically to platelets of host or donor (immunizing) type, in contrast to the species specificity demonstrated for IgG eluted from platelets that had been reacted in vitro with specific antibody. Platelets in a few normal, nonimmunized marmosets were found to have signficant amounts of IgG on their surface, comparable to that observed in the immunized animal; interestingly, such IgG-positive platelets were found among imported but not laboratory-bred marmosets.

  20. Expansion of the neonatal platelet mass is achieved via an extension of platelet lifespan

    OpenAIRE

    Liu, Zhi-Jian; Hoffmeister, Karin M.; Hu, Zhongbo; Mager, Donald E.; Ait-Oudhia, Sihem; Debrincat, Marlyse A.; Pleines, Irina; Josefsson, Emma C.; Benjamin T Kile; Italiano, Joseph; Ramsey, Haley; Grozovsky, Renata; Veng-Pedersen, Peter; Chavda, Chaitanya; Sola-Visner, Martha

    2014-01-01

    Rapid growth and rising platelet counts result in a significant expansion of platelet mass during neonatal life.The rise in platelet counts is mediated by a prolongation in the neonatal platelet lifespan.

  1. Yeast lysates carrying the nucleoprotein from measles virus vaccine as a novel subunit vaccine platform to deliver Plasmodium circumsporozoite antigen

    National Research Council Canada - National Science Library

    Daria Jacob; Claude Ruffie; Chantal Combredet; Pauline Formaglio; Rogerio Amino; Robert Menard; Frederic Tangy; Monica Sala

    2017-01-01

    .... In this study, Pichia pastoris yeast lysates carrying the nucleoprotein (N) from the measles vaccine virus were evaluated as a novel subunit vaccine platform to deliver the circumsporozoite surface antigen (CS) of Plasmodium...

  2. Treatment of established colon carcinoma-bearing mice by dendritic cells pulsed with lysates of heat-treated tumor cells

    Institute of Scientific and Technical Information of China (English)

    YING MinGang; ZHEN QiuHong; LIU Sheng; GONG FuSheng; XIE YunQing

    2009-01-01

    To investigate the therapeutic effect of dendritic cells pulsed with lysates of heat-treated CT26 colon carcinoma cells. Bone marrow-derived DCs were pulsed with lysates of heat-treated tumor cells and were used to immunize BALB/c mice with established colon carcinoma. Cytotoxic T lymphocyte (CTL) response was detected. The therapeutic effect induced by DCs was observed by tumor weight and survival time. DCs pulsed with lysates of heat-treated tumor cells markedly induced specific cytotoxic activity of CTLs. Tumor growth in the immunized BALB/c mice was significantly inhibited and the survival time of the tumor-bearing mice was prolonged, DCs pulsed with lysates of heat-treated tumor cells have an observable therapeutic effect on established colon carcinoma-bearing mice.

  3. Effective biotic elicitation of Ruta graveolens L. shoot cultures by lysates from Pectobacterium atrosepticum and Bacillus sp.

    Science.gov (United States)

    Orlita, A; Sidwa-Gorycka, M; Malinski, E; Czerwicka, M; Kumirska, J; Golebiowski, M; Lojkowska, E; Stepnowski, P

    2008-03-01

    Growth of Ruta graveolens shoots was induced when Bacillus sp. cell lysates were added to the culture medium. Elicitation of coumarin by this lysate was also very effective; the concentrations of isopimpinelin, xanthotoxin and bergapten increased to 610, 2120 and 1460 microg g(-1) dry wt, respectively. It also had a significant effect on the production of psoralen and rutamarin (680 and 380 microg g(-1) dry wt) and induced the biosynthesis of chalepin, which was not detected in the control sample, up to 47 microg g(-1) dry wt With lysates of the Pectobacterium atrosepticum, their effect on growth was not so significant and had no effect on the induction of coumarin accumulation. But elicitation with this lysate was much more effective for inducing the production of furoquinolone alkaloids; the concentrations of gamma-fagarine, skimmianine, dictamnine and kokusaginine rose to 99, 680, 172 and 480 microg g(-1) dry wt, respectively.

  4. Treatment of established colon carcinoma-bearing mice by dendritic cells pulsed with lysates of heat-treated tumor cells

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    To investigate the therapeutic effect of dendritic cells pulsed with lysates of heat-treated CT26 colon carcinoma cells. Bone marrow-derived DCs were pulsed with lysates of heat-treated tumor cells and were used to immunize BALB/c mice with established colon carcinoma. Cytotoxic T lymphocyte (CTL) response was detected. The therapeutic effect induced by DCs was observed by tumor weight and survival time. DCs pulsed with lysates of heat-treated tumor cells markedly induced specific cytotoxic activity of CTLs. Tumor growth in the immunized BALB/c mice was significantly inhibited and the survival time of the tumor-bearing mice was prolonged. DCs pulsed with lysates of heat-treated tumor cells have an observable therapeutic effect on established colon carcinoma-bearing mice.

  5. PREGNANCY WITH PLATELET FUNCTION DISORDER

    Directory of Open Access Journals (Sweden)

    Sheila K

    2014-01-01

    Full Text Available latelets play a vital role in haemostasis . Antenatal patients with platelet function disorders should be managed in tertiary care centres that are well equipped to tackle any obstetric haemorrhage that can ensue during labour and delivery . Primi gravida was admitted for safe confinement . She had been evaluated earlier for complaints of multiple episodes of mucosal bleeding . On evaluation she had nor mal platelet counts and coagulation factor assay was normal . Platelet aggregometry revealed mild disorder of platelet aggregation . She was planned for induction of labour after arranging enough blood and blood products . She got into active labour and was p ut on syntocinon augmentation . She had emergency Caesarean section for foetal distress . Oxytocics were given proactively . Intraoperatively platelet transfusions and tranexamic acid infusion were given . Complete haemostasis was achieved . She had an uneventf ul postoperative period . Patients with functional platelet disorders can be successfully managed with local application of antifibrinolytic agents like tranexamic acid , in case of minor bleeds . Platelet transfusions are very effective in tackling major ble eds , especially during surgeries and for obstetric indications . If a patient has the history of clinically significant bleeding suggestive of platelet dysfunction , appropriate platelet function tests should be obtained so that the risk of bleeding can be adequately assessed and therapy chosen more rationally . . In obstetric practice the response of such patients to platelet transfusions has been excellent

  6. Platelet audit: Assessment and utilization of this precious resource from a tertiary care hospital

    Directory of Open Access Journals (Sweden)

    Saluja K

    2007-01-01

    Full Text Available Background: To assess the appropriate utilization of platelet transfusions [random donor platelets (RDP and single donor platelets (SDP]; a six-month retrospective audit was carried out in a tertiary care hospital. Materials and Methods: A six-month retrospective platelet audit was carried out from May to October 2005 to estimate its preparation, appropriate utilization and wastage rate. Patient′s demographics, transfusion triggers and episodes and ABO and Rh (D group specific or non-group specific transfusions were also assessed. Results: About 5525 units of platelets [PRP-PC, 3,813 (69%; BC-PC, 983 (17.8%; PRP, 648 (11.7% and SDP 81 (1.5%] were prepared and transfused to 853 patients (RDP to 814 patients and SDP to 39 patients in 2,093 transfusion episodes. Adult and pediatric hemato-oncology were the main user specialties utilizing 39.1 and 87.6% of the RDPs and SDPs prepared. Of the patients receiving RDPs, 95% were transfused ABO and Rh (D group specific platelets whereas 100% SDPs transfusions were of group specific platelets. 88% of prophylactic platelet transfusions were appropriate as per the recommended BCSH guidelines. However, 12% of the prophylactic platelets were transfused inappropriately in cardiopulmonary bypass (CPB surgeries with normal platelet counts and no evidence of bleeding related to platelets. Out of 5,444 RDPs prepared 1,585 (29.11% units were not utilized. Conclusions: Regular audit of blood and blood components is a must so that necessary remedial measures can be taken to maximize appropriate and judicious utilization of each component.

  7. Effects of hormones on platelet aggregation.

    Science.gov (United States)

    Farré, Antonio López; Modrego, Javier; Zamorano-León, José J

    2014-04-01

    Platelets and their activation/inhibition mechanisms play a central role in haemostasis. It is well known agonists and antagonists of platelet activation; however, during the last years novel evidences of hormone effects on platelet activation have been reported. Platelet functionality may be modulated by the interaction between different hormones and their platelet receptors, contributing to sex differences in platelet function and even in platelet-mediated vascular damage. It has suggested aspects that apparently are well established should be reviewed. Hormones effects on platelet activity are included among them. This article tries to review knowledge about the involvement of hormones in platelet biology and activity.

  8. Platelet aggregation following trauma

    DEFF Research Database (Denmark)

    Windeløv, Nis A; Sørensen, Anne M; Perner, Anders

    2014-01-01

    We aimed to elucidate platelet function in trauma patients, as it is pivotal for hemostasis yet remains scarcely investigated in this population. We conducted a prospective observational study of platelet aggregation capacity in 213 adult trauma patients on admission to an emergency department (ED......). Inclusion criteria were trauma team activation and arterial cannula insertion on arrival. Blood samples were analyzed by multiple electrode aggregometry initiated by thrombin receptor agonist peptide 6 (TRAP) or collagen using a Multiplate device. Blood was sampled median 65 min after injury; median injury...... severity score (ISS) was 17; 14 (7%) patients received 10 or more units of red blood cells in the ED (massive transfusion); 24 (11%) patients died within 28 days of trauma: 17 due to cerebral injuries, four due to exsanguination, and three from other causes. No significant association was found between...

  9. Murine monoclonal antibody to platelet factor 4/heparin complexes as a potential reference standard for platelet activation assays in heparin-induced thrombocytopenia.

    Science.gov (United States)

    Asada, Reiko; Wanaka, Keiko; Walenga, Jeanine; Prechel, Margaret; Miyashita, Kumiko; Escalante, Vicki; Kaneko, Chieko; Hoshino, Nobuhiro; Oosawa, Mitsuru; Matsuo, Miyako

    2013-01-01

    Quality control of the platelet activation assays to diagnose heparin-induced thrombocytopenia (HIT), (14)C-serotonin release assay (SRA) and platelet aggregation test (PAT) has yet to be established due to lack of reference standards and the difficulty of obtaining significant amounts of HIT antibodies from patients with HIT. We prepared a murine monoclonal antibody to human platelet factor 4 (hPF4)/heparin complexes (HIT-MoAb) and investigated the platelet activating action of HIT-MoAb by using SRA and PAT. The HIT-MoAb activated human platelets at low heparin concentration and the platelet activations were inhibited at high heparin concentration in both SRA and PAT. The HIT-MoAb produced a concentration-dependent effect. Moreover, the platelet activation at low heparin concentration was inhibited by anti-FcγRIIa antibody. These results indicated that HIT-MoAb has characteristics similar to human HIT antibodies regarding heparin-dependent platelet activation. Therefore, it is suggested that HIT-MoAb has the potential to be a positive control or reference standard in platelet activation assays.

  10. Platelets in inflammation and immunity.

    Science.gov (United States)

    Herter, J M; Rossaint, J; Zarbock, A

    2014-11-01

    The paradigm of platelets as mere mediators of hemostasis has long since been replaced by a dual role: hemostasis and inflammation. Now recognized as key players in innate and adaptive immune responses, platelets have the capacity to interact with almost all known immune cells. These platelet-immune cell interactions represent a hallmark of immunity, as they can potently enhance immune cell functions and, in some cases, even constitute a prerequisite for host defense mechanisms such as NETosis. In addition, recent studies have revealed a new role for platelets in immunity: They are ubiquitous sentinels and rapid first-line immune responders, as platelet-pathogen interactions within the vasculature appear to precede all other host defense mechanisms. Here, we discuss recent advances in our understanding of platelets as inflammatory cells, and provide an exemplary review of their role in acute inflammation.

  11. Estrogen, inflammation, and platelet phenotype.

    Science.gov (United States)

    Miller, Virginia M; Jayachandran, Muthuvel; Hashimoto, Kazumori; Heit, John A; Owen, Whyte G

    2008-01-01

    Although exogenous estrogenic therapies increase the risk of thrombosis, the effects of estrogen on formed elements of blood are uncertain. This article examines the genomic and nongenomic actions of estrogen on platelet phenotype that may contribute to increased thrombotic risk. To determine aggregation, secretion, protein expression, and thrombin generation, platelets were collected from experimental animals of varying hormonal status and from women enrolled in the Kronos Early Estrogen Prevention Study. Estrogen receptor beta predominates in circulating platelets. Estrogenic treatment in ovariectomized animals decreased platelet aggregation and adenosine triphosphate (ATP) secretion. However, acute exposure to 17beta-estradiol did not reverse decreases in platelet ATP secretion invoked by lipopolysaccharide. Thrombin generation was positively correlated to the number of circulating microvesicles expressing phosphatidylserine. Assessing the effect of estrogen treatments on blood platelets may lead to new ways of identifying women at risk for adverse thrombotic events with such therapies.

  12. Differential capacity of chaperone-rich lysates in cross-presenting human endogenous and exogenous melanoma differentiation antigens.

    Science.gov (United States)

    Bleifuss, Elke; Bendz, Henriette; Sirch, Birgit; Thompson, Sylvia; Brandl, Anna; Milani, Valeria; Graner, Michael W; Drexler, Ingo; Kuppner, Maria; Katsanis, Emmanuel; Noessner, Elfriede; Issels, Rolf-Dieter

    2008-12-01

    The goal of immune-based tumor therapies is the activation of immune cells reactive against a broad spectrum of tumor-expressed antigens. Vaccines based on chaperone proteins appear promising as these proteins naturally exist as complexes with various protein fragments including those derived from tumor-associated antigens. Multi-chaperone systems are expected to have highest polyvalency as different chaperones can carry distinct sets of antigenic fragments. A free-solution isoelectric focusing (FS-IEF) technique was established to generate chaperone-rich cell lysates (CRCL). Results from murine systems support the contention that CRCL induce superior anti-tumor responses than single chaperone vaccines. We established an in vitro model for human melanoma to evaluate the capacity of CRCL to transfer endogenously expressed tumor antigens to the cross-presentation pathway of dendritic cells (DC) for antigen-specific T cell stimulation. CRCL prepared from human melanoma lines contained the four major chaperone proteins Hsp/Hsc70, Hsp90, Grp94/gp96 and calreticulin. The chaperones within the melanoma cell-derived CRCL were functionally active in that they enhanced cross-presentation of exogenous peptides mixed into the CRCL preparation. Superior activity was observed for Hsp70-rich CRCL obtained from heat-stressed melanoma cells. Despite the presence of active chaperones, melanoma cell-derived CRCL failed to transfer endogenously expressed melanoma-associated antigens to DC for cross-presentation and cytotoxic T cell (CTL) recognition, even after increasing intracellular protein levels of tumor antigen or chaperones. These findings reveal limitations of the CRCL approach regarding cross-presentation of endogenously expressed melanoma-associated antigens. Yet, CRCL may be utilized as vehicles to enhance the delivery of exogenous antigens for DC-mediated cross-presentation and T cell stimulation.

  13. Complement Activation Alters Platelet Function

    Science.gov (United States)

    2015-12-01

    Award Number: W81XWH-12-1-0523 TITLE: Complement Activation Alters Platelet Function PRINCIPAL INVESTIGATOR: George Tsokos, M.D. CONTRACTING...Activation Alters Platelet Function 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-12-1-0523 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) George Tsokos, M.D...a decreased level of disease. Further studies will expand upon these observations better outlining the function of platelets in the injury associated

  14. One size doesn’t fit all: Should we reconsider the introduction of cold-stored platelets in blood bank inventories?

    Science.gov (United States)

    Berzuini, Alessandra; Spreafico, Marta; Prati, Daniele

    2017-01-01

    Platelet concentrates are universally prepared with a standard method and stored for 5 days at room temperature (20–24°C) in gentle agitation. Currently, there is a renewed interest in the possibility of storing platelet concentrates below the standard temperatures. In fact, cold platelets might be more effective in bleeding patients and have a lower risk of bacterial transmission. Inventories including platelets at different temperatures may favour patient-centred strategies for prophylactic or therapeutic transfusions.

  15. Platelet effects on ovarian cancer

    Science.gov (United States)

    Davis, Ashley; Afshar-Kharghan, Vahid; Sood, Anil K.

    2014-01-01

    Growing understanding of the role of thrombocytosis, high platelet turnover, and the presence of activated platelets in the circulation in cancer progression and metastasis has brought megakaryocytes into focus. Platelet biology is essential to hemostasis, vascular integrity, angiogenesis, inflammation, innate immunity, wound healing, and cancer biology. However, before megakaryocyte/platelet-directed therapies can be considered for clinical use, understanding of the mechanism and biology of paraneoplastic thrombocytosis in malignancy is required. Here, we provide an overview of the clinical implications, biological significance, and mechanisms of paraneoplastic thrombocytosis in the context of ovarian cancer. PMID:25023353

  16. Novel aspects of platelet aggregation

    Directory of Open Access Journals (Sweden)

    Roka-Moya Y. M.

    2014-01-01

    Full Text Available The platelet aggregation is an important process, which is critical for the hemostatic plug formation and thrombosis. Recent studies have shown that the platelet aggregation is more complex and dynamic than it was previously thought. There are several mechanisms that can initiate the platelet aggregation and each of them operates under specific conditions in vivo. At the same time, the influence of certain plasma proteins on this process should be considered. This review intends to summarize the recent data concerning the adhesive molecules and their receptors, which provide the platelet aggregation under different conditions.

  17. Overview of platelet physiology and laboratory evaluation of platelet function.

    Science.gov (United States)

    Rodgers, G M

    1999-06-01

    Appropriate laboratory testing for the platelet-type bleeding disorders hinges on an adequate assessment in the history and physical examination. Patients with histories and screening laboratory results consistent with coagulation disorders (hemophilia, disseminated intravascular coagulation) are not appropriate candidates for platelet function testing. In contrast, patients with a lifelong history of platelet-type bleeding symptoms and perhaps a positive family history of bleeding would be appropriate for testing. Figure 6 depicts one strategy to evaluate these patients. Platelet morphology can easily be evaluated to screen for two uncommon qualitative platelet disorders: Bernard-Soulier syndrome (associated with giant platelets) and gray platelet syndrome, a subtype of storage pool disorder in which platelet granulation is morphologically abnormal by light microscopy. If the bleeding disorder occurred later in life (no bleeding with surgery or trauma early in life), the focus should be on acquired disorders of platelet function. For those patients thought to have an inherited disorder, testing for vWD should be done initially because approximately 1% of the population has vWD. The complete vWD panel (factor VIII coagulant activity, vWf antigen, ristocetin cofactor activity) should be performed because many patients will have abnormalities of only one particular panel component. Patients diagnosed with vWD should be classified using multimeric analysis to identify the type 1 vWD patients likely to respond to DDAVP. If vWD studies are normal, platelet aggregation testing should be performed, ensuring that no antiplatelet medications have been ingested at least 1 week before testing. If platelet aggregation tests are normal and if suspicion for an inherited disorder remains high, vWD testing should be repeated. The evaluation of thrombocytopenia may require bone marrow examination to exclude primary hematologic disorders. If future studies with thrombopoietin assays

  18. Quality evaluation of red blood cell suspension and platelet concentrate separated and prepared from whole blood stored at 22℃ for 24 hours%全血于22℃保存24h分离制备悬浮红细胞及浓缩血小板的质量评价

    Institute of Scientific and Technical Information of China (English)

    黄彦; 符雪丽; 李诗敏; 梁若鹄; 高艳; 陈龙菊

    2016-01-01

    细胞混入量、储存期末pH值均符合《全血及成分血质量要求》(GB18469-2012)的相关规定;两组浓缩血小板的血小板含量、红细胞混入量、FHb水平、血小板黏附率、血小板聚集率,储存期末pH值,以及K+、Na+、Cl-浓度比较,差异均无统计学意义(t=0.17、0.16、0.56、2.43、0.36、2.50、1.85、1.75、0.32,P>0.05).结论 22℃保存24 h制备的悬浮红细胞、浓缩血小板的质量符合国家标准,全血22℃保存过夜分离制备悬浮红细胞、浓缩血小板的方法可行.%Objective To evaluate the quality of red blood cell suspension and platelet concentrate separated and prepared from whole blood stored at 22℃ for 24 h.Methods From May 2014 to February 2015,a total of 60 voluntary blood donors who were recruited by Maoming Central Blood Station of Guangdong Province were included into this study,using simple random sampling method.The inclusion criterion for subjects was that the physical examination and related hematology tests results of all blood donors before blood donation met the relevant regulations in the Whole Blood and Blood Component Donor Selection Requirements (GB18467-2011).All the 60 donors were randomly divided into study group (n =30) and control group (n=30).A total of 60 bags of whole blood (400 mL) were collected from the blood donors in the two groups,using quintuple blood collection bags.In the study group,the whole blood was stored and transported at 22 ℃,and prepared into red blood cell suspension and platelet concentrate 24 h after blood collection.In the control group,the whole blood was also stored and transported at 22 ℃,and prepared into red blood cell suspension and platelet concentrate within 8 h after blood collection.The preparation of red blood cell suspension and platelet concentrate in both groups was conducted by the buffy coat method.Red blood cell suspension was stored at 4 ℃ until the end of the storage period (35 d after preparation),and its

  19. Comparative Analysis of Click Chemistry Mediated Activity-Based Protein Profiling in Cell Lysates

    Directory of Open Access Journals (Sweden)

    Yinliang Yang

    2013-10-01

    Full Text Available Activity-based protein profiling uses chemical probes that covalently attach to active enzyme targets. Probes with conventional tags have disadvantages, such as limited cell permeability or steric hindrance around the reactive group. A tandem labeling strategy with click chemistry is now widely used to study enzyme targets in situ and in vivo. Herein, the probes are reacted in live cells, whereas the ensuing detection by click chemistry takes place in cell lysates. We here make a comparison of the efficiency of the activity-based tandem labeling strategy by using Cu(I-catalyzed and strain-promoted click chemistry, different ligands and different lysis conditions.

  20. Platelet Concentrates: Past, Present and Future

    OpenAIRE

    2011-01-01

    Platelets play a crucial role in hemostasis and wound healing, platelet growth factors are well known source of healing cytokines. Numerous techniques of autologous platelet concentrates have been developed and applied in oral and maxillofacial surgery. This review describes the evolution of the first and second generation of platelet concentrates (platelet rich plasma and platelet rich fibrin respectively) from their fore runner-fibrin sealants.

  1. Studies on megakaryopoiesis and platelet function

    OpenAIRE

    Meinders, M.

    2015-01-01

    Platelets are blood circulating specialized subcellular fragments, which are produced by megakaryocytes. Platelets are essential for hemostasis and wound healing but also play a role in non-hemostatic processes such as the immune response or cancer metastasis. Considering the immediate precursors of platelets, normal megakaryocyte development is essential for normal platelet function. Although much is known about platelet development, some aspects of platelet production remain poorly understo...

  2. [STRUCTURAL CHARACTERIZATION OF PLATELETS AND PLATELET-DERIVED MICROVESICLES].

    Science.gov (United States)

    Ponomareva, A A; Nevzorova, T A; Mordakhanova, E R; Andrianova, I A; Litvinov, R I

    2016-01-01

    Platelets are the anucleated blood cells, wich together with the fibrin stop bleeding (hemostasis). Cellular microvesicles are membrane-surrounded microparticles released into extracellular space upon activation and/or apoptosis of various cells. Platelet-derived macrovesicles from the major population of circulating blood microparticles that play an important role in hemostasis and thrombosis. Despite numerous studies on the pathophysiology of platelet-derived macrovesicles, mechanisms of their formation and structural details remain poorly understood. Here we investigated the ultrastructure of parental platelets and platelet-derived microvesicles formed in vitro by quiescent cells as well as by cells stimulated with one of the following activators: arachidonic acid, ADP, thrombin, calcium ionophore A23187. Using transmission electron microscopy of human platelets and isolated microvesicles, we analyzed the intracellular origin, steps of formation, structural diversity, and size distributions of the subcellular particles. We have revealed that thrombin, unlike other stimuli, not only induced vesiculation of the plasma membrane but also caused break-up of the cells followed by formation of microparticles that are comparable with microvesicles by size. A fraction of these microparticles contained cellular organelles surrounded by a thin membrane. The size of platelet-derived macrovesicles varied from 30 nm to 500 nm, however, the size distributions depended on the nature of a cell-activating stimulus. The results obtained provide new information about the formation of platelet-derived macrovesicles and their structural diversity, wich is important to understand their multiple functions in normal and disease states.

  3. Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination.

    Science.gov (United States)

    Bosschem, Iris; Bayry, Jagadeesh; De Bruyne, Ellen; Van Deun, Kim; Smet, Annemieke; Vercauteren, Griet; Ducatelle, Richard; Haesebrouck, Freddy; Flahou, Bram

    2015-01-01

    Helicobacter suis (H. suis) is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund's Complete and Incomplete, Cholera toxin), administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund's complete (FC)/lysate and intranasal immunization with Cholera toxin (CT)/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually) is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.

  4. Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination.

    Directory of Open Access Journals (Sweden)

    Iris Bosschem

    Full Text Available Helicobacter suis (H. suis is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund's Complete and Incomplete, Cholera toxin, administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund's complete (FC/lysate and intranasal immunization with Cholera toxin (CT/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.

  5. Characterization and ATPase activity of human platelet actomyosin

    NARCIS (Netherlands)

    Lindemans, J.; Bouma, B.N.; Sixma, J.J.

    1974-01-01

    Platelet actomyosin, partially purified by successive precipitation had a specific viscosity of 0,15 and a sensitivity to ATP of 60 %. The enzyme preparation was separated into the actin and myosin components and some myosin fragments by SDS-polyacrylamide gel electrophoresis. The ATPase activity of

  6. Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation.

    Science.gov (United States)

    Frelinger, A L; Torres, A S; Caiafa, A; Morton, C A; Berny-Lang, M A; Gerrits, A J; Carmichael, S L; Neculaes, V B; Michelson, A D

    2016-01-01

    Therapeutic use of activated platelet-rich plasma (PRP) has been explored for wound healing, hemostasis and antimicrobial wound applications. Pulse electric field (PEF) stimulation may provide more consistent platelet activation and avoid complications associated with the addition of bovine thrombin, the current state of the art ex vivo activator of therapeutic PRP. The aim of this study was to compare the ability of PEF, bovine thrombin and thrombin receptor activating peptide (TRAP) to activate human PRP, release growth factors and induce cell proliferation in vitro. Human PRP was prepared in the Harvest SmartPreP2 System and treated with vehicle, PEF, bovine thrombin, TRAP or Triton X-100. Platelet activation and procoagulant markers and microparticle generation were measured by flow cytometry. Released growth factors were measured by ELISA. The releasates were tested for their ability to stimulate proliferation of human epithelial cells in culture. PEF produced more platelet-derived microparticles, P-selectin-positive particles and procoagulant annexin V-positive particles than bovine thrombin or TRAP. These differences were associated with higher levels of released epidermal growth factor after PEF than after bovine thrombin or TRAP but similar levels of platelet-derived, vascular-endothelial, and basic fibroblast growth factors, and platelet factor 4. Supernatant from PEF-treated platelets significantly increased cell proliferation compared to plasma. In conclusion, PEF treatment of fresh PRP results in generation of microparticles, exposure of prothrombotic platelet surfaces, differential release of growth factors compared to bovine thrombin and TRAP and significant cell proliferation. These results, together with PEF's inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications.

  7. [Application of platelet rich plasma (PRP) and its derivatives in dental implantologie and plastic surgery].

    Science.gov (United States)

    González, Maczy; Arteaga-Vizcaíno, Melvis; Benito, Marisol; Benito, Mariluz

    2012-12-01

    The platelet rich plasma (PRP), as its name implies, has a high concentration of thrombocytes; is a non-toxic and non-allergenic autologous preparation, obtained by blood centrifugation at low speed. Its function is directly linked to the release of growth factors (FC) by platelets. These factors have properties of induction of tissue regeneration. For the preparation of the present work, a literature search was performed in different documentary sources using the following descriptors: platelet rich plasma, PRP, growth factors, GF, dentistry and cosmetic surgery. This article is a description of some relevant aspects of the PRP and its application in the areas of dentistry and cosmetic surgery.

  8. Platelets, inflammation and tissue regeneration.

    Science.gov (United States)

    Nurden, Alan T

    2011-05-01

    Blood platelets have long been recognised to bring about primary haemostasis with deficiencies in platelet production and function manifesting in bleeding while upregulated function favourises arterial thrombosis. Yet increasing evidence indicates that platelets fulfil a much wider role in health and disease. First, they store and release a wide range of biologically active substances including the panoply of growth factors, chemokines and cytokines released from a-granules. Membrane budding gives rise to microparticles (MPs), another active participant within the blood stream. Platelets are essential for the innate immune response and combat infection (viruses, bacteria, micro-organisms). They help maintain and modulate inflammation and are a major source of pro-inflammatory molecules (e.g. P-selectin, tissue factor, CD40L, metalloproteinases). As well as promoting coagulation, they are active in fibrinolysis; wound healing, angiogenesis and bone formation as well as in maternal tissue and foetal vascular remodelling. Activated platelets and MPs intervene in the propagation of major diseases. They are major players in atherosclerosis and related diseases, pathologies of the central nervous system (Alzheimers disease, multiple sclerosis), cancer and tumour growth. They participate in other tissue-related acquired pathologies such as skin diseases and allergy, rheumatoid arthritis, liver disease; while, paradoxically, autologous platelet-rich plasma and platelet releasate are being used as an aid to promote tissue repair and cellular growth. The above mentioned roles of platelets are now discussed.

  9. Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

    Science.gov (United States)

    Marchenko, N Yu; Sikorskaya, E V; Marchenkov, V V; Kashparov, I A; Semisotnov, G V

    2016-03-01

    Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme.

  10. Naegleria fowleri lysate induces strong cytopathic effects and pro-inflammatory cytokine release in rat microglial cells.

    Science.gov (United States)

    Lee, Yang-Jin; Park, Chang-Eun; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Jung, Suk-Yul; Shin, Ho-Joon

    2011-09-01

    Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A (51)Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.

  11. Naegleria fowleri Lysate Induces Strong Cytopathic Effects and Pro-inflammatory Cytokine Release in Rat Microglial Cells

    Science.gov (United States)

    Lee, Yang-Jin; Park, Chang-Eun; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Jung, Suk-Yul

    2011-01-01

    Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A 51Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response. PMID:22072830

  12. Peptide bond cleavage site determination of novel proteolytic enzymes found in ROS 17/2.8 cell lysates.

    Science.gov (United States)

    Guidon, P T; Perrin, D; Harrison, P

    1996-02-01

    We have identified proteolytic activities in the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation (PSPKC, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys). Using polyacrylamide gel electrophoresis conditions similar to those used to resolve small molecular weight proteins, the peptide bonds of PSPKC which are cleaved by the proteolytic activities present in ROS 17/2.8 cell lysates have been determined. These activities cleave the Ser-Arg, Thr-Leu, and Ser-Val peptide bonds. To date, no proteolytic activities present in osteoblast cell lysates have been described with the aforementioned peptide bond specificities, suggesting that these activities are novel. The PSPKC-cleaved peptide fragment pattern generated was similar for several different osteoblast cell lysates. Lysates generated from different rat tissues were also able to cleave PSPKC, but the peptide fragment pattern generated by ROS 17/2.8 cell lysates appeared to be unique amongst these tissues.

  13. Analyzing the platelet proteome.

    Science.gov (United States)

    García, Angel; Zitzmann, Nicole; Watson, Steve P

    2004-08-01

    During the last 10 years, mass spectrometry (MS) has become a key tool for protein analysis and has underpinned the emerging field of proteomics. Using high-throughput tandem MS/MS following protein separation, it is potentially possible to analyze hundreds to thousands of proteins in a sample at a time. This technology can be used to analyze the protein content (i.e., the proteome) of any cell or tissue and complements the powerful field of genomics. The technology is particularly suitable for platelets because of the absence of a nucleus. Cellular proteins can be separated by either gel-based methods such as two-dimensional gel electrophoresis or one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by liquid chromatography (LC) -MS/MS or by multidimensional LC-MS/MS. Prefractionation techniques, such as subcellular fractionations or immunoprecipitations, can be used to improve the analysis. Each method has particular advantages and disadvantages. Proteomics can be used to compare the proteome of basal and diseased platelets, helping to reveal information on the molecular basis of the disease.

  14. Determination of Immunogenic Relevant Antigens in the Excretory-Secretory (ES Products and the Lysates of Ascaridia galli Larvae

    Directory of Open Access Journals (Sweden)

    S Saffi

    2008-04-01

    Full Text Available Background: Ascaridia galli, the largest nematode of small intestine of birds, especially the native poultry, may give rise to serious illness, pathological defects and economical losses even in modern poultry production systems. Although various measures have been undertaken to vaccinate poultry against A.galli, no satisfactory results were obtained so far. However, there is no report on the efficacy of excretory-secretory (ES proteins of A.galli larvae in immunization of poultry. Thus, the aim of the present research project was based on the use of the ES products of the larvae, in order to find the protective anti­gens.Methods: Five hundred native poultry were autopsied and adult A.galli was removed form their intestines. The eggs were harvested form the uterus of female worms and cultured at 25 ˚C in water containing 0.1 N sulphuric acid for almost a fort­night. The larvae were then freed mechanically and kept in Earl's salt solution for a few days. The supernatant solution of alive larvae containing the ES products of the larvae, as well as the sonicated alive and dead larvae, was analyzed by SDS-PAGE.Result: Many protein fractions of 15 kDa up to 200 kDa were demonstrated in lysate of these larvae. Using the serum of a hen, infected with a high numbers of A.galli, an immunogenic antigen was identified between 55 kDa to 72 kDa by Western blotting procedure.Conclusion: Finding the protein band between 55 and 72 kDa can be promising for preparation of vaccine, though more investigations are needed to prove the protective ability of this antigen.

  15. Human platelets as a model for the binding and degradation of thrombopoietin.

    Science.gov (United States)

    Fielder, P J; Hass, P; Nagel, M; Stefanich, E; Widmer, R; Bennett, G L; Keller, G A; de Sauvage, F J; Eaton, D

    1997-04-15

    Recent studies have shown that plasma thrombopoietin (TPO) levels appear to be directly regulated by platelet mass and that removal of plasma TPO by platelets via binding to the c-Mpl receptor is involved in the clearance of TPO in rodents. To help elucidate the role of platelets in the clearance of TPO in humans, we studied the in vitro specific binding of recombinant human TPO (rhTPO) to human platelet-rich plasma (PRP), washed platelets (WP), and cloned c-Mpl. Using a four-parameter fit and/or Scatchard analysis, the approximate affinity of rhTPO for its receptor, which was calculated from multiple experiments using different PRP preparations, was between 128 and 846 pmol/L, with approximately 25 to 224 receptors per platelet. WP preparations gave an affinity of 260 to 540 pmol/L, with approximately 25 to 35 receptors per platelet, and erythropoietin failed to compete with 125I-rhTPO for binding to WP. Binding and dissociation studies conducted with a BiaCore apparatus yielded an affinity of 350 pmol/L for rhTPO binding to cloned c-Mpl receptors. The ability of PRP to bind and degrade 125I-rhTPO was both time- and temperature-dependent and was blocked by the addition of excess cold rhTPO. Analysis of platelet pellets by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that 125I-rhTPO was degraded into a major fragment of approximately 45 to 50 kD. When 125I-rhTPO was incubated with a platelet homogenate at pH = 7.4, a degradation pattern similar to intact platelets was observed. Together, these data show that human platelets specifically bind rhTPO with high affinity, internalize, and then degrade the rhTPO.

  16. Platelet adhesion studies on nanostructured poly(lactic-co-glycolic-acid)-carbon nanotube composite.

    Science.gov (United States)

    Koh, Li Buay; Rodriguez, Isabel; Zhou, Jijie

    2008-08-01

    Design of blood-compatible surfaces is required to minimize platelet-surface interactions and increase the thromboresistance of foreign surfaces. Poly(lactic-co-glycolic-acid)-carbon nanotube (PLGA-CNT) composite is studied as a building material to fabricate artificial blood prostheses. This nanocomposite-based biomaterial is prepared by an electrostatic Layer-by-Layer (LbL) deposition technique, in which layers of CNTs are adsorbed onto a PLGA film. Before incubation in nonstimulated platelet-rich plasma (PRP) for platelet studies, fibrinogen is immobilized on PLGA-CNT composite. Interactions between the plasma proteins, e.g. fibrinogen and PRP, are investigated on the prepared PLGA-CNT composite. Contact angle measurements on the PLGA-CNT composite displayed a good resistance of platelets adhesion on a hydrophilic surface with an angle of 64.94 degrees as compared to pristine PLGA control with an angle of 93.43 degrees . A significant reduction of adhesion is observed on the PLGA-CNT composite, as well as the absence of platelet activation. On the contrary, both platelet adhesion and activation are observed on control samples. We inferred this suppression in secretion of granule contents in the platelet by the presence of the CNTs that resulted in the absence of platelet activation and its subsequent inhibition in the release of adhesive membrane receptors on the PLGA-CNT composite.

  17. Antibacterial Effect of Autologous Platelet-Rich Gel Derived from Subjects with Diabetic Dermal Ulcers In Vitro

    OpenAIRE

    Lihong Chen; Chun Wang; Hengchuan Liu; Guanjian Liu; Xingwu Ran

    2013-01-01

    Background. Autologous platelet-rich gel (APG) is an effective method to improve ulcer healing. However, the mechanisms are not clear. This study aimed to investigate the antibacterial effect of APG in vitro. Methods. Platelet-rich plasma (PRP), platelet-poor plasma (PPP) and APG were prepared from whole blood of sixteen diabetic patients with dermal ulcers. Antibacterial effects against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were evaluated by bacteriostasis assay...

  18. Encapsulation of Platelet in Kefiran Polymer and Detection of Bioavailability of Immobilized Platelet in Probiotic Kefiran as A New Drug for Surface Bleeding

    Directory of Open Access Journals (Sweden)

    Anahita Jenab

    2015-11-01

    Full Text Available Background : Kefir contains lactic acid bacteria (Lactobacillus, Lactococcus, Leuconostoc, Acetobacter and Streptococcus and yeasts (Kluyveromyces, Torula, Candida, Saccharomyces .Kefiran is the polysaccharide produced by lactic acid bacteria in kefir.Methods : Kefiran was prepared from milk containing 0.5% fat and 10 grams kefir grains and was separated from kefir by ethanol (0.02 gram following entrapping the platelets to this polymer. Ligand of the platelet-polysaccharide was studied by FTIR.Results : FTIR results showed that the bands of C-O and C-O-C connections were formed and the polysaccharides had been attached to the receptors of the platelet glycoproteins (GP Ib,GPIIb / IIIa. Stability and encapsulation of the platelet and kefiran were assessed by Coulter Counter. Encapsulation of the platelets by polysaccharide at the beginning caused to reduce the number of platelets following by releasing of 50% of the platelets after 3 hours.Conclusion : The platelets were encapsulated in kefiran polymer and detected for bioavailability as new drug for surface bleeding. Also, kefiran has antimicrobial and antifungal properties. On the other hand, the existence of nisin in kefiran could be useful as an antibacterial lantibiotic. 

  19. Platelet-neutrophil complex formation-a detailed in vitro analysis of murine and human blood samples.

    Science.gov (United States)

    Mauler, Maximilian; Seyfert, Julia; Haenel, David; Seeba, Hannah; Guenther, Janine; Stallmann, Daniela; Schoenichen, Claudia; Hilgendorf, Ingo; Bode, Christoph; Ahrens, Ingo; Duerschmied, Daniel

    2016-05-01

    Platelets form complexes with neutrophils during inflammatory processes. These aggregates migrate into affected tissues and also circulate within the organism. Several studies have evaluated platelet-neutrophil complexes as a marker of cardiovascular diseases in human and mouse. Although multiple publications have reported platelet-neutrophil complex counts, we noticed that different methods were used to analyze platelet-neutrophil complex formation, resulting in significant differences, even in baseline values. We established a protocol for platelet-neutrophil complex measurement with flow cytometry in murine and human whole blood samples. In vitro platelet-neutrophil complex formation was stimulated with ADP or PMA. We tested the effect of different sample preparation steps and cytometer settings on platelet-neutrophil complex detection and noticed false-positive counts with increasing acquisition speed. Platelet-neutrophil complex formation depends on platelet P-selectin expression, and antibody blocking of P-selectin consequently prevented ADP-induced platelet-neutrophil complex formation. These findings may help generating more comparable data among different research groups that examine platelet-neutrophil complexes as a marker for cardiovascular disease and novel therapeutic interventions.

  20. Platelets in inflammation and infection.

    Science.gov (United States)

    Jenne, Craig N; Kubes, Paul

    2015-01-01

    Although platelets are traditionally recognized for their central role in hemostasis, many lines of research clearly demonstrate these rather ubiquitous blood components are potent immune modulators and effectors. Platelets have been shown to directly recognize, sequester and kill pathogens, to activated and recruit leukocytes to sites of infection and inflammation, and to modulate leukocyte behavior, enhancing their ability to phagocytose and kill pathogens and inducing unique effector functions, such as the production of Neutrophil Extracellular Traps (NETs). This multifaceted response to infection and inflammation is due, in part, to the huge array of soluble mediators and cell surface molecules expressed by platelets. From their earliest origins as primordial hemocytes in invertebrates to their current form as megakaryocyte-derived cytoplasts, platelets have evolved to be one of the key regulators of host intravascular immunity and inflammation. In this review, we present the diverse roles platelets play in immunity and inflammation associated with autoimmune diseases and infection. Additionally, we highlight recent advances in our understanding of platelet behavior made possible through the use of advanced imaging techniques that allow us to visualize platelets and their interactions, in real-time, within the intact blood vessels of a living host.

  1. [Murine models of platelet diseases].

    Science.gov (United States)

    Lanza, F

    2007-05-01

    Platelet-related diseases correspond to functional defects or abnormal production (thrombopoiesis) of hereditary and immunological origins. Recent progress in the manipulation of the mouse genome (transgenesis, gene inactivation or insertion) has resulted in the generation of numerous strains exhibiting defective platelet function or production. Some strains reproduce known hereditary diseases affecting haemostasis (Glanzmann thrombasthenia, Bernard-Soulier syndrome (BSS) or thrombopoiesis (Wiscott-Aldrich or May-Hegglin syndrome). More often the mutated strains have no human equivalent and represent useful models to study: (i) the role of adhesive or signalling receptors or of signalling proteins in platelet-dependent haemostasis and thrombosis or; (ii) to study the poorly characterized mechanisms of thrombopoiesis, which implicate transcription factors (GATA, Fli1), growth factors and receptors (TPO, cMPL), and cytoskeletal or contractile proteins (tubulin, myosin). Additional mouse strains result from the selection of spontaneous mutants many of which affect intracellular platelet granules, representing models of storage pool diseases (SPD) such as the Gray platelet syndrome (alphaSPD) or Hermansky-Pudlack syndrome (deltaSPD). More recently, a systematic chemical mutagenesis approach has also identified genes involved in thrombopoiesis and platelet survival. Finally, mouse models of auto- or allo-immune thrombocytopenia have been developed to study the mechanisms of platelet destruction or removal.

  2. Platelet scintigraphy in atherothrombotic disease

    Energy Technology Data Exchange (ETDEWEB)

    Isaka, Yoshinari (Osaka National Hospital (Japan))

    1993-01-01

    Indium-111 platelet scintigraphy for the measurement of in vivo thrombogenicity is a useful noninvasive technique with a number of applications. From 1982 to 1989, we explored clinical relevance of this method for 576 consecutive patients with atherothrombotic disease. There was a disease-related difference in the percentage of positive platelet accumulation; 85% in patients with Dacron bifurcation graft, 75% in abdominal or thoracic aneurysm, 40% in intra-cardiac thrombi, 33% in arteriosclerosis obliterans and 25% in ischemic cerebrovascular disease. Labelled platelets accumulated frequently in the lesion with severe arteriographic abnormality. Aspirin clearly inhibited platelet accumulation on carotid atheroma but the effect of ticlopidine has been less conclusive. Short-term orally active PGI[sub 2] analogue had inhibitory effects on platelet accumulation in carotid atheroma and platelet aggregability, but did not cause significant reduction in plaque size. The results suggest the usefulness of platelet scintigraphy for monitoring the thrombogenicity in various atherothrombotic diseases. It will be necessary, however, to simplify the labelling procedures and to develop a new [sup 99m]Tc-labelled thrombus imaging agent, if thrombus imaging is to be considered for more generall use for patients with atherosclerosis. (author).

  3. Cyclosporine A enhances platelet aggregation.

    Science.gov (United States)

    Grace, A A; Barradas, M A; Mikhailidis, D P; Jeremy, J Y; Moorhead, J F; Sweny, P; Dandona, P

    1987-12-01

    In view of the reported increase in thromboembolic episodes following cyclosporine A (CyA) therapy, the effect of this drug on platelet aggregation and thromboxane A2 release was investigated. The addition of CyA, at therapeutic concentrations to platelet rich plasma from normal subjects in vitro was found to increase aggregation in response to adrenaline, collagen and ADP. Ingestion of CyA by healthy volunteers was also associated with enhanced platelet aggregation. The CyA-mediated enhancement of aggregation was further enhanced by the addition in vitro of therapeutic concentrations of heparin. Platelets from renal allograft recipients treated with CyA also showed hyperaggregability and increased thromboxane A2 release, which were most marked at "peak" plasma CyA concentration and less so at "trough" concentrations. Platelet hyperaggregability in renal allograft patients on long-term CyA therapy tended to revert towards normal following the replacement of CyA with azathioprine. Hypertensive patients with renal allografts on nifedipine therapy had normal platelet function and thromboxane release in spite of CyA therapy. These observations suggest that CyA-mediated platelet activation may contribute to the pathogenesis of the thromboembolic phenomena associated with the use of this drug. The increased release of thromboxane A2 (a vasoconstrictor) may also play a role in mediating CyA-related nephrotoxicity.

  4. Platelet-rich-fibrin: A novel root coverage approach

    Directory of Open Access Journals (Sweden)

    Anilkumar K

    2009-01-01

    Full Text Available Treatment of gingival recession has become an important therapeutic issue due to increasing cosmetic demand. Multiple surgical procedures have been developed to obtain predictable esthetic root coverage. More specifically, after periodontal regenerative surgery, the aim is to achieve complete wound healing and regeneration of the periodontal unit. A recent innovation in dentistry is the preparation and use of platelet-rich plasma (PRP, a concentrated suspension of the growth factors, found in platelets. These growth factors are involved in wound healing and postulated as promoters of tissue regeneration. This paper reports the use of PRF membrane for root coverage on the labial surfaces of the mandibular anterior teeth. This was accomplished using laterally displaced flap technique with platelet rich fibrin (PRF membrane at the recipient site.

  5. Platelet-rich plasma: the PAW classification system.

    Science.gov (United States)

    DeLong, Jeffrey M; Russell, Ryan P; Mazzocca, Augustus D

    2012-07-01

    Platelet-rich plasma (PRP) has been the subject of hundreds of publications in recent years. Reports of its effects in tissue, both positive and negative, have generated great interest in the orthopaedic community. Protocols for PRP preparation vary widely between authors and are often not well documented in the literature, making results difficult to compare or replicate. A classification system is needed to more accurately compare protocols and results and effectively group studies together for meta-analysis. Although some classification systems have been proposed, no single system takes into account the multitude of variables that determine the efficacy of PRP. In this article we propose a simple method for organizing and comparing results in the literature. The PAW classification system is based on 3 components: (1) the absolute number of Platelets, (2) the manner in which platelet Activation occurs, and (3) the presence or absence of White cells. By analyzing these 3 variables, we are able to accurately compare publications.

  6. Platelet enzyme abnormalities in leukemias

    Directory of Open Access Journals (Sweden)

    S Sharma

    2011-01-01

    Full Text Available Aim of the Study: The aim of this study was to evaluate platelet enzyme activity in cases of leukemia. Materials and Methods: Platelet enzymes glucose-6-phosphate dehydrogenase (G6PD, pyruvate kinase (PK and hexokinase (HK were studied in 47 patients of acute and chronic leukemia patients, 16 patients with acute myeloid leukemia (AML(13 relapse, three in remission, 12 patients with acute lymphocytic leukemia (ALL (five in relapse, seven in remission, 19 patients with chronic myeloid leukemia (CML. Results: The platelet G6PD activity was significantly low in cases of AML, ALL and also in CML. G6PD activity was normalized during AML remission. G6PD activity, although persistently low during ALL remission, increased significantly to near-normal during remission (P < 0.05 as compared with relapse (P < 0.01. Platelet PK activity was high during AML relapse (P < 0.05, which was normalized during remission. Platelet HK however was found to be decreased during all remission (P < 0.05. There was a significant positive correlation between G6PD and PK in cases of AML (P < 0.001 but not in ALL and CML. G6PD activity did not correlate with HK activity in any of the leukemic groups. A significant positive correlation was however seen between PK and HK activity in cases of ALL remission (P < 0.01 and CML (P < 0.05. Conclusions: Both red cell and platelet enzymes were studied in 36 leukemic patients and there was no statistically significant correlation between red cell and platelet enzymes. Platelet enzyme defect in leukemias suggests the inherent abnormality in megakaryopoiesis and would explain the functional platelet defects in leukemias.

  7. Platelet surface glutathione reductase-like activity.

    Science.gov (United States)

    Essex, David W; Li, Mengru; Feinman, Richard D; Miller, Anna

    2004-09-01

    We previously found that reduced glutathione (GSH) or a mixture of GSH/glutathione disulfide (GSSG) potentiated platelet aggregation. We here report that GSSG, when added to platelets alone, also potentiates platelet aggregation. Most of the GSSG was converted to GSH by a flavoprotein-dependent platelet surface mechanism. This provided an appropriate redox potential for platelet activation. The addition of GSSG to platelets generated sulfhydryls in the beta subunit of the alpha(IIb)beta(3) fibrinogen receptor, suggesting a mechanism for facilitation of agonist-induced platelet activation.

  8. BACTERIAL LYSATES FOR TOPICAL APPLICATION IN PREVENTION AND TREATMENT OF CHRONIC TONSILLITIS AMONG CHILDREN

    Directory of Open Access Journals (Sweden)

    T.I. Garashchenko

    2006-01-01

    Full Text Available The authors have carried on the research of the influence Imudon exerts (during topical application on the run of chronic tonsillitis among 48 children aged between 5 and 10 years old, being dispensary registrants. The sublingual application of a medicine 6 pills daily within 20 days demonstrated the frequency reduction of chronic tonsillitis acerbations by 2.9 times, as well as the reduction of total need in systemic antibacterial treatment by 10 times. Apart from that, the frequency of S. pyogenes group a exposure reduced by 3 times. The researchers noticed the tendency to normalization of pharynx biocenosis. Thus, Imudon may be recommended for the daily courses of treatment to the people, suffering from chronic tonsillitis, palatine tonsil auxesis and recurrent tonsillo-pharyngites.Key words: chronic tonsillitis, children, prevention, bacterial lysates.

  9. BACTERIAL LYSATES MIXTURE FOR THE TOPICAL ADMINISTRATION IN PROPHYLAXIS AND TREATMENT OF CHRONIC TONSILLITIS IN CHILDREN

    Directory of Open Access Journals (Sweden)

    T.I. Garashchenko

    2009-01-01

    Full Text Available Chronic tonsillitis is the infectious and allergic disease; the main clinical symptom of this disease is chronic inflammation of palatine tonsils. The prevalence of chronic tonsillitis is up to 10% among adults and 15% — among children. Undoubtedly, the prevention of exacerbations of chronic tonsillitis is difficult task. Its significant component is immunocorrection with elements of vaccinal process, because tonsils themselves are the part of forming immune system of a child. Imudon can be recommended for the inclusion to the yearly prophylactic treatment courses in patients with chronic tonsillitis, hypertrophy of palatine tonsils and recurrent tonsillopharyngitis.Key words: children, chronic tonsillitis, prophylaxis, treatment, bacterial lysates mixture.(Voprosy sovremennoi pediatrii — Current Pediatrics. 2009;8(6:109-112

  10. Routine limulus amoebocyte lysate (LAL) test for endotoxin determination in milk using a Toxinometer ET-201.

    Science.gov (United States)

    Mottar, J; De Block, J; Merchiers, M; Vantomme, K; Moermans, R

    1993-05-01

    A rapid method of performing the Limulus amoebocyte lysate (LAL) test in milk is proposed using the Toxinometer ET-201. This instrument measured the increase in turbidity due to the interaction between the endotoxins of the Gram-negative bacteria and the LAL reagent, monitored the ratio Rt of the sequential to the initial transmission at 12 s intervals and quantified endotoxins by determination of the reaction time Tr required to obtain a 5% decrease in Rt. There was a good correlation between the toxinometrically determined endotoxin concentrations and the number of Gram-negative bacteria (SD, 0.18 log(plate count units)), and the repeatability (CV, 6-10%) was high. The assay may be useful for screening raw materials for UHT milk production, as the endotoxin content of the raw material is related to the rest proteinase activity in the UHT milk.

  11. Platelet rich fibrin: a new paradigm in periodontal regeneration.

    Science.gov (United States)

    Kumar, R Vinaya; Shubhashini, N

    2013-09-01

    Among the great challenges facing clinical research is the development of bioactive surgical additives regulating inflammation and increasing healing. Although the use of fibrin adhesives and platelet-rich plasma (PRP) is well documented, they have their own limitations. Hence, reconstructive dental surgeons are looking for an "edge" that jump starts the healing process to maximize predictability as well as the volume of regenerated bone. Overcoming the restrictions related to the reimplantation of blood-derived products, a new family of platelet concentrate, which is neither a fibrin glue nor a classical platelet concentrate, was developed in France. This second generation platelet concentrate called platelet-rich fibrin (PRF), has been widely used to accelerate soft and hard tissue healing. Its advantages over the better known PRP include ease of preparation/application, minimal expense, and lack of biochemical modification (no bovine thrombin or anticoagulant is required). This article serves as an introduction to the PRF "concept" and its potential clinical applications with emphasis on periodontal regeneration.

  12. Mechanisms of platelet-mediated liver regeneration.

    Science.gov (United States)

    Lisman, Ton; Porte, Robert J

    2016-08-04

    Platelets have multiple functions beyond their roles in thrombosis and hemostasis. Platelets support liver regeneration, which is required after partial hepatectomy and acute or chronic liver injury. Although it is widely assumed that platelets stimulate liver regeneration by local excretion of mitogens stored within platelet granules, definitive evidence for this is lacking, and alternative mechanisms deserve consideration. In-depth knowledge of mechanisms of platelet-mediated liver regeneration may lead to new therapeutic strategies to treat patients with failing regenerative responses.

  13. Platelets as delivery systems for disease treatments

    OpenAIRE

    Shi, Qizhen; Montgomery, Robert R.

    2010-01-01

    Platelets are small, anucleate, discoid shaped blood cells that play a fundamental role in hemostasis. Platelets contain a large number of biologically active molecules within cytoplasmic granules that are critical to normal platelet function. Because platelets circulate in blood through out the body, release biological molecules and mediators on demand, and participate in hemostasis as well as many other pathophysiologic processes, targeting expression of proteins of interest to platelets an...

  14. Electrical protein detection in cell lysates using high-density peptide-aptamer microarrays

    Directory of Open Access Journals (Sweden)

    Evans David

    2008-01-01

    Full Text Available Abstract Background The dissection of biological pathways and of the molecular basis of disease requires devices to analyze simultaneously a staggering number of protein isoforms in a given cell under given conditions. Such devices face significant challenges, including the identification of probe molecules specific for each protein isoform, protein immobilization techniques with micrometer or submicrometer resolution, and the development of a sensing mechanism capable of very high-density, highly multiplexed detection. Results We present a novel strategy that offers practical solutions to these challenges, featuring peptide aptamers as artificial protein detectors arrayed on gold electrodes with feature sizes one order of magnitude smaller than existing formats. We describe a method to immobilize specific peptide aptamers on individual electrodes at the micrometer scale, together with a robust and label-free electronic sensing system. As a proving proof of principle experiment, we demonstrate the specific recognition of cyclin-dependent protein kinases in whole-cell lysates using arrays of ten electrodes functionalized with individual peptide aptamers, with no measurable cross-talk between electrodes. The sensitivity is within the clinically relevant range and can detect proteins against the high, whole-cell lysate background. Conclusion The use of peptide aptamers selected in vivo to recognize specific protein isoforms, the ability to functionalize each microelectrode individually, the electronic nature and scalability of the label-free detection and the scalability of the array fabrication combine to yield the potential for highly multiplexed devices with increasingly small detection areas and higher sensitivities that may ultimately allow the simultaneous monitoring of tens or hundreds of thousands of protein isoforms.

  15. The use of autologous platelet-leukocyte gels to enhance the healing process in surgery, a review

    NARCIS (Netherlands)

    Everts, P. A.; Overdevest, E. P.; Jakimowicz, J. J.; Oosterbos, C. J.; Schonberger, J. P.; Knape, J. T.; van Zundert, A.

    2007-01-01

    Background: The therapeutic use of autologously prepared, platelet-leukocyte-enriched gel (PLG) is a relatively new technology for the stimulation and acceleration of soft tissue and bone healing. The effectiveness of this procedure lies in the delivery of a wide range of platelet growth factors mim

  16. [Protein kinase C activation induces platelet apoptosis].

    Science.gov (United States)

    Zhao, Li-Li; Chen, Meng-Xing; Zhang, Ming-Yi; Dai, Ke-Sheng

    2013-10-01

    Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelets, or in the development of some platelet-related diseases. However, up to now, a little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase C (PKC) is highly expressed in platelets and plays central roles in regulating platelet functions. Although there is evidence indicating that PKC is involved in the regulation of apoptosis of nucleated cells, it is still unclear whether PKC plays a role in platelet apoptosis. The aim of this study was to investigate the role of PKC in platelet apoptosis. The effects of PKC on mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 activation of platelets were analyzed by flow cytometry and Western blot. The results showed that the ΔΨm depolarization in platelets was induced by PKC activator in time-dependent manner, and the caspase-3 activation in platelets was induced by PKC in concentration-dependent manner. However, the platelets incubated with PKC inhibitor did not results in ΔΨm depolarization and PS exposure. It is concluded that the PKC activation induces platelet apoptosis through influencing the mitochondrial functions and activating caspase 3. The finds suggest a novel mechanism for PKC in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.

  17. Inherited platelet disorders and oral health.

    Science.gov (United States)

    Valera, Marie-Cécile; Kemoun, Philippe; Cousty, Sarah; Sie, Pierre; Payrastre, Bernard

    2013-02-01

    Platelets play a key role in thrombosis and hemostasis. Accumulation of platelets at the site of vascular injury is the first step in the formation of hemostatic plugs, which play a pivotal role in preventing blood loss after injury. Platelet adhesion at sites of injury results in spreading, secretion, recruitment of additional platelets, and formation of platelet aggregates. Inherited platelet disorders are rare causes of bleeding syndromes, ranging from mild bruising to severe hemorrhage. The defects can reflect deficiency or dysfunction of platelet surface glycoproteins, granule contents, cytoskeletal proteins, platelet pro-coagulant function, and signaling pathways. For instance, Bernard-Soulier syndrome and Glanzmann thrombasthenia are attributed to deficiencies of glycoprotein Ib/IX/V and GPIIb/IIIa, respectively, and are rare but severe platelet disorders. Inherited defects that impair platelet secretion and/or signal transduction are among the most common forms of mild platelet disorders and include gray platelet syndrome, Hermansky-Pudlak syndrome, and Chediak-Higashi syndrome. When necessary, desmopressin, antifibrinolytic agents, and transfusion of platelets remain the most common treatment of inherited platelet disorders. Alternative therapies such as recombinant activated factor VII are also available for a limited number of situations. In this review, we will discuss the management of patients with inherited platelet disorders in various clinical situations related to dental cares, including surgical intervention. © 2012 John Wiley & Sons A/S.

  18. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part II: platelet-related biologic features.

    Science.gov (United States)

    Dohan, David M; Choukroun, Joseph; Diss, Antoine; Dohan, Steve L; Dohan, Anthony J J; Mouhyi, Jaafar; Gogly, Bruno

    2006-03-01

    Platelet-rich fibrin (PRF) belongs to a new generation of platelet concentrates, with simplified processing and without biochemical blood handling. In this second article, we investigate the platelet-associated features of this biomaterial. During PRF processing by centrifugation, platelets are activated and their massive degranulation implies a very significant cytokine release. Concentrated platelet-rich plasma platelet cytokines have already been quantified in many technologic configurations. To carry out a comparative study, we therefore undertook to quantify PDGF-BB, TGFbeta-1, and IGF-I within PPP (platelet-poor plasma) supernatant and PRF clot exudate serum. These initial analyses revealed that slow fibrin polymerization during PRF processing leads to the intrinsic incorporation of platelet cytokines and glycanic chains in the fibrin meshes. This result would imply that PRF, unlike the other platelet concentrates, would be able to progressively release cytokines during fibrin matrix remodeling; such a mechanism might explain the clinically observed healing properties of PRF.

  19. Platelet-containing tantalum powders

    Energy Technology Data Exchange (ETDEWEB)

    Schiele, E.K.

    1988-04-26

    A method of forming platelet tantalum powders is described comprising the steps of: (a) providing an ingot-derived precursor tantalum powder, and (b) ball-milling the precursor powder for a time sufficient to form a platelet powder having an average FSSS of less than about 2 micrometers, a Scott density not greater than about 30 g/in/sup 3/ and a BET surface area of at least about 0.7 in/sup 2//g.

  20. Activated platelets release sphingosine 1-phosphate and induce hypersensitivity to noxious heat stimuli in vivo

    Directory of Open Access Journals (Sweden)

    Daniela eWeth

    2015-04-01

    Full Text Available At the site of injury activated platelets release various mediators, one of which is sphingosine 1-phosphate (S1P. It was the aim of this study to explore whether activated human platelets had a pronociceptive effect in an in vivo mouse model and whether this effect was based on the release of S1P and subsequent activation of neuronal S1P receptors 1 or 3. Human platelets were prepared in different concentrations (105/µl, 106/µl, 107/µl and assessed in mice with different genetic backgrounds (WT, S1P1fl/fl, SNS-S1P1-/-, S1P3-/-. Intracutaneous injections of activated human platelets induced a significant, dose-dependent hypersensitivity to noxious thermal stimulation. The degree of heat hypersensitivity correlated with the platelet concentration as well as the platelet S1P content and the amount of S1P released upon platelet activation as measured with LC MS/MS. Despite the significant correlations between S1P and platelet count, no difference in paw withdrawal latency (PWL was observed in mice with a global null mutation of the S1P3 receptor or a conditional deletion of the S1P1 receptor in nociceptive primary afferents. Furthermore, neutralisation of S1P with a selective anti-S1P antibody did not abolish platelet induced heat hypersensitivity. Our results suggest that activated platelets release S1P and induce heat hypersensitivity in vivo. However, the platelet induced heat hypersensitivity was caused by mediators other than S1P.

  1. In vitro cytokine profiles and viability of different human cells treated with whole cell lysate of Mycobacterium avium subsp. paratuberculosis

    Directory of Open Access Journals (Sweden)

    Rani Pittu

    2012-09-01

    Full Text Available Abstract Mycobacterium avium subsp. paratuberculosis (MAP is a zoonotic pathogen, a very slow growing bacterium which is difficult to isolate and passage in conventional laboratory culture. Although its association with Johne’s disease or paratuberculosis of cattle is well established, it has been only putatively linked to Crohn’s disease in humans. Further, MAP has been recently suggested to be a trigger for other autoimmune diseases such as type-1 diabetes mellitus (T1DM. Recently, some studies have indicated that exposure to MAP is associated with elevated levels of antibodies against MAP lysate although the exact mechanism and significance of the same remains unclear. Further, the cytokine profiles relevant in MAP associated diseases of humans and their exact role in the pathophysiology are not clearly known. We performed in vitro cytokine analyses after exposing different cultured human cells to the whole cell lysate of MAP and found that MAP lysate induces secretion of cytokines IL-1β, IL-6, IL-8, IL-10 and TNF-α by human peripheral blood mononuclear cells (PBMCs. Also, it induces secretion of IL-8 by cultured human stomach adenocarcinoma cells (AGS and PANC-1(human pancreatic carcinoma cell line cells. We also found that MAP lysate induced cytotoxicity in PANC-1cells. Collectively, these results provide a much needed base-line data set of cytokines broadly signifying a MAP induced cellular response by human cells.

  2. Quantitative electrochemical detection of cathepsin B activity in complex tissue lysates using enhanced AC voltammetry at carbon nanofiber nanoelectrode arrays.

    Science.gov (United States)

    Swisher, Luxi Z; Prior, Allan M; Shishido, Stephanie; Nguyen, Thu A; Hua, Duy H; Li, Jun

    2014-06-15

    The proteolytic activity of a cancer-related enzyme cathepsin B is measured with alternating current voltammetry (ACV) using ferrocene (Fc) labeled tetrapeptides attached to nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). This combination enables the use of high AC frequencies (~1kHz) with enhanced electrochemical signals. The specific proteolysis of the Fc-peptide by cathepsin B produces decay in the ACV peak current versus the reaction time. The exponential component of the raw data can be extracted and defined as the "extracted proteolytic signal" which allows consistent quantitative analyses using a heterogeneous Michaelis-Menten model. A "specificity constant" kcat/KM = (3.68 ± 0.50) × 10(4)M(-1)s(-1) for purified cathepsin B was obtained. The detections of cathepsin B activity in different concentrations of whole lysate of human breast tissue, tissue lysate spiked with varied concentrations of cathepsin B, and the tissue lysate after immunoprecipitation showed that there is ~13.4 nM higher cathepsin B concentration in 29.1 µg mL(-1) of whole tissue lysate than the immunoprecipitated sample. The well-defined regular VACNF NEAs by e-beam lithography show a much faster kinetics for cathepsin B proteolysis with kcat/KM = 9.2 × 10(4)M(-1)s(-1). These results illustrate the potential of this technique as a portable multiplex electronic system for cancer diagnosis by rapid protease profiling of serum or blood samples.

  3. A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing

    DEFF Research Database (Denmark)

    Moesby, Lise; Jensen, S; Hansen, E W

    1999-01-01

    Pyrogen induced secretion of interleukin 6 (IL-6) in Mono Mac 6 (MM6) cells was measured. The ability of the MM6 cell culture to detect pyrogens was compared to the Limulus amoebocyte lysate (LAL) test and isolated mononuclear cells (MNC). The detection limit of MM6 for lipopolysaccharide (LPS...

  4. A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing

    DEFF Research Database (Denmark)

    Moesby, Lise; Jensen, S; Hansen, E W;

    1999-01-01

    Pyrogen induced secretion of interleukin 6 (IL-6) in Mono Mac 6 (MM6) cells was measured. The ability of the MM6 cell culture to detect pyrogens was compared to the Limulus amoebocyte lysate (LAL) test and isolated mononuclear cells (MNC). The detection limit of MM6 for lipopolysaccharide (LPS...

  5. Quantitative analysis of antiretroviral drugs in lysates of peripheral blood mononuclear cells using MALDI-triple quadrupole mass spectrometry.

    NARCIS (Netherlands)

    Kampen, JJ van; Burgers, P.C.; Gruters, R.A.; Osterhaus, A.D.; Groot, R. de; Luider, T.M.; Volmer, D.A.

    2008-01-01

    We report here on the use of a prototype matrix-assisted laser desorption/ionization (MALDI)-triple quadrupole mass spectrometer for quantitative analysis of six antiretroviral drugs in lysates of peripheral blood mononuclear cells (PBMC). Of the five investigated MALDI matrixes, 2,5-dihydroxybenzoi

  6. Adjusting MtDNA Quantification in Whole Blood for Peripheral Blood Platelet and Leukocyte Counts.

    Science.gov (United States)

    Hurtado-Roca, Yamilee; Ledesma, Marta; Gonzalez-Lazaro, Monica; Moreno-Loshuertos, Raquel; Fernandez-Silva, Patricio; Enriquez, Jose Antonio; Laclaustra, Martin

    2016-01-01

    Alterations of mitochondrial DNA copy number (mtDNAcn) in the blood (mitochondrial to nuclear DNA ratio) appear associated with several systemic diseases, including primary mitochondrial disorders, carcinogenesis, and hematologic diseases. Measuring mtDNAcn in DNA extracted from whole blood (WB) instead of from peripheral blood mononuclear cells or buffy coat may yield different results due to mitochondrial DNA present in platelets. The aim of this work is to quantify the contribution of platelets to mtDNAcn in whole blood [mtDNAcn(WB)] and to propose a correction formula to estimate leukocytes' mtDNAcn [mtDNAcn(L)] from mtDNAcn(WB). Blood samples from 10 healthy adults were combined with platelet-enriched plasma and saline solution to produce artificial blood preparations. Aliquots of each sample were combined with five different platelet concentrations. In 46 of these blood preparations, mtDNAcn was measured by qPCR. MtDNAcn(WB) increased 1.07 (95%CI 0.86, 1.29; p<0.001) per 1000 platelets present in the preparation. We proved that leukocyte count should also be taken into account as mtDNAcn(WB) was inversely associated with leukocyte count; it increased 1.10 (95%CI 0.95, 1.25, p<0.001) per unit increase of the ratio between platelet and leukocyte counts. If hematological measurements are available, subtracting 1.10 the platelets/leukocyte ratio from mtDNAcn(WB) may serve as an estimation for mtDNAcn(L). Both platelet and leukocyte counts in the sample are important sources of variation if comparing mtDNAcn among groups of patients when mtDNAcn is measured in DNA extracted from whole blood. Not taking the platelet/leukocyte ratio into account in whole blood measurements, may lead to overestimation and misclassification if interpreted as leukocytes' mtDNAcn.

  7. Adjusting MtDNA Quantification in Whole Blood for Peripheral Blood Platelet and Leukocyte Counts

    Science.gov (United States)

    Gonzalez-Lazaro, Monica; Moreno-Loshuertos, Raquel; Fernandez-Silva, Patricio; Enriquez, Jose Antonio; Laclaustra, Martin

    2016-01-01

    Alterations of mitochondrial DNA copy number (mtDNAcn) in the blood (mitochondrial to nuclear DNA ratio) appear associated with several systemic diseases, including primary mitochondrial disorders, carcinogenesis, and hematologic diseases. Measuring mtDNAcn in DNA extracted from whole blood (WB) instead of from peripheral blood mononuclear cells or buffy coat may yield different results due to mitochondrial DNA present in platelets. The aim of this work is to quantify the contribution of platelets to mtDNAcn in whole blood [mtDNAcn(WB)] and to propose a correction formula to estimate leukocytes' mtDNAcn [mtDNAcn(L)] from mtDNAcn(WB). Blood samples from 10 healthy adults were combined with platelet-enriched plasma and saline solution to produce artificial blood preparations. Aliquots of each sample were combined with five different platelet concentrations. In 46 of these blood preparations, mtDNAcn was measured by qPCR. MtDNAcn(WB) increased 1.07 (95%CI 0.86, 1.29; p<0.001) per 1000 platelets present in the preparation. We proved that leukocyte count should also be taken into account as mtDNAcn(WB) was inversely associated with leukocyte count; it increased 1.10 (95%CI 0.95, 1.25, p<0.001) per unit increase of the ratio between platelet and leukocyte counts. If hematological measurements are available, subtracting 1.10 the platelets/leukocyte ratio from mtDNAcn(WB) may serve as an estimation for mtDNAcn(L). Both platelet and leukocyte counts in the sample are important sources of variation if comparing mtDNAcn among groups of patients when mtDNAcn is measured in DNA extracted from whole blood. Not taking the platelet/leukocyte ratio into account in whole blood measurements, may lead to overestimation and misclassification if interpreted as leukocytes' mtDNAcn. PMID:27736919

  8. Direct quantification of mRNA and miRNA from cell lysates using reverse transcription real time PCR: a multidimensional analysis of the performance of reagents and workflows.

    Directory of Open Access Journals (Sweden)

    Yoon Khei Ho

    Full Text Available Substantial efforts have been devoted to in vitro testing of candidate chemotherapeutics by profiling transcriptional changes across the collection of NCI-60 cell-lines. A work-flow with reagents that enable the direct quantification of RNA of different molecular sizes simultaneously in the same sample without laborious total RNA isolation will invariably increase the throughput and accuracy of the study. MicroRNAs (miRNAs are known to regulate most cellular functions, acting post-transcriptionally by repressing numerous eukaryotic mRNAs. Recent findings on the remarkable stability of miRNA prompted us to investigate the feasibility of quantifying the expression levels of both mRNA and miRNA directly from cell lysates (cell-to-Ct. Multidimensional analyses of the expressions of mRNA and miRNA across seven NCI-60 cell lines and multiple reagents were conducted to assess the performances of these reagents and workflows for cell-to-Ct measurements using reverse transcription-quantitative polymerase chain reaction (RT-qPCR. Quantification of RNA species using lysates prepared from an in-house and one of the commercial reagents demonstrated comparable performance to those prepared by the more laborious and conventional method of using guanidinium-phenol-chloroform. Additionally, miRNA was found to be highly stable in the cell lysates when incubated at room temperature for prolonged period of time and subjected to multiple freeze-thaw cycles. In summary, this study demonstrated significant differences in pre-analytical performance of a variety of commercially available reagents and described a cost-effective reagent useful for rapid, scalable, and high-throughput workflow for the detection of mRNA and miRNA from the same biological sample.

  9. Morphology of platelet Golgi apparatus and their significance after acute cerebral infarction.

    Science.gov (United States)

    Lu, Wei; Xu, Dong; Tu, Ranran; Hu, Zhiping

    2013-08-15

    Blood samples were harvested from the antecubital vein of 20 fasting patients with acute cerebral infarction at 1, 7 and 15 days after onset to prepare blood platelet suspension. Fasting antecubital vein blood was collected from an additional 20 normal adults as controls. Under transmission tron microscope, platelet Golgi tubules and vesicles became significantly thickened, enlarged, and irregular after acute cerebral infarction. Alpha granules in platelets significantly reduced in number, especially 1 day after cerebral infarction. Under immunoelectron microscopy, a few alpha granules aggregated around Golgi tubules and vesicles after infarction. These results suggested that platelet Golgi apparatus displayed significant morphological changes, which were possibly associated with enhanced synthetic and secretory functions of activated platelets after acute cerebral infarction. This study used Golgi apparatus blocking agent Brefeldin A to block Golgi apparatus in an aim to study the effects of Golgi apparatus on CD40L expression on the surface of activated platelets. Flow cytometry revealed that CD40L expression on activated platelet surfaces decreased significantly when Golgi apparatus was blocked, which indicated that Golgi apparatus participated in the synthesis and transport of CD40L to the platelet surface.

  10. Morphology of platelet Golgi apparatus and their significance after acute cerebral infarction***

    Institute of Scientific and Technical Information of China (English)

    Wei Lu; Dong Xu; Ranran Tu; Zhiping Hu

    2013-01-01

    Blood samples were harvested from the antecubital vein of 20 fasting patients with acute cerebral infarction at 1, 7 and 15 days after onset to prepare blood platelet suspension. Fasting antecubital vein blood was col ected from an additional 20 normal adults as controls. Under transmission tron microscope, platelet Golgi tubules and vesicles became significantly thickened, enlarged, and irregular after acute cerebral infarction. Alpha granules in platelets significantly reduced in number, especial y 1 day after cerebral infarction. Under immunoelectron microscopy, a few alpha granules aggregated around Golgi tubules and vesicles after infarction. These results suggested that platelet Golgi apparatus displayed significant morphological changes, which were possibly associated with enhanced synthetic and secretory functions of activated platelets after acute cerebral infarction. This study used Golgi apparatus blocking agent Brefeldin A to block Golgi apparatus in an aim to study the effects of Golgi apparatus on CD40L expression on the surface of activated platelets. Flow cytometry revealed that CD40L expression on activated platelet surfaces decreased significantly when Golgi apparatus was blocked, which indicated that Golgi apparatus participated in the syn-thesis and transport of CD40L to the platelet surface.

  11. Extract of a spice--omum (Trachyspermum ammi)-shows antiaggregatory effects and alters arachidonic acid metabolism in human platelets.

    Science.gov (United States)

    Srivastava, K C

    1988-07-01

    An ethereal extract of omum (Trachyspermum ammi; Hindustani: ajwan)--a frequently consumed spice--was found to inhibit platelet aggregation induced by arachidonic acid (AA), epinephrine and collagen; in this respect it was most effective against AA-induced aggregation. Inhibition of aggregation by omum could be explained by its effect on platelet thromboxane production as suggested by the following experimental observation. (i) Omum reduced TxB2 formation in intact platelet preparations from added arachidonate, and (ii) it reduced the formation of TxB2 from AA-labelled platelets after stimulation with Ca2+-ionophore A23187 by a direct action on cyclooxygenase as it did not affect the release of AA from labelled platelets. An increased formation of lipoxygenase-derived products from exogenous AA in omum-treated platelets was apparently due to redirection of AA from cyclooxygenase to the lipoxygenase pathway.

  12. Evidence of platelet activation in multiple sclerosis

    Directory of Open Access Journals (Sweden)

    Alexander J Steven

    2008-06-01

    Full Text Available Abstract Objective A fatality in one multiple sclerosis (MS patient due to acute idiopathic thrombocytopenic purpura (ITP and a near fatality in another stimulated our interest in platelet function abnormalities in MS. Previously, we presented evidence of platelet activation in a small cohort of treatment-naive MS patients. Methods In this report, 92 normal controls and 33 stable, untreated MS patients were studied. Platelet counts, measures of platelet activation [plasma platelet microparticles (PMP, P-selectin expression (CD62p, circulating platelet microaggragtes (PAg], as well as platelet-associated IgG/IgM, were carried out. In addition, plasma protein S activity was measured. Results Compared to controls, PMP were significantly elevated in MS (p Conclusion Platelets are significantly activated in MS patients. The mechanisms underlying this activation and its significance to MS are unknown. Additional study of platelet activation and function in MS patients is warranted.

  13. Platelet-rich fibrin matrix improves wound angiogenesis via inducing endothelial cell proliferation.

    Science.gov (United States)

    Roy, Sashwati; Driggs, Jason; Elgharably, Haytham; Biswas, Sabyasachi; Findley, Muna; Khanna, Savita; Gnyawali, Urmila; Bergdall, Valerie K; Sen, Chandan K

    2011-11-01

    The economic, social, and public health burden of chronic ulcers and other compromised wounds is enormous and rapidly increasing with the aging population. The growth factors derived from platelets play an important role in tissue remodeling including neovascularization. Platelet-rich plasma (PRP) has been utilized and studied for the last four decades. Platelet gel and fibrin sealant, derived from PRP mixed with thrombin and calcium chloride, have been exogenously applied to tissues to promote wound healing, bone growth, hemostasis, and tissue sealing. In this study, we first characterized recovery and viability of as well as growth factor release from platelets in a novel preparation of platelet gel and fibrin matrix, namely platelet-rich fibrin matrix (PRFM). Next, the effect of PRFM application in a delayed model of ischemic wound angiogenesis was investigated. The study, for the first time, shows the kinetics of the viability of platelet-embedded fibrin matrix. A slow and steady release of growth factors from PRFM was observed. The vascular endothelial growth factor released from PRFM was primarily responsible for endothelial mitogenic response via extracellular signal-regulated protein kinase activation pathway. Finally, this preparation of PRFM effectively induced endothelial cell proliferation and improved wound angiogenesis in chronic wounds, providing evidence of probable mechanisms of action of PRFM in healing of chronic ulcers.

  14. Platelet counting with the BD Accuri(TM) C6 flow cytometer.

    Science.gov (United States)

    Masters, Andrew; Harrison, Paul

    2014-01-01

    The Accuri™ C6 is a compact flow cytometer that uses a peristaltic pump with a laminar flow fluidic system and can measure absolute cell counts. In this study we have evaluated this method with the International Reference Method (IRM) simultaneously measured on both the Accuri™ C6 and a reference flow cytometer. After optimisation of sample labelling conditions, final dilutions and flow cytometer settings, a comparison of the absolute fluorescent platelet count with the RBC/platelet ratio on the C6 and the IRM was then performed in 144 patient samples with a full range of platelet counts (range 2-650 × 10(9)/l). The platelet/RBC ratio method determined on the Accuri™ agreed well with the IRM (R(2)=0.99, bias=2.3 (Bland Altman) and R(2)=0.96, bias=1.02 at counts <50 × 10(9)/l). The absolute platelet count also agreed well with the IRM (R(2)=0.97, bias=-0.16 and R(2)=0.91, bias=3.7 at <50 × 10(9)/l). The C6 absolute platelet count and RBC/platelet ratio methods also agreed well (R(2)=0.99, bias=-2.5 and R(2)=0.95, bias=2.71 at counts <50 × 10(9)/l). Reproducibility studies on the C6 gave CVs of <5% for the RB/platelet ratio and <12% for the absolute cell counts. The C6 also demonstrated excellent linearity on diluted samples with both volume and ratio methods (R(2)=0.99). As one might expect, the absolute platelet count is therefore slightly more inaccurate than the RBC/platelet ratio particularly at platelet counts <50 × 10(9)/l as it is likely to be more sensitive to pipetting error. The Accuri™ C6 provides a simple, rapid and reliable method for measuring platelet counts by either the RBC/platelet or direct volume methods. The direct volume method can also be used to determine platelet counts within purified platelet preparations or concentrates in the absence of RBC.

  15. Effects of platelet-rich plasma gel on skin healing in surgical wound in horses

    OpenAIRE

    DeRossi, Rafael; Coelho,Anna Carolina Anciliero de Oliveira; Mello,Gisele Silveira de; Frazílio,Fabrício de Oliveira; Leal,Cássia Rejane Brito; Facco,Gilberto Gonçalves; Brum,Karine Bonucielli

    2009-01-01

    PURPOSE: To establish a low-cost method to prepare platelet-rich plasma (PRP) and evaluates the potential of platelet derived factors to enhance wound healing in the surgical wounds in equine. METHODS: To obtain a PRP gel, calcium gluconate and autologous thrombin were added to platelet-rich plasma. For the tests six saddle horses were used and two surgical incisions were made in each animal. Wounds were treated with PRP gel or untreated. Sequential wound biopsies collected at Treatment 1: at...

  16. Impact of reticulated platelets on antiplatelet response to thienopyridines is independent of platelet turnover.

    Science.gov (United States)

    Stratz, Christian; Nührenberg, Thomas; Amann, Michael; Cederqvist, Marco; Kleiner, Pascal; Valina, Christian M; Trenk, Dietmar; Neumann, Franz-Josef; Hochholzer, Willibald

    2016-10-28

    Reticulated platelets are associated with impaired antiplatelet response to thienopyridines. It is uncertain whether this interaction is caused by a decreased drug exposure due to high platelet turnover reflected by elevated levels of reticulated platelets or by intrinsic properties of reticulated platelets. This study sought to investigate if the impact of reticulated platelets on early antiplatelet response to thienopyridines is mainly caused by platelet turnover as previously suggested. Elective patients undergoing coronary intervention were randomised to loading with clopidogrel 600 mg or prasugrel 60 mg (n=200). Adenosine diphosphate (ADP)-induced platelet reactivity was determined by impedance aggregometry before, at 30, 60, 90, and 120 minutes and at day 1 after loading. Immature platelet count was assessed as marker of reticulated platelets by flow cytometry. Platelet reactivity increased with rising levels of immature platelet count in both groups. This effect was more distinctive in patients on clopidogrel as compared to patients on prasugrel. Overall, immature platelet count correlated well with on-treatment platelet reactivity at all time-points (p < 0.001). These correlations did not change over time in the entire cohort as well as in patients treated with clopidogrel or prasugrel indicating an effect independent of platelet turnover (comparison of correlations 120 minutes/day 1: p = 0.64). In conclusion, the association of immature platelet count with impaired antiplatelet response to thienopyridines is similar early and late after loading. This finding suggests as main underlying mechanism another effect of reticulated platelets on thienopyridines than platelet turnover.

  17. Adjuvant treatment with the bacterial lysate (OM-85) improves management of atopic dermatitis: A randomized study

    Science.gov (United States)

    Bodemer, Christine; Guillet, Gerard; Cambazard, Frederic; Boralevi, Franck; Ballarini, Stefania; Milliet, Christian; Bertuccio, Paola; La Vecchia, Carlo; Bach, Jean-François; de Prost, Yves

    2017-01-01

    Background Environmental factors play a major role on atopic dermatitis (AD) which shows a constant rise in prevalence in western countries over the last decades. The Hygiene Hypothesis suggesting an inverse relationship between incidence of infections and the increase in atopic diseases in these countries, is one of the working hypothesis proposed to explain this trend. Objective This study tested the efficacy and safety of oral administration of the bacterial lysate OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®), in the treatment of established AD in children. Methods Children aged 6 months to 7 years, with confirmed AD diagnosis, were randomized in a double-blind, placebo-controlled trial to receive, in addition to conventional treatment with emollients and topical corticosteroids, 3.5mg of the bacterial extract OM-85 or placebo daily for 9 months. The primary end-point was the difference between groups in the occurrence of new flares (NF) during the study period, evaluated by Hazard Ratio (HR) derived from conditional Cox proportional hazard regression models accounting for repeated events. Results Among the 179 randomized children, 170 were analysed, 88 in the OM-85 and 82 in the placebo group. As expected most children in both treatment groups experienced at least 1 NF during the study period (75 (85%) patients in the OM-85 group and 72 (88%) in the placebo group). Patients treated with OM-85 as adjuvant therapy had significantly fewer and delayed NFs (HR of repeated flares = 0.80; 95% confidence interval (CI): 0.67–0.96), also when potential confounding factors, as family history of atopy and corticosteroids use, were taken into account (HR = 0.82; 95% CI: 0.69–0.98). No major side effect was reported, with comparable and good tolerability for OM-85 and placebo. Conclusions Results show an adjuvant therapeutic effect of a well standardized bacterial lysate OM-85 on established AD. PMID:28333952

  18. Platelets and infection — an emerging role of platelets in viral infection

    Directory of Open Access Journals (Sweden)

    Alice eAssinger

    2014-12-01

    Full Text Available Platelets are anucleate blood cells that play a crucial role in the maintenance of hemostasis. While platelet activation and elevated platelet counts (thrombocytosis are associated with increased risk of thrombotic complications, low platelet counts (thrombocytopenia and several platelet function disorders increase the risk of bleeding. Over the last years more and more evidence has emerged that platelets and their activation state can also modulate innate and adaptive immune responses and low platelet counts have been identified as a surrogate marker for poor prognosis in septic patients.Viral infections often coincide with platelet activation. Host inflammatory responses result in the release of platelet activating mediators and a pro-oxidative and pro-coagulant environment, which favours platelet activation. However, viruses can also directly interact with platelets and megakaryocytes and modulate their function. Furthermore, platelets can be activated by viral antigen-antibody complexes and in response to some viruses B-lymphocytes also generate anti-platelet antibodies.All these processes contributing to platelet activation result in increased platelet consumption and removal and often lead to thrombocytopenia, which is frequently observed during viral infection. However, virus-induced platelet activation does not only modulate platelet count, but also shapes immune responses. Platelets and their released products have been reported to directly and indirectly suppress infection and to support virus persistence in response to certain viruses, making platelets a double-edged sword during viral infections. This review aims to summarize the current knowledge on platelet interaction with different types of viruses, the viral impact on platelet activation and platelet-mediated modulations of innate and adaptive immune responses.

  19. Platelets and infection - an emerging role of platelets in viral infection.

    Science.gov (United States)

    Assinger, Alice

    2014-01-01

    Platelets are anucleate blood cells that play a crucial role in the maintenance of hemostasis. While platelet activation and elevated platelet counts (thrombocytosis) are associated with increased risk of thrombotic complications, low platelet counts (thrombocytopenia) and several platelet function disorders increase the risk of bleeding. Over the last years, more and more evidence has emerged that platelets and their activation state can also modulate innate and adaptive immune responses and low platelet counts have been identified as a surrogate marker for poor prognosis in septic patients. Viral infections often coincide with platelet activation. Host inflammatory responses result in the release of platelet activating mediators and a pro-oxidative and pro-coagulant environment, which favors platelet activation. However, viruses can also directly interact with platelets and megakaryocytes and modulate their function. Furthermore, platelets can be activated by viral antigen-antibody complexes and in response to some viruses B-lymphocytes also generate anti-platelet antibodies. All these processes contributing to platelet activation result in increased platelet consumption and removal and often lead to thrombocytopenia, which is frequently observed during viral infection. However, virus-induced platelet activation does not only modulate platelet count but also shape immune responses. Platelets and their released products have been reported to directly and indirectly suppress infection and to support virus persistence in response to certain viruses, making platelets a double-edged sword during viral infections. This review aims to summarize the current knowledge on platelet interaction with different types of viruses, the viral impact on platelet activation, and platelet-mediated modulations of innate and adaptive immune responses.

  20. Allogeneic Platelet Releasate Preparations Derived via a Novel Rapid Thrombin Activation Process Promote Rapid Growth and Increased BMP-2 and BMP-4 Expression in Human Adipose-Derived Stem Cells.

    Science.gov (United States)

    McLaughlin, Michael; Gagnet, Paul; Cunningham, Elizabeth; Yeager, Randi; D'Amico, Michael; Guski, Katie; Scarpone, Michael; Kuebler, Daniel

    2016-01-01

    The administration of human adipose-derived stem cells (ASCs) represents a promising regenerative therapy for the treatment of orthopedic injuries. While ASCs can be easily isolated from liposuction-derived adipose tissue, most clinical applications will likely require in vitro culture expansion of these cells using nonxenogeneic components. In this study, platelet releasate was generated using a novel rapid thrombin activation method (tPR). ASCs grown in media supplemented with tPR proliferated much faster than ASCs grown in media supplemented with 10% fetal bovine serum. The cells also retained the ability to differentiate along chondrogenic, adipogenic, and osteogenic lineages. The tPR cultured ASCs displayed elevated expression of BMP-4 (5.7 ± 0.97-fold increase) and BMP-2 (4.7 ± 1.3-fold increase) and decreased expression of PDGF-B (4.0 ± 1.4-fold decrease) and FGF-2 (33 ± 9.0-fold decrease). No significant changes in expression were seen with TGF-β and VEGF. This pattern of gene expression was consistent across different allogeneic tPR samples and different ASC lines. The use of allogeneic rapidly activated tPR to culture ASCs is associated with both an increased cell yield and a defined gene expression profile making it an attractive option for cell expansion prior to cell-based therapy for orthopedic applications.

  1. Allogeneic Platelet Releasate Preparations Derived via a Novel Rapid Thrombin Activation Process Promote Rapid Growth and Increased BMP-2 and BMP-4 Expression in Human Adipose-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Michael McLaughlin

    2016-01-01

    Full Text Available The administration of human adipose-derived stem cells (ASCs represents a promising regenerative therapy for the treatment of orthopedic injuries. While ASCs can be easily isolated from liposuction-derived adipose tissue, most clinical applications will likely require in vitro culture expansion of these cells using nonxenogeneic components. In this study, platelet releasate was generated using a novel rapid thrombin activation method (tPR. ASCs grown in media supplemented with tPR proliferated much faster than ASCs grown in media supplemented with 10% fetal bovine serum. The cells also retained the ability to differentiate along chondrogenic, adipogenic, and osteogenic lineages. The tPR cultured ASCs displayed elevated expression of BMP-4 (5.7 ± 0.97-fold increase and BMP-2 (4.7 ± 1.3-fold increase and decreased expression of PDGF-B (4.0 ± 1.4-fold decrease and FGF-2 (33 ± 9.0-fold decrease. No significant changes in expression were seen with TGF-β and VEGF. This pattern of gene expression was consistent across different allogeneic tPR samples and different ASC lines. The use of allogeneic rapidly activated tPR to culture ASCs is associated with both an increased cell yield and a defined gene expression profile making it an attractive option for cell expansion prior to cell-based therapy for orthopedic applications.

  2. Platelet satellitism in infectious disease?

    Science.gov (United States)

    Laskaj, Renata; Sikiric, Dubravka; Skerk, Visnja

    2015-01-01

    Background Platelet satellitism is a phenomenon of unknown etiology of aggregating platelets around polymorphonuclear neutrophils and other blood cells which causes pseudothrombocytopenia, visible by microscopic examination of blood smears. It has been observed so far in about a hundred cases in the world. Case subject and methods Our case involves a 73-year-old female patient with a urinary infection. Biochemical serum analysis (CRP, glucose, AST, ALT, ALP, GGT, bilirubin, sodium, potassium, chloride, urea, creatinine) and blood cell count were performed with standard methods on autoanalyzers. Serum protein fractions were examined by electrophoresis and urinalysis with standard methods on autoanalyzer together with microscopic examination of urine sediment. Erythrocyte sedimentation rate, blood culture and urine culture tests were performed with standard methods. Results Due to typical pathological values for bacterial urinary infection, the patient was admitted to the hospital. Blood smear examination revealed phenomenon, which has persisted for three weeks after the disease has been cured. Blood smears with EDTA as an anticoagulant had platelet satellitism whereas the phenomenon was not observed in tubes with different anticoagulants (Na, Li-heparin) and capillary blood. Discussion We hypothesize that satellitism was induced by some immunological mechanism through formation of antibodies which have mediated platelets binding to neutrophil membranes and vice versa. Unfortunately we were unable to determine the putative trigger for this phenomenon. To our knowledge this is the second case of platelet satellitism ever described in Croatia. PMID:26110042

  3. Proteomic analysis using 2-D liquid separations of intact proteins from whole-cell lysates.

    Science.gov (United States)

    Zhu, Kan; Yan, Fang; O'Neil, Kimberly A; Hamler, Rick; Lubman, David M; Lin, Linda; Barder, Timothy J

    2004-02-01

    This unit describes procedures for 2-D liquid separations of proteins from whole-cell lysates. Protocols for protein isoelectric point (pI) fractionation in the first dimension include the use of liquid isoelectric focusing (IEF) and chromatofocusing. The liquid IEF provides a pI-based fractionation using a batch-phase electrophoretic method, while chromatofocusing uses a column-based chromatographic method to generate the pH gradient. Using either method, a second-dimension fractionation is provided in the liquid phase using nonporous silica-based reversed-phase HPLC (NPS-RP-HPLC) to generate a 2-D liquid map of the protein content of the cell. The eluate of the 2-D liquid fractionation is directly coupled to a mass spectrometer for on-line detection of the intact molecular weights of proteins. As a result, a multidimensional map of protein expression is obtained that characterizes cellular proteins by pI, hydrophobicity, and intact molecular weight. Such expression maps are useful for differential proteomic comparison between different cell samples.

  4. Immunological Characterization of Whole Tumour Lysate-Loaded Dendritic Cells for Cancer Immunotherapy.

    Directory of Open Access Journals (Sweden)

    Veronica Rainone

    Full Text Available Dendritic cells play a key role as initiators of T-cell responses, and even if tumour antigen-loaded dendritic cells can induce anti-tumour responses, their efficacy has been questioned, suggesting a need to enhance immunization strategies.We focused on the characterization of bone marrow-derived dendritic cells pulsed with whole tumour lysate (TAA-DC, as a source of known and unknown antigens, in a mouse model of breast cancer (MMTV-Ras. Dendritic cells were evaluated for antigen uptake and for the expression of MHC class I/II and costimulatory molecules and markers associated with maturation.Results showed that antigen-loaded dendritic cells are characterized by a phenotypically semi-mature/mature profile and by the upregulation of genes involved in antigen presentation and T-cell priming. Activated dendritic cells stimulated T-cell proliferation and induced the production of high concentrations of IL-12p70 and IFN-γ but only low levels of IL-10, indicating their ability to elicit a TH1-immune response. Furthermore, administration of Antigen loaded-Dendritic Cells in MMTV-Ras mice evoked a strong anti-tumour response in vivo as demonstrated by a general activation of immunocompetent cells and the release of TH1 cytokines.Data herein could be useful in the design of antitumoral DC-based therapies, showing a specific activation of immune system against breast cancer.

  5. An efficient method for refolding the extracellular portion of CD147 from the total bacterial lysate

    Institute of Scientific and Technical Information of China (English)

    Fei Song; Xin Zhang; Yifei Li; Qiang Ru; Xiaobai Ren; Bin Xia; Zhi-Nan Chen

    2011-01-01

    CD147 is a widely expressed transmembrane protein that mediates signal transduction,and it plays important roles in many physiological and pathological processes,such as tumor invasion and metastasis.The extracellular portion of CD147 (CD147EC) is responsible for its functional interactions with different signaling molecules.Due to the existence of two disulfide bonds,CD147EC is mainly expressed as an inclusion body in Escherichia coli.Here,we report a convenient rapid-dilution refolding protocol that enables the refolding of CD147EC efficiently from total bacterial lysate instead of pure inclusion bodies.Using this method,over 25 mg of CD147EC can be purified from 1 1 of bacterial culture in M9 medium.The refolded CD147EC is well folded as characterized by nuclear magnetic resonance (NMR),and it can induce the expression of matrix metalloproteinase-9 in fibroblast cells.The described protocol is also applicable to the refolding of two immunoglobulin domains of CD147EC individually.Interestingly,we noticed that little protein was produced for the C-terminal immunoglobulin (Ig) domain of CD147EC by bacteria in M9 medium,even though it was overexpressed in LuriaBertani (LB) medium.However,when the pH of the bacterial culture in M9 medium was adjusted in accordance with that in LB medium during growth,comparable expression level could be achieved.

  6. Interaction of inorganic nanoparticles of lunar soil simulant with blood platelets

    Science.gov (United States)

    Borisova, Tatiana; Kasatkina, Ludmila; Krisanova, Natalia; Sivko, Roman; Borisov, Arseniy; Slenzka, Klaus

    Blood platelets play a central role in the physiology of primary hemostasis and in the patholog-ical processes of arterial thrombosis. Also, blood platelets contain neuronal high-affinity Na+-dependent glutamate transporters (EAAT 1 -3) and are able to uptake glutamate, thereby playing possible physiological role in extracellular glutamate homeostasis in the mammalian CNS as an additional powerful target for excessive neurotoxic glutamate accumulation and storage. The health effects of lunar soil exposure are almost completely unknown, whereas the observations suggest that it can be deleterious to human physiology. It is important that the components of lunar soil may be internalized with lipid fractions of the lung epithelium, which in turn may help ions to overcome the blood-brain barrier. The study focused on the effects of JSC-1a Lunar Soil Simulant (LSS) (Orbital Technologies Corporation, Madison, USA) on platelets isolated from rabbit blood. We revealed that platelets were not indifferent to the expo-sure to LSS. Flow cytometric analysis showed that the incubation of platelets with LSS resulted in an upper shift of platelet spot in histograms presenting cell size (FS) and granularity (SS) as x and y coordinates, thereby demonstrating apparent increase in platelet granularity. Analysis of control platelet preparation did not reveal the alterations in platelet size and granularity during the same incubation period. LSS scatter per se did not cover area of platelet prepara-tion in histogram. Using Zetasizer Nanosystem (Malvern Instruments) with helium-neon laser for dynamic light scattering (DLS), the platelet size before and after the addition of LSS was measured. We have found the increase in the mean size of the population of platelets from 2.45 ±0.09 µm in control to 3.0 ± 0.25 µm in the presence of LSS. Thus, we report that inorganic nanoparticles of LSS bind to blood platelets and this fact may have considerable harmful conse-quences to human

  7. Platelet adhesion onto wettability gradient surfaces in the absence and presence of plasma proteins.

    Science.gov (United States)

    Lee, J H; Lee, H B

    1998-08-01

    A wettability gradient was prepared on lowdensity polyethylene (PE) sheets by treating them in air with a corona from a knife-type electrode the power of which increased gradually along the sample length. The PE surfaces oxidized gradually with the increasing corona power and a wettability gradient was created on the surfaces, as evidenced by the measurement of water contact angles, Fourier transform infrared spectroscopy in the attenuated total reflectance mode, and electron spectroscopy for chemical analysis. The wettability gradient surfaces prepared were used to investigate the adhesion behavior of platelets in the absence and presence of plasma proteins in terms of the surface hydrophilicity/hydrophobicity of polymeric materials. The platelets adhered to the wettability gradient surfaces along the sample length were counted and examined by scanning electron microscopy (SEM). It was observed that the platelet adhesion in the absence of plasma proteins increased gradually as the surface wettability increased along the sample length. The platelets adhered to the hydrophilic positions of the gradient surface also were more activated (possessed more pseudo pods as examined by SEM) than on the more hydrophobic ones. However, platelet adhesion in the presence of plasma proteins decreased gradually with the increasing surface wettability; the platelets adhered to the surface also were more activated on the hydrophobic positions of the gradient surface. This result is closely related to plasma protein adsorption on the surface. Plasma protein adsorption on the wettability gradient surface increased with the increasing surface wettability. More plasma protein adsorption on the hydrophilic positions of the gradient surface caused less platelet adhesion, probably due to platelet adhesion inhibiting proteins, such as high-molecular-weight kininogen, which preferably adsorbs onto the surface by the so-called Vroman effect. It seems that both the presence of plasma proteins

  8. CD8+ T cells induce platelet clearance in the liver via platelet desialylation in immune thrombocytopenia

    Science.gov (United States)

    Qiu, Jihua; Liu, Xuena; Li, Xiaoqing; Zhang, Xu; Han, Panpan; Zhou, Hai; Shao, Linlin; Hou, Yu; Min, Yanan; Kong, Zhangyuan; Wang, Yawen; Wei, Yu; Liu, Xinguang; Ni, Heyu; Peng, Jun; Hou, Ming

    2016-01-01

    In addition to antiplatelet autoantibodies, CD8+ cytotoxic T lymphocytes (CTLs) play an important role in the increased platelet destruction in immune thrombocytopenia (ITP). Recent studies have highlighted that platelet desialylation leads to platelet clearance via hepatocyte asialoglycoprotein receptors (ASGPRs). Whether CD8+ T cells induce platelet desialylation in ITP remains unclear. Here, we investigated the cytotoxicity of CD8+ T cells towards platelets and platelet desialylation in ITP. We found that the desialylation of fresh platelets was significantly higher in ITP patients with positive cytotoxicity of CD8+ T cells than those without cytotoxicity and controls. In vitro, CD8+ T cells from ITP patients with positive cytotoxicity induced significant platelet desialylation, neuraminidase-1 expression on the platelet surface, and platelet phagocytosis by hepatocytes. To study platelet survival and clearance in vivo, CD61 knockout mice were immunized and their CD8+ splenocytes were used. Platelets co-cultured with these CD8+ splenocytes demonstrated decreased survival in the circulation and increased phagocytosis in the liver. Both neuraminidase inhibitor and ASGPRs competitor significantly improved platelet survival and abrogated platelet clearance caused by CD8+ splenocytes. These findings suggest that CD8+ T cells induce platelet desialylation and platelet clearance in the liver in ITP, which may be a novel mechanism of ITP. PMID:27321376

  9. An overview of platelet indices and methods for evaluating platelet function in thrombocytopenic patients

    DEFF Research Database (Denmark)

    Vinholt, Pernille Just; Hvas, Anne-Mette; Nybo, Mads

    2014-01-01

    in thrombocytopenia. Flow cytometry, platelet aggregometry and platelet secretion tests are used to diagnose specific platelet function defects. The flow cytometric activation marker P-selectin and surface coverage by the Cone and Plate[let] analyser™ predict bleeding in selected thrombocytopenic populations...

  10. The nature of interactions between tissue-type plasminogen activator and platelets

    Energy Technology Data Exchange (ETDEWEB)

    Torr, S.R.; Winters, K.J.; Santoro, S.A.; Sobel, B.E. (Washington Univ., St. Louis, MO (USA))

    1990-07-15

    To elucidate interactions responsible for inhibition of aggregation of platelets in platelet-rich plasma (PRP) harvested from whole blood preincubated with t-PA, experiments were performed with PRP and washed platelets under diverse conditions of preincubation. Both ADP and collagen induced aggregation were inhibited in PRP unless aprotinin had been added to the preincubated whole blood concomitantly with t-PA. However, in washed platelets prepared after the same exposure aggregation was intact. When washed platelets were supplemented with fibrinogen degradation products (FDPs) in concentrations simulating those in whole blood preincubated with t-PA, aggregation induced with either ADP or collagen was inhibited. Thus, the inhibition in PRP depended on generation of FDPs by activated plasminogen. The functional integrity of surface glycoprotein (GP) IIb/IIIa receptors in washed platelets was documented by autoradiography after SDS-PAGE of surface labeled GPs and by fibrinogen binding despite preincubation of the whole blood or washed platelets themselves with t-PA and plasminogen as long as exogenous calcium (greater than or equal to 0.1 microM) was present. In contrast, when calcium was absent, the platelet GP IIb/IIIa receptor was rendered susceptible to degradation by plasmin, and aggregation was inhibited by preincubation at 37 degrees C even if aprotinin was present when aggregation was being assayed. These observations reconcile disparate results in the literature from studies in vivo and in vitro by demonstrating that inhibition of aggregation of platelets in PRP and in whole blood reflects indirect effects of plasminogen activation rather than direct effects of t-PA or plasmin on the platelets themselves.

  11. Grain and bean lysates improve function of endothelial progenitor cells from human peripheral blood: involvement of the endogenous antioxidant defenses.

    Directory of Open Access Journals (Sweden)

    Daniela Lucchesi

    Full Text Available Increased oxidative stress contributes to the functional impairment of endothelial progenitor cells (EPCs, the pivotal players in the servicing of the endothelial cell lining. Several evidences suggest that decreasing oxidative stress by natural compounds with antioxidant properties may improve EPCs bioactivity. Here, we investigated the effects of Lisosan G (LG, a Triticum Sativum grain powder, and Lady Joy (LJ, a bean lysate, on function of EPCs exposed to oxidative stress. Peripheral blood mononuclear cells were isolated and plated on fibronectin-coated culture dishes; adherent cells, identified as early EPCs, were pre-treated with different concentrations of LG and LJ and incubated with hydrogen peroxide (H2O2. Viability, senescence, adhesion, ROS production and antioxidant enzymes gene expression were evaluated. Lysate-mediated Nrf-2 (nuclear factor (erythroid-derived 2-like 2/ARE (antioxidant response element activation, a modulator of oxidative stress, was assessed by immunocytochemistry. Lady Joy 0.35-0.7 mg/ml increases EPCs viability; pre-treatment with either LG 0.7 mg/ml and LJ 0.35-0.7 mg/ml protect EPCs viability against H2O2-induced injury. LG 0.7 and LJ 0.35-0.7 mg/ml improve EPCs adhesion; pre-treatment with either LG 0.35 and 0.7 mg/ml or LJ 0.35, 0.7 and 1.4 mg/ml preserve adhesiveness of EPCs exposed to H2O2. Senescence is attenuated in EPCs incubated with lysates 0.35 mg/ml. After exposure to H2O2, LG pre-treated cells show a lower senescence than untreated EPCs. Lysates significantly decrease H2O2-induced ROS generation. Both lysates increase glutathione peroxidase-1 and superoxide dismutase-2 (SOD-2 expression; upon H2O2 exposure, pre-treatment with LJ allows higher SOD-2 expression. Heme oxigenase-1 increases in EPCs pre-treated with LG even upon H2O2 exposure. Finally, incubation with LG 0.7 mg/ml results in Nrf-2 translocation into the nucleus both at baseline and after the oxidative challenge. Our data suggest a

  12. Platelet antigens and antibodies. Literature review

    Directory of Open Access Journals (Sweden)

    N. V. Mineeva

    2013-01-01

    Full Text Available Platelet antigens structure, role of platelet antibodies in the pathogenesis of various clinical conditions, characteristic of modern antibodies detection methods are presented in this article.

  13. Platelet antigens and antibodies. Literature review

    Directory of Open Access Journals (Sweden)

    N. V. Mineeva

    2014-07-01

    Full Text Available Platelet antigens structure, role of platelet antibodies in the pathogenesis of various clinical conditions, characteristic of modern antibodies detection methods are presented in this article.

  14. Platelet Disorders: MedlinePlus Health Topic

    Science.gov (United States)

    ... Article: Erythropoietin and thrombopoietin mimetics: Natural alternatives to erythrocyte and platelet... Article: Detection of CALR Mutation in Clonal and Nonclonal Hematologic Diseases... Platelet Disorders -- see more articles Thrombocytopenias -- see more ...

  15. [Platelet-rich plasma (platelet gel) in secondary alveoloplasty in cleft patients].

    Science.gov (United States)

    Méndez, R; López-Cedrún, J L; Patiño, B; Vázquez, I; Martín-Sastre, R; Tellado, M G; Vela, D

    2006-01-01

    The use of Platelet-rich plasma (platelet gel--PRP--) was introduced in the oral and maxilofacial surgery 10 years ago. Its good results are due to the quickly generation of new bone and the acceleration of the period of surgical scar formation. Its employment in the alveolar reconstruction of the cleft patient is not still consistent and the works published in the literature are infrequent. The objective of this preliminary study is presenting our experience with the use of PRP in the reconstruction of the alveolar congenital defects of cleft patients. Between July 2002 and January 2004 were operated 14 patients with congenital alveolar cleft carrying out an standard secondary alveoloplasty. In 12 cases was employed cancellous bone of iliac crest and in two cases the donor area was tibial. The laboratory of Hematology of our Hospital prepared the plasma gel rich in platelets by means of a double centrifuge of autologous serum of the patient. After adding calcium a rich gel in platelets in approximate quantities of 1-2 ml was obtained. In this preliminary report we have studied the aspect of the surgical injury the 3er postoperative day, and the first and second weeks after intervention. These results were compared carried out previously with the Standard alveoloplasty without use of the PRP. Likewise we evaluated the bony density by means of intraoral Rx at 3 and 6 months postoperative. The injury of the alveoloplasty healed more quickly in the patients in which plasma enriched gel was employed. The patients referred less pain and edema in the first days of the postoperative period. The alveolar bony regeneration was faster to the 3 months, though the result was similar in the intraoral Rx to the 6 months. The necessary quantities of cancellous bone were smaller in all cases in which the plasma enriched gel was used (30% less). The use of Platelet-rich plasma (PRP) is a valid protocol for the reconstruction in patients with congenital alveolar clefts. Its low

  16. Dengue platelets meet Sir Arthur Conan Doyle.

    Science.gov (United States)

    Bray, Paul F

    2013-11-14

    In this issue of Blood, Hottz et al provide compelling evidence that dengue virus (DV) induces (1) platelet synthesis of interleukin-1b (IL-1b); (2) platelet-derived IL-1b–containing microvesicles (MVs) that increase vascular permeability; and (3) DV-triggered inflammasome activation in platelets.

  17. Molecular Basis Linking Platelet to Inflammation

    Institute of Scientific and Technical Information of China (English)

    马丽萍

    2010-01-01

    @@ Introduction Blood platelets not only play an important role in hemostasis and thrombosis,but increasing evidence show that they participate in the induction of inflammation.Firstly,platelets contain and release cytokines and immune mediators.And platelets are able to modulate and regulate the function of surrounding cells by adhesion molecules or by the release of various factors.

  18. Platelet regulating properties of insulin revisited

    NARCIS (Netherlands)

    Andrade Ferreira, I. (Irlando)

    2005-01-01

    Disturbances in platelet responsiveness in diabetes mellitus (DM) lead to platelet-dependent complications in the vasculature. Our studies showed that insulin inhibits platelet activation by inhibiting ADP- and thrombin-induced Ca2+ levels. Ca2+ is under control of cAMP that is a potent endogenous p

  19. Treatment of chronic venous leg ulcers by platelet gel.

    Science.gov (United States)

    Ficarelli, Elena; Bernuzzi, Gino; Tognetti, Elena; Bussolati, Ovidio; Zucchi, Alfredo; Adorni, Daniela; De Panfilis, Giuseppe

    2008-07-01

    Chronic venous leg ulcers (CVLU) are chronic wounds, associated with long-standing venous hypertension, which have a poor prognosis for healing. In the process of wound healing the first step is represented by platelet aggregation and subsequent release of growth factors and other mediators, which play a key role in the repair response. Platelet gel (PG), a hemocomponent obtained by mixing platelets, thrombin, and calcium, is able, when applied topically, to release platelet mediators that likely favor CVLU healing. However, unstandardized protocols have been described in studies utilizing PG for the regeneration of a number of tissues, including CVLU; the relative clinical outcomes were hence highly variable. In our experience the topical use of PG, together with the strict adherence to the principles of good wound care, quickly promoted increased granulation tissue, followed by a complete CVLU epithelization. Although further studies and trials are needed to establish the major outcome affecting rules for optimal indications, preparation, and use of PG for CVLU treatment, PG can be undoubtedly considered a useful tool, able to improve the management of CVLU.

  20. Classification of platelet concentrates (Platelet-Rich Plasma-PRP, Platelet-Rich Fibrin-PRF) for topical and infiltrative use in orthopedic and sports medicine: current consensus, clinical implications and perspectives.

    Science.gov (United States)

    Dohan Ehrenfest, David M; Andia, Isabel; Zumstein, Matthias A; Zhang, Chang-Qing; Pinto, Nelson R; Bielecki, Tomasz

    2014-01-01

    Platelet concentrates for topical and infiltrative use - commonly termed Platetet-Rich Plasma (PRP) or Platelet-Rich Fibrin (PRF) - are used or tested as surgical adjuvants or regenerative medicine preparations in most medical fields, particularly in sports medicine and orthopaedic surgery. Even if these products offer interesting therapeutic perspectives, their clinical relevance is largely debated, as the literature on the topic is often confused and contradictory. The long history of these products was always associated with confusions, mostly related to the lack of consensual terminology, characterization and classification of the many products that were tested in the last 40 years. The current consensus is based on a simple classification system dividing the many products in 4 main families, based on their fibrin architecture and cell content: Pure Platelet-Rich Plasma (P-PRP), such as the PRGF-Endoret technique; Leukocyte- and Platelet-Rich Plasma (LPRP), such as Biomet GPS system; Pure Platelet-Rich Fibrin (P-PRF), such as Fibrinet; Leukocyte- and Platelet-Rich Fibrin (L-PRF), such as Intra-Spin L-PRF. The 4 main families of products present different biological signatures and mechanisms, and obvious differences for clinical applications. This classification serves as a basis for further investigations of the effects of these products. Perspectives of evolutions of this classification and terminology are also discussed, particularly concerning the impact of the cell content, preservation and activation on these products in sports medicine and orthopaedics.

  1. Human platelet antigen genotyping of platelet donors in southern Brazil.

    Science.gov (United States)

    Merzoni, J; Fagundes, I S; Lunardi, L W; Lindenau, J D-R; Gil, B C; Jobim, M; Dias, V G; Merzoni, L; Sekine, L; Onsten, T G H; Jobim, L F

    2015-10-01

    Human platelet antigens (HPA) are immunogenic structures that result from single nucleotide polymorphisms (SNPs) leading to single amino acid substitutions. This study sought to determine the allele and genotype frequencies of HPA-1, HPA-2, HPA-3, HPA-4, HPA-5 and HPA-15 in platelet donors from the state of Rio Grande do Sul (RS), Brazil, and compare their allele frequencies to those observed in other populations. HPA genotyping was performed by PCR-SSP method. The study sample comprised 201 platelet donors (167 Caucasians and 34 non-Caucasians). Allele 'a' was that most commonly found for HPA-1 to 5 in both groups. The HPA-15ab genotype predominated over homozygous genotypes of this system. Fisher's exact test revealed statistically significant differences for the HPA-5 system, with a greater prevalence of the HPA-5b allele in non-Caucasians. The neighbour-joining method and principal components analysis revealed genetic proximity between our Caucasian group and European populations. We conclude that the allele frequencies of HPA-1 to 5 and HPA-15 found in our Caucasian sample are similar to those reported for European populations. These findings corroborate the ethnic makeup of the population of RS. The higher frequency of the HPA-5b allele found in the non-Caucasian group of our sample suggests the possibility of allosensitization in patients who receive platelet transfusions from genetically incompatible donors.

  2. Estrogen receptor α determination in serum, cell lysates and breast cancer cells using an amperometric magnetoimmunosensing platform

    Directory of Open Access Journals (Sweden)

    U. Eletxigerra

    2016-03-01

    Full Text Available An electrochemical magnetoimmunosensor for the determination of estrogen receptor α (ERα protein in complex samples (serum and cell lysates able to discriminate between ERα positive and negative breast cancer cells is reported. Specifically functionalized magnetic microbeads with sandwich immunocomplexes and amperometric detection at disposable screen-printed carbon electrodes (SPCEs resulted in highly selective and sensitive ERα detection with a detection limit of 19 pg mL−1. This magnetoimmunosensing platform was successfully applied to the quantitation of ERα in spiked human serum and cell lysates samples without any matrix effect with an advantageous performance in terms of simplicity and assay times over commercial ELISA assays. The biosensor capability for assessing ERα in intact breast cancer cells makes it competitive with conventional strategies providing rapidly quantitative and reliable results on this relevant biomarker currently used in the clinical practice for diagnosis, follow-up and monitoring of metastatic breast cancer.

  3. Clinical Benefit of Allogeneic Melanoma Cell Lysate-Pulsed Autologous Dendritic Cell Vaccine in MAGE-Positive Colorectal Cancer Patients

    DEFF Research Database (Denmark)

    Toh, Han Chong; Wang, Who-Whong; Chia, Whay Kuang

    2009-01-01

    PURPOSE: We evaluated the clinical benefit of an allogeneic melanoma cell lysate (MCL)-pulsed autologous dendritic cell (DC) vaccine in advanced colorectal cancer patients expressing at least one of six MAGE-A antigens overexpressed by the cell line source of the lysate. EXPERIMENTAL DESIGN: DCs...... patients received a total of 161 vaccinations. Treatment was well tolerated and quality of life measurements did not vary much across time. One patient experienced partial response [5%; 95% confidence interval (CI), 1-24%] and seven achieved stable disease (35%; 95% CI, 18-57%), one of whom also achieved......-free for >27 and >37 months, respectively. This result is particularly meaningful as all patients had progressive disease before treatment. Overall, DC vaccination was associated with a serial decline in regulatory T cells. Using an antibody array, we characterized plasma protein profiles in responding...

  4. Platelet count and platelet indices in women with preeclampsia

    Directory of Open Access Journals (Sweden)

    AlSheeha MA

    2016-11-01

    Full Text Available Muneera A AlSheeha,1 Rafi S Alaboudi,1 Mohammad A Alghasham,1 Javed Iqbal,2 Ishag Adam1 1Department of Obstetrics and Gynaecology, College of Medicine, Qassim University, Buriadah, 2Department of Obstetrics and Gynecology, Maternity and Children’s Hospital, Qassim, Kingdom of Saudi Arabia Background: Although the exact pathophysiology of preeclampsia is not completely understood, the utility of different platelets indices can be utilized to predict preeclampsia.Objective: To compare platelet indices, namely platelet count (PC, mean platelet volume (MPV, platelet distribution width (PDW, and PC to MPV ratio in women with preeclampsia compared with healthy controls.Setting: Qassim Hospital, Kingdom of Saudi Arabia.Design: A case–control study. Sixty preeclamptic women were the cases and an equal number of healthy pregnant women were the controls.Results: There was no significant difference in age, parity, and body mass index between the study groups. Sixteen and 44 of the cases were severe and mild preeclampsia, respectively. There was no significant difference in PDW and MPV between the preeclamptic and control women. Both PC and PC to MPV ratios were significantly lower in the women with preeclampsia compared with the controls. There was no significant difference in the PC, PDW, MPV, and PC to MPV ratio when women with mild and severe preeclampsia were compared. Using receiver operating characteristic (ROC curves, the PC cutoff was 248.0×103/µL for diagnosis of preeclampsia (P=0.019; the area under the ROC curve was 62.4%. Binary regression suggests that women with PC <248.010×103/µL were at higher risk of preeclampsia (odds ratio =2.2, 95% confidence interval =1.08–4.6, P=0.03. The PC/MPV cutoff was 31.2 for diagnosis of preeclampsia (P=0.035, the area under the ROC curve was 62.2%.Conclusion: PC <248.010×103/µL and PC to MPV ratio 31.2 are valid predictors of preeclampsia. Keywords: preeclampsia, platelets, PDW, mean platelet

  5. ASP-56, a new actin sequestering protein from pig platelets with homology to CAP, an adenylate cyclase-associated protein from yeast.

    Science.gov (United States)

    Gieselmann, R; Mann, K

    1992-02-24

    A new 56 kDa actin-binding protein (ASP-56) was isolated from pig platelet lysate. In falling ball viscosimetry it caused a reduction in viscosity that could be attributed to a decrease in the concentration of polymeric actin. Fluorescence measurements with NBD-labelled actin showed reduction of polymeric actin, too. These results could be explained by sequestering of actin in a non-polymerizable 1:1 ASP-56/actin complex. Sequencing of about 20 tryptic peptides of ASP-56 and comparison with known sequences revealed about 60% homology to the adenylate cyclase-associated protein (CAP) from yeast.

  6. Physical and functional association of the Src family kinases Fyn and Lyn with the collagen receptor glycoprotein VI-Fc receptor gamma chain complex on human platelets.

    Science.gov (United States)

    Ezumi, Y; Shindoh, K; Tsuji, M; Takayama, H

    1998-07-20

    We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor gamma chain (FcRgamma) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRgamma, Syk, and phospholipase Cgamma2 (PLCgamma2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI-FcRgamma complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI-FcRgamma complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRgamma, Syk, and PLCgamma2 and recruited tyrosine-phosphorylated Syk to the GPVI-FcRgamma complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRgamma and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI-FcRgamma complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family-specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRgamma, Syk, and PLCgamma2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI-FcRgamma complex.

  7. Physical and Functional Association of the Src Family Kinases Fyn and Lyn with the Collagen Receptor Glycoprotein VI-Fc Receptor γ Chain Complex on Human Platelets

    Science.gov (United States)

    Ezumi, Yasuharu; Shindoh, Keisuke; Tsuji, Masaaki; Takayama, Hiroshi

    1998-01-01

    We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor γ chain (FcRγ) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRγ, Syk, and phospholipase Cγ2 (PLCγ2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI–FcRγ complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI–FcRγ complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRγ, Syk, and PLCγ2 and recruited tyrosine-phosphorylated Syk to the GPVI–FcRγ complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRγ and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI–FcRγ complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family–specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRγ, Syk, and PLCγ2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI–FcRγ complex. PMID:9670039

  8. Platelet count and platelet indices in women with preeclampsia.

    Science.gov (United States)

    AlSheeha, Muneera A; Alaboudi, Rafi S; Alghasham, Mohammad A; Iqbal, Javed; Adam, Ishag

    2016-01-01

    Although the exact pathophysiology of preeclampsia is not completely understood, the utility of different platelets indices can be utilized to predict preeclampsia. To compare platelet indices, namely platelet count (PC), mean platelet volume (MPV), platelet distribution width (PDW), and PC to MPV ratio in women with preeclampsia compared with healthy controls. Qassim Hospital, Kingdom of Saudi Arabia. A case-control study. Sixty preeclamptic women were the cases and an equal number of healthy pregnant women were the controls. There was no significant difference in age, parity, and body mass index between the study groups. Sixteen and 44 of the cases were severe and mild preeclampsia, respectively. There was no significant difference in PDW and MPV between the preeclamptic and control women. Both PC and PC to MPV ratios were significantly lower in the women with preeclampsia compared with the controls. There was no significant difference in the PC, PDW, MPV, and PC to MPV ratio when women with mild and severe preeclampsia were compared. Using receiver operating characteristic (ROC) curves, the PC cutoff was 248.0×10(3)/µL for diagnosis of pre-eclampsia (P=0.019; the area under the ROC curve was 62.4%). Binary regression suggests that women with PC preeclampsia (odds ratio =2.2, 95% confidence interval =1.08-4.6, P=0.03). The PC/MPV cutoff was 31.2 for diagnosis of preeclampsia (P=0.035, the area under the ROC curve was 62.2%). PC preeclampsia.

  9. CORRECTION OF THE DISORDERS OF THE MOUTH CAVITY BIOCENOSIS AMONG THE CHILDREN WITH CHRONIC TONSILLITIS, AIDED BY THE TOPICAL BACTERIAL LYSATE

    Directory of Open Access Journals (Sweden)

    E.B. Foshina

    2007-01-01

    Full Text Available The authors studied the impact of the Imudon bacterial lysate on the status of the tonsil microbiocenosis among the children with chronic tonsillitis in remission. They revealed a certain reduction in the level of pathogenic and opportunistic pathogenic microflora carriage, as well as intensity of the tonsil colonization after 20-day sublingual application of topical bacterial lysate. The medication may be recommended for the preventive practices among the children with chronic tonsillitis to reduce the bacterial contamination of the pharyngeal tonsils.Key words: chronic tonsillitis, children, bacterial lysates, microbiocenosis of the mouth cavity.

  10. Effect of ionizing radiation on platelet function in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kalovidouris, A.E.; Papayannis, A.G. (Evangelismos Hospital, Athens (Greece))

    1981-01-01

    The effect of ionizing radiation on platelet function was investigated in vitro. Platelet-rich plasma (300x10/sup 9//l) was irradiated with doses of 1, 4, 10, 20 and 50 Gy. Platelet function tests were performed on both irradiated and control (non-irradiated) platelet samples. The platelet function tests were (1) platelet aggregation by ADP (1, 2, 4 ..mu..mol final concentration), adrenaline and collagen, (2) ADP-release from platelets, (3) clot retraction and (4) platelet factor-3 availability. It was found that roentgen irradiation of platelets in vitro did not affect these platelet function tests.

  11. Effect of hyperbaric oxygen therapy combined with autologous platelet concentrate applied in rabbit fibula fraction healing

    Directory of Open Access Journals (Sweden)

    Paulo Cesar Fagundes Neves

    2013-09-01

    Full Text Available OBJECTIVES: The purpose is to study the effects of hyperbaric oxygen therapy and autologous platelet concentrates in healing the fibula bone of rabbits after induced fractures. METHODS: A total of 128 male New Zealand albino rabbits, between 6-8 months old, were subjected to a total osteotomy of the proximal portion of the right fibula. After surgery, the animals were divided into four groups (n = 32 each: control group, in which animals were subjected to osteotomy; autologous platelet concentrate group, in which animals were subjected to osteotomy and autologous platelet concentrate applied at the fracture site; hyperbaric oxygen group, in which animals were subjected to osteotomy and 9 consecutive daily hyperbaric oxygen therapy sessions; and autologous platelet concentrate and hyperbaric oxygen group, in which animals were subjected to osteotomy, autologous platelet concentrate applied at the fracture site, and 9 consecutive daily hyperbaric oxygen therapy sessions. Each group was divided into 4 subgroups according to a pre-determined euthanasia time points: 2, 4, 6, and 8 weeks postoperative. After euthanasia at a specific time point, the fibula containing the osseous callus was prepared histologically and stained with hematoxylin and eosin or picrosirius red. RESULTS: Autologous platelet concentrates and hyperbaric oxygen therapy, applied together or separately, increased the rate of bone healing compared with the control group. CONCLUSION: Hyperbaric oxygen therapy and autologous platelet concentrate combined increased the rate of bone healing in this experimental model.

  12. Role of platelets in maintenance of pulmonary vascular permeability to protein

    Energy Technology Data Exchange (ETDEWEB)

    Lo, S.K.; Burhop, K.E.; Kaplan, J.E.; Malik, A.B. (Albany Medical College of Union Univ., NY (USA))

    1988-04-01

    The authors examined the role of platelets in maintenance of pulmonary vascular integrity by inducing thrombocytopenia in sheep using antiplatelet serum (APS). A causal relationship between thrombocytopenia and increase in pulmonary vascular permeability was established by platelet repletion using platelet-rich plasma (PRP). Sheep were chronically instrumented and lung lymph fistulas prepared to monitor pulmonary lymph flow (Q{sub lym}). A balloon catheter was positioned in the left atrium to assess pulmonary vascular permeability to protein after raising the left atrial pressure (P{sub la}). Thrombocytopenia was maintained for 3 days by daily intramuscular APS injections. In studies using cultured bovine pulmonary artery endothelial monolayers, transendothelia permeability of {sup 125}I-labeled albumin was reduced 50 and 95%, respectively, when 2.5 {times} 10{sup 7} or 5 {times} 10{sup 7} platelets were added onto endothelial monolayers. However, addition of 5 {times} 10{sup 6} platelets or 5 {times} 10{sup 7} red blood cells did not reduce endothelial monolayer albumin permeability. Results indicate that platelets are required for the maintenance of pulmonary vascular permeability. Reduction in permeability appears to involve an interaction of platelets with the endothelium.

  13. Rapid and selective extraction, isolation, preconcentration, and quantitation of small RNAs from cell lysate using on-chip isotachophoresis.

    Science.gov (United States)

    Schoch, Reto B; Ronaghi, Mostafa; Santiago, Juan G

    2009-08-07

    We present a technique which enables the separation of small RNAs-such as microRNAs (miRNAs), short interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs)-from >or=66 nucleotide RNAs and other biomolecules contained in a cell lysate. In particular, the method achieves separation of small RNAs from precursor miRNAs (pre-miRNAs) in less than 3 min. We use on-chip isotachophoresis (ITP) for the simultaneous extraction, isolation, preconcentration and quantitation of small RNAs (approximately 22 nucleotides) and employ the high-efficiency sieving matrix Pluronic F-127; a thermo-responsive triblock copolymer which allows convenient microchannel loading at low temperature. We present the isolation of small RNAs from the lysate of 293A human kidney cells, and quantitate the number of short RNA molecules per cell to be 2.9x10(7). We estimate this quantity is an aggregate of roughly 500 types of short RNA molecules per 293A cell. Currently, the minimal cell number for small RNA extraction and detection with our method is approximately 900 (from a 5 microL sample volume), and we believe that small RNA analysis from tens of cells is realizable. Techniques for rapid and sensitive extraction and isolation of small RNAs from cell lysate are much-needed to further uncover their full range and functionality, including RNA interference studies.

  14. Applicability of bacterial endotoxins test to various blood products by the use of endotoxin-specific lysates.

    Science.gov (United States)

    Ochiai, Masaki; Yamamoto, Akihiko; Naito, Seishiro; Maeyama, Jun-Ichi; Masumi, Atsuko; Hamaguchi, Isao; Horiuchi, Yoshinobu; Yamaguchi, Kazunari

    2010-11-01

    Endotoxin contamination is a serious threat to the safety of parenteral drugs, and the rabbit pyrogen test has played a crucial role in controlling this contamination. Although the highly sensitive endotoxin test has replaced the pyrogen test for various pharmaceuticals, the pyrogen test is still implemented as the control test for most blood products in Japan. We examined the applicability of the endotoxin test to blood products for reliable detection and quantification of endotoxin. Nineteen types of blood products were tested for interfering factors based on spike/recovery of endotoxin by using 2 types of endotoxin-specific lysate reagents for photometric techniques. Interfering effects on the endotoxin test by the products could be eliminated by diluting from 1/2 to 1/16, with the exception of antithrombin III. However, conventional lysate reagents that also react with non-pyrogenic substances, such as (1-3)-β-D-glucan, produced results that were not relevant to endotoxin content or pyrogenicity. Our results showed that the endotoxin test would be applicable to most blood products if used with appropriate endotoxin-specific lysate reagents.

  15. Platelets: crossroads of immunity and hemostasis.

    Science.gov (United States)

    Jenne, Craig N

    2014-07-31

    In this issue of Blood, Koupenova and colleagues report that platelets express functional TOLL-like receptor 7 (TLR7) and contribute to host survival during viral infection. Through a series of experiments utilizing mice deficient for TLR7 together with adoptive transfer of wild-type platelets, Koupenova et al demonstrate that platelets specifically respond to viral analogs and intact virus, leading to platelet activation and binding to various leukocyte subsets. Perhaps most importantly, this platelet activation appears absolutely essential for host survival during infection with some viral pathogens such as encephalomyocarditis virus (EMCV).

  16. Evidence that platelet buoyant density, but not size, correlates with platelet age in man.

    Science.gov (United States)

    Mezzano, D; Hwang, K; Catalano, P; Aster, R H

    1981-01-01

    Following infusion of 51Cr-labeled autologous platelets into normal subjects, high-density (HD) and low-density (LD) platelet cohorts were isolated by prolonged centrifugation in isosmotic arabino-galactan (Stractan). Specific radio-activity of LD platelets declined rapidly post-infusion (T1/2 = 1.5 days), but specific radioactivity of HD platelets remained constant or increased over a 3--4-day period and gradually declined for 6--7 days thereafter. These differences were exaggerated when platelet cohorts enriched in LD or HD cells by slow centrifugation in high-density albumin were labeled and transfused. Mean survival of a platelet cohort enriched with HD cells was significantly (P less than 0.02) shorter (7.73 days) than that of a cohort enriched with LD cells (9.33) days). In normal subjects treated with aspirin, capacity for thromboxane synthesis was regained more rapidly (P less than 0.05) in LD than in HD platelets. HD and LD platelets differed only slightly in mean volume (HD platelets = 7.57 mu3, LD platelets = 6.87 mu3, 0.05 less than P less than 0.01). We believe the most logical interpretation of these findings is that under normal conditions in man, newly formed platelets are less dense on the average than total platelets and become more dense as they age in the circulation. Thus, specific radioactivity of LD platelets declines rapidly as these platelets move into a more dense compartment and are replaced by newly formed, unlabelled cells; specific radioactivity of HD platelets remains constant or increases as labelled platelets enter this compartment in numbers equal to or greater than the number leaving it at the end of their life span. The similarity in mean volumes of LD and HD platelets suggests that platelet size is unrelated to platelet age under normal conditions.

  17. Evidence that platelet buoyant density, but not size, correlates with platelet age in man

    Energy Technology Data Exchange (ETDEWEB)

    Mezzano, D.; Hwang, K.; Catalano, P.; Aster, R.H.

    1981-01-01

    Following infusion of 51Cr-labeled autologous platelets into normal subjects, high-density (HD) and low-density (LD) platelet cohorts were isolated by prolonged centrifugation in isosmotic arabino-galactan (Stractan). Specific radio-activity of LD platelets declined rapidly post-infusion (T1/2 . 1.5 days), but specific radioactivity of HD platelets remained constant or increased over a 3--4-day period and gradually declined for 6--7 days thereafter. These differences were exaggerated when platelet cohorts enriched in LD or HD cells by slow centrifugation in high-density albumin were labeled and transfused. Mean survival of a platelet cohort enriched with HD cells was significantly (P less than 0.02) shorter (7.73 days) than that of a cohort enriched with LD cells (9.33) days). In normal subjects treated with aspirin, capacity for thromboxane synthesis was regained more rapidly (P less than 0.05) in LD than in HD platelets. HD and LD platelets differed only slightly in mean volume (HD platelets . 7.57 mu3, LD platelets . 6.87 mu3, 0.05 less than P less than 0.01). We believe the most logical interpretation of these findings is that under normal conditions in man, newly formed platelets are less dense on the average than total platelets and become more dense as they age in the circulation. Thus, specific radioactivity of LD platelets declines rapidly as these platelets move into a more dense compartment and are replaced by newly formed, unlabelled cells; specific radioactivity of HD platelets remains constant or increases as labelled platelets enter this compartment in numbers equal to or greater than the number leaving it at the end of their life span. The similarity in mean volumes of LD and HD platelets suggests that platelet size is unrelated to platelet age under normal conditions.

  18. Platelets: bridging hemostasis, inflammation, and immunity.

    Science.gov (United States)

    Jenne, C N; Urrutia, R; Kubes, P

    2013-06-01

    Although the function of platelets in the maintenance of hemostasis has been studied in great detail, more recent evidence has highlighted a central role for platelets in the host inflammatory and immune responses. Platelets by virtue of their large numbers and their ability to rapidly release a broad spectrum of immunomodulatory cytokines, chemokines, and other mediators act as circulating sentinels. Upon detection of a pathogen, platelets quickly activate and begin to drive the ensuing inflammatory response. Platelets have the ability to directly modulate the activity of neutrophils (phagocytosis, oxidative burst), endothelium (adhesion molecule and chemokine expression), and lymphocytes. Due to their diverse array of adhesion molecules and preformed chemokines, platelets are able to adhere to leukocytes and facilitate their recruitment to sites of tissue damage or infection. Furthermore, platelets directly participate in the capture and sequestration of pathogens within the vasculature. Platelet-neutrophil interactions are known to induce the release of neutrophil extracellular traps (NETs) in response to either bacterial or viral infection, and platelets have been shown to internalize pathogens, sequestering them in engulfment vacuoles. Finally, emerging data indicate that platelets also participate in the host immune response by directly killing infected cells. This review will highlight the central role platelets play in the initiation and modulation of the host inflammatory and immune responses.

  19. Calpain Activator Dibucaine Induces Platelet Apoptosis

    Directory of Open Access Journals (Sweden)

    Jun Liu

    2011-03-01

    Full Text Available Calcium-dependent calpains are a family of cysteine proteases that have been demonstrated to play key roles in both platelet glycoprotein Ibα shedding and platelet activation and altered calpain activity is associated with thrombotic thrombocytopenic purpura. Calpain activators induce apoptosis in several types of nucleated cells. However, it is not clear whether calpain activators induce platelet apoptosis. Here we show that the calpain activator dibucaine induced several platelet apoptotic events including depolarization of the mitochondrial inner transmembrane potential, up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation and phosphatidylserine exposure. Platelet apoptosis elicited by dibucaine was not affected by the broad spectrum metalloproteinase inhibitor GM6001. Furthermore, dibucaine did not induce platelet activation as detected by P-selectin expression and PAC-1 binding. However, platelet aggregation induced by ristocetin or α-thrombin, platelet adhesion and spreading on von Willebrand factor were significantly inhibited in platelets treated with dibucaine. Taken together, these data indicate that dibucaine induces platelet apoptosis and platelet dysfunction.

  20. Platelet-mediated cytotoxicity and its enhancement by platelet activating factor.

    Science.gov (United States)

    Bykovskaya, S N; Bolvacheva, A V; Kiselevsky, M V; Khaylenko, V A; Bykovsky, A F

    1991-01-01

    Platelet cytotoxicity was assessed in 70 cancer patients with various tumor localizations and in 30 normal donors. The data presented reveal that the ACL cell line displays the highest sensitivity to platelet cytotoxicity. Using the ACL cells, we discovered that platelets from oncological patients and normal donors display comparable cytotoxicity. The level of platelet lytic activity is irrelevant to tumor localisation; however, it appears to be dependent on the stage of tumor growth. Incubation of platelets, both from donors and patients, with PAF (concentration range 10 pM to 10 nM) results in a significant rise of the killing activity of platelets. PAF induces greater cytotoxicity enhancement for platelets with lower initial activity, this pattern appearing to be the specific feature of the PAF mediated effect. Hence, platelets can be considered as effector cells relevant to antitumor immunity; PAF-mediated enhancement of platelet cytotoxicity can appear to be useful in the search for new immunotherapeutic drugs.

  1. Platelet function tests: a comparative review

    Directory of Open Access Journals (Sweden)

    Paniccia R

    2015-02-01

    Full Text Available Rita Paniccia,1,2 Raffaella Priora,1,2 Agatina Alessandrello Liotta,2 Rosanna Abbate1,2 1Department of Experimental and Clinical Medicine, Thrombosis Center, University of Florence, Florence, Italy; 2Department of Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy Abstract: In physiological hemostasis a prompt recruitment of platelets on the vessel damage prevents the bleeding by the rapid formation of a platelet plug. Qualitative and/or quantitative platelet defects promote bleeding, whereas the high residual reactivity of platelets in patients on antiplatelet therapies moves forward thromboembolic complications. The biochemical mechanisms of the different phases of platelet activation – adhesion, shape change, release reaction, and aggregation – have been well delineated, whereas their complete translation into laboratory assays has not been so fulfilled. Laboratory tests of platelet function, such as bleeding time, light transmission platelet aggregation, lumiaggregometry, impedance aggregometry on whole blood, and platelet activation investigated by flow cytometry, are traditionally utilized for diagnosing hemostatic disorders and managing patients with platelet and hemostatic defects, but their use is still limited to specialized laboratories. To date, a point-of-care testing (POCT dedicated to platelet function, using pertinent devices much simpler to use, has now become available (ie, PFA-100, VerifyNow System, Multiplate Electrode Aggregometry [MEA]. POCT includes new methodologies which may be used in critical clinical settings and also in general laboratories because they are rapid and easy to use, employing whole blood without the necessity of sample processing. Actually, these different platelet methodologies for the evaluation of inherited and acquired bleeding disorders and/or for monitoring antiplatelet therapies are spreading and the study of platelet function is strengthening. In this review, well

  2. Platelet Function Tests in Bleeding Disorders.

    Science.gov (United States)

    Lassila, Riitta

    2016-04-01

    Functional disorders of platelets can involve any aspect of platelet physiology, with many different effects or outcomes. These include platelet numbers (thrombocytosis or thrombocytopenia); changes in platelet production or destruction, or capture to the liver (Ashwell receptor); altered adhesion to vascular injury sites and/or influence on hemostasis and wound healing; and altered activation or receptor functions, shape change, spreading and release reactions, procoagulant and antifibrinolytic activity. Procoagulant membrane alterations, and generation of thrombin and fibrin, also affect platelet aggregation. The above parameters can all be studied, but standardization and quality control of assay methods have been limited despite several efforts. Only after a comprehensive clinical bleeding assessment, including family history, information on drug use affecting platelets, and exclusion of coagulation factor, and tissue deficits, should platelet function testing be undertaken to confirm an abnormality. Current diagnostic tools include blood cell counts, platelet characteristics according to the cell counter parameters, peripheral blood smear, exclusion of pseudothrombocytopenia, whole blood aggregometry (WBA) or light transmission aggregometry (LTA) in platelet-rich plasma, luminescence, platelet function analysis (PFA-100) for platelet adhesion and deposition to collagen cartridges under blood flow, and finally transmission electron microscopy to exclude rare structural defects leading to functional deficits. The most validated test panels are included in WBA, LTA, and PFA. Because platelets are isolated from their natural environment, many simplifications occur, as circulating blood and interaction with vascular wall are omitted in these assays. The target to reach a highly specific platelet disorder diagnosis in routine clinical management can be exhaustive, unless needed for genetic counseling. The elective overall assessment of platelet function disorder

  3. Platelet receptors and patient responses: The contributions of Professor Stan Heptinstall to platelet research.

    Science.gov (United States)

    Clemetson, Kenneth J

    2015-01-01

    Stan Heptinstall's contributions to platelet research covered organising meetings at the national and European level as well as starting and maintaining the journal "Platelets". The major part of his research addressed problems of inhibition of platelet receptors and the effects of this on patient health. In particular, the effects of P2Y12 inhibitors on patients with acute cardiovascular problems were a major focus. Other studies included the effects of feverfew (Tanacetum parthenium) extracts on platelets, of direct anti-IIb/IIIa receptor (αIIbβ3) inhibitors and of prostanoids on platelet function. Recently, methods for assessing the effectiveness of platelet inhibition were investigated.

  4. Platelet count and platelet indices in women with preeclampsia

    OpenAIRE

    AlSheeha MA; Alaboudi RS; Alghasham MA; Iqbal J; Adam I

    2016-01-01

    Muneera A AlSheeha,1 Rafi S Alaboudi,1 Mohammad A Alghasham,1 Javed Iqbal,2 Ishag Adam1 1Department of Obstetrics and Gynaecology, College of Medicine, Qassim University, Buriadah, 2Department of Obstetrics and Gynecology, Maternity and Children’s Hospital, Qassim, Kingdom of Saudi Arabia Background: Although the exact pathophysiology of preeclampsia is not completely understood, the utility of different platelets indices can be utilized to predict preeclampsia.Obj...

  5. Pathogen-Reduced, Platelet Additive Solution, Extended Stored Platelets (PREPS)

    Science.gov (United States)

    2015-10-01

    associated sepsis remains the principal lethal risk associated with platelet transfusion. Cold storage (4°C) is known to reduce post transfusion...and no residual radiation is detectable . *P-selectin samples will be prepped on end of storage day and batch tested. **Bacterial Culture sample...temperature controlled room until such time as they have no detectable residual radiation. This is generally about 3-4 months. At that point they are

  6. Detection of microbial contamination in platelets

    Science.gov (United States)

    Berg, Tracy L.; Leparc, German; Huffman, Debra E.; Gennaccaro, Angela L.; Garcia-Lopez, Alicia; Klungness, Greta; Stephans, Christie; Garcia-Rubio, Luis H.

    2005-03-01

    In the United States, approximately 100 patients develop fatal sepsis associated with platelet transfusions every year. Current culture methods take 24-48 hours to acquire results, which in turn decrease the shelf life of platelets. Many of the microorganisms that contaminate platelets can replicate easily at room temperature, which is the necessary storage temperature to keep platelets functional. Therefore, there is a need for in-situ quality control assessment of the platelet quality. For this purpose, a real time spectrophotometric technique has been developed. The Spectral Acquisition Processing Detection (SAPD) method, comprised of a UV-vis spectrophotometer and modeling algorithms, is a rapid method that can be performed prior to platelet transfusion to decrease the risk of bacterial infection to patients. The SAPD method has been used to determine changes in cell suspensions, based on size, shape, chemical composition and internal structure. Changes in these cell characteristics can in turn be used to determine microbial contamination, platelet aging and other physiologic changes. Detection limits of this method for platelet suspensions seeded with bacterial contaminants were identified to be less than 100 cfu/ml of sample. Bacterial counts below 1000 cfu/ml are not considered clinically significant. The SAPD method can provide real-time identification of bacterial contamination of platelets affording patients an increased level of safety without causing undue strain on laboratory budgets or personnel while increasing the time frame that platelets can be used by dramatically shortening contaminant detection time.

  7. Influence of Oxidative Stress on Stored Platelets

    Directory of Open Access Journals (Sweden)

    K. Manasa

    2016-01-01

    Full Text Available Platelet storage and its availability for transfusion are limited to 5-6 days. Oxidative stress (OS is one of the causes for reduced efficacy and shelf-life of platelets. The studies on platelet storage have focused on improving the storage conditions by altering platelet storage solutions, temperature, and materials. Nevertheless, the role of OS on platelet survival during storage is still unclear. Hence, this study was conducted to investigate the influence of storage on platelets. Platelets were stored for 12 days at 22°C. OS markers such as aggregation, superoxides, reactive oxygen species, glucose, pH, lipid peroxidation, protein oxidation, and antioxidant enzymes were assessed. OS increased during storage as indicated by increments in aggregation, superoxides, pH, conjugate dienes, and superoxide dismutase and decrements in glucose and catalase. Thus, platelets could endure OS till 6 days during storage, due to the antioxidant defense system. An evident increase in OS was observed from day 8 of storage, which can diminish the platelet efficacy. The present study provides an insight into the gradual changes occurring during platelet storage. This lays the foundation towards new possibilities of employing various antioxidants as additives in storage solutions.

  8. Effects of Physical (Inactivity on Platelet Function

    Directory of Open Access Journals (Sweden)

    Stefan Heber

    2015-01-01

    Full Text Available As platelet activation is closely related to the liberation of growth factors and inflammatory mediators, platelets play a central role in the development of CVD. Virtually all cardiovascular risk factors favor platelet hyperreactivity and, accordingly, also physical (inactivity affects platelet function. Within this paper, we will summarize and discuss the current knowledge on the impact of acute and habitual exercise on platelet function. Although there are apparent discrepancies regarding the reported effects of acute, strenuous exercise on platelet activation, a deeper analysis of the available literature reveals that the applied exercise intensity and the subjects’ cardiorespiratory fitness represent critical determinants for the observed effects. Consideration of these factors leads to the summary that (i acute, strenuous exercise can lead to platelet activation, (ii regular physical activity and/or physical fitness diminish or prevent platelet activation in response to acute exercise, and (iii habitual physical activity and/or physical fitness also favorably modulate platelet function at physical rest. Notably, these effects of exercise on platelet function show obvious similarities to the well-recognized relation between exercise and the risk for cardiovascular events where vigorous exercise transiently increases the risk for myocardial infarction and a physically active lifestyle dramatically reduces cardiovascular mortality.

  9. Small RNAs as potential platelet therapeutics.

    Science.gov (United States)

    Edelstein, Leonard C; Bray, Paul F

    2012-01-01

    MicroRNAs (miRNAs) are 21-23 nucleotide RNAs that regulate more than 60% of mammalian protein coding genes. miRNAs play critical roles in hematopoiesis and megakaryocyte function and development. Platelets, in addition to possessing functional miRNA processing machinery, have miRNA levels that have been correlated with platelet reactivity, and these miRNAs have been shown to target mRNAs that encode proteins that alter platelet function. There are potential uses of platelet miRNA as biomarkers and therapeutic agents. Due to the ability of platelets to release miRNA-containing microparticles at sites of activation, including angiogenic regions, tumors, and atherosclerotic plaques, there is the possibility of engineering platelets to deliver miRNA-based therapies to these sites. Cellpreferential expression of miRNAs could be exploited to restrict transgene expression in hematopoietic stem cell gene therapy to the desired lineage, including megakaryocytes and platelets. Finally, manipulation of gene expression in stored platelets may allow more effective platelet storage. Although much work remains to be done, there is great potential in miRNA-based platelet therapies.

  10. Platelet MicroRNAs: An Overview.

    Science.gov (United States)

    Dahiya, Neetu; Sarachana, Tewarit; Vu, Long; Becker, Kevin G; Wood, William H; Zhang, Yongqing; Atreya, Chintamani D

    2015-10-01

    MicroRNAs (miRNAs) are short ~22-nucleotide noncoding RNA that have been found to influence the expression of many genes and cellular processes by either repressing translation or degrading messenger RNA transcripts. Platelet miRNA expression has been shown to be perturbed during ex vivo storage of platelets and in platelet-associated disorders. Although bioinformatics-based miRNA target predictions have been established, direct experimental validation of the role of miRNAs in platelet biology has been rather slow. Target prediction studies are, nonetheless, valuable in directing the design of appropriate experiments to test specific miRNA:messenger RNA interactions relevant to the underlying mechanisms of platelet function in general and in disease as well as in ex vivo storage-associated "storage lesions," a collective term used to include physiologic, biochemical, and morphologic changes that occur in stored platelets. This brief review will focus on emerging human platelet miRNA studies to emphasize their potential role relevant to transfusion medicine field in terms of regulating platelet signaling pathways, markers of platelet associated disorders, and remote impactors of gene expression (intercellular biomodulators) as well as potential platelet quality markers of storage and pathogen reduction treatments.

  11. Understanding platelet function through signal transduction.

    Science.gov (United States)

    Lazarus, Alan H; Song, Seng; Crow, Andrew R

    2003-01-01

    Platelets are activated by a number of stimuli resulting in the expression and/or activation of surface receptors, secretion of vasoactive substances, adhesion, aggregation, and finally thrombus formation. These events are propagated by a process known as transmembrane signaling, which relays the activating signal from the platelet membrane (eg, von Willebrand Factor binding to glycoprotein Ib) to the inside of the platelet which then serves to activate the platelet via a cascade of biochemical interactions. Inhibition of these transmembrane signaling molecules with a variety of available inhibitors or antagonists can in many cases prevent the platelet from becoming activated. An awareness of the mechanisms involved in platelet transmembrane signaling and the recent availability of new reagents to inhibit signaling may provide us with additional means to prevent platelet activation and perhaps even ameliorate the platelet storage lesion. This review will provide an introduction to the field of platelet transmembrane signaling and give an overview of some of the platelet signaling mechanisms that are relevant to transfusion medicine. Copyright 2003, Elsevier Science (USA). All rights reserved.

  12. Platelet destruction in autoimmune thrombocytopenic purpura: kinetics and clearance of indium-111-labeled autologous platelets

    Energy Technology Data Exchange (ETDEWEB)

    Stratton, J.R.; Ballem, P.J.; Gernsheimer, T.; Cerqueira, M.; Slichter, S.J.

    1989-05-01

    Using autologous /sup 111/In-labeled platelets, platelet kinetics and the sites of platelet destruction were assessed in 16 normal subjects (13 with and three without spleens), in 17 studies of patients with primary autoimmune thrombocytopenic purpura (AITP), in six studies of patients with secondary AITP, in ten studies of patients with AITP following splenectomy, and in five thrombocytopenic patients with myelodysplastic syndromes. In normal subjects, the spleen accounted for 24 +/- 4% of platelet destruction and the liver for 15 +/- 2%. Untreated patients with primary AITP had increased splenic destruction (40 +/- 14%, p less than 0.001) but not hepatic destruction (13 +/- 5%). Compared with untreated patients, prednisone treated patients did not have significantly different spleen and liver platelet sequestration. Patients with secondary AITP had similar platelet counts, platelet survivals, and increases in splenic destruction of platelets as did patients with primary AITP. In contrast, patients with myelodysplastic syndromes had a normal pattern of platelet destruction. In AITP patients following splenectomy, the five nonresponders all had a marked increase (greater than 45%) in liver destruction compared to five responders (all less than 40%). Among all patients with primary or secondary AITP, there was an inverse relationship between the percent of platelets destroyed in the liver plus spleen and both the platelet count (r = 0.75, p less than 0.001) and the platelet survival (r = 0.86, p less than 0.001). In a stepwise multiple linear regression analysis, total liver plus spleen platelet destruction, the platelet survival and the platelet turnover were all significant independent predictors of the platelet count. Thus platelet destruction is shifted to the spleen in primary and secondary AITP. Failure of splenectomy is associated with a marked elevation in liver destruction.

  13. Differential regulation of protein-tyrosine phosphatases by integrin alpha IIb beta 3 through cytoskeletal reorganization and tyrosine phosphorylation in human platelets.

    Science.gov (United States)

    Ezumi, Y; Takayama, H; Okuma, M

    1995-05-19

    The major platelet integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) has been implicated in the regulation of tyrosine phosphorylation and dephosphorylation in activated platelets. To investigate the mechanisms of the alpha IIb beta 3-dependent tyrosine dephosphorylation, normal platelets or thrombasthenic platelets lacking alpha IIb beta 3 were stimulated with thrombin and fractionated into Triton X-100-soluble or -insoluble subcellular matrices. We then examined the kinetics of the tyrosine-phosphorylated proteins and distribution of protein-tyrosine phosphatases in these fractions and whole cell lysates. First, alpha IIb beta 3-dependent tyrosine dephosphorylation was recovered mainly in the cytoskeleton with similar kinetics to the whole cell lysate. Second, protein-tyrosine phosphatase (PTP) 1B and its cleaved 42-kDa form were associated with the cytoskeleton in an aggregation-dependent manner, whereas association of PTP1C with the cytoskeleton was regulated differentially both by thrombin stimulation and by alpha IIb beta 3-mediated aggregation. Several calpain inhibitors did not affect either tyrosine phosphorylation and dephosphorylation or relocation of PTP1B, but they did inhibit cleavage of PTP1B. Cytochalasin D blocked relocation of both PTP1B and PTP1C but not PTP1B cleavage. SH-PTP2 was distributed in the other fractions than the cytoskeleton and showed no relocation on thrombin stimulation. Finally, the cytoskeleton-associated PTP1C became tyrosine-phosphorylated in an alpha IIb beta 3-mediated aggregation-dependent manner. Thus, integrin alpha IIb beta 3 was involved differentially in the regulation of PTP1B and PTP1C.

  14. Platelet activation patterns in platelet size sub-populations: differential responses to aspirin in vitro.

    Science.gov (United States)

    Mangalpally, Kiran Kumar R; Siqueiros-Garcia, Alan; Vaduganathan, Muthiah; Dong, Jing-Fei; Kleiman, Neal S; Guthikonda, Sasidhar

    2010-10-01

    Circulating platelets are heterogeneous in size and structure. Whether this translates into differences in platelet function and efficacy of antiplatelet therapy is unclear. Hence, we decided to investigate the activation patterns among different platelet populations differentiated by size, and to compare the inhibitory effects of aspirin in these populations. Circulating platelets from 9 healthy volunteers were separated by size and stratified into the largest and smallest quintiles. Platelets were stimulated with 75 μM arachidonic acid (AA), 10 μM ADP or 25 μM TRAP. Alpha-granule protein secretion and expression (P-selectin, VWF, fibrinogen), surface-protein activation (activated integrin αIIbβ3) were assessed. Platelet thromboxane B(2) (TxB(2)) synthesis following AA stimulation was measured in vitro before and after incubation with 265 μM aspirin. Reticulated (juvenile) platelets were assessed using thiazole orange staining. A greater number of large platelets in the largest quintile were reticulated compared with the smallest quintile (6.1 ± 2.8% vs. 1.2 ± 1.5% respectively, p aspirin (1029 ± 190 pg/mL vs. 851 ± 159 pg/mL, respectively, p = 0.03). After stimulation with each agonist, a greater proportion of large platelets bound fibrinogen, VWF, P-selectin and activated integrin αIIbβ3 than small platelets both in the presence and in the absence of in vitro aspirin. In an in vitro setting, large platelets appear to be more active than small platelets and continue to be more active even after in vitro aspirin. Platelets exhibit heterogeneity in size and structure. Whether this translates into platelet function and efficacy of antiplatelet therapy is unclear. We evaluated platelet functional properties and the effects of aspirin on separated platelet subpopulations in an in vitro setting. Platelets were sorted into the largest and smallest size quintiles using flow cytometry forward scatter. Alpha-granule protein release, dense granule content

  15. Losartan inhibits the adhesion of rat platelets to fibrillar collagen--a potential role of nitric oxide and prostanoids.

    Science.gov (United States)

    Matys, T; Chabielska, E; Pawlak, R; Kucharewicz, I; Buczko, W

    2000-12-01

    The aim of the study was to evaluate the effect of losartan on rat platelet adhesion to fibrillar collagen. Washed platelets were counted before and after 15 minutes incubation with collagen (50 microg/ml) and the percentage of adhering platelets was calculated as the index of their adhesion. When the platelets were incubated with collagen 40.8 +/- 0.3% of the platelets adhered. Losartan produced a dose dependent decrease in a number of adhering platelets both when the drug was administered to the animals ex vivo at doses of 3, 10 and 30 mg/kg (p < 0.01-0.001) or was added to the preparation of washed platelets in vitro in concentrations of 10(-8)-10(-5) M (p < 0.01-0.001). In the next step of the study we assessed the influence of L-NAME (10 mg/kg ex vivo, 30 microM in vitro) and indomethacin (2.5 mg/kg ex vivo, 30 microM in vitro) on the antiadhesive effect of losartan (10 mg/kg ex vivo, 10(-6) M in vitro). Blockade of nitric oxide synthase with L-NAME partially reversed the antiadhesive effect of losartan both ex vivo and in vitro. Indomethacin diminished the inhibitory effect of losartan on platelet adhesion when administered ex vivo, but it failed to modify this parameter when added to the suspension of platelets in vitro. In conclusion, losartan reduces platelet adhesion to fibrillar collagen in a dose-dependent manner. The observed action of losartan seems to be mediated mainly by endothelium- and platelet-derived nitric oxide.

  16. Tendon Graft and Platelet Concentrate for Chronic Achilles Tendon Rupture. A Case Report

    Directory of Open Access Journals (Sweden)

    Yovanny Ferrer Lozano

    2015-12-01

    Full Text Available Achilles tendon is third most common tendon torn, following the rotator cuff and the quadriceps extensor mechanism. Ruptures can be partial or complete and their etiology is multifactorial. We present the case of a 63-year-old woman who attended the Orthopedics service because of discomfort when walking that had been present for several months and persistent pain in his right heel related to an acetonide triamcinolone injection into the back of the calcaneus. The gap in the tendon (hatchet strike defect and a Thompson test confirmed the Achilles tendon rupture. The tendon was repaired using a peroneus brevis tendon graft and the repair was reinforced with gastrocnemius aponeurosis. The skin flap necrosis led to conservative debridement, graft exposure, and daily application of a platelet lysate, which was subsequently alternated due to the successful formation of the scar tissue. Epithelialization was reached in the fourth postoperative week. Twelve weeks after surgery, the patient began to resume her normal life. The use of platelet concentrates as adjuvant therapy is rare in these patients. For this reason, we decided to publish this case.

  17. Estimation of platelet count in unstained peripheral blood smears in comparison with stained smears and evaluation of its efficacy.

    Science.gov (United States)

    Umashankar, T; Thomas, B M; Sahana, P

    2014-12-01

    Assessment of platelet count is an important diagnostic parameter in haematology. Automated blood cell counters have largely replaced the manual method. However, all abnormal platelet counts are verified in Leishman's stained peripheral blood smear. Platelets also can be identified in the unstained blood smears. The objective of this study is to compare the unstained peripheral blood smears with the stained smears and determination of the effectiveness of unstained smears in the estimation of platelet count. 250 Venous blood samples sent for blood cell counts were analyzed. Platelets were counted in the unstained smear under 100× objective in 10 fields without placing immersion oil and the average number of platelets was calculated. Same smear was stained by Leishman's stain and platelets were counted under 100× objective after placing a drop of immersion oil. Collected data were analyzed for intra-class correlation coefficient (ICC). ICC showed excellent agreement (ICC > 0.85). The unstained smears were found to be as effective as stained smears for platelet count in most of the cases. However, in case of doubt a stained preparation has to be made to confirm the count. The turnaround time was 3-5 minutes compared to 15-20 minutes by stained smear technique, thus this technique may be used as an initial screening method whenever there is large sample load.

  18. TRA-418, a novel compound having both thromboxane A(2) receptor antagonistic and prostaglandin I(2) receptor agonistic activities: its antiplatelet effects in human and animal platelets.

    Science.gov (United States)

    Yamada, N; Miyamoto, M; Isogaya, M; Suzuki, M; Ikezawa, S; Ohno, M; Otake, A; Umemura, K

    2003-08-01

    TRA-418 is a novel compound that has been found in our screening for compounds having both thromboxane A2 (TP) receptor antagonistic and prostaglandin I2 (IP) receptor agonistic activities. In the binding assays, TRA-418 showed a 10-fold higher affinity to TP-receptors than IP-receptors. TRA-418 inhibited platelet aggregation induced by the TP-receptor agonist, U-46619 and by arachidonic acid at concentrations lower than those required for inhibition of ADP-induced aggregations. Furthermore, TRA-418 inhibited not only platelet aggregation induced by ADP alone, but also that induced by ADP in the presence of the TP-receptor antagonist, SQ-29548. When the IC50 values of TRA-418 for platelet aggregation were estimated in platelet preparations from monkeys, dogs, cats, and rats using ADP and arachidonic acid as the platelet stimulating agents, it was found that the values estimated in monkey platelets were quite similar to those estimated in human platelets. In ex vivo platelet aggregation in monkeys, TRA-418 exhibited significant inhibitory effects on arachidonic acid-induced aggregation in platelet preparations from monkeys treated at 3 micro g kg min-1 or higher doses, where neither a significant decrease in blood pressure nor a significant increase in heart rate was observed. These results are consistent with the fact that TRA-418 has a relatively potent TP-receptor antagonistic activity together with a relatively weak IP-receptor agonistic activity.

  19. The inhibition of oxygen radical release from human neutrophils by resting platelets is reversed by administration of acetylsalicylic acid or clopidogrel.

    Science.gov (United States)

    Reinisch, C M; Dunzendorfer, S; Pechlaner, C; Ricevuti, G; Wiedermann, C J

    2001-05-01

    Resting platelets inhibit oxygen radical release from neutrophils. Antiplatelet therapy may support this function by preventing platelet activation. Whether antiplatelet agents affect the antioxidative action of resting platelets in the absence of platelet activation is unknown. The effect of acetylsalicylic acid or clopidogrel administration on the antioxidative action of resting platelets was therefore studied in ten healthy volunteers. Preparations of resting platelets were obtained from 5 subjects each - before, during and after an eight-day course of daily treatment with 100 mg of acetylsalicylic acid or 75 mg of the thienopyridine clopidogrel. Human peripheral blood neutrophils were pretreated with the platelets at a ratio of (1/5)0 for 45 min; then formyl-Met-Leu-Phe-triggered oxygen radical release was measured fluorometrically. The inhibitory effect of platelets on oxygen radical release from neutrophils which was seen before treatment was abolished by antiplatelet therapy with either of the drugs, and inhibition was restored gradually after discontinuing acetlsalicylic acid/ clopidogrel intake. Results suggest that the protective role of resting platelets in controlling oxygen radical release from neutrophils in the absence of platelet activation may be impaired by antiplatelet therapy.

  20. Mean platelet volume in acute rheumatic fever.

    Science.gov (United States)

    Sert, Ahmet; Aypar, Ebru; Odabas, Dursun

    2013-01-01

    Acute rheumatic fever (ARF) is still an endemic disease, especially among school-aged children in developing countries. Mean platelet volume (MPV), which is commonly used as a measure of platelet size, indicates the rate of platelet production and platelet activation. We aimed to investigate MPV in children with ARF. The study population consisted of 40 children with ARF (32 patients with carditis and 8 patients without carditis) and 40 healthy control subjects. White blood cell (WBC) and platelet counts were significantly higher and MPV values were significantly lower in patients with ARF during the acute stage when compared to controls. Erythrocyte sedimentation rate (ESR) and C-reactive protein values significantly decreased in patients with ARF after the treatment when compared to baseline, whereas MPV values increased. MPV values were negatively correlated with ESR and WBC, and platelet counts. In conclusion, during the acute stage of ARF, MPV values were lower when compared to controls.

  1. Therapeutic platelet reduction: Use in postsplenectomy thrombocytosis

    Directory of Open Access Journals (Sweden)

    Gita Negi

    2015-01-01

    Full Text Available Therapeutic platelet reduction is an effective modality for the reduction of platelet count in patients with treatment of extreme thrombocytosis resulting from a variety of primary and secondary causes of thrombocytosis, which may be associated with thrombotic or hemorrhagic complications of varying degrees. These cases when symptomatic fall into the ASFA Category II indication for therapeutic platelet apheresis procedure. Here, we report a case of postsplenectomy secondary thrombocytosis presenting with extremely high platelet counts and subsequent thrombosis in the shunt and successful treatment after therapeutic platelet reduction. The case is being presented to bring forth the fact that therapeutic platelet reduction is an easy procedure that gives quick and good results and also to bring to the attention of transfusion specialists an associated but as yet unreported procedural finding.

  2. Laboratory testing for platelet function disorders.

    Science.gov (United States)

    Israels, S J

    2015-05-01

    Platelet function testing is both complex and labor intensive. A stepwise approach to the evaluation of patients with suspected platelet disorders will optimize the use of laboratory resources, beginning with an appropriate clinical evaluation to determine whether the bleeding is consistent with a defect of primary hemostasis. Bleeding assessment tools, evaluation of platelet counts, and review of peripheral blood cell morphology can aid the initial assessment. For patients requiring further laboratory testing, platelet aggregometry, secretion assays, and von Willebrand factor assays are the most useful next steps and will direct further specialized testing including flow cytometry, electron microscopy, and molecular diagnostics. Guidelines and recommendations for standardizing platelet function testing, with a particular focus on light transmission aggregometry, are available and can provide a template for clinical laboratories in establishing procedures that will optimize diagnosis and assure quality results. This review outlines an approach to platelet function testing and reviews testing methods available to clinical laboratories.

  3. Differential effects of platelets and platelet inhibition by ticagrelor on TLR2- and TLR4-mediated inflammatory responses

    NARCIS (Netherlands)

    Tunjungputri, R.N.; Ven, A.J.A.M. van der; Riksen, N.P.; Rongen, G.A.P.J.M.; Tacke, S.; Berg, T.N.A. van den; Fijnheer, R.; Gomes, M.E.; Dinarello, C.A.; Veerdonk, F.L. van de; Gasem, M.H.; Netea, M.G.; Joosten, L.A.B.; Groot, P.G. de; Mast, Q. de

    2015-01-01

    Platelets and platelet-monocyte interaction play an important role in inflammation. Both pro- and anti-inflammatory effects of platelet inhibition have been reported in animal models. This study aimed to investigate the effect of platelets and platelet inhibition by the new P2Y12 receptor antagonist

  4. Rhizodeposition flux of competitive versus conservative graminoid: contribution of exudates and root lysates as affected by N loading

    Science.gov (United States)

    Kastovska, Eva; Edwards, Keith; Santruckova, Hana

    2017-04-01

    Carbon allocation pattern represents the plant strategy for growth and nutrient capture. Plants exhibit high plasticity in their allocation pattern and belowground C partitioning in response to changes in the availability of nutrients limiting their production, namely nitrogen (N). Any shift in the belowground C fluxes and partitioning between root production, exudation and other rhizodeposits could affect the soil microbial activity and soil organic matter turnover. We studied the influence of N availability on plant allocation patterns with emphasis on belowground C fluxes of two wetland graminoids, the competitive Glyceria maxima and the conservative Carex acuta. Plants were grown in pots under two levels of N availability. We combined pulse-labeling of plants with 13CO2 to track recent assimilates with estimation of the root death rate calculated from the difference between gross and net root growth rates for assessing the rhizodeposition flux to soil, and the contribution of root exudates and lysates from root turnover. We found that higher N supply enhanced root biomass and, subsequently, the total rhizodeposition. Both species shifted partitioning of belowground C towards higher mass-specific root production and turnover, with lower investments into root exudation. Therefore, the rhizodeposition flux was enriched in root-derived lysates over soluble exudates. Root exudates accounted for 50-70% of the rhizodeposition flux in conditions of low N availability, while it was only 20-40% under high N availability. The N fertilization induced changes in belowground C fluxes were species-specific, with more pronounced changes in the conservative Carex than the competitive Glyceria. In summary, soil N loading enhanced total C rhizodeposition and, simultaneously, the proportion of predominantly more complex root lysates over soluble root exudates, with potential implications for soil organic matter dynamics. Our results further stress the importance of species

  5. Dendritic cells loaded with pancreatic Cancer Stem Cells (CSCs lysates induce antitumor immune killing effect in vitro.

    Directory of Open Access Journals (Sweden)

    Tao Yin

    Full Text Available According to the cancer stem cells (CSCs theory, malignant tumors may be heterogeneous in which a small population of CSCs drive the progression of cancer. Because of their intrinsic abilities, CSCs may survive a variety of treatments and then lead to therapeutic resistance and cancer recurrence. Pancreatic CSCs have been reported to be responsible for the malignant behaviors of pancreatic cancer, including suppression of immune protection. Thus, development of immune strategies to eradicate pancreatic CSCs may be of great value for the treatment of pancreatic cancer. In this study, we enriched pancreatic CSCs by culturing Panc-1 cells under sphere-forming conditions. Panc-1 CSCs expressed low levels of HLA-ABC and CD86, as measured by flow cytometry analysis. We further found that the Panc-1 CSCs modulate immunity by inhibiting lymphocyte proliferation which is promoted by phytohemagglutinin (PHA and anti-CD3 monoclonal antibodies. The monocyte derived dendritic cells (DCs were charged with total lysates generated from Panc-1 CSCs obtained from tumor sphere culturing. After co-culturing with lymphocytes at different ratios, the Panc-1 CSCs lysates modified DC effectively promoted lymphocyte proliferation. The activating efficiency reached 72.4% and 74.7% at the ratios of 1∶10 and 1∶20 with lymphocytes. The activated lymphocytes secreted high levels of INF-γ and IL-2, which are strong antitumor cytokines. Moreover, Panc-1 CSCs lysates modified DC induced significant cytotoxic effects of lymphocytes on Panc-1 CSCs and parental Panc-1 cells, respectively, as shown by lactate dehydrogenase (LDH assay. Our study demonstrates that the development of CSCs-based vaccine is a promising strategy for treating pancreatic cancer.

  6. The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays

    Science.gov (United States)

    Barnard, Sunelle A.; Loots, Du Toit; Rijken, Dingeman C.

    2017-01-01

    Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of

  7. LDL oxidation by platelets propagates platelet activation via an oxidative stress-mediated mechanism.

    Science.gov (United States)

    Carnevale, Roberto; Bartimoccia, Simona; Nocella, Cristina; Di Santo, Serena; Loffredo, Lorenzo; Illuminati, Giulio; Lombardi, Elisabetta; Boz, Valentina; Del Ben, Maria; De Marco, Luigi; Pignatelli, Pasquale; Violi, Francesco

    2014-11-01

    Platelets generate oxidized LDL (ox-LDL) via NOX2-derived oxidative stress. We investigated if once generated by activated platelets ox-LDL can propagate platelet activation. Experiments were performed in platelets from healthy subjects (HS), hyper-cholesterolemic patients and patients with NOX2 hereditary deficiency. Agonist-stimulated platelets from HS added with LDL were associated with a dose-dependent increase of reactive oxidant species and ox-LDL. Agonist-stimulated platelets from HS added with a fixed dose of LDL (57.14 μmol/L) or added with homogenized human atherosclerotic plaque showed enhanced ox-LDL formation (approximately +50% and +30% respectively), which was lowered by a NOX2 inhibitor (approximately -35% and -25% respectively). Compared to HS, ox-LDL production was more pronounced in agonist-stimulated platelet rich plasma (PRP) from hyper-cholesterolemic patients but was almost absent in PRP from NOX2-deficient patients. Platelet aggregation and 8-iso-PGF2α-ΙΙΙ formation increased in LDL-treated washed platelets (+42% and +53% respectively) and PRP (+31% and +53% respectively). Also, LDL enhanced platelet-dependent thrombosis at arterial shear rate (+33%) but did not affect platelet activation in NOX2-deficient patients. Platelet activation by LDL was significantly inhibited by CD36 or LOX1 blocking peptides, two ox-LDL receptor antagonists, or by a NOX2 inhibitor. LDL-added platelets showed increased p38MAPK (+59%) and PKC (+51%) phosphorylation, p47(phox) translocation to platelet membrane (+34%) and NOX2 activation (+30%), which were inhibited by ox-LDL receptor antagonists. Platelets oxidize LDL, which in turn amplify platelet activation via specific ox-LDL receptors; both effects are mediated by NOX2 activation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  8. Bone marrow-derived dendritic cells pulsed with tumor lysates induce anti-tumor immunity against gastric cancer ex vivo

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    AIM: To investigate whether bone marrow-derived denritic cells pulsed with tumor lysates induce immunity against gastric cancer ex vivo. METHODS: c-kit+ hematopoietic progenitor cells were magnetically isolated with a MiniMACS separator from BALB/c mice bone marrow cells. These cells were cultured with cytokines GM-CSF, IL-4, and TNFα to induce their maturation. They were analysed by morphological observation, phenotype analysis, and mixed lymphocyte reaction (MLR). Bone marrow-derived DCs (BM-DCs) were pulsed with tumor cell lysate obtained by rapid freezing and thawing at a 1:3 DC:tumor cell ratio. Finally, cytotoxic T lymphocyte (CTL) activity and interferon gamma (IFNy) secretion was evaluated ex vivo.RESULTS: c-kit+ hematopoietic progenitor cells from mice bone marrow cells cultured with cytokines for 8 d showed the character of typical mature DCs. Norphologically, observed by light microscope, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed.high levels of Ia, DEC-205, CD11b, CD80 and CD86 antigen, moderate levels of CD40, and negative for F4/80. Functionally, these ceils gained the capacity to stimulate allogeneic T cells in MLR assay. However, immature DCs cultured with cytokines for 5 d did not have typical DCs phenotypic markers and could not stimulate allogeneic T cells. Ex vivo primed T cells with SGC-7901 tumor cell lysate-pulsed (TP) DCs were able to induce effective CTL activity against SGC-7901 tumor cells (E:T = 100:1, 69.55% ± 6.05% specific lysis), but not B16 tumor cells, and produced higher levels of IFNγ, when stimulated with SGC-7901 tumor cells but not when stimulated with B16 tumor cells (1575.31 ± 60.25 pg/mL in SGC-7901 group vs 164.11 ± 18.52 pg/mL in B16 group, P < 0.01).CONCLUSION: BM-derived DCs pulsed with tumor lysates can induce anti-tumor immunity specific to gastric cancer ex vivo.

  9. Identification of ligand-target pairs from combined libraries of small molecules and unpurified protein targets in cell lysates.

    Science.gov (United States)

    McGregor, Lynn M; Jain, Tara; Liu, David R

    2014-02-26

    We describe the development and validation of interaction determination using unpurified proteins (IDUP), a method that selectively amplifies DNA sequences identifying ligand+target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. By operating in cell lysates, IDUP preserves native post-translational modifications and interactions with endogenous binding partners, thereby enabling the study of difficult-to-purify targets and increasing the potential biological relevance of detected interactions compared with methods that require purified proteins. In IDUP, target proteins are associated with DNA oligonucleotide tags either non-covalently using a DNA-linked antibody or covalently using a SNAP-tag. Ligand-target binding promotes hybridization of a self-priming hairpin that is extended by a DNA polymerase to create a DNA strand that contains sequences identifying both the target and its ligand. These sequences encoding ligand+target pairs are selectively amplified by PCR and revealed by high-throughput DNA sequencing. IDUP can respond to the effect of affinity-modulating adaptor proteins in cell lysates that would be absent in ligand screening or selection methods using a purified protein target. This capability was exemplified by the 100-fold amplification of DNA sequences encoding FRB+rapamycin or FKBP+rapamycin in samples overexpressing both FRB and FKBP (FRB·rapamycin+FKBP, Kd ≈ 100 fM; FKBP·rapamycin+FRB, Kd = 12 nM). In contrast, these sequences were amplified 10-fold less efficiently in samples overexpressing either FRB or FKBP alone (rapamycin+FKBP, Kd ≈ 0.2 nM; rapamcyin+FRB, Kd = 26 μM). Finally, IDUP was used to process a model library of DNA-linked small molecules and a model library of cell lysates expressing SNAP-target fusions combined in a single sample. In this library×library experiment, IDUP resulted in enrichment of sequences corresponding to five known ligand+target pairs ranging in binding

  10. Platelet aggregation and quality control of platelet concentrates produced in the Amazon Blood Bank

    Directory of Open Access Journals (Sweden)

    Maria José Dantas Coêlho

    2011-01-01

    Full Text Available BACKGROUND: The study of platelet aggregation is essential to assess in vitro platelet function by different platelet activation pathways. OBJECTIVE: To assess aggregation and biochemical parameters of random platelet concentrates produced at the Fundação HEMOAM using the quality control tests defined by law. METHODS: Whole blood samples from 80 donors and the respective platelet concentrate units were tested. Platelet concentrates were tested (platelet count, aggregation and pH on days 1, 3 and 5 of storage. Additionally a leukocyte count was done only on day 1 and microbiological tests on day 5 of storage. Collagen and adenosine diphosphate were used as inducing agonists for platelet aggregation testing. RESULTS: Donor whole blood had normal aggregation (aggregation with adenosine diphosphate = 67% and with collagen = 78%. The median aggregation in platelet concentrates with adenosine diphosphate was low throughout storage (18% on day 1, 7% on day 3 and 6% on day 5 and the median aggregation with collagen was normal only on day 1 and low thereafter (54.4% on day 1, 20.5% on day 3 and 9% on day 5. CONCLUSION: Although the results were within the norms required by law, platelet concentrates had low aggregation rates. We suggest the inclusion of a functional assessment test for the quality control of platelet concentrates for a more effective response to platelet replacement therapy.

  11. The basic science of platelet-rich plasma (PRP): what clinicians need to know.

    Science.gov (United States)

    Arnoczky, Steven P; Sheibani-Rad, Shahin; Shebani-Rad, Shahin

    2013-12-01

    Platelet-rich plasma (PRP) has been advocated for the biological augmentation of tissue healing and regeneration through the local introduction of increased levels (above baseline) of platelets and their associated bioactive molecules. In theory, the increased levels of autologous growth factors and secretory proteins provided by the concentrated platelets may enhance the wound healing process, especially in degenerative tissues or biologically compromised individuals. Although PRP has been increasingly utilized in the treatment of a variety of sports-related injuries, improvements in healing and clinical outcomes have not been universally reported. One reason for this may be the fact that all PRP preparations are not the same. Variations in the volume of whole blood taken, the platelet recovery efficacy, the final volume of plasma in which the platelets are suspended, and the presence or absence of white blood cells, and the addition of exogenous thrombin to activate the platelets or calcium chloride to induce fibrin formation, can all affect the character and potential efficacy of the final PRP product. This article will review the basic principles involved in creating PRP and examine the potential basic scientific significance of the individual blood components contained in the various forms of PRP currently used in sports medicine.

  12. A STUDY ON EFFICACY OF AUTOLOGOUS PLATELET RICH PLASMA IN MYRINGOPLASTY

    Directory of Open Access Journals (Sweden)

    Sankaranarayanan Gopalakrishnan

    2013-06-01

    Full Text Available Objective: To study the efficacy and advantage in using autologus platelet rich plasma during myringoplasty in closure of tympanic membrane perforation preventing the graft displacement, promoting quicker healing & improving overall outcome.Methods : Platelet rich plasma(PRP is an autologus platelet rich concentrate prepared from patients own blood with growth factors up to 8 times that of normal serum and its efficacy when used during myringoplasty is studied.50 patients with chronic otitis media inactive mucosal disease were randomly chosen and 25 of them was the study group and other 25 were the control group. Both group patients underwent myringoplasty and PRP was used in the study group and the results were evaluated.Results: In our study among 25 cases that underwent myringoplasty with use of platelet rich fibrin, 24 had complete tympanic membrane closure and only one failure has been noticed. In controls 5 out of 25 cases had failure. The graft take up rate in our study is comparable with the reference studies. Use of PRP accelerates graft uptake.Conclusion: Platelet rich plasma is a cheap and cost effective platelet concentrate with enriched growth factors. It accelerates the tympanic membrane closure following myringoplasty.

  13. Freeze-dried platelet-rich plasma shows beneficial healing properties in chronic wounds.

    Science.gov (United States)

    Pietramaggiori, Giorgio; Kaipainen, Arja; Czeczuga, Joshua M; Wagner, Christopher T; Orgill, Dennis P

    2006-01-01

    Fresh platelet concentrates are used in many centers to treat recalcitrant wounds. To extend the therapeutic shelf-life of platelets, we analyzed the wound-healing effects of fresh-frozen and freeze-dried (FD) platelet-rich plasma (PRP) using a diabetic mouse model. Db/db mice with 1.0 cm2 dorsal excisional wounds (n = 15/group) were treated with a single application of FD PRP (1.2 x 10(6) platelets/microL) with or without a stabilization solution, and compared with wounds treated with fresh-frozen, sonicated PRP, and untreated wounds. Granulation tissue area, thickness, and wound size were analyzed 9 days posttreatment. Immunostained sections were quantified for vascularity and proliferation using antiplatelet endothelial cell adhesion molecule I and antiproliferating cell nuclear antigen antibodies. The results showed that all PRP preparations increased granulation tissue formation as assessed by surface coverage, thickness, and angiogenic response, when compared with untreated wounds. In addition, wounds treated with FD PRP, and biochemically stabilized FD PRP, exhibited higher proliferative levels. The possibility to deliver growth factors using platelets, and the potential to extend the shelf-life of platelet concentrates makes freeze-drying methods particularly suitable for enhanced wound care.

  14. Reduced platelet-mediated and enhanced leukocyte-mediated fibrinolysis in experimentally induced diabetes in rats

    Energy Technology Data Exchange (ETDEWEB)

    Winocour, P.D.; Colwell, J.A.

    1985-05-01

    Studies of fibrinolytic activity in diabetes mellitus have produced conflicting results. This may be a result of methodologic insensitivity or of variable contributions of the different blood components to whole blood fibrinolysis. To explore these two possibilities, the authors used a sensitive solid-phase radiometric assay to examine the fibrinolytic activity of whole blood, platelet-rich plasma, leukocytes, and platelet- and leukocyte-poor plasma prepared from control rats and rats with streptozocin-induced diabetes at various times after induction of diabetes. Fibrinolytic activity of whole blood from diabetic rats after 7 days was significantly reduced, and remained reduced after longer durations of diabetes up to 28 days. Platelet-rich plasma from diabetic rats had decreased fibrinolytic activity, which followed the same time course of changes as in whole blood. The platelet contribution to whole blood fibrinolysis was further reduced in vivo after 14 days of diabetes by a reduced whole blood platelet count. In contrast, fibrinolytic activity of leukocytes from diabetic rats became enhanced after 7 days of diabetes. After 49 days of diabetes, the whole blood leukocyte count was reduced, and in vivo would offset the enhanced activity. Plasma fibrinolytic activity was small compared with that of whole blood and was unaltered in diabetic rats. The authors conclude that altered platelet function contributes to decreased fibrinolytic activity of whole blood in diabetic rats, and that this may be partially offset by enhanced leukocyte-mediated fibrinolysis.

  15. Ultrastructural studies of the gray platelet syndrome.

    Science.gov (United States)

    White, J G

    1979-05-01

    The gray platelet syndrome (GPS) is a rare inherited disorder in which peripheral blood platelets are relatively large, vacuolated, and almost devoid of cytoplasmic granulation. In the present study we have evaluated the ultrastructure and cytochemistry of platelets from 2 patients with the GPS to determine precisely which organelles are missing from their cells. The findings indicate that gray platelets contain normal numbers of mitochondria, dense bodies, peroxisomes, and lysosomes but specifically lack alpha-granules. Preliminary studies of megakaryocytes from 1 of the 2 patients suggest that the defect in granule formation may lie at the level of the Golgi zone.

  16. Identification of platelet refractoriness in oncohematologic patients

    Directory of Open Access Journals (Sweden)

    Aline Aparecida Ferreira

    2011-01-01

    Full Text Available OBJECTIVES: To identify the occurrence and the causes of platelet refractoriness in oncohematologic patients. INTRODUCTION: Platelet refractoriness (unsatisfactory post-transfusion platelet increment is a severe problem that impairs the treatment of oncohematologic patients and is not routinely investigated in most Brazilian services. METHODS: Forty-four episodes of platelet concentrate transfusion were evaluated in 16 patients according to the following parameters: corrected count increment, clinical conditions and detection of anti-platelet antibodies by the platelet immunofluorescence test (PIFT and panel reactive antibodies against human leukocyte antigen class I (PRA-HLA. RESULTS: Of the 16 patients evaluated (median age: 53 years, nine (56% were women, seven of them with a history of pregnancy. An unsatisfactory increment was observed in 43% of the transfusion events, being more frequent in transfusions of random platelet concentrates (54%. Platelet refractoriness was confirmed in three patients (19%, who presented immunologic and non-immunologic causes. Alloantibodies were identified in eight patients (50% by the PIFT and in three (19% by the PRA-HLA. Among alloimmunized patients, nine (64% had a history of transfusion, and three as a result of pregnancy (43%. Of the former, two were refractory (29%. No significant differences were observed, probably as a result of the small sample size. CONCLUSION: The high rate of unsatisfactory platelet increment, refractoriness and alloimmunization observed support the need to set up protocols for the investigation of this complication in all chronically transfused patients, a fundamental requirement for the guarantee of adequate management.

  17. Does bipolar pacemaker current activate blood platelets?

    DEFF Research Database (Denmark)

    Gjesdal, Grunde; Hansen, Annebirthe Bo; Brandes, Axel

    2009-01-01

    OBJECTIVE: The aim of this study was to investigate whether bipolar pacemaker current lead can activate blood platelets. The null hypothesis was that 1 minute of electrical stimulation of platelets would not influence their subsequent reactivity to adenosine diphosphate (ADP). BACKGROUND: Both...... platelets and muscle cells contain actin and myosin filaments, and both cells are activated following calcium influx. Muscle cells open their calcium channels and contract when exposed to an electric current. Current through a bipolar pacemaker lead will expose a small volume of blood, including platelets...

  18. Platelet cytoskeleton and its hemostatic role.

    Science.gov (United States)

    Cerecedo, Doris

    2013-12-01

    Upon vascular injury, platelets adhere to the exposed extracellular matrix, which triggers the platelet activation and aggregation to form a hemostatic plug to seal the wound. All of these events involve dramatic changes in shape because of the cytoskeleton reorganization. The versatility of the cytoskeleton's main elements depends on the biochemical nature of the elements, as well as on the associated proteins that confer multiple functions within the cell. The list of these associated proteins grows actively, increasing our knowledge concerning the complexity of platelet cytoskeleton machinery. The present review evidences the recently described platelet proteins that promote characteristic modifications in their cytoskeleton organization, with special focus on the dystrophin-glycoprotein complex.

  19. Platelet function alterations in dengue are associated with plasma leakage

    NARCIS (Netherlands)

    Michels, M.; Alisjahbana, B.; Groot, P.G. de; Indrati, A.R.; Fijnheer, R.; Puspita, M.; Dewi, I.M.; Wijer, L. van de; Boer, E.M. de; Roest, M.; Ven, A.J. van der; Mast, Q. de

    2014-01-01

    Severe dengue is characterised by thrombocytopenia, plasma leakage and bleeding. Platelets are important for preservation of endothelial integrity. We hypothesised that platelet activation with secondary platelet dysfunction contribute to plasma leakage. In adult Indonesian patients with acute dengu

  20. Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma

    Science.gov (United States)

    Frelinger, Andrew L.; Gerrits, Anja J.; Garner, Allen L.; Torres, Andrew S.; Caiafa, Antonio; Morton, Christine A.; Berny-Lang, Michelle A.; Carmichael, Sabrina L.; Neculaes, V. Bogdan; Michelson, Alan D.

    2016-01-01

    Background Activated autologous platelet-rich plasma (PRP) used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF) to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF) pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma. Aims To determine whether sub-microsecond duration, low electric field (SMLEF) bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity. Methods PRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and platelet factor 4 (PF4), and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin. Results PRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the

  1. Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma (PRP), plasma rich in growth factors (PRGF), advanced platelet-rich fibrin (A-PRF), and concentrated growth factors (CGF).

    Science.gov (United States)

    Masuki, Hideo; Okudera, Toshimitsu; Watanebe, Taisuke; Suzuki, Masashi; Nishiyama, Kazuhiko; Okudera, Hajime; Nakata, Koh; Uematsu, Kohya; Su, Chen-Yao; Kawase, Tomoyuki

    2016-12-01

    The development of platelet-rich fibrin (PRF) drastically simplified the preparation procedure of platelet-concentrated biomaterials, such as platelet-rich plasma (PRP), and facilitated their clinical application. PRF's clinical effectiveness has often been demonstrated in pre-clinical and clinical studies; however, it is still controversial whether growth factors are significantly concentrated in PRF preparations to facilitate wound healing and tissue regeneration. To address this matter, we performed a comparative study of growth factor contents in PRP and its derivatives, such as advanced PRF (A-PRF) and concentrated growth factors (CGF). PRP and its derivatives were prepared from the same peripheral blood samples collected from healthy donors. A-PRF and CGF preparations were homogenized and centrifuged to produce extracts. Platelet and white blood cell counts in A-PRF and CGF preparations were determined by subtracting those counts in red blood cell fractions, supernatant acellular serum fractions, and A-PRF/CGF exudate fractions from those counts of whole blood samples. Concentrations of growth factors (TGF-β1, PDGF-BB, VEGF) and pro-inflammatory cytokines (IL-1β, IL-6) were determined using ELISA kits. Compared to PRP preparations, both A-PRF and CGF extracts contained compatible or higher levels of platelets and platelet-derived growth factors. In a cell proliferation assay, both A-PRF and CGF extracts significantly stimulated the proliferation of human periosteal cells without significant reduction at higher doses. These data clearly demonstrate that both A-PRF and CGF preparations contain significant amounts of growth factors capable of stimulating periosteal cell proliferation, suggesting that A-PRF and CGF preparations function not only as a scaffolding material but also as a reservoir to deliver certain growth factors at the site of application.

  2. Induction of interleukin-8 by Naegleria fowleri lysates requires activation of extracellular signal-regulated kinase in human astroglial cells.

    Science.gov (United States)

    Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Sang-Hee; Kwon, Daeho; Shin, Ho-Joon

    2012-08-01

    Naegleria fowleri is a pathogenic free-living amoeba which causes primary amoebic meningoencephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astroglial cells was investigated following treatment with N. fowleri lysates. We demonstrated that N. fowleri are potent inducers for the expression of interleukin-8 (IL-8) genes in human astroglial cells which was preceded by activation of extracellular signal-regulated kinase (ERK). In addition, N. fowleri lysates induces the DNA binding activity of activator protein-1 (AP-1), an important transcription factor for IL-8 induction. The specific mitogen-activated protein kinase kinase/ERK inhibitor, U0126, blocks N. fowleri-mediated AP-1 activation and subsequent IL-8 induction. N. fowleri-induced IL-8 expression requires activation of ERK in human astroglial cells. These findings indicate that treatment of N. fowleri on human astroglial cells leads to the activation of AP-1 and subsequent expression of IL-8 which are dependent on ERK activation. These results may help understand the N. fowleri-mediated upregulation of chemokine and cytokine expression in the astroglial cells.

  3. Multicenter phase II study of matured dendritic cells pulsed with melanoma cell line lysates in patients with advanced melanoma

    Directory of Open Access Journals (Sweden)

    Hernandez Jackie

    2010-09-01

    Full Text Available Abstract Background Several single center studies have provided evidence of immune activation and antitumor activity of therapeutic vaccination with dendritic cells (DC in patients with metastatic melanoma. The efficacy of this approach in patients with favorable prognosis metastatic melanoma limited to the skin, subcutaneous tissues and lung (stages IIIc, M1a, M1b was tested in a multicenter two stage phase 2 study with centralized DC manufacturing. Methods The vaccine (IDD-3 consisted 8 doses of autologous monocyte-derived matured DC generated in serum-free medium with granulocyte macrophage colony stimulating factor (GM-CSF and interleukin-13 (IL-13, pulsed with lysates of three allogeneic melanoma cell lines, and matured with interferon gamma. The primary endpoint was antitumor activity. Results Among 33 patients who received IDD-3 there was one complete response (CR, two partial responses (PR, and six patients had stable disease (SD lasting more than eight weeks. The overall prospectively defined tumor growth control rate was 27% (90% confidence interval of 13-46%. IDD-3 administration had minimal toxicity and it resulted in a high frequency of immune activation to immunizing melanoma antigens as assessed by in vitro immune monitoring assays. Conclusions The administration of matured DC loaded with tumor lysates has significant immunogenicity and antitumor activity in patients with limited metastatic melanoma. Clinical trial registration NCT00107159.

  4. Hereditary sideroblastic anemia with associated platelet abnormalities.

    Science.gov (United States)

    Soslau, G; Brodsky, I

    1989-12-01

    A 62 year old male (R.H.) presented with a mild anemia (Hb 11-12 gm%) and a history of multiple hemorrhagic episodes. The marrow had 40-50% sideroblasts. Marrow chromosomes were normal. His wife was hematologically normal, while one daughter, age 30 years, had a sideroblastic anemia (Hb 11-12 gm%) with 40-50% sideroblasts in the marrow. Her anemia was first noted at age 15 years. Administration of vitamin B6 did not correct the anemia in either the father or daughter. Platelet abnormalities inherited jointly with this disorder are described for the first time. Both R.H. and his daughter had prolonged bleeding times, with normal PTT, PT times, fVIII:C, fVIII:Ag levels, and vWF multimers, which may rule out a von Willebrand's disease. They have normal platelet numbers but abnormally low platelet adhesiveness and greatly depressed ADP, collagen, and epinephrine responsiveness. Response to ristocetin was in the low normal range, and aggregation with thrombin was normal. While desmopressin completely normalized R.H.'s bleeding time, none of these platelet parameters were improved. No differences in the SDS PAGE protein patterns of RH platelets could be detected in comparison to normal samples. His platelets took up and released serotonin (5HT) normally, and electron micrographs defined no morphological abnormalities. However, no ATP was released from platelets activated with collagen, and when followed by thrombin about fourfold greater ATP was released by control platelets as compared to RH platelets. The dense granule fraction derived from RH platelets contained about 20% the level of ATP, 40% the level of ADP, and 50% the level of 5HT detected in a normal sample. The results indicate that the bleeding disorder is related to a non-classical heritable storage pool defect. The connection between the inherited sideroblastic anemia and platelet defects is obscure.

  5. Design and Synthesis of New Arylsulfonamide and Arylamide Derivatives for the Platelet Aggression Inhibitor

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A series of arylsulfonamide and arylamide derivatives have been prepared from anisolein good yields. The structures of those compounds were confirmed by 1H-NMR and MS analysis.Their activities against platelet aggregation were tested in vitro by using the Born test on rabbits.

  6. Equine autologous platelet concentrates : A comparative study between different available systems

    NARCIS (Netherlands)

    Hessel, L N; Bosch, G; van Weeren, P R; Ionita, J-C

    2015-01-01

    REASONS FOR PERFORMING STUDY: Autologous platelet concentrates (APCs) are being used increasingly in horses to enhance regeneration in tissues that have poor natural healing capabilities. Numerous APC systems, which are based on different preparation techniques and were originally developed for huma

  7. Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets

    DEFF Research Database (Denmark)

    Pleines, Irina; Eckly, Anita; Elvers, Margitta;

    2010-01-01

    formation and exocytosis in various cell types, but its exact function in platelets is not established. Here, we show that the megakaryocyte/platelet-specific loss of Cdc42 leads to mild thrombocytopenia and a small increase in platelet size in mice. Unexpectedly, Cdc42-deficient platelets were able to form...... reduced, suggesting increased clearing of the cells under physiologic conditions. These data point to novel multiple functions of Cdc42 in the regulation of platelet activation, granule organization, degranulation, and a specific role in GPIb signaling....

  8. Human platelets antigens influence the viral load of platelets after the interaction of the platelets with HCV and HIV in vitro

    Directory of Open Access Journals (Sweden)

    Rejane Maria Tommasini Grotto

    Full Text Available Abstract: INTRODUCTION: In this study, we evaluated hepatitis C virus (HCV and human immunodeficiency virus (HIV - platelet interactions in vitro as well as human platelets antigen (HPA polymorphisms. METHODS: Platelets were obtained from 100 healthy HPA-genotyped volunteer donors and incubated with HIV or HCV. The viral load after in vitro exposure was detected. RESULTS: The viral load in the platelets after exposure to the virus was higher in the HIV exposure than in the HCV exposure. CONCLUSIONS: HIV-platelet ligation could be more efficient than HCV-platelet interaction. Further, the HPA-1b allele seems to influence the interaction of platelets with HCV.

  9. Microarray R-based analysis of complex lysate experiments with MIRACLE

    DEFF Research Database (Denmark)

    List, Markus; Block, Ines; Pedersen, Marlene Lemvig

    2014-01-01

    MOTIVATION: Reverse-phase protein arrays (RPPAs) allow sensitive quantification of relative protein abundance in thousands of samples in parallel. Typical challenges involved in this technology are antibody selection, sample preparation and optimization of staining conditions. The issue of combin......: Project URL: http://www.nanocan.org/miracle/ CONTACT: : mlist@health.sdu.dk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online....

  10. A preliminary comparison of dendritic cell maturation by total cellular RNA to total cellular lysate derived from breast cancer stem cells

    Directory of Open Access Journals (Sweden)

    Phong Minh Le

    2016-06-01

    Full Text Available Introduction: Dendritic cells (DCs have been widely considered as the most potent antigen-presenting cells. As such, DC-based vaccines are regarded as a promising strategy in cancer vaccination and therapy. This study compared the maturation of DCs induced by total cellular RNA and cell lysate (i.e. nucleic acid and protein. Methods: Both total RNA and cell lysate were isolated from breast cancer stem cells (BCSCs. The lysates were used to incubate with monocyte-derived immature DCs. To track the transfection efficiency, the BCSCs were stably transfected with green fluorescent protein (GFP. The maturation of DCs was evaluated by expression of costimulatory molecules including CD40, CD80, and CD86. Transfections were confirmed by evaluating GFP expression in DCs at 24 hours post transfection. Results: The results of this study showed that GFP is expressed in DCs after both total RNA and lysate incubation. The expression of costimulatory molecules (CD40, CD80, and CD86 was significantly higher in RNA-transfected DCs than in cell lysate-primed DCs. Conclusion: Our findings suggest that total RNA primed BCSCs can be a suitable platform for DC-based vaccine therapy of breast cancer. [Biomed Res Ther 2016; 3(6.000: 679-686

  11. Expansion of the neonatal platelet mass is achieved via an extension of platelet lifespan.

    Science.gov (United States)

    Liu, Zhi-Jian; Hoffmeister, Karin M; Hu, Zhongbo; Mager, Donald E; Ait-Oudhia, Sihem; Debrincat, Marlyse A; Pleines, Irina; Josefsson, Emma C; Kile, Benjamin T; Italiano, Joseph; Ramsey, Haley; Grozovsky, Renata; Veng-Pedersen, Peter; Chavda, Chaitanya; Sola-Visner, Martha

    2014-05-29

    The fetal/neonatal hematopoietic system must generate enough blood cells to meet the demands of rapid growth. This unique challenge might underlie the high incidence of thrombocytopenia among preterm neonates. In this study, neonatal platelet production and turnover were investigated in newborn mice. Based on a combination of blood volume expansion and increasing platelet counts, the platelet mass increased sevenfold during the first 2 weeks of murine life, a time during which thrombopoiesis shifted from liver to bone marrow. Studies applying in vivo biotinylation and mathematical modeling showed that newborn and adult mice had similar platelet production rates, but neonatal platelets survived 1 day longer in circulation. This prolonged lifespan fully accounted for the rise in platelet counts observed during the second week of murine postnatal life. A study of pro-apoptotic and anti-apoptotic Bcl-2 family proteins showed that neonatal platelets had higher levels of the anti-apoptotic protein Bcl-2 and were more resistant to apoptosis induced by the Bcl-2/Bcl-xL inhibitor ABT-737 than adult platelets. However, genetic ablation or pharmacologic inhibition of Bcl-2 alone did not shorten neonatal platelet survival or reduce platelet counts in newborn mice, indicating the existence of redundant or alternative mechanisms mediating the prolonged lifespan of neonatal platelets. © 2014 by The American Society of Hematology.

  12. [Platelet allo-antibodies identification strategies for preventing and managing platelet refractoriness].

    Science.gov (United States)

    Basire, A; Picard, C

    2014-11-01

    Platelet refractoriness is a serious complication for patients receiving recurrent platelet transfusions, which can be explained by non-immune and immune causes. Human Leukocyte Antigens (HLA) allo-immunization, especially against HLA class I, is the major cause for immune platelet refractoriness. To a lesser extent, allo-antibodies against specific Human Platelet Antigen (HPA) are also involved. Pregnancy, transplantation and previous transfusions can lead to allo-immune reaction against platelet antigens. After transfusion, platelet count is decreased by accelerated platelet destruction related to antibodies fixation on incompatible platelet antigens. New laboratory tests for allo-antibodies identification were developed to improve sensibility and specificity, especially with the LUMINEX(®) technology. The good use and interpretation of these antibodies assays can improve strategies for platelet refractoriness prevention and management with a patient adapted response. Compatible platelets units can be selected according to their identity with recipient typing or immune compatibility regarding HLA or HPA antibodies or HLA epitope compatibility. Prospective studies are needed to further confirm the clinical benefit of new allo-antibodies identification methods and consensus strategies for immune platelet refractoriness management.

  13. Platelet antibodies, activated platelets and serum leptin in childhood immune thrombocytopenic purpura.

    Science.gov (United States)

    Badrawy, Hosny; Elsayh, Khalid I; Zahran, Asmaa M; El-Ghazali, Mohamad Hamdy

    2013-01-01

    The aim of this study was to evaluate the levels of platelet-associated antibodies (PAIgG and PAIgM), activated platelets and serum leptin in children with acute immune thrombocytopenic purpura (ITP). The study included 40 patients with ITP and 40 healthy age- and sex-matched controls. PAIgG, PAIgM and activated platelet levels were estimated by flow cytometry, and serum leptin levels were estimated by ELISA. Activated platelets and serum leptin were significantly higher in the ITP patients than in the controls. The percentage and mean fluorescence intensity of PAIgG and PAIgM staining were significantly higher in the patients than in the controls. Serum leptin and activated platelet levels in patients with thrombocytopenia of brief duration were significantly lower than those in patients with thrombocytopenia of prolonged duration. The levels of activated platelets, serum leptin and PAIgG were positively correlated, and the levels of serum leptin, activated platelets and platelet counts were negatively correlated. The increased levels of activated platelets, serum leptin and platelet-associated antibodies in children with acute ITP suggest that these factors could play a role in ITP pathogenesis. Additionally, activated platelets and serum leptin could have prognostic significance in paediatric acute ITP. Copyright © 2013 S. Karger AG, Basel.

  14. Determination of platelet-activating factor by reverse phase high-performance liquid chromatography and its application in viral hepatitis

    Institute of Scientific and Technical Information of China (English)

    Hong-Cui Cao; Xiao-Ming Chen; Wei Xu

    2005-01-01

    AIM: To detect the platelet-activating factor (PAF) and the plasma or serum levels of tumor necrosis factor-α (TNF-α) malondialdehyde (MDA), endotoxin (ET) and to discuss their significance in various types of viral hepatitis.METHODS: PAF, TNF-α, MDA, and ET levels in 60 controls, 16 cases of acute viral hepatitis, 71 cases of chronic viral hepatitis, 19 cases of severe viral hepatitis were detected by reverse phase high-performance liquid chromatography (rHPLC), bio-assay, ELISA, thiobarbituric acid (TBA), and limulus lysate test (LLT), respectively.RESULTS: The rHPLC was more sensitive and specific than bio-assay (r = 0.912, P<0.01). The plasma levels of PAF, TNF-α, MDA, and ET in patients with viral hepatitis were higher than those in controls (P<0.01).CONCLUSION: rHPLC is more reliable and accurate for the detection of PAF.

  15. Platelet-rich fibrin application in dentistry: a literature review.

    Science.gov (United States)

    Borie, Eduardo; Oliví, Daniel García; Orsi, Iara Augusta; Garlet, Katia; Weber, Benjamín; Beltrán, Víctor; Fuentes, Ramón

    2015-01-01

    The development of bioactive surgical additives to regulate the inflammation and increase the speed of healing process is one of the great challenges in clinical research. In this sense, platelet rich fibrin (PRF) appears as a natural and satisfactory alternative with favorable results and low risks. The following review attempts to summarize the relevant literature regarding the technique of using PRF, focusing on its preparation, advantages, and disadvantages of using it in clinical applications. PRF alone or in combination with other biomaterials seems to have several advantages and indications both for medicine and dentistry, due it is a minimally invasive technique with low risks and satisfactory clinical results.

  16. Application of platelet-rich plasma and platelet-rich fibrin in fat grafting: basic science and literature review.

    Science.gov (United States)

    Liao, Han-Tsung; Marra, Kacey G; Rubin, J Peter

    2014-08-01

    Due to the natural properties of fat, fat grafting remains a popular procedure for soft tissue volume augmentation and reconstruction. However, clinical outcome varies and is technique dependent. Platelet-rich plasma (PRP) contains α-granules, from which multiple growth factors such as platelet-derived growth factor, transforming growth factor-β, vascular endothelial growth factor, and epidermal growth factor can be released after activation. In recent years, the scope of PRP therapies has extended from bone regeneration, wound healing, and healing of musculoskeletal injuries, to enhancement of fat graft survival. In this review, we focus on the definition of PRP, the different PRP preparation and activation methods, and growth factor concentrations. In addition, we discuss possible mechanisms for the role of PRP in fat grafting by reviewing in vitro studies with adipose-derived stem cells, preadipocytes, and adipocytes, and preclinical and clinical research. We also review platelet-rich fibrin, a so-called second generation PRP, and its slow-releasing biology and effects on fat grafts compared to PRP in both animal and clinical research. Finally, we provide a general foundation on which to critically evaluate earlier studies, discuss the limitations of previous research, and direct plans for future experiments to improve the optimal effects of PRP in fat grafting.

  17. Bulk fluid phase behaviour of colloidal platelet-sphere and platelet-polymer mixtures.

    Science.gov (United States)

    de las Heras, Daniel; Schmidt, Matthias

    2013-04-13

    Using a geometry-based fundamental measure density functional theory, we calculate bulk fluid phase diagrams of colloidal mixtures of vanishingly thin hard circular platelets and hard spheres. We find isotropic-nematic phase separation, with strong broadening of the biphasic region, upon increasing the pressure. In mixtures with large size ratio of platelet and sphere diameters, there is also demixing between two nematic phases with differing platelet concentrations. We formulate a fundamental measure density functional for mixtures of colloidal platelets and freely overlapping spheres, which represent ideal polymers, and use it to obtain phase diagrams. We find that, for low platelet-polymer size ratio, in addition to isotropic-nematic and nematic-nematic phase coexistence, platelet-polymer mixtures also display isotropic-isotropic demixing. By contrast, we do not find isotropic-isotropic demixing in hard-core platelet-sphere mixtures for the size ratios considered.

  18. IgG platelet antibodies in EDTA-dependent pseudothrombocytopenia bind to platelet membrane glycoprotein IIb.

    Science.gov (United States)

    Fiorin, F; Steffan, A; Pradella, P; Bizzaro, N; Potenza, R; De Angelis, V

    1998-08-01

    EDTA-dependent pseudothrombocytopenia (PTCP) consists of an inappropriate low platelet count caused by autoantibodies present in the serum samples reacting with platelets only in EDTA-anticoagulated blood. By using immunoprecipitation and Western blot techniques, we studied the immunochemical specificity of platelet agglutinating autoantibodies in the serum samples of 10 patients with PTCP. Furthermore, to evaluate a possible role of PTCP-associated IgG autoantibodies in increased platelet turnover, we assayed the plasma glycocalicin (GC) level and calculated the GC index for every patient. Our results provide direct evidence that an epitope located on platelet membrane glycoprotein IIb is recognized by PTCP-associated IgG antibodies; moreover GC levels in patients with EDTA-dependent PTCP were similar to control levels, thus excluding an increased platelet turnover. We conclude that antiplatelet antibodies directed against platelet cryptantigens are unlikely to have a major role in the increased removal of cells from circulation.

  19. Sodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate.

    Science.gov (United States)

    Bonturi, Nemailla; Radke, Vanessa Soraia Cortez Oliveira; Bueno, Sônia Maria Alves; Freitas, Sindélia; Azzoni, Adriano Rodrigues; Miranda, Everson Alves

    2013-03-01

    The number of studies on gene therapy using plasmid vectors (pDNA) has increased in recent years. As a result, the demand for preparations of pDNA in compliance with recommendations of regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is often obtained through fermentation of transformed Escherichia coli and purification by a series of unit operations, including chromatography. Hydrophobic interaction chromatography (HIC) and thiophilic aromatic chromatography (TAC), both using ammonium sulfate buffers, are commonly employed with success. This work was aimed at studying the feasibility of utilizing alternative salts in the purification of pDNA from neutralized lysate with phenyl-agarose (HIC) and mercaptopyrimidine-agarose (TAC) adsorbents. Their selectivity toward sc pDNA was evaluated through adsorption studies using 1.5 mol/L sodium citrate and 2.0 mol/L potassium phosphate as adsorption buffers. Chromatography with mercaptopyrimidine-agarose adsorbent and 1.5 mol/L sodium citrate was able to recover 91.1% of the pDNA with over 99.0% removal of gDNA and endotoxin. This represents a potential alternative for the primary recovery of sc pDNA. However, the most promising result was obtained using 2.0 mol/L potassium phosphate buffer and a mercaptopyrimidine-agarose column. In a single chromatographic step, this latter buffer/adsorbent system recovered 68.5% of the pDNA with 98.8% purity in accordance with the recommendations of regulatory agencies with regard to RNA and endotoxin impurity.

  20. Fractal and Euclidean descriptors of platelet shape.

    Science.gov (United States)

    Kraus, Max-Joseph; Neeb, Heiko; Strasser, Erwin F

    2014-01-01

    Platelet shape change is a dynamic membrane surface process that exhibits remarkable morphological heterogeneity. Once the outline of an irregular shape is identified and segmented from a digital image, several mathematical descriptors can be applied to numerical characterize the irregularity of the shapes surface. 13072 platelet outlines (PLO) were segmented automatically from 1928 microscopic images using a newly developed algorithm for the software product Matlab R2012b. The fractal dimension (FD), circularity, eccentricity, area and perimeter of each PLO were determined. 972 PLO were randomly assigned for computer-assisted manual measurement of platelet diameter as well as number, width and length of filopodia per platelet. FD can be used as a surrogate parameter for determining the roughness of the PLO and circularity can be used as a surrogate to estimate the number and length of filopodia. The relationship between FD and perimeter of the PLO reveals the existence of distinct groups of platelets with significant structural differences which may be caused by platelet activation. This new method allows for the standardized continuous numerical classification of platelet shape and its dynamic change, which is useful for the analysis of altered platelet activity (e.g. inflammatory diseases, contact activation, drug testing).

  1. Clinica use of platelet additive solutions.

    Science.gov (United States)

    van Rhenen, Dick J

    2007-12-01

    Randomised clinical trial (RCT) to study the clinical efficacy and safety of new platelet products using platelet additive solutions are scarce. In this paper a number of recent RCT's is discussed. It can be the start of a development where new transfusion products enter a RCT before the product is applied in clinical practice.

  2. Platelets in liver transplantation : Friend or foe?

    NARCIS (Netherlands)

    Pereboom, Ilona T. A.; Lisman, Ton; Porte, Robert J.

    2008-01-01

    Apart from the well-known role of blood platelets in hemostasis, there is emerging evidence that platelets have various nonhemostatic properties that play a critical role in inflammation, angiogenesis, tissue repair and regeneration, and ischemia/reperfusion (I/R) injury. All these processes may be

  3. Studies on megakaryopoiesis and platelet function

    NARCIS (Netherlands)

    Meinders, M.

    2015-01-01

    Platelets are blood circulating specialized subcellular fragments, which are produced by megakaryocytes. Platelets are essential for hemostasis and wound healing but also play a role in non-hemostatic processes such as the immune response or cancer metastasis. Considering the immediate precursors of

  4. Erythrocyte-platelet interaction in uncomplicated pregnancy.

    Science.gov (United States)

    Swanepoel, Albe C; Pretorius, Etheresia

    2014-12-01

    Maternal and fetal requirements during uncomplicated pregnancy are associated with changes in the hematopoietic system. Platelets and erythrocytes [red blood cells (RBCs)], and especially their membranes, are involved in coagulation, and their interactions may provide reasons for the changed hematopoietic system during uncomplicated pregnancy. We review literature regarding RBC and platelet membrane structure and interactions during hypercoagulability and hormonal changes. We then study interactions between RBCs and platelets in uncomplicated pregnancy, as their interactions may be one of the reasons for increased hypercoagulability during uncomplicated pregnancy. Scanning electron microscopy was used to study whole blood smears from 90 pregnant females in different phases of pregnancy. Pregnancy-specific interaction was seen between RBCs and platelets. Typically, one or more platelets interacted through platelet spreading and pseudopodia formation with a single RBC. However, multiple interactions with RBCs were also shown for a single platelet. Specific RBC-platelet interaction seen during uncomplicated pregnancy may be caused by increased estrogen and/or increased fibrinogen concentrations. This interaction may contribute to the hypercoagulable state associated with healthy and uncomplicated pregnancy and may also play a fundamental role in gestational thrombocytopenia.

  5. BETA-ADRENOBLOCKERS AND PLATELET AGGREGATION. CARVEDILOL

    Directory of Open Access Journals (Sweden)

    A. N. Zakirova

    2008-01-01

    Full Text Available Approaches evolution to studying of beta-blockers influence on platelet aggregation is reviewed. The current view on of beta-blocker antiplatelet effects is presented on the basis of physical and chemical drug properties (water repellency, dipole moment, molecular mass. Trail results on carvedilol influence on platelet aggregation are focused.

  6. Novel agents for anti-platelet therapy

    Directory of Open Access Journals (Sweden)

    Ji Xuebin

    2011-11-01

    Full Text Available Abstract Anti-platelet therapy plays an important role in the treatment of patients with thrombotic diseases. The most commonly used anti-platelet drugs, namely, aspirin, ticlopidine, and clopidogrel, are effective in the prevention and treatment of cardio-cerebrovascular diseases. Glycoprotein IIb/IIIa antagonists (e.g., abciximab, eptifibatide and tirofiban have demonstrated good clinical benefits and safety profiles in decreasing ischemic events in acute coronary syndrome. However, adverse events related to thrombosis or bleeding have been reported in cases of therapy with glycoprotein IIb/IIIa antagonists. Cilostazol is an anti-platelet agent used in the treatment of patients with peripheral ischemia, such as intermittent claudication. Presently, platelet adenosine diphosphate P2Y(12 receptor antagonists (e.g., clopidogrel, prasugrel, cangrelor, and ticagrelor are being used in clinical settings for their pronounced protective effects. The new protease-activated receptor antagonists, vorapaxar and atopaxar, potentially decrease the risk of ischemic events without significantly increasing the rate of bleeding. Some other new anti-platelet drugs undergoing clinical trials have also been introduced. Indeed, the number of new anti-platelet drugs is increasing. Consequently, the efficacy of these anti-platelet agents in actual patients warrants scrutiny, especially in terms of the hemorrhagic risks. Hopefully, new selective platelet inhibitors with high anti-thrombotic efficiencies and low hemorrhagic side effects can be developed.

  7. Performance evaluation of PL-11 platelet analyzer

    Institute of Scientific and Technical Information of China (English)

    张有涛

    2013-01-01

    Objective To evaluate and report the performance of PL-11 platelet analyzer. Methods Intravenous blood sam-ples anticoagulated with EDTA-K2 and sodium citrate were tested by the PL-11 platelet analyzer to evaluate the intra-assay and interassay coefficient of variation(CV),

  8. Platelet affinity for burro aorta collagen

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, M.D.

    1977-10-01

    Despite ingenious concepts, there are no unequivocal clues as to what, when, and how some undefined biochemical factor(s) or constituent(s) that localizes in the arterial wall can precipitate a thromboatheromatous lesion or arterial disease. The present study focused on the extraction, partial purification, and characterization of a collagen-active platelet stimulator from the aortas of aged burros. The aggregator moiety in the aorta extracts invariably had a higher affinity for platelets in citrated platelet-rich plasma of human beings than for platelets of homologous burros. The platelet-aggregating factor(s) in the aorta extract was retained by incubation with ..cap alpha..-chymotrypsin. Platelet-aggregating activity was rapidly abolished after incubation with collagenase, as determined by platelet-aggregometry tests. Evidence based on light microscope and polysaccharide histochemical reactions indicates a probability that the intracellular amorphous matrix (PAS-positive) and filamentous components (PTAH-positive) expelled from smooth muscle cells disrupted during homogenization of the aorta may be a principal source of a precursor collagen species which is a potent inducer of platelet aggregation.

  9. The origin and function of platelet glycosyltransferases

    DEFF Research Database (Denmark)

    Wandall, Hans H; Rumjantseva, Viktoria; Sørensen, Anne Louise Tølbøll;

    2012-01-01

    Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver pro- and anti-angiogenic factors throughout the vascular system. Platelets are anucleated cells and lack a complex secretory apparatus with distinct Golgi/endoplasmic reticulum compartme...

  10. Function of eltrombopag-induced platelets compared to platelets from control patients with immune thrombocytopenia.

    Science.gov (United States)

    Haselboeck, Johanna; Kaider, Alexandra; Pabinger, Ingrid; Panzer, Simon

    2013-04-01

    Data on the in vivo function of platelets induced by the thrombopoietin receptor agonist eltrombopag are scarce. To assess a possible influence of eltrombopag we compared platelet function of eltrombopag-treated immune thrombocytopenia (ITP) patients (group 1; n=10) after treatment response to that from control ITP patients (group 2; n=12). We further analysed platelet function at baseline and after one, three, and four weeks of eltrombopag treatment and estimated daily changes of platelet function during the eltrombopag-induced platelet rise. The formation of platelet-monocyte aggregates (PMA), P-selectin expression [MFI], and platelet adhesion under high shear conditions (surface coverage, SC) in vivo and after in vitro addition of agonists (ADP, TRAP-6, Collagen) were similar between both groups after response to eltrombopag treatment. Only TRAP-6 induced a lower SC in the eltrombopag group (p=0.03). All platelet function parameters except for Collagen-induced P-selectin expression changed significantly during treatment with eltrombopag. PMA, naïve and after addition of ADP or TRAP-6 increased with increasing platelet counts. P-selectin expression decreased, when measured without and upon addition of ADP, increased in the presence of TRAP-6, and remained unchanged after addition of Collagen. SC increased during the eltrombopag-induced platelet rise. All significant changes of platelet function correlated to changes in platelet counts. Two patients developed venous thromboses during eltrombopag treatment, but no association with any distinct single platelet function parameter or combinations thereof was identifiable. Thus, eltrombopag-induced platelets function similar to those from control ITP patients without discernible increased hyper-reactivity.

  11. A platelet monoclonal antibody inhibition assay for detection of glycoprotein IIb/IIIa-related platelet alloantibodies.

    Science.gov (United States)

    Reiner, A P; Teramura, G; Nelson, K A; Slichter, S J

    1995-08-18

    Post-transfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAT) result from formation of alloantibodies to platelet membrane glycoprotein-associated antigens. The detection and identification of platelet-specific alloantibodies in patient sera is often complicated by the presence of co-existing HLA antibodies and/or more than one platelet specificity in the same serum. We describe a solid phase assay that specifically detects antibodies to platelet membrane associated alloantigens by measuring the ability of patient antisera to inhibit the binding of glycoprotein GPIIb or GPIIIa monoclonal antibodies to intact platelets. When tested in the GPIIIa assay against a panel of random platelet donors, the reactivities of two known PLAI antisera that also contained different HLA antibodies were highly correlated (r = 0.99) and allowed PLA phenotyping of the population. A standard direct binding platelet ELISA, on the other hand, was unable to accurately PLA phenotype the same population. The reactivities of two known Baka antisera (one containing additional anti-PLA2 and the other anti-Brb specificities) were highly correlated (r = 0.95) in the GPIIb assay, and Bak phenotype determination was similarly accomplished for a random platelet panel. Furthermore, a comparison of platelet phenotype results (using the monoclonal inhibition assay) and genotype results (using DNA analysis) for the PLA and Bak systems showed a concordance of 98% for 146 alleles tested. In conclusion, the platelet monoclonal antibody inhibition assay: (1) allows determination of platelet-specific alloantibodies in the presence of contaminating HLA antibodies and/or in sera containing multiple platelet alloantibodies; (2) allows accurate platelet phenotyping for the GPIIIa-associated PLA and GPIIb-associated Bak antigen systems; and (3) may be applicable to the detection of other known or even novel platelet glycoprotein-associated antigens.

  12. Modulatory effect of coffee on platelet function.

    Science.gov (United States)

    Bhaskar, Shobha; Rauf, Arun A

    2010-01-01

    Blood platelets play a major role in cardiovascular disease (CVD) and thrombosis. Conflicting information exists regarding the effect of coffee consumption on the cardiovascular system. We have investigated whether the consumption of moderate amount of coffee affect platelet functions and primary hemostasis in vivo in normal and high fat diet fed rats. Coffee fed group showed significant (P production from membrane arachidonic acid and it was decreased in coffee treated group. Platelet aggregation studies with ADP, collagen, arachidonic acid and epinephrine showed significant (P coffee fed group. Scanning electron microscopic studies revealed that platelet aggregation tendency increased in HFD group and was reduced in coffee fed group. These results indicate that coffee is active in inhibiting platelet aggregation, a critical step involved in thrombosis.

  13. Platelet mitochondrial function and dysfunction: physiological consequences

    Energy Technology Data Exchange (ETDEWEB)

    Popov, D.

    2015-07-01

    There is a general trend in revisiting mitochondria using the up-to-date technologies that uncovered novel attributes of this organelle, such as the intracellular displacement to locations where an energy supply is needed, the dynamic shape changes and turnover, the initiation of signaling to the rest of the cell, and the ability to crosstalk with other cellular organelles. The in-depth scrutiny of platelet mitochondria role in health and pathology is included within this ongoing revisiting trend. The current article puts into a nutshell the most recent data on platelet mitochondria function and disease-related ion, focusing on generation of stress- and apoptosis-related signaling molecules, overproduction of reactive oxygen species during activation and disease, on the biomarker potential of platelets mitochondria, and their prospective exploitation in translational applications. These novel findings complete the physiological profile of platelets and could have potential therapeutic effectiveness in platelet-associated disorders.

  14. Platelets as immune cells in infectious diseases.

    Science.gov (United States)

    Speth, Cornelia; Löffler, Jürgen; Krappmann, Sven; Lass-Flörl, Cornelia; Rambach, Günter

    2013-11-01

    Platelets have been shown to cover a broad range of functions. Besides their role in hemostasis, they have immunological functions and thus participate in the interaction between pathogens and host defense. Platelets have a broad repertoire of receptor molecules that enable them to sense invading pathogens and infection-induced inflammation. Consequently, platelets exert antimicrobial effector mechanisms, but also initiate an intense crosstalk with other arms of the innate and adaptive immunity, including neutrophils, monocytes/macrophages, dendritic cells, B cells and T cells. There is a fragile balance between beneficial antimicrobial effects and detrimental reactions that contribute to the pathogenesis, and many pathogens have developed mechanisms to influence these two outcomes. This review aims to highlight aspects of the interaction strategies between platelets and pathogenic bacteria, viruses, fungi and parasites, in addition to the subsequent networking between platelets and other immune cells, and the relevance of these processes for the pathogenesis of infections.

  15. Platelet bioreactor-on-a-chip

    Science.gov (United States)

    Mazutis, Linas; Wu, Stephen; Sylman, Joanna L.; Ehrlicher, Allen; Machlus, Kellie R.; Feng, Qiang; Lu, Shijiang; Lanza, Robert; Neeves, Keith B.; Weitz, David A.; Italiano, Joseph E.

    2014-01-01

    Platelet transfusions total >2.17 million apheresis-equivalent units per year in the United States and are derived entirely from human donors, despite clinically significant immunogenicity, associated risk of sepsis, and inventory shortages due to high demand and 5-day shelf life. To take advantage of known physiological drivers of thrombopoiesis, we have developed a microfluidic human platelet bioreactor that recapitulates bone marrow stiffness, extracellular matrix composition, micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts, and it supports high-resolution live-cell microscopy and quantification of platelet production. Physiological shear stresses triggered proplatelet initiation, reproduced ex vivo bone marrow proplatelet production, and generated functional platelets. Modeling human bone marrow composition and hemodynamics in vitro obviates risks associated with platelet procurement and storage to help meet growing transfusion needs. PMID:25606631

  16. Platelet-rich plasma in otolaryngology.

    Science.gov (United States)

    Stavrakas, M; Karkos, P D; Markou, K; Grigoriadis, N

    2016-12-01

    Platelet-rich plasma is a novel material that is being used more frequently in many surgical specialties. A literature review on the current and potential uses of platelet-rich plasma in otolaryngology was performed. There is limited evidence on the use of platelet-rich plasma in otolaryngology compared with other specialties: only 11 studies on various subspecialties (otology, rhinology and laryngology) were included in the final review. Based on the limited number of studies, we cannot draw safe conclusions about the value of platelet-rich plasma in otolaryngology. Nevertheless, the available literature suggests that platelet-rich plasma holds promise for future research and may have a number of clinical applications.

  17. Calidad del plasma rico en plaquetas: estudio de la activación plaquetaria Platelet-rich plasma quality: a study on platelet activation

    Directory of Open Access Journals (Sweden)

    C. Sáez-Torres Barroso

    2007-08-01

    Full Text Available Objetivo. El plasma rico en plaquetas (PRP es utilizado de forma cada vez más frecuente en técnicas quirúrgicas de regeneración tisular. No obstante, el procesamiento de la sangre hasta obtener PRP puede desencadenar la activación prematura de las plaquetas y la pérdida de los factores bioactivos. En este trabajo estudiamos la calidad de los concentrados de plaquetas obtenidos siguiendo la técnica de doble centrifugación en tubo. Método. Se someten 50 ml de sangre a una primera centrifugación a 200g 10 minutos, se recoge el sobrenadante y se centrifuga a 700g 15 minutos. Posteriormente, tras eliminar las 2/3 partes del plasma, se resuspenden las plaquetas y se analiza el grado de enriquecimiento, el estado de activación y la reserva funcional de las plaquetas. Resultados. El enriquecimiento en plaquetas del PRP fue de 364±177% (n=45 respecto de los niveles presentes en sangre total. Mediante el estudio de la expresión de CD62 por citometría de flujo se determinó el porcentaje de plaquetas activadas en las muestras de 8 donantes. Mientras que en la sangre no procesada se detectó un 2,7% de plaquetas activadas, tras la preparación del PRP éste era sólo de 3,6%, aumentando hasta el 16% en el concentrado almacenado toda la noche a 22º C. Tras la estimulación con trombina el porcentaje de plaquetas activadas fue de 96,2%. Conclusión. Este protocolo de preparación de PRP no produce una activación significativa de las plaquetas. La respuesta a la estimulación con trombina de los concentrados indica un buen estado de reserva plaquetaria.Objective. Platelet Rich Plasma (PRP is an autologous preparation currently used in oral and maxillofacial reconstructive surgery. Blood collection and preparation of platelet concentrates may lead to platelet activation and the premature loss of their granular load. In this study, we have analyzed the quality of the PRP obtained from a small volume of whole blood through a double centrifugation

  18. Tumor cell lysate-pulsed dendritic cells induce a T cell response against colon cancer in vitro and in vivo.

    Science.gov (United States)

    Wu, Yu-gang; Wu, Guang-zhou; Wang, Liang; Zhang, Yan-Yun; Li, Zhong; Li, De-Chun

    2010-09-01

    To investigate whether tumor cell lysate-pulsed (TP) dendritic cells (DCs) induce cytotoxic T lymphocyte (CTL) activity against colon cancer in vitro and in vivo. Hematopoietic progenitor cells were magnetically isolated from BALB/c mice bone marrow cells. These cells were cultured with cytokines GM-CSF, IL-4, and TNFalpha to induce their maturation. They were analyzed by morphological observation and phenotype analysis. DCs were pulsed with tumor cell lysate obtained by rapid freezing and thawing at a 1:3 DC:tumor cell ratio. CTL activity and interferon gamma (IFNgamma) secretion was evaluated ex vivo. In order to determine whether or not vaccination with CT26 TP DCs induce the therapeutic potential in the established colon tumor model, CT26 colon tumor cells were implanted subcutaneously (s.c.) in the midflank of naïve BALB/c mice. Tumor-bearing mice were injected with vaccination with CT26 TP DCs on days 3 and 10. Tumor growth was assessed every 2-3 days. Finally, CTL activity and IFNgamma secretion were evaluated in immunized mice. Hematopoietic progenitor cells from mice bone marrow cells cultured with cytokines for 8 days showed the character of typical mature DCs. Morphologically, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed high levels of MHC II, CD11b, CD80, and CD86 antigen, and were negative for CD8alpha. However, immature DCs cultured with cytokines for 5 days did not have typical DCs phenotypic markers. Ex vivo primed T cells with CT26 TP DCs were able to induce effective CTL activity against CT26 tumor cells, but not B16 tumor cells (E:T = 100:1, 60.36 +/- 7.11% specific lysis in CT26 group vs. 17.36 +/- 4.10% specific lysis in B16 group), and produced higher levels of IFNgamma when stimulated with CT26 tumor cells but not when stimulated with B16 tumor cells (1210.33 +/- 72.15 pg/ml in CT26 group vs. 182.25 +/- 25.51 pg/ml in B16 group, P models

  19. Evaluation of ToxA and Vibrio parahaemolyticus lysate on humoral immune response and immune-related genes in Pacific red snapper.

    Science.gov (United States)

    Reyes-Becerril, Martha; Maldonado-García, Minerva; Guluarte, Crystal; León-Gallo, Amalia; Rosales-Mendoza, Sergio; Ascencio, Felipe; Hirono, Ikuo; Angulo, Carlos

    2016-09-01

    Immunogenicity of ToxA and Vibrio parahaemolyticus lysate was evaluated in a double immunostimulation scheme in Pacific red snapper after V. parahaemolyticus infection. Three groups of Pacific red snapper were intraperitonealy (i.p.) injected with phosphate-buffered saline (PBS group), ToxA of V. parahaemolyticus (ToxA-Vp group) or V. parahaemolyticus lysate (lysate-Vp group) (first injection, day 1; second injection, day 7). Fish were subsequently infected with live V. parahaemolyticus. Humoral immune parameters in skin mucus and serum were evaluated on days 1, 7, 8 and 14 days post-immunostimulation and 7 days post-infection. Moreover expression of immune-related genes was quantified by real time PCR in head-kidney leukocytes, spleen, liver, and intestine. The ToxA-Vp-treated group showed a higher anti-protease and catalase activity in skin mucus when compared with the PBS group. Measurements of SOD and CAT activities showed an increment in both activities a day after the second boost with ToxA-Vp or lysate-Vp. Interestingly, IgM levels in mucus and transcripts were enhanced followed the ToxA-Vp treatment even after challenge. Furthermore, IL-1β was strongly expressed in all analyzed cell or tissues followed ToxA-Vp or Vp-lysate treatments. Finally, SOD and CAT gene expression was up-regulated in fish immunostimulated with either treatment ToxA-Vp or lysate-Vp, mainly after infection in head-kidney leukocytes and intestine. This is the first study where the effects of ToxA from V. parahaemolyticus in the immune system of Pacific red snapper was evaluated. These results suggest that ToxA-Vp would positively affect humoral immune response and up-regulate expression of genes involved in the immune system function; and could help in the control of V. parahaemolyticus infection in Pacific red snapper Lutjanus peru, an economic important fish in Mexico.

  20. Protective Mechanisms of Guanosine from Solanum lycopersicum on Agonist-Induced Platelet Activation: Role of sCD40L

    Directory of Open Access Journals (Sweden)

    Iván Palomo

    2013-07-01

    Full Text Available In the past 30 years, only three natural products have been sources of new drugs with antiplatelet activity. In this study, we have demonstrated for the first time that guanosine from Solanum lycopersicum possesses antiplatelet (secretion, spreading, adhesion and aggregation activity in vitro and inhibition of platelet inflammatory mediator of atherosclerosis (sCD40L. According to ADP-induced platelet aggregation inhibiting, the total extract residue was fractionated by liquid chromatography/phase separation, affording an aqueous fraction. This fraction was subjected to repeated permeation over Sephadex LH-20 and semi-preparative TLC. The isolated compound finally obtained was identified as guanosine on the basis of its UV-spectra, HPLC and 1H-NMR data. Guanosine concentration dose-dependently (1 to 4 mmol/L inhibited platelet secretion and aggregation induced by ADP and collagen. Spread of human platelets on collagen in the presence of guanosine was fully inhibited. After incubation of whole blood with guanosine, the platelet adhesion and aggregation under flow conditions was inhibited concentration dependently (0.2 to 2 mmol/L. At the same concentrations that guanosine inhibits platelet aggregation, levels of sCD40L were significantly decreased. Guanosine is thus likely to exert significant protective effects in thromboembolic-related disorders by inhibiting platelet aggregation.

  1. Platelet responses to dynamic biomaterial surfaces with different poly(ethylene glycol) and polyrotaxane molecular architectures constructed on gold substrates.

    Science.gov (United States)

    Kakinoki, Sachiro; Yui, Nobuhiko; Yamaoka, Tetsuji

    2013-11-01

    Four different dynamic biomaterial surfaces with different molecular architectures were prepared using two hydrophilic polymers: poly(ethylene glycol) and polyrotaxanes containing α-cyclodextrin. Either one or both terminals of the poly(ethylene glycol) or polyrotaxanes were immobilized onto a gold substrate via Au-S bonds, resulting in poly(ethylene glycol)-graft, polyrotaxanes-graft, poly(ethylene glycol)-loop, and polyrotaxanes-loop structures. Human platelet adhesion was suppressed more effectively on the graft surfaces than on the loop surfaces for both poly(ethylene glycol) and polyrotaxanes due to the high mobility of graft polymer chains with a free terminal. Moreover, the platelets adhered to the polyrotaxane surfaces much less than the poly(ethylene glycol) surfaces, possibly because of the mobile nature of the α-cyclodextrin molecules that were threaded on the poly(ethylene glycol) chain. Actin filament assembly in adherent platelets was also greatly prevented on the poly(ethylene glycol)/polyrotaxanes-graft surfaces in comparison with the corresponding loop surfaces. A clear correlation between the numbers and areas of adherent platelets on these surfaces suggests that platelet adhesion and activation were dominated by the platelet GPIIb/IIIa-adsorbed fibrinogen interaction. These results indicate that both of the different modes of dynamic features, sliding/rotation of α-cyclodextrin and polymer chain mobility, effectively suppressed platelet adhesion in spite of the similar hydrophilicity. This research affords a novel chemical strategy for designing hemocompatible biomaterial surfaces.

  2. Systemic effects of locally injected platelet rich plasma in a rat model: an analysis on muscle and bloodstream.

    Science.gov (United States)

    Borrione, P; Grasso, L; Racca, S; Abbadessa, G; Carriero, V; Fagnani, F; Quaranta, F; Pigozzi, F

    2015-01-01

    Abundant evidence suggests that growth factors, contained in platelets alpha granules, may play a key role in the early stages of the muscle healing process with particular regard to the inflammatory phase. Although the contents of the platelet-rich plasma preparations have been extensively studied, the biological mechanisms involved as well as the systemic effects and the related potential doping implications of this approach are still largely unknown. The aim of the present study was to investigate whether local platelet-rich plasma administration may modify the levels of specific cytokines and growth factors both in treated muscle and bloodstream in rats. An additional aim was to investigate more deeply whether the local platelet-rich plasma administration may exert systemic effects by analyzing contralateral lesioned but untreated muscles. The results showed that platelet-rich plasma treatment induced a modification of certain cytokines and growth factor levels in muscle but not in the bloodstream, suggesting that local platelet-rich plasma treatment influenced directly or, more plausibly, indirectly the synthesis or recruitment of cytokines and growth factors at the site of injury. Moreover, the observed modifications of cytokine and growth factor levels in contralateral injured but not treated muscles, strongly suggested a systemic effect of locally injected platelet-rich plasma.

  3. Decreased TGF-β1 and VEGF Release in Cystic Fibrosis Platelets: Further Evidence for Platelet Defects in Cystic Fibrosis

    Science.gov (United States)

    Maloney, James P.; Narasimhan, Jayashree; Biller, Julie

    2016-01-01

    Purpose Cystic fibrosis (CF) patients suffer from chronic lung inflammation. This inflammation may activate platelets. There are limited data on the role of platelet-secreted cytokines in CF. Platelet cytokines with inflammatory effects include vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1). As levels of these cytokines are tenfold greater in serum than plasma due to platelet release, serum levels may be one index of platelet content; but a more specific index is release during the aggregation of isolated platelets. We postulated that altered release of these platelet cytokines occurs in CF. Methods We obtained sera and plasma from CF outpatients (n=21) and from healthy controls (n=20), measured VEGF and TGF-β1, assessed for correlations with platelet number, analyzed cytokine release during platelet aggregation to collagen, and analyzed differences in maximal platelet aggregation. Results Platelet number and maximal aggregation levels were higher in CF. Plasma and serum levels of TGF-β1 and VEGF were higher in CF, but these levels were similar after adjusting for platelet number (serum cytokines correlated with platelet count). The release of VEGF and TGF-β1 during aggregation was decreased in CF platelets (by 52% and 29%, respectively). Conclusion Platelet release is not a source of the elevated blood proinflammatory cytokines TGF-β1 and VEGF in CF, as platelets from CF patients actually release less of these cytokines. These data provide further evidence for platelet defects in CF. PMID:27423781

  4. Breaking the mold: transcription factors in the anucleate platelet and platelet-derived microparticles

    Directory of Open Access Journals (Sweden)

    Katie L Lannan

    2015-02-01

    Full Text Available Platelets are small anucleate blood cells derived from megakaryocytes. In addition to their pivotal roles in hemostasis, platelets are the smallest, yet most abundant, immune cell and regulate inflammation, immunity, and disease progression. Although platelets lack DNA, and thus no functional transcriptional activities, they are nonetheless rich sources of RNAs, possess an intact spliceosome, and are thus capable of synthesizing proteins. Previously, it was thought that platelet RNAs and translational machinery were remnants from the megakaryocyte. We now know that the initial description of platelets as cellular fragments is an antiquated notion, as mounting evidence suggests otherwise. Therefore, it is reasonable to hypothesize that platelet transcription factors are not vestigial remnants from megakaryoctes, but have important, if only partly understood functions. Proteins play multiple cellular roles to minimize energy expenditure for maximum cellular function; thus, the same can be expected for transcription factors. In fact, numerous transcription factors have non-genomic roles, both in platelets and in nucleated cells. Our lab and others have discovered the presence and nongenomic roles of transcription factors in platelets, such as the nuclear factor kappa β (NFκB family of proteins and peroxisome proliferator activated receptor gamma (PPARγ. In addition to numerous roles in regulating platelet activation, functional transcription factors can be transferred to vascular and immune cells through platelet microparticles. This method of transcellular delivery of key immune molecules may be a vital mechanism by which platelet transcription factors regulate inflammation and immunity. At the very least, platelets are an ideal model cell to dissect out the nongenomic roles of transcription factors in nucleated cells. There is abundant evidence to suggest that transcription factors in platelets play key roles in regulating inflammatory and

  5. Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA

    Directory of Open Access Journals (Sweden)

    Malathi Narayan

    2016-06-01

    Full Text Available Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA on a mouse microglial (N9 cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier http://www.ebi.ac.uk/pride/archive/projects/PXD003032.

  6. Vaccination with melanoma lysate-pulsed dendritic cells, of patients with advanced colorectal carcinoma: report from a phase I study

    DEFF Research Database (Denmark)

    Burgdorf, S K; Fischer, A; Claesson, M H;

    2006-01-01

    Immune therapy have shown new and exciting perspectives for cancer treatment. Aim of our study was to evaluate toxicity and possible adverse effects from vaccination of patients with advanced colorectal cancer with autologous dendritic cells (DC) pulsed with lysate from a newly developed melanoma...... and selected melanoma cell line enriched in expression of MAGE-A antigens and deficient in expression of melanoma differentiation antigens: tyrosinase, MART-1 and gp100. Vaccinations were administered intradermally on the proximal thigh with a total of five given vaccines at 2 weeks intervals. Each vaccine...... contained 3-5 x 10(6) DCs. Five of the six patients received all five vaccines. The treatment was well tolerated in all patients without any observed vaccine-correlated adverse effects. Treatment with this DC-based cancer vaccine proved safe and non-toxic....

  7. Detection of gram-negative bacteremia by limulus amebocyte lysate assay: evaluation in a rat model of peritonitis.

    Science.gov (United States)

    du Moulin, G C; Lynch, S E; Hedley-Whyte, J; Broitman, S A

    1985-01-01

    A spectrophotometric Limulus amebocyte lysate assay using lysis filtration and centrifugation has been developed for the detection of gram-negative bacteria in blood. The assay is directed at detection of endotoxin in viable and nonviable bacteria present in the blood-stream and not detection of free endotoxin in plasma. The assay was evaluated in a model of peritonitis in which rats were challenged with an inoculum consisting of sterilized human feces, barium sulfate, and one of eight species of bacteria. This assay was able to detect gram-negative bacteremia due to Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Proteus mirabilis, and Klebsiella pneumoniae in the rat model when compared with sham-inoculated uninfected rats. The assay failed to detect bacteremia due to Bacteroides fragilis or Staphylococcus aureus, nor was there a significant rise in absorbance when a pellet containing sterilized feces was implanted in the rat.

  8. Platelet-TLR7 mediates host survival and platelet count during viral infection in the absence of platelet-dependent thrombosis.

    Science.gov (United States)

    Koupenova, Milka; Vitseva, Olga; MacKay, Christopher R; Beaulieu, Lea M; Benjamin, Emelia J; Mick, Eric; Kurt-Jones, Evelyn A; Ravid, Katya; Freedman, Jane E

    2014-07-31

    Viral infections have been associated with reduced platelet counts, the biological significance of which has remained elusive. Here, we show that infection with encephalomyocarditis virus (EMCV) rapidly reduces platelet count, and this response is attributed to platelet Toll-like receptor 7 (TLR7). Platelet-TLR7 stimulation mediates formation of large platelet-neutrophil aggregates, both in mouse and human blood. Intriguingly, this process results in internalization of platelet CD41-fragments by neutrophils, as assessed biochemically and visualized by microscopy, with no influence on platelet prothrombotic properties. The mechanism includes TLR7-mediated platelet granule release, translocation of P-selectin to the cell surface, and a consequent increase in platelet-neutrophil adhesion. Viral infection of platelet-depleted mice also led to increased mortality. Transfusion of wild-type, TLR7-expressing platelets into TLR7-deficient mice caused a drop in platelet count and increased survival post EMCV infection. Thus, this study identifies a new link between platelets and their response to single-stranded RNA viruses that involves activation of TLR7. Finally, platelet-TLR7 stimulation is independent of thrombosis and has implications to the host immune response and survival.

  9. Platelet-like hexagonal SrFe12O19 particles: Hydrothermal synthesis and their orientation in a magnetic field

    Science.gov (United States)

    Zhang, Tao; Peng, Xiaoling; Li, Jing; Yang, Yanting; Xu, Jingcai; Wang, Panfeng; Jin, Dingfeng; Jin, Hongxiao; Hong, Bo; Wang, Xinqing; Ge, Hongliang

    2016-08-01

    Platelet-like hexagonal SrFe12O19 particles were prepared by hydrothermal synthesis, and the effects of molar ratio of Fe/Sr (RFe/Sr) on the phase compositions, morphologies and magnetic properties of as-prepared samples were investigated. The optimum RFe/Sr is identified as 8:1. The hexagonal platelet-like particles are nano-scale in thickness and micro-scale in diameter. The low coercivity is a consequence of the large shape anisotropy of the as-synthesized particles. The platelet-like hexagonal SrFe12O19 particles were then dispersed in epoxy resin and formed ordered arrangement structure which took root in the curing epoxy matrix under an external magnetic field of 8000 Oe. The microstructures, morphologies and magnetic properties of the bulk samples orientated and nonaligned were studied. The platelet-like particles arrange with the platelet perpendicular to the magnetic field direction in the orientated samples. This demonstrates that the easy axis of the particle is perpendicular to the platelet, and that the magnetocrystalline anisotropy still plays a leading role in the changing effective anisotropy with the rapidly growing shape anisotropy. The remanence (Mr) of the bulk samples is changed obviously after orientation, while the coercivity nearly remains constant. That is, the maximum energy products (BH)max can be effectively adjusted by given a suitable magnetic field.

  10. Blood platelets in the progression of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Nina S Gowert

    Full Text Available Alzheimer's disease (AD is characterized by neurotoxic amyloid-ß plaque formation in brain parenchyma and cerebral blood vessels known as cerebral amyloid angiopathy (CAA. Besides CAA, AD is strongly related to vascular diseases such as stroke and atherosclerosis. Cerebrovascular dysfunction occurs in AD patients leading to alterations in blood flow that might play an important role in AD pathology with neuronal loss and memory deficits. Platelets are the major players in hemostasis and thrombosis, but are also involved in neuroinflammatory diseases like AD. For many years, platelets were accepted as peripheral model to study the pathophysiology of AD because platelets display the enzymatic activities to generate amyloid-ß (Aß peptides. In addition, platelets are considered to be a biomarker for early diagnosis of AD. Effects of Aß peptides on platelets and the impact of platelets in the progression of AD remained, however, ill-defined. The present study explored the cellular mechanisms triggered by Aß in platelets. Treatment of platelets with Aß led to platelet activation and enhanced generation of reactive oxygen species (ROS and membrane scrambling, suggesting enhanced platelet apoptosis. More important, platelets modulate soluble Aß into fibrillar structures that were absorbed by apoptotic but not vital platelets. This together with enhanced platelet adhesion under flow ex vivo and in vivo and platelet accumulation at amyloid deposits of cerebral vessels of AD transgenic mice suggested that platelets are major contributors of CAA inducing platelet thrombus formation at vascular amyloid plaques leading to vessel occlusion critical for cerebrovascular events like stroke.

  11. Brief Report: Platelet-Poor Plasma Serotonin in Autism

    Science.gov (United States)

    Anderson, George M.; Hertzig, Margaret E.; McBride, P. A.

    2012-01-01

    Possible explanations for the well-replicated platelet hyperserotonemia of autism include an alteration in the platelet's handling of serotonin (5-hydroxyserotonin, 5-HT) or an increased exposure of the platelet to 5-HT. Measurement of platelet-poor plasma (PPP) levels of 5-HT appears to provide the best available index of in vivo exposure of the…

  12. 21 CFR 864.5700 - Automated platelet aggregation system.

    Science.gov (United States)

    2010-04-01

    ... addition of an aggregating reagent to a platelet-rich plasma. (b) Classification. Class II (performance... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Automated platelet aggregation system. 864.5700... § 864.5700 Automated platelet aggregation system. (a) Identification. An automated platelet...

  13. Platelet thrombosis in cardiac-valve prostheses

    Energy Technology Data Exchange (ETDEWEB)

    Dewanjee, M.K.

    1989-01-01

    The contribution of platelets and clotting factors in thrombosis on cardiovascular prostheses had been quantified with several tracers. Thrombus formation in vivo could be measured semiquantitatively in animal models and patients with indium-111, Technetium-99m labeled platelets, iodine-123, iodine-131 labeled fibrinogen, and In-111 and Tc-99m labeled antibody to the fibrinogen-receptor on the platelet- membrane, or fibrin. The early studies demonstrated that certain platelet-inhibitors, e.g. sulfinpyrazone, aspirin or aspirin- persantine increased platelet survival time with mechanical valves implanted in the baboon model and patients. Thrombus localization by imaging is possible for large thrombus on thrombogenic surface of prosthesis in the acute phase. The majority of thrombus was found in the sewing ring (Dacron) in the acute phase in both the mechanical and tissue valves. The amount of retained thrombus in both mechanical and tissue valves in our one-day study in the dog model was similar (< 1% if injected In-111 platelets = 5 billion platelets). As the fibrous ingrowth covered the sewing ring, the thrombus formation decreased significantly. Only a small amount of thrombus was found on the leaflets at one month in both the dog and calf models. 38 refs., 9 figs., 5 tabs.

  14. Changes in platelet parameters in leukocytosis.

    Science.gov (United States)

    Ozturk, Nurinnisa; Baygutalp, Nurcan Kilic; Bakan, Ebubekir; Altas, Gulsum Feyza; Polat, Harun; Dorman, Emrullah

    2016-01-01

    In recent years, platelets are known to have a large variety of functions in many pathophysiological processes and their interaction with endothelial cells and leukocytes is known to play an important role in the pathophysiology of vascular inflammation. The aim of this study was to investigate the relationship between white blood cell count in conditions resulting in leukocytosis and platelet count and platelet parameters including mean platelet volume, platelet distribution width, and plateletcrit. White blood cell counts count and all platelet parameters were evaluated in 341 results of normal complete blood count (of which the white blood cell counts were within reference range, group 1) and 327 results of elevated white blood cell counts count (group 2). There was a significant difference between these two groups in PLT counts and PCT values, being higher in Group 2. However, there was no statistically significant difference between two groups in MPV and PDW values. On the other hand, there were statistically significant, but weak, correlations between the WBC and platelet counts in both groups (p<0.01, r=0.235 for group 1, p<0.05, r=0.116 for group 2). As a conclusion PLT count and PCT values increase in infectious conditions. This study and previous studies show that PLTs are employed in infectious conditions but the exact mechanism and the exact clinical importance of this response remains to be cleared by further studies.

  15. Purification of plasmid DNA from clarified and non-clarified Escherichia coli lysates by berenil pseudo-affinity chromatography.

    Science.gov (United States)

    Caramelo-Nunes, C; Gabriel, M F; Almeida, P; Marcos, J C; Tomaz, C T

    2012-09-01

    In this study, berenil was tested as a ligand, specifically to purify plasmids of different sizes pVAX1-LacZ (6.05 Kbp) and pCAMBIA-1303 (12.361 Kbp) from clarified Escherichia coli alkaline lysates. For this purpose, chromatographic experiments were performed using Sepharose derivatized with berenil. The results showed that both pDNA molecules are completely purified using lower amounts of salt in the eluent than those previously reported for other pseudo-affinity and hydrophobic interaction chromatography based processes. Total retention of all lysate components was achieved with 1.3M ammonium sulphate in the eluent buffer and pDNA elution was obtained by decreasing the salt concentration to 0.55 M. All impurities were eluted after decreasing the concentration to 0M. The recovery yield for pCAMBIA-1303 (45%) was lower than that obtained for pVAX1-LacZ (85%), however the larger pDNA showed a higher purity level. Purification of pVAX1-LacZ was also performed using non-clarified E. coli process streams, replacing the clarification step with a second chromatographic run on the berenil-Sepharose. Using the same binding and elution conditions as before, a pure plasmid sample was obtained with a 33% yield and with all host impurity levels in accordance with the requirements established by the regulatory agencies. These results suggest that this chromatographic method is a promising alternative to purify pDNA for therapeutic use. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. (/sup 125/I) radioiodinated metaraminol: A new platelet-specific labeling agent

    Energy Technology Data Exchange (ETDEWEB)

    Ohmomo, Y.; Yokoyama, A.; Kawai, K.; Arano, Y.; Horiuchi, K.; Tanaka, C.; Saji, H.; Torizuka, K.

    1985-03-01

    In our search for a platelet-specific labeling agent, metaraminol (MA), a low-toxic pharmaceutical for the treatment of hypotension and cardiogenic shock, attracted our attention. Its active incorporation and accumulation by platelets have been recognized. At first, the preparation of /sup 125/I radioiodinated metaraminol (/sup 125/I-MA) was carried out using the chloramine-T method. Then, upon the harvest of platelets as platelet-rich plasma (PRP), their labeling with this new radiopharmaceutical was easily performed by incubation for 10 min at 37/sup 0/C. The cell-labeling efficiency was dependent on cell density, reaching 63.0%+-3.1% at 2.4x10/sup 9/ cells/ml. The specific incorporation of /sup 125/I-MA by an active transport system similar to that of 5-hydroxytryptamine (5-HT) as well as by passive diffusion was demonstrated. In vitro studies, the unaltered state of /sup 125/I-MA-labeled platelets with their cellular functions fully retained was estimated. In vivo studies carried out in rabbits with induced thrombi in the femoral artery showed a rather rapid disappearance of the radioactivity from circulating blood, reaching a high thrombus-to-blood activity ratio of 19.8+-4.3 within 30 min of the administration of /sup 125/I-MA-labeled autologous platelets. Thus, with the potential availability of /sup 123/I, /sup 123/I-MA-labeled platelets appear to be a promising agent for thrombus imaging using single-emission computed tomography (CT) studies.

  17. Uniform patchy and hollow rectangular platelet micelles from crystallizable polymer blends

    Science.gov (United States)

    Qiu, Huibin; Gao, Yang; Boott, Charlotte E.; Gould, Oliver E. C.; Harniman, Robert L.; Miles, Mervyn J.; Webb, Stephen E. D.; Winnik, Mitchell A.; Manners, Ian

    2016-05-01

    The preparation of colloidally stable, self-assembled materials with tailorable solid or hollow two-dimensional (2D) structures represents a major challenge. We describe the formation of uniform, monodisperse rectangular platelet micelles of controlled size by means of seeded-growth methods that involve the addition of blends of crystalline-coil block copolymers and the corresponding crystalline homopolymer to cylindrical micelle seeds. Sequential addition of different blends yields solid platelet block comicelles with concentric rectangular patches with distinct coronal chemistries. These complex nano-objects can be subject to spatially selective processing that allows their disassembly to form perforated platelets, such as well-defined hollow rectangular rings. The solid and hollow 2D micelles provide a tunable platform for further functionalization and potential for a variety of applications.

  18. Homogeneous precipitation of α-phase Co-Ni hydroxides hexagonal platelets

    Institute of Scientific and Technical Information of China (English)

    Juanjuan Liu; Xuyang Wang; Xiayin Yao; Jun wang; Zhaoping Liu

    2012-01-01

    This paper describes the synthesis of α-phase Co-Ni hydroxides hexagonal platelets through homogeneous precipitation,using hexamethylenetetramine (HMT) or urea as a hydrolytic agent.In the CoCl2-NiCl2-HMT system,pure α-phase can be synthesized at the concentrations of both metal ions higher than 20 mM,while in the CoCl2-NiCl2-urea system,the formation of pure α-phase is independent of the concentrations of the metal ions.When using HMT,monodisperse hexagonal platelets of α-phase Co-Ni hydroxides can be produced in the presence of polyvinylpyrrolidone (PVP).Cyclic voltammogram curve of the hexagonal platelets prepared with HMT demonstrates electrochemical performance superior to that of urea.

  19. Analysis of aggregation of platelets in thrombosis

    Science.gov (United States)

    Ahuja, Suresh

    Platelets are key players in thrombus formation by first rolling over collagen bound von Willebrand factor followed by formation of a stable interaction with collagen. The first adhered platelets bind additional platelets until the whole injury is sealed off by a platelet aggregate. The coagulation system stabilizes the formed platelet plug by creating a tight fibrin network, and then wound contraction takes place because of morphological changes in platelets. Coagulation takes place by platelet activation and aggregation mainly through fibrinogen polymerization into fibrin fibers. The process includes multiple factors, such as thrombin, plasmin, and local shear-rate which regulate and control the process. Coagulation can be divided into two pathways: the intrinsic pathway and the extrinsic pathway. The intrinsic pathway is initiated by the exposure of a negatively charged. It is able to activate factor XII, using a complex reaction that includes prekallikrein and high-molecular-weight kininogen as cofactors.. Thrombin is the final enzyme that is needed to convert fibrinogen into fibrin. The extrinsic pathway starts with the exposure of tissue factor to the circulating blood, which is the major initiator of coagulation. There are several feedback loops that reinforce the coagulation cascade, resulting in large amounts of thrombin. It is dependent on the presence of pro-coagulant surfaces of cells expressing negatively charged phospholipids--which include phosphatidylserine (PS)--on their outer membrane. PS-bearing surfaces are able to increase the efficiency of the reactions by concentrating and co-localizing coagulation factors.. Aggregation of platelets are analyzed and compared to adhesion of platelet to erythrocyte and to endothelial cells. This abstract is replacing MAR16-2015-020003.

  20. Evaluation of platelet function using multiple electrode platelet aggregometry in dogs with septic peritonitis.

    Science.gov (United States)

    Li, Ronald H L; Chan, Daniel L

    2016-09-01

    To assess platelet function via multiple electrode platelet aggregometry (MEPA) in dogs with septic peritonitis and in healthy dogs. The secondary aim was to determine if there is prognostic significance to changes in platelet function observed in septic dogs. Prospective, observational cohort study conducted from January 2012 to March 2014. University teaching hospital. Twenty dogs with septic peritonitis and 23 healthy dogs. None. MEPA using arachidonic acid, adenosine diphosphate, and collagen (COL) as agonists was measured within 24 hours of diagnosis of sepsis. Compared to healthy dogs, platelet aggregation was reduced in dogs with septic peritonitis for all agonists (P peritonitis. Circulating platelets from dogs with septic peritonitis have diminished aggregation in response to multiple platelet agonists. MEPA may serve as an assessment tool for illness severity in this patient population. © Veterinary Emergency and Critical Care Society 2016.

  1. [Glycoproteins, inherited diseases of platelets, and the role of platelets in wound healing].

    Science.gov (United States)

    Nurden, Alan T; Nurden, Paquita

    2013-02-01

    Recognition that platelets have a glycocalyx rich in membrane glycoproteins prompted the discovery in France that inherited bleeding syndromes due to defects of platelet adhesion and aggregation were caused by deficiencies in major receptors at the platelet surface. Identification of the alpha IIb beta3 integrin prompted the development of powerful anti-thrombotic drugs that have gained worldwide use. Since these discoveries, the genetic causes of many other defects of platelet function and production have been elucidated, with the identification of an ADP receptor, P2 Y12, another widespread target for anti-thrombotic drugs. Discovery of the molecular basis of a rare disease of storage of biologically active proteins in platelet alpha-granules has been accompanied by the recognition of the roles of platelets in inflammation, the innate immune system and tissue repair, opening new avenues for therapeutic advances.

  2. Decrease in platelet activating factor stimulated phosphoinositide turnover during storage of human platelets in plasma

    Energy Technology Data Exchange (ETDEWEB)

    Carter, M.G.; Shukla, S.D. (Univ. of Missouri School of Medicine, Columbia (USA))

    1987-05-01

    Human platelet concentrate from the American Red Cross Blood Center was stored at 24{degree}C in a shaker and aliquots were taken out at time intervals aseptically. Platelet activating factor (PAF) stimulated turnover of phosphoinositide (PPI) was monitored by assaying {sup 32}P incorporation into phosphoinositides using platelet rich plasma (PRP). Platelets in PRP were incubated with 1 {times} 10{sup {minus}7} M PAF at 37{degree}C with gentle shaking and after 5 min their lipids were extracted and analysed by TLC for {sup 32}P-phosphoinositides. The percent stimulation of {sup 32}P incorporation by PAF (over control) into PPI was approximately 250, 100, 60, 25 and 20 on days 1, 2, 3, 5 and 6, respectively. This indicated a dramatic decrease in PAF responsive turnover of platelet PPI during storage. These findings have important implications in relation to PAF receptor activity and viability of platelets at different periods of storage.

  3. Platelets: at the nexus of antimicrobial defence.

    Science.gov (United States)

    Yeaman, Michael R

    2014-06-01

    Platelets have traditionally been viewed as fragmentary mediators of coagulation. However, recent molecular and cellular evidence suggests that they have multiple roles in host defence against infection. From first-responders that detect pathogens and rapidly deploy host-defence peptides, to beacons that recruit and enhance leukocyte functions in the context of infection, to liaisons that facilitate the T cell-B cell crosstalk that is required in adaptive immunity, platelets represent a nexus at the intersection of haemostasis and antimicrobial host defence. In this Review, I consider recent insights into the antimicrobial roles of platelets, which are mediated both directly and indirectly to integrate innate and adaptive immune responses to pathogens.

  4. Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia.

    Science.gov (United States)

    Żmigrodzka, M; Guzera, M; Winnicka, A

    2016-01-01

    Platelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/l and group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogs in vitro varied depending on the platelet count and platelet activator.

  5. Identification of canine platelet proteins separated by differential detergent fractionation for nonelectrophoretic proteomics analyzed by Gene Ontology and pathways analysis

    Directory of Open Access Journals (Sweden)

    Trichler SA

    2014-01-01

    Full Text Available Shauna A Trichler,1,* Sandra C Bulla,1,* Nandita Mahajan,1 Kari V Lunsford,2 Ken Pendarvis,3 Bindu Nanduri,4,5 Fiona M McCarthy,3 Camilo Bulla1 1Department of Pathobiology and Population Medicine, 2Department of Clinical Sciences and Animal Health Center, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, 3Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ, 4Department of Biological Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, 5Institute for Genomics, Biocomputing and Biotechnology, Starkville, MS, USA *These authors contributed equally to this work Abstract: During platelet development, proteins necessary for the many functional roles of the platelet are stored within cytoplasmic granules. Platelets have also been shown to take up and store many plasma proteins into granules. This makes the platelet a potential novel source of biomarkers for many disease states. Approaches to sample preparation for proteomic studies for biomarkers search vary. Compared with traditional two-dimensional polyacrylamide gel electrophoresis systems, nonelectrophoretic proteomics methods that employ offline protein fractionation methods such as the differential detergent fractionation method have clear advantages. Here we report a proteomic survey of the canine platelet proteome using differential detergent fractionation coupled with mass spectrometry and functional modeling of the canine platelet proteins identified. A total of 5,974 unique proteins were identified from platelets, of which only 298 (5% had previous experimental evidence of in vivo expression. The use of offline prefractionation of canine proteins by differential detergent fractionation resulted in greater proteome coverage as compared with previous reports. This initial study contributes to a broader understanding of canine platelet biology and aids functional research

  6. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

    Science.gov (United States)

    Chou, Ming-Li; Burnouf, Thierry; Wang, Tsung-Jen

    2014-01-01

    Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml) and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml) neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices and regulatory

  7. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

    Directory of Open Access Journals (Sweden)

    Ming-Li Chou

    Full Text Available Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices

  8. Experience of buffy coat pooling of platelets as a supportive care in thrombocytopenic dengue patients: A prospective study

    Directory of Open Access Journals (Sweden)

    Kabita Chatterjee

    2014-01-01

    Full Text Available Random donor platelet (RDP is not sufficient to improve the platelet count in most thrombocytopenic patients. Single donor platelet (SDP or buffy coat pooled platelet (BCPP are the two choices to provide a full therapeutic dose of platelets. However, there are constraints in the preparation of SDP due to stringent donor selection procedure, time required for procedure, and need of special expensive equipments and kits. BCPP is widely practiced, especially in the European countries, since 1995. In India, we decided to adopt the procedure of buffy coat pooling of platelets, especially for economically backward patients and for emergencies. This study was prospectively conducted from September 2009 to September 2010. A total of 129 units of BCPP [tested prior for viral markers by enzyme-linked immunosorbent assay (ELISA and individual donor nucleic acid amplification test (ID-NAT] were issued to 129 patients suffering from dengue and were included in this study. For comparison between efficacy of SDP and BCCP, patients were divided into two groups of 50 each. The post-transfusion platelet counts of the patients were noted after 2 hours of transfusion for each type of component. The platelet yield varied from 2.5 to 4.4 Χ 10ΉΉ in BCPP samples. The samples analyzed were sterile without any contamination. The different biochemical parameters were analyzed in detail. The observed post-transfusion platelet recovery and corrected count increment (CCI at 1 hour and 24 hours after BCPP transfusion were similar to that after SDP transfusion. Hence, we concluded that BCPP can be a low cost alternative to SDP in the times of emergencies like dengue and non-affordability by the patient for SDP.

  9. The Effect of Ginger (Zingiber officinale on Platelet Aggregation: A Systematic Literature Review.

    Directory of Open Access Journals (Sweden)

    Wolfgang Marx

    Full Text Available The potential effect of ginger on platelet aggregation is a widely-cited concern both within the published literature and to clinicians; however, there has been no systematic appraisal of the evidence to date.Using the PRISMA guidelines, we systematically reviewed the results of clinical and observational trials regarding the effect of ginger on platelet aggregation in adults compared to either placebo or baseline data. Studies included in this review stipulated the independent variable was a ginger preparation or isolated ginger compound, and used measures of platelet aggregation as the primary outcome.Ten studies were included, comprising eight clinical trials and two observational studies. Of the eight clinical trials, four reported that ginger reduced platelet aggregation, while the remaining four reported no effect. The two observational studies also reported mixed findings.Many of the studies appraised for this review had moderate risks of bias. Methodology varied considerably between studies, notably the timeframe studied, dose of ginger used, and the characteristics of subjects recruited (e.g. healthy vs. patients with chronic diseases.The evidence that ginger affects platelet aggregation and coagulation is equivocal and further study is needed to definitively address this question.

  10. The Pasteur effect in human platelets: implications for storage and metabolic control.

    Science.gov (United States)

    Guppy, M; Abas, L; Arthur, P G; Whisson, M E

    1995-11-01

    The Pasteur effect and the associated acidosis have long been considered a major cause of platelet death during storage. We have investigated this phenomenon using a defined platelet preparation and a system whereby the oxidative and glycolytic contributions to total ATP production can be measured over a range of oxygen concentrations from saturating (pO2 = 158 mmHg) to anoxic (pO2 = 0 mmHg). Platelets do not show a Pasteur effect until the pO2 decreases to Pasteur effect is therefore not a likely cause of platelet death during storage where pO2 in a storage bag typically drops to no less than 50 mmHg. The data also have implications for the role of oxygen diffusion in oxidative metabolism, and for the compensatory nature of the Pasteur effect. As platelets are relatively small cells, and the onset of the Pasteur effect occurs at a relatively low oxygen concentration, diffusion may limit the rate of oxygen consumption in most other (larger) cells. The Pasteur effect is only fully compensative if the P/O2 ratio used for the calculations is lower than the conventional one. Since recent research strongly suggests that the conventional P/O2 ratio is too high, examples of fully compensative Pasteur effects may be more common than the literature suggests.

  11. Pathogen-Reduced, Extended Platelet Storage in Platelet Additive Solution (PAS)

    Science.gov (United States)

    2016-10-01

    concentrations will be performed to ensure the desired concentration was achieved. Platelet Additive Solutions are isotonic solutions used to replace a...Sherrill J. Slichter, MD CONTRACTING ORGANIZATION: Bloodworks Northwest Seattle, WA 98104 REPORT DATE: October 2016 TYPE OF REPORT: Annual...TITLE AND SUBTITLE Pathogen-Reduced, Extended Platelet Storage in Platelet Additive Solution (PAS) 5a. CONTRACT NUMBER W81XWH-12-1-0441 5b. GRANT

  12. Xanthohumol, a Prenylated Flavonoid from Hops (Humulus lupulus), Prevents Platelet Activation in Human Platelets

    OpenAIRE

    Ye-Ming Lee; Kuo-Hsien Hsieh; Wan-Jung Lu; Hsiu-Chu Chou; Duen-Suey Chou; Li-Ming Lien; Joen-Rong Sheu; Kuan-Hung Lin

    2012-01-01

    Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulus L.). Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation...

  13. Revascularization of Immature Necrotic Teeth: Platelet rich Fibrin an Edge over Platelet rich Plasma

    OpenAIRE

    Neelam Mittal; Isha Narang

    2012-01-01

    Introduction: Revascularization is one such entity that has found its clinical application in the field of endodontics for the manage-ment of immature permanent necrotic teeth. The protocols for revascularization of such teeth focus especially on delivery of stem cells and scaffolds in a nonsurgical manner rather than concentrated growth micro molecules.The hypothesis: This article proposes the role of platelet concentrates such as platelet rich fibrin (PRF) and platelet rich plasma (PRP) in ...

  14. Platelet lipidomics: a modern day perspective on lipid discovery and characterization in platelets

    OpenAIRE

    O’Donnell, Valerie B.; Murphy, Robert C.; Watson, Steve P.

    2014-01-01

    Lipids are diverse families of biomolecules that perform essential structural and signaling roles in platelets. Their formation and metabolism is tightly controlled by enzymes and signal transduction pathways, and their dysregulation leads to significant defects in platelet function and disease. Platelet activation is associated with significant changes to membrane lipids, and formation of diverse bioactive lipids that play essential roles in hemostasis. In recent years, new generation mass s...

  15. Platelet interaction with bacterial toxins and secreted products.

    Science.gov (United States)

    Shannon, Oonagh

    2015-01-01

    Bacteria that enter the bloodstream will encounter components of the cellular and soluble immune response. Platelets contribute to this response and have emerged as an important target for bacterial pathogens. Bacteria produce diverse extracellular proteins and toxins that have been reported to modulate platelet function. These interactions can result in complete or incomplete platelet activation or inhibition of platelet activation, depending on the bacteria and bacterial product. The nature of the platelet response may be highly relevant to disease pathogenesis.

  16. Platelet-rich plasma regenerative medicine sports medicine, orthopedic, and recovery of musculoskeletal injuries

    CERN Document Server

    Santana, Maria; Belangero, William; Luzo, Angela

    2014-01-01

    Platelet-Rich Plasma (PRP) has gained tremendous popularity in recent years as a treatment option for specialties including Orthopedics, Dentistry, Sports Medicine, Otorhinolaryngology, Neurosurgery, Ophthalmology, Urology, Vascular, Cardiothoracic and Maxillofacial Surgery, and Veterinarian Medicine. Nowadays, PRP and Stem Cell Science have added an exciting dimension to tissue repair. This book begins by giving the reader a broad overview of current progress as well as a discussion of the technical aspects of preparation and therapeutic use of autologous PRP. It is followed by a review of platelet structure, function and major growth factors in PRP (PDGF and TGFβ).The third chapter outlines the basic principles of biochemical cellular metabolism that increases the efficacy of PRP. Analogous to the preparation of soil for a garden, restoring cellular health should be the first consideration in Regenerative Medicine. Standardization of PRP preparation to clinical use still remains a challenging prospect. In ...

  17. Evaluation of platelet thromboxane radioimmunoassay method to measure platelet life-span: Comparison with /sup 111/indium-platelet method

    Energy Technology Data Exchange (ETDEWEB)

    Vallabhajosula, S.; Machac, J.; Badimon, L.; Lipszyc, H.; Goldsmith, S.J.; Fuster, V.

    1985-05-01

    The platelet activation during radiolabeling in vitro with Cr-51 and In-111 may affect the platelet life-span (PLS) in vivo. A new RIA method to measure PLS is being evaluated. Aspirin inhibits platelet thromboxane (TxA/sub 2/) by acetylating cyclooxygenase. The time required for the TxA/sub 2/ levels to return towards control values depends on the rate of new platelets entering circulation and is a measure of PLS. A single dose of aspirin (150mg) was given to 5 normal human subjects. Blood samples were collected for 2 days before aspirin and daily for 10 days. TxA/sub 2/ production in response to endogenous thrombin was studied by allowing 1 ml blood sample to clot at 37/sup 0/C for 90 min. Serum TxB/sub 2/ (stable breakdown product of Tx-A/sub 2/) levels determined by RIA technique. The plot of TxB/sub 2/ levels (% control) against time showed a gradual increase. The PLS calculated by linear regression analysis assuming a 2-day lag period before cyclooxygenase recovery is 9.7 +- 2.37. In the same 5 subjects, platelets from a 50ml blood sample were labeled with /sup 111/In-tropolone in 2 ml autologous plasma. Starting at 1 hr after injection of labeled platelets, 10 blood samples were obtained over a 8 day period. The PLS calculated based on a linear regression analysis is 10.2 +. 1.4. The PLS measured from the rate of platelet disappearance from circulation and the rate of platelet regeneration into circulation are quite comparable in normal subjects. TxA/sub 2/ regeneration RIA may provide a method to measure PLS without administering radioactivity to patient.

  18. Thrombopoietin induces p-selectin expression on platelets and subsequent platelet/leukocyte interactions.

    Science.gov (United States)

    Tibbles, Heather E; Navara, Christopher S; Hupke, Michael A; Vassilev, Alexei O; Uckun, Fatih M

    2002-04-12

    Ligation of thrombopoietin (TPO) to the platelet c-Mpl receptor induces numerous biochemical pathways in the absence of aggregation. Two forms of recombinant TPO are currently in clinical trials for the treatment of thrombocytopenia. This study focuses on the effects of the full-length recombinant human TPO (rhTPO) on platelets in a whole blood system. Platelet-leukocyte associations (PLAs) were visualized following rhTPO stimulation as CD42b/CD 45 double positive clusters by FACS analysis. Treatment of washed platelets with rhTPO induced granule release and expression of the leukocyte adhesion receptor P-selectin (CD 62P) in the absence of aggregation and calcium mobilization. RhTPO also induced platelet-leukocyte interactions in whole blood. Following stimulation, leukocytes were recruited by platelets through P-selectin in a calcium-dependent manner. rhTPO stimulates platelet-leukocyte associations in whole blood through expression of platelet P-selectin. To our knowledge, this is the first report that identifies TPO as a promoter of platelet-leukocyte interactions.

  19. Platelets and erythrocyte-bound platelets bind infectious HIV-1 in plasma of chronically infected patients.

    Science.gov (United States)

    Beck, Zoltan; Jagodzinski, Linda L; Eller, Michael A; Thelian, Doris; Matyas, Gary R; Kunz, Anjali N; Alving, Carl R

    2013-01-01

    Chronic HIV-1 infection is associated with persistent viremia in most patients, but it remains unclear how free virus may survive the potential hostile effects of plasma. We investigated whether sites might exist on the surfaces of circulating blood cells for protection of infectious HIV-1 particles. Red blood cells (RBC) either from blood of uninfected normal individuals, or from blood obtained without EDTA from chronically infected HIV-1 patients, invariably contained a small number of RBC having attached platelets as determined by flow cytometry, light microscopy, and immunofluorescence microscopy. After mixing normal RBC with platelet-rich plasma, discrete populations of RBC, platelets, and complexes of platelets attached to RBC were purified by fluorescence-activated cell sorting. Upon incubation of purified cells or platelets with HIV-1 followed by washing and co-incubation with CD4-positive peripheral blood mononuclear cells (PBMC), platelets, and platelet-RBC complexes, but not platelet-free RBC, caused infection of PBMC. Infection was prevented by pre-treating the platelet-RBC complexes with EDTA. Plasma and RBC (comprising a RBC/platelet-RBC mixture) from chronically infected patients with low viral loads were also co-incubated with PBMC ex vivo to determine the presence of infectious HIV-1. All freshly isolated plasmas from the HIV-1-infected donors, obtained in the absence of anticoagulant, were noninfectious. Interestingly, the RBC from most of the patients caused cell-cell infection of PBMC that was prevented by stripping the RBC with EDTA. A monoclonal antibody to DC-SIGN partially inhibited cell-cell HIV-1 infection of PBMC by normal RBC pre-incubated with platelets and HIV-1. We conclude: (a) platelet-free EDTA-free plasma from chronically infected HIV-1 patients, although containing viral RNA, is an environment that lacks detectable infectious HIV-1; (b) platelets and platelet-RBC complexes, but not purified RBC, bind infectious HIV-1; (c) DC

  20. Platelets and erythrocyte-bound platelets bind infectious HIV-1 in plasma of chronically infected patients.

    Directory of Open Access Journals (Sweden)

    Zoltan Beck

    Full Text Available Chronic HIV-1 infection is associated with persistent viremia in most patients, but it remains unclear how free virus may survive the potential hostile effects of plasma. We investigated whether sites might exist on the surfaces of circulating blood cells for protection of infectious HIV-1 particles. Red blood cells (RBC either from blood of uninfected normal individuals, or from blood obtained without EDTA from chronically infected HIV-1 patients, invariably contained a small number of RBC having attached platelets as determined by flow cytometry, light microscopy, and immunofluorescence microscopy. After mixing normal RBC with platelet-rich plasma, discrete populations of RBC, platelets, and complexes of platelets attached to RBC were purified by fluorescence-activated cell sorting. Upon incubation of purified cells or platelets with HIV-1 followed by washing and co-incubation with CD4-positive peripheral blood mononuclear cells (PBMC, platelets, and platelet-RBC complexes, but not platelet-free RBC, caused infection of PBMC. Infection was prevented by pre-treating the platelet-RBC complexes with EDTA. Plasma and RBC (comprising a RBC/platelet-RBC mixture from chronically infected patients with low viral loads were also co-incubated with PBMC ex vivo to determine the presence of infectious HIV-1. All freshly isolated plasmas from the HIV-1-infected donors, obtained in the absence of anticoagulant, were noninfectious. Interestingly, the RBC from most of the patients caused cell-cell infection of PBMC that was prevented by stripping the RBC with EDTA. A monoclonal antibody to DC-SIGN partially inhibited cell-cell HIV-1 infection of PBMC by normal RBC pre-incubated with platelets and HIV-1. We conclude: (a platelet-free EDTA-free plasma from chronically infected HIV-1 patients, although containing viral RNA, is an environment that lacks detectable infectious HIV-1; (b platelets and platelet-RBC complexes, but not purified RBC, bind infectious HIV

  1. The expression levels of platelet adhesive receptors in PRP derived platelet concentrates during storage

    Directory of Open Access Journals (Sweden)

    Fatemeh Nassaji

    2016-04-01

    Full Text Available Background: Major platelet adhesive receptors that contribute significantly to thrombus formation include platelet receptor glycoprotein Ibα (GPIbα of the GPIb-IX-V complex and platelet glycoprotein VI (GPVI. GPIbα plays a crucial role in platelet tethering to sub-endothelial matrix, which initiates thrombus formation at arterial shear rates, whereas GPVI is critically involved in platelets firm adhesion to the site of injury regardless of shear condition. During storage, platelets experience some changes that deleteriously affect the expression levels of platelet receptors, which in turn can alter platelet functional behaviors. Considering the important roles of GPIbα and GPVI in platelet adhesion, it seems that any dramatic changes in the expression levels of these receptors can influence adhesive function of transfused platelets. Thereby examining GPIbα and GPVI expression during the storage of platelet concentrates may provide some useful information about the functional quality of these products after transfusion. Methods: In our experimental study, 5 PRP-platelet concentrates were randomly obtained from Iranian Blood Transfusion Organization (IBTO. All the platelet products met the standard quality assessment based on AABB (American Association of Blood Banks guidelines. Washed platelets were subjected to flowcytometry analysis for the evaluation of GPIbα and GPVI receptor expression in day 1, 3 and 5 after storage. Data were presented as mean fluorescence intensity (MFI and analyzed by Kruskal-Wallis test with Dunn’s multiple comparison test. Results: The GPIbα expression on first day (MFI=86±5.9 was reduced three days after storage (MFI= 69±6.9. The expression levels continued to reduce until day 5 in which GPIbα expression was markedly decreased to (MFI= 61±7.7 (P= 0.0094. GPVI expression on the days 1, 3 and 5 after storage were 20.6±3.3, 24±2.5 and 14±4.9, respectively. The results showed a significant decrease of

  2. Understanding platelet generation from megakaryocytes: implications for in vitro-derived platelets.

    Science.gov (United States)

    Sim, Xiuli; Poncz, Mortimer; Gadue, Paul; French, Deborah L

    2016-03-10

    Platelets are anucleate cytoplasmic discs derived from megakaryocytes that circulate in the blood and have major roles in hemostasis, thrombosis, inflammation, and vascular biology. Platelet transfusions are required to prevent the potentially life-threatening complications of severe thrombocytopenia seen in a variety of medical settings including cancer therapy, trauma, and sepsis. Platelets used in the clinic are currently donor-derived which is associated with concerns over sufficient availability, quality, and complications due to immunologic and/or infectious issues. To overcome our dependence on donor-derived platelets for transfusion, efforts have been made to generate in vitro-based platelets. Work in this area has advanced our understanding of the complex processes that megakaryocytes must undergo to generate platelets both in vivo and in vitro. This knowledge has also defined the challenges that must be overcome to bring in vitro-based platelet manufacturing to a clinical reality. This review will focus on our understanding of committed megakaryocytes and platelet release in vivo and in vitro, and how this knowledge can guide the development of in vitro-derived platelets for clinical application.

  3. Donor demographic and laboratory predictors of single donor platelet yield

    Directory of Open Access Journals (Sweden)

    R. Arun

    2013-10-01

    Full Text Available Background: Platelet transfusions are essential to prevent morbidity and mortality in patients who are severely thrombocytopenic and are at risk of spontaneous bleeding. Platelets are currently obtained either by fractionation of whole blood or by platelet apheresis. The quality of single donor platelets (SDP in terms of yield influences platelet recovery in the recipient and allows prolonging intervals between transfusions. Material and Methods: Donor demographic and laboratory data were analyzed prior to performing plateletpheresis to identify donor factors that influence platelet yield. The study was conducted on 130 healthy, first-time plateletpheresis donors over a period of 4 years. The plateletpheresis procedures were performed using Fresenius Kabi COM.TEC and Hemonetics MCS plus separator. A relationship between pre-donation donor variables and yield of platelets was studied using the Pearson correlation. Results: The mean platelet yield was 3.160.62x1011 per unit. A positive correlation was observed between platelet yield and pre-donation platelet count, body mass index (BMI; Kg/m2 of the donor, while a negative correlation was observed between age and the platelet yield. Conclusion: Donor pre-donation platelet count, BMI and donor age influence platelet yield. Young healthy donors with a high platelet count and better BMI can give a better platelet yield in the SDP.

  4. Propranolol modifies platelet serotonergic mechanisms in rats.

    Science.gov (United States)

    Zółtowski, R; Pawlak, R; Matys, T; Pietraszek, M; Buczko, W

    2002-06-01

    Though the mechanisms for the vascular actions of vasodilatory beta-blockers are mostly determined, some of their interactions with monoaminergic systems are not elucidated. Because there are evidences supporting a possible involvement of serotonin (5-HT) in the actions of beta-blockers, we studied the effect of propranolol on peripheral serotonergic mechanisms in normotensive and Goldblatt two-kidney - one clip (2K1C) hypertensive rats. In both groups of animals propranolol decreased systolic blood pressure, significantly increased whole blood serotonin concentration and at the same time it decreased platelet serotonin level. The uptake of the amine by platelets from hypertensive animals was lower than that of normotensive animals and it was decreased by propranolol only in the latter. In both groups propranolol inhibited potentiation of ADP-induced platelet aggregation by serotonin. In conclusion, this study provides evidence that propranolol modifies platelet serotonergic mechanisms in normotensive and renal hypertensive rats.

  5. Activation of human platelets by misfolded proteins

    NARCIS (Netherlands)

    Herczenik, E.; Bouma, B.; Korporaal, J.A.; Strangi, R.; Zeng, Q.; Gros, P.; van Eck, M.; van Berkel, T.J.C.; Gebbink, M.F.B.G.; Akkerman, J.W.N.

    2007-01-01

    Objective: Protein misfolding diseases result from the deposition of insoluble protein aggregates that often contain fibrils called amyloid. Amyloids are found in Alzheimer disease, atherosclerosis, diabetes mellitus, and systemic amyloidosis,which are diseases where platelet activation might be

  6. Mapuche herbal medicine inhibits blood platelet aggregation.

    Science.gov (United States)

    Falkenberg, Susan Skanderup; Tarnow, Inge; Guzman, Alfonso; Mølgaard, Per; Simonsen, Henrik Toft

    2012-01-01

    12 plant species traditionally used by the Mapuche people in Chile to treat wounds and inflammations have been evaluated for their direct blood platelet inhibition. Seven of the 12 tested plant species showed platelet inhibitory effect in sheep blood, and four of these were also able to inhibit the ADP- (5.0 μM) and collagen- (2.0 μg/mL) induced aggregations in human blood. These four species in respective extracts (in brackets) were Blechnum chilense (MeOH), Luma apiculata (H(2)O), Amomyrtus luma (DCM : MeOH 1 : 1) and Cestrum parqui (DCM : MeOH 1 : 1). The platelet aggregating inhibitory effects of A. luma (DCM : MeOH 1 : 1), and L. apiculata (H(2)O) were substantial and confirmed by inhibition of platelet surface activation markers.

  7. Mapuche Herbal Medicine Inhibits Blood Platelet Aggregation

    Directory of Open Access Journals (Sweden)

    Susan Skanderup Falkenberg

    2012-01-01

    Full Text Available 12 plant species traditionally used by the Mapuche people in Chile to treat wounds and inflammations have been evaluated for their direct blood platelet inhibition. Seven of the 12 tested plant species showed platelet inhibitory effect in sheep blood, and four of these were also able to inhibit the ADP- (5.0 μM and collagen- (2.0 μg/mL induced aggregations in human blood. These four species in respective extracts (in brackets were Blechnum chilense (MeOH, Luma apiculata (H2O, Amomyrtus luma (DCM : MeOH 1 : 1 and Cestrum parqui (DCM : MeOH 1 : 1. The platelet aggregating inhibitory effects of A. luma (DCM : MeOH 1 : 1, and L. apiculata (H2O were substantial and confirmed by inhibition of platelet surface activation markers.

  8. Relationship between platelet parameters and sudden ...

    African Journals Online (AJOL)

    Relationship between platelet parameters and sudden sensorineural hearing loss: a ... Data source: A PubMed, Science Direct, Scopus, OVID, EMBASE and ... relationship of PDW and SSNHL but due to the limited studies on this subject more ...

  9. Effects of drugs on platelet function.

    Science.gov (United States)

    Morse, E E

    1977-01-01

    Numerous drugs and chemicals affect the function of human blood platelets. The mechanism of action of some medications is partly understood. Aspirin is the most frequently involved drug. It appears to interfere with the platelet release reaction by acetylation of a platelet membrane protein which may be involved in the synthesis of prostaglandins. Other anti-inflammatory drugs, including indomethacin, phenylbutazone, ibuprophen (Motrin) and clonixin, also interfere with the release reaction but have a shorter acting course than aspirin. Some drugs stimulate adenylcyclase (gliclazide) or block phosphodiesterase, (dipyridamole, caffeine) both of which actions lead to an increase in adenosine cyclic 3':5' monophosphate (cAMP) and decrease aggregation by adenosine diphosphate (ADP). These interactions should be known to clinical scientists since patients using these medicaments may manifest abnormal platelet function tests in the laboratory and mild hemorrhagic syndromes in the clinic.

  10. Physiopathology of blood platelets and development of platelet substitutes. Progress report, August 1, 1975--July 31, 1976

    Energy Technology Data Exchange (ETDEWEB)

    Baldini, M G

    1976-04-28

    Progress is reported on studies on the physiology of blood platelets in thrombocytopenic patients and rabbits. Methods for the detection of platelet antibodies and the preservation of platelets in vitro were investigated. Studies on the effect of low doses of x irradiation (up to 1000 R) on platelet function indicate that platelets exposed to ionizing radiation have increased functional activity. A list is included of publications that report the results of the studies in detail.

  11. PELA microspheres loaded H. pylori lysates and their mucosal immune response

    Institute of Scientific and Technical Information of China (English)

    Jian-Min Ren; Ouan-Ming Zou; Fu-Kun Wang; Qiang He; Wei Chen; Wen-Kun Zen

    2002-01-01

    AIM: To prepare poly (D,L-lactide)-polyethylene glycol copolymer (PELA) microspheres loaded H.pylorilysates or CyStografin and observe their targeting in gastrointestinal mucous membrane or analyze the mucosal immune responses by oral administration. METHODS: PELA microspheres loaded H. Pylorilysates or Cystografin were preparedby double emulsion evaporation method. Their distribution in gastrointestinal mucous membrane was observed by CT. Balb/c mice orally immunized in mucosal immune responses, whose antibody production in salivary and gut washing and antibody secreting cells in Peyer's patches (PP) were estimated by ELISA and ELISPOT, respectively. The microspheres' physical properties, such as particle size, protein level and morphology were investigated.RESULTS: All prepared microspheres were found to have a smooth surface morphology from 3.20-4.05 μm in diameter and high encapsulation efficiency from 74.9-82.2 %. No significant correlation in their physical properties was shown, depending on their molecular weight at the similar composition ratio. Immunization with all types of PELA-Hp microspheres elevated the saliva sIgA level at week 3 by approximately 3-4 times that with soluble antigen, which was greatly enhanced after boosting. At one week after last immunization with all types of PELA-Hp microspheres (week 8), the specific sIgA-ASCs, IgG-ASCs and stgA in salivary rose obviously. In intestinal Peyer's patches, the specific sIgA-ASCs were 5.92-6.98× 104/ml cell and IgG-ASCs were 3.47-4.02 × 104/ml cell, about 5-9 times higher than those with soluble antigen (P<0.01). ASCs in intestine were more than those in stomach and the majority of the ASCs were sIgA-ASCs. The sIgA in gut washing fluid was 1.62-1.85 OD, about 3-6 times tthat of those with soluble antigen. There were significant differences of the ASCs and sIgA in gut washing fluid as compared with those of PBS and MS-0 (P<0.05). There appeared to be good correlation between sIgA level in gut

  12. Preparation of tungsten oxide

    Science.gov (United States)

    Bulian, Christopher J.; Dye, Robert C.; Son, Steven F.; Jorgensen, Betty S.; Perry, W. Lee

    2009-09-22

    Tungsten trioxide hydrate (WO.sub.3.H.sub.2O) was prepared from a precursor solution of ammonium paratungstate in concentrated aqueous hydrochloric acid. The precursor solution was rapidly added to water, resulting in the crash precipitation of a yellow white powder identified as WO.sub.3.H.sub.2O nanosized platelets by x-ray diffraction and scanning electron microscopy. Annealing of the powder at 200.degree. C. provided cubic phase WO.sub.3 nanopowder, and at 400.degree. C. provided WO.sub.3 nanopowder as a mixture of monoclinic and orthorhombic phases.

  13. Platelet-rich plasma, plasma rich in growth factors and simvastatin in the regeneration and repair of alveolar bone.

    Science.gov (United States)

    Rivera, César; Monsalve, Francisco; Salas, Juan; Morán, Andrea; Suazo, Iván

    2013-12-01

    Platelet preparations promote bone regeneration by inducing cell migration, proliferation and differentiation in the area of the injury, which are essential processes for regeneration. In addition, several studies have indicated that simvastatin (SIMV), widely used for the treatment of hypercholesterolemia, stimulates osteogenesis. The objective of this study was to evaluate the effects of treatment with either platelet-rich plasma (PRP) or plasma rich in growth factors (PRGF) in combination with SIMV in the regeneration and repair of alveolar bone. The jaws of Sprague Da