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

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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.

Releasing growth factors from activated human platelets after chitosan stimulation: a possible bio-material for platelet-rich plasma preparation.

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Shen, E-Chin; Chou, Tz-Chong; Gau, Ching-Hwa; Tu, Hsiao-Pei; Chen, Yen-Teen; Fu, Earl

2006-10-01

Thrombin is commonly used for activating the platelets and releasing the growth factors on the application of platelet-rich plasma (PRP). We have previously reported that chitosan can enhance rabbit platelet aggregation. In this study, the effects of chitosan on the subsequent growth factors release after human platelets activation were examined to evaluate the possibility of chitosan being used as a substitute for thrombin during PRP preparation. Human platelet activation was determined by aggregation, adhesion and alpha-granule membrane glycoprotein expression. Platelet aggregation was measured by the turbidimetric method, the adhesion was directly examined on chitosan-coated glass plates under light microscope and scanning electron microscope (SEM), and the alpha-granule membrane glycoprotein was detected by fluorescent isothiocyanate (FITC)-conjugated anti-CD61 antibody through flow cytometry. The subsequent epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets were assayed by ELISA after mixing with chitosan. The enhancing effects on the platelet adhesion and the aggregation from chitosan were observed. Under both microscopes, the adhesive platelets on the chitosan-coated plates were not only greater in number but also earlier in activation than those on the control plates. With flow cytometry, increased glycoprotein IIIa expression in platelets was detected after chitosan treatment. Greater concentrations of growth factors were measured from PRP after chitosan treatment than after the solvent treatment. Because of the observations of growth factors releasing from activated human platelets after chitosan stimulation, we suggest that chitosan may be an appropriate substitute for thrombin in PRP preparation.

Enhancement by platelets of oxygen radical responses of human neutrophils

Energy Technology Data Exchange (ETDEWEB)

McCulloch, K.K.; Powell, J.; Johnson, K.J.; Ward, P.A.

1986-03-01

When human blood neutrophils were incubated with immune complexes (consisting of IgG antibody) in the presence of platelets, there was a 2 to 10 fold enhancement in the generation of O-/sub 2/ and H/sub 2/O/sub 2/. This enhancement phenomenon was proportional to the dose of immune complex added and the number of platelets present. The response was not agonist specific since similar enhancement also occurred with the following agonists: phorbol myristate acetate, opsonized zymosan particles and the chemotactic peptide N-formyl-met-leu-phe. The platelet related phenomenon of enhanced O-/sub 2/ generation could not be reproduced by the addition of serotonin, histamine or platelet-derived growth factor and was not affected by prior treatment of platelets with cyclooxygenase inhibitors (indomethacin, piroxicam) or lipoxygenase inhibitors (nafazatrom, BW755C or nordihydroguaiaretic acid). However, activation of platelets by thrombin caused release into the platelet supernatant fluid of a factor that, only in the presence of immune complexes, caused enhanced O-/sub 2/ responses to neutrophils. These data indicate that platelets potentiate oxygen radical responses of human neutrophils and suggest a mechanisms by which platelets may participate in tissue injury which is mediated by oxygen radical products from activated neutrophils.

Radioimmune assay of human platelet prostaglandin synthetase

International Nuclear Information System (INIS)

Roth, G.J.; Machuga, E.T.

1982-01-01

Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH 2 from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and [ 125 I]-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the [ 125 I]antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10 9 platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency

Platelets are a possible regulator of human endometrial re-epithelialization during menstruation.

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Suginami, Koh; Sato, Yukiyasu; Horie, Akihito; Matsumoto, Hisanori; Kyo, Satoru; Araki, Yoshihiko; Konishi, Ikuo; Fujiwara, Hiroshi

2017-01-01

The human endometrium periodically breaks down and regenerates. As platelets have been reported to contribute to the tissue remodeling process, we examined the possible involvement of platelets in endometrial regeneration. The distribution of extravasating platelets throughout the menstrual cycle was immunohistochemically examined using human endometrial tissues. EM-E6/E7/hTERT cells, a human endometrial epithelial cell-derived immortalized cell line, were co-cultured with platelets, and the effects of platelets on the epithelialization response of EM-E6/E7/hTERT cells were investigated by attachment and permeability assays, immunohistochemical staining, and Western blot analysis. Immunohistochemical study showed numerous extravasated platelets in the subluminar stroma during the menstrual phase. The platelets promoted the cell-to-matrigel attachment of EM-E6/E7/hTERT cells concomitantly with the phosphorylation of focal adhesion kinase. They also promoted cell-to-cell contact among EM-E6/E7/hTERT cells in parallel with E-cadherin expression. These results indicate the possible involvement of platelets in the endometrial epithelial re-epithelialization process. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The Use of Recombinant Human Platelet-Derived Growth Factor for Maxillary Sinus Augmentation.

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Kubota, Atsushi; Sarmiento, Hector; Alqahtani, Mohammed Saad; Llobell, Arturo; Fiorellini, Joseph P

The maxillary sinus augmentation procedure has become a predictable treatment to regenerate bone for implant placement. The purpose of this study was to evaluate the effect of recombinant human platelet-derived growth factor BB (rhPDGF-BB) combined with a deproteinized cancellous bovine bone graft for sinus augmentation. The lateral window approach was used for maxillary sinuses with minimal residual bone. After a healing period of 4 months, dental implants were placed and then restored following a 2-month osseointegration period. The result demonstrated increased bone height and ISQ values and a 100% survival rate. This study indicates that the addition of rhPDGF-BB to deproteinized cancellous bovine bone accelerated the healing period in maxillary sinuses with minimal native bone.

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression

International Nuclear Information System (INIS)

Nygaard, Gyrid; Herfindal, Lars; Kopperud, Reidun; Aragay, Anna M.; Holmsen, Holm; Døskeland, Stein Ove; Kleppe, Rune; Selheim, Frode

2014-01-01

Highlights: • We investigated the impact of cyclic nucleotide analogues on platelet activation. • Different time dependence were found for inhibition of platelet activation. • Additive effect was found using PKA- and PKG-activating analogues. • Our results may explain some of the discrepancies reported for cNMP signalling. - Abstract: In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation

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.

Glycoprotein biosynthesis by human normal platelets

International Nuclear Information System (INIS)

Rodriguez, P.; Bello, O.; Apitz-Castro, R.

1987-01-01

Incorporation of radioactive Man, Gal, Fuc, Glc-N, and NANA into washed human normal platelets and endogenous glycoproteins has been found. Both parameters were time dependent. Analysis of hydrolyzed labeled glycoproteins by paper chromatography revealed that the radioactive monosaccharide incubated with the platelets had not been converted into other sugars. Acid hydrolysis demonstrates the presence of a glycosidic linkage. All the effort directed to the demonstration of the existence of a lipid-sugar intermediate in intact human platelets yielded negative results for Man and Glc-N used as precursors. The incorporation of these sugars into glycoproteins is insensitive to bacitracin, suggesting no involvement of lipid-linked saccharides in the synthesis of glycoproteins in human blood platelets. The absence of inhibition of the glycosylation process in the presence of cycloheximide suggests that the sugars are added to proteins present in the intact platelets. These results support the contention that glycoprotein biosynthesis in human blood platelets observed under our experimental conditions is effected through direct sugar nucleotide glycosylation

Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis

OpenAIRE

Takagi, Satoshi; Takemoto, Ai; Takami, Miho; Oh-hara, Tomoko; Fujita, Naoya

2014-01-01

The interactions of tumor cells with platelets contribute to the progression of tumor malignancy, and the expression levels of platelet aggregation-inducing factors positively correlate with the metastatic potential of osteosarcoma cells. However, it is unclear how tumor-platelet interaction contributes to the proliferation of osteosarcomas. We report here that osteosarcoma-platelet interactions induce the release of platelet-derived growth factor (PDGF) from platelets, which promotes the pro...

Niacin and biosynthesis of PGD2 by platelet COX-1 in mice and humans

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Song, Wen-Liang; Stubbe, Jane; Ricciotti, Emanuela; Alamuddin, Naji; Ibrahim, Salam; Crichton, Irene; Prempeh, Maxwell; Lawson, John A.; Wilensky, Robert L.; Rasmussen, Lars Melholt; Puré, Ellen; FitzGerald, Garret A.

2012-01-01

The clinical use of niacin to treat dyslipidemic conditions is limited by noxious side effects, most commonly facial flushing. In mice, niacin-induced flushing results from COX-1–dependent formation of PGD2 and PGE2 followed by COX-2–dependent production of PGE2. Consistent with this, niacin-induced flushing in humans is attenuated when niacin is combined with an antagonist of the PGD2 receptor DP1. NSAID-mediated suppression of COX-2–derived PGI2 has negative cardiovascular consequences, yet little is known about the cardiovascular biology of PGD2. Here, we show that PGD2 biosynthesis is augmented during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis. Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1–derived PGD2 biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD2 was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD2, like PGI2, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular relevance as a constraint on platelets during niacin therapy. PMID:22406532

Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis.

Science.gov (United States)

Takagi, Satoshi; Takemoto, Ai; Takami, Miho; Oh-Hara, Tomoko; Fujita, Naoya

2014-08-01

The interactions of tumor cells with platelets contribute to the progression of tumor malignancy, and the expression levels of platelet aggregation-inducing factors positively correlate with the metastatic potential of osteosarcoma cells. However, it is unclear how tumor-platelet interaction contributes to the proliferation of osteosarcomas. We report here that osteosarcoma-platelet interactions induce the release of platelet-derived growth factor (PDGF) from platelets, which promotes the proliferation of osteosarcomas. Co-culture of platelets with MG63 or HOS osteosarcoma cells, which could induce platelet aggregation, enhanced the proliferation of each cell line in vitro. Analysis of phospho-antibody arrays revealed that co-culture of MG63 cells with platelets induced the phosphorylation of platelet derived growth factor receptor (PDGFR) and Akt. The addition of supernatants of osteosarcoma-platelet reactants also increased the growth of MG63 and HOS cells as well as the level of phosphorylated-PDGFR and -Akt. Sunitinib or LY294002, but not erlotinib, significantly inhibited the platelet-induced proliferation of osteosarcoma cells, indicating that PDGF released from platelets plays an important role in the proliferation of osteosarcomas by activating the PDGFR and then Akt. Our results suggest that inhibitors that specifically target osteosarcoma-platelet interactions may eradicate osteosarcomas. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Signal transduction by the platelet-derived growth factor receptor

International Nuclear Information System (INIS)

Williams, L.T.; Escobedo, J.A.; Keating, M.T.; Coughlin, S.R.

1988-01-01

The mitogenic effects of platelet-derived growth factor (PDGF) are mediated by the PDGF receptor. The mouse PDGF receptor was recently purified on the basis of its ability to become tyrosine phosphorylated in response to the A-B human platelet form of PDGF, and the receptor amino acid sequence was determined from a full-length cDNA clone. Both the human and mouse receptor cDNA sequences have been expressed in Chinese hamster ovary fibroblast (CHO) cells that normally lack PDGF receptors. This paper summarizes recent results using this system to study signal transduction by the PDGF receptor. Some of the findings show that the KI domain of the PDGF receptor plays an important role in the stimulation of DNA synthesis by PDGF. Surprisingly, the kinase insert region is not essential for PDGF stimulation of PtdIns turnover, pH change, increase in cellular calcium, and receptor autophosphorylation. In addition, PDGF stimulates a conformational change in the receptor

Superoxide dismutase of human platelets

International Nuclear Information System (INIS)

Kimura, Akiro; Fujimura, Kingo; Kuramoto, Atsushi

1979-01-01

Superoxide dismutase (S.O.D.) is the enzyme to protect from destructive effect of superoxide (O 2 -) produced in many metabolic pathways related to oxygen. The purpose of this study was to investigate the possibility that S.O.D. may play an important role in the platelet function. The cytoplasmic and mitochondrial S.O.D. has been investigated spectrophotometrically and gel electrophoretically in human platelets from eleven patients of chronic myelogenous leukemia (CML) and three patients of primary thrombocythemia (P.Th.). Neither deficiency nor abnormality of cytoplasmic and mitochondrial S.O.D. has been found electrophoretically in any case compared to normal platelets. However, the total activity from three of the CML patients and one of the P.Th. patients were above 3 unit/mg platelet protein (normal subject: 2.11 - 2.70 unit/mg protein), suggesting the possibility either that more O 2 -production occurs in the platelets or that rather little O 2 -production due to much O 2 -deprivation by the increased S.O.D. The S.O.D. activity of human platelets has been also investigated in several conditions, where much O 2 -generation might occur in platelets. Sodium fluoride (2 mM), which increases platelet O 2 -production about 3 fold, had no effect on platelet S.O.D. The aggregated platelets induced by ADP (10 -5 M), epinephrin (50 μg/ml), ristocetin (1.5 mg/ml) or collagen (1 - 20 μg/ml) had no increase of S.O.D. activity compared to that from non aggregated platelets. X-ray irradiation (1,000 - 20,000R) had not induced its activity increase or decrease. These findings indicated the induction of platelet S.O.D. was not brought about under these conditions. (author)

Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support

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Anita Muraglia

2017-11-01

Full Text Available Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i an heparin-free human platelet lysate (PL devoid of serum or plasma components (v-PL and (ii an heparin-free human serum derived from plasma devoid of PL components (Pl-s and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment, but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79 regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype.

Breaking the mold: transcription factors in the anucleate platelet and platelet-derived microparticles

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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

Apheresis platelet concentrates contain platelet-derived and endothelial cell-derived microparticles

NARCIS (Netherlands)

Rank, A.; Nieuwland, R.; Liebhardt, S.; Iberer, M.; Grützner, S.; Toth, B.; Pihusch, R.

2011-01-01

Background and Objectives Microparticles (MP) are membrane vesicles with thrombogenic and immunomodulatory properties. We determined MP subgroups from resting platelets, activated platelets and endothelial cells in donors and apheresis platelet concentrates (PC). Material and Methods MP were double

Comparison of cDNA-derived protein sequences of the human fibronectin and vitronectin receptor α-subunits and platelet glycoprotein IIb

International Nuclear Information System (INIS)

Fitzgerald, L.A.; Poncz, M.; Steiner, B.; Rall, S.C. Jr.; Bennett, J.S.; Phillips, D.R.

1987-01-01

The fibronectin receptor (FnR), the vitronectin receptor (VnR), and the platelet membrane glycoprotein (GP) IIb-IIIa complex are members of a family of cell adhesion receptors, which consist of noncovalently associated α- and β-subunits. The present study was designed to compare the cDNA-derived protein sequences of the α-subunits of human FnR, VnR, and platelet GP IIb. cDNA clones for the α-subunit of the FnR (FnR/sub α/) were obtained from a human umbilical vein endothelial (HUVE) cell library by using an oligonucleotide probe designed from a peptide sequence of platelet GP IIb. cDNA clones for platelet GP IIb were isolated from a cDNA expression library of human erythroleukemia cells by using antibodies. cDNA clones of the VnR α-subunit (VnR/sub α/) were obtained from the HUVE cell library by using an oligonucleotide probe from the partial cDNA sequence for the VnR/sub α/. Translation of these sequences showed that the FNR/sub α/, the VnR/sub α/, and GP IIb are composed of disulfide-linked large (858-871 amino acids) and small (137-158 amino acids) chains that are posttranslationally processed from a single mRNA. A single hydrophobic segment located near the carboxyl terminus of each small chain appears to be a transmembrane domain. The large chains appear to be entirely extracellular, and each contains four repeated putative Ca 2+ -binding domains of about 30 amino acids that have sequence similarities to other Ca 2+ -binding proteins. The identity among the protein sequences of the three receptor α-subunits ranges from 36.1% to 44.5%, with the Ca 2+ -binding domains having the greatest homology. These proteins apparently evolved by a process of gene duplication

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.

Lea blood group antigen on human platelets

International Nuclear Information System (INIS)

Dunstan, R.A.; Simpson, M.B.; Rosse, W.F.

1985-01-01

One- and two-stage radioligand assays were used to determine if human platelets possess the Lea antigen. Goat IgG anti-Lea antibody was purified by multiple adsorptions with Le(a-b-) human red blood cells, followed by affinity chromatography with synthetic Lea substance and labeling with 125 I. Human IgG anti-Lea antibody was used either in a two stage radioassay with 125 I-labeled mouse monoclonal IgG anti-human IgG as the second antibody or, alternatively, purified by Staph protein A chromatography, labeled with 125 I, and used in a one-stage radioassay. Platelets from donors of appropriate red blood cell phenotypes were incubated with the antisera, centrifuged through phthalate esters, and assayed in a gamma scintillation counter. Dose response and saturation curve analysis demonstrate the presence of Lewis a antigen on platelets from Lea+ donors. Furthermore, platelets from an Le(a-b-) donor incubated in Le (a+b-) plasma adsorb Lea antigen in a similar manner to red blood cells. The clinical significance of these antigens in platelet transfusion remains undefined

Platelet-Rich Blood Derivatives for Stem Cell-Based Tissue Engineering and Regeneration

NARCIS (Netherlands)

Masoudi, E.A.; Ribas, J.; Kaushik, G.; Leijten, Jeroen Christianus Hermanus; Khademhosseini, A.

2016-01-01

Platelet-rich blood derivatives have been widely used in different fields of medicine and stem cell-based tissue engineering. They represent natural cocktails of autologous growth factors, which could provide an alternative for recombinant protein-based approaches. Platelet-rich blood derivatives,

Effect of losartan on human platelet activation.

Science.gov (United States)

Guerra-Cuesta, J I; Montón, M; Rodríguez-Feo, J A; Jiménez, A M; González-Fernández, F; Rico, L A; García, R; Gómez, J; Farré, J; Casado, S; López-Farré, A

1999-03-01

Previous studies have demonstrated that losartan can block the thromboxane A2 receptor on the vascular wall. The aim of the present study was to assess the effect of losartan on human platelet activation. Platelets were obtained from 15 healthy men, aged 26-40 years. Platelet activation was measured by changes in the light transmission of platelet-rich plasma stimulated by the thromboxane A2 analog U46619 (5 x 10(-6) mol/l) or ADP (10(-5) mol/l). U46619-stimulated platelet aggregation was significantly inhibited by losartan in a dose-dependent manner. Only a high dose of EXP 3174 (5 x 10(-5) mol/l), the in vivo active metabolite of losartan, was able to attenuate U46619-induced platelet activation. Captopril, an angiotensin I converting inhibitor, failed to modify U46619-induced platelet aggregation. Furthermore, the binding of [3H]-U46619 to platelets was competitively inhibited by losartan, whereas only a high dose of EXP 3174 reduced the binding of [3H]-U46619. Captopril failed to modify the binding of [3H]-U46619 to platelets. Losartan also reduced the platelet activation induced by ADP (10(-5) mol/l), a platelet agonist partially dependent on thromboxane A2. In addition, when thromboxane A2 generation was blocked by aspirin, ADP-induced platelet aggregation was inhibited to a similar degree to the inhibition induced by losartan. Exogenous angiotensin II did not elicit any modification of either U46619- or ADP-stimulated platelet aggregation. Losartan decreased platelet aggregation by a thromboxane A2-dependent mechanism. EXP 3174 was less potent than losartan in reducing thromboxane A2-dependent platelet activation. Captopril and exogenous angiotensin II had no effect on human platelet activation. These results suggest that losartan reduced thromboxane A2-dependent platelet activation independently of its effect on angiotensin II.

Differences in levels of platelet-derived microparticles in platelet components prepared using the platelet rich plasma, buffy coat, and apheresis procedures.

Science.gov (United States)

Noulsri, Egarit; Udomwinijsilp, Prapaporn; Lerdwana, Surada; Chongkolwatana, Viroje; Permpikul, Parichart

2017-04-01

There has been an increased interest in platelet-derived microparticles (PMPs) in transfusion medicine. Little is known about PMP status during the preparation of platelet concentrates for transfusion. The aim of this study is to compare the PMP levels in platelet components prepared using the buffy coat (BC), platelet-rich plasma platelet concentrate (PRP-PC), and apheresis (AP) processes. Platelet components were prepared using the PRP-PC and BC processes. Apheresis platelets were prepared using the Trima Accel and Amicus instruments. The samples were incubated with annexin A5-FITC, CD41-PE, and CD62P-APC. At day 1 after processing, the PMPs and activated platelets were determined using flow cytometry. Both the percentage and number of PMPs were higher in platelet components prepared using the Amicus instrument (2.6±1.8, 32802±19036 particles/μL) than in platelet components prepared using the Trima Accel instrument (0.5±0.4, 7568±5298 particles/μL), BC (1.2±0.6, 12,920±6426 particles/μL), and PRP-PC (0.9±0.6, 10731±5514 particles/μL). Both the percentage and number of activated platelets were higher in platelet components prepared using the Amicus instrument (33.2±13.9, 427553±196965 cells/μL) than in platelet components prepared using the Trima Accel instrument (16.2±6.1, 211209±87706 cells/μL), BC (12.9±3.2, 140624±41003 cells/μL), and PRP-PC (21.1±6.3, 265210±86257 cells/μL). The study suggests high variability of PMPs and activated platelets in platelet components prepared using different processes. This result may be important in validating the instruments involved in platelet blood collection and processing. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stage specific requirement of platelet-derived growth factor receptor-α in embryonic development.

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Chen Qian

Full Text Available Platelet-derived growth factor receptor alpha (PDGFRα is a cell-surface receptor tyrosine kinase for platelet-derived growth factors. Correct timing and level of Pdgfra expression is crucial for embryo development, and deletion of Pdgfra caused developmental defects of multiple endoderm and mesoderm derived structures, resulting in a complex phenotypes including orofacial cleft, spina bifida, rib deformities, and omphalocele in mice. However, it is not clear if deletion of Pdgfra at different embryonic stages differentially affects these structures.To address the temporal requirement of Pdgfra in embryonic development.We have deleted the Pdgfra in Pdgfra-expressing tissues at different embryonic stages in mice, examined and quantified the developmental anomalies.Current study showed that (i conditional deletion of Pdgfra at different embryonic days (between E7.5 and E10.5 resulted in orofacial cleft, spina bifida, rib cage deformities, and omphalocele, and (ii the day of Pdgfra deletion influenced the combinations, incidence and severities of these anomalies. Deletion of Pdgfra caused apoptosis of Pdgfra-expressing tissues, and developmental defects of their derivatives.Orofacial cleft, spina bifida and omphalocele are among the commonest skeletal and abdominal wall defects of newborns, but their genetic etiologies are largely unknown. The remarkable resemblance of our conditional Pdgfra knockout embryos to theses human congenital anomalies, suggesting that dysregulated PDGFRA expression could cause these anomalies in human. Future work should aim at defining (a the regulatory elements for the expression of the human PDGFRA during embryonic development, and (b if mutations / sequence variations of these regulatory elements cause these anomalies.

Platelet-Derived Microvesicles in Cardiovascular Diseases

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Maria T. K. Zaldivia

2017-11-01

Full Text Available Microvesicles (MVs circulating in the blood are small vesicles (100–1,000 nm in diameter derived from membrane blebs of cells such as activated platelets, endothelial cells, and leukocytes. A growing body of evidence now supports the concept that platelet-derived microvesicles (PMVs, the most abundant MVs in the circulation, are important regulators of hemostasis, inflammation, and angiogenesis. Compared with healthy individuals, a large increase of circulating PMVs has been observed, particularly in patients with cardiovascular diseases. As observed in MVs from other parent cells, PMVs exert their biological effects in multiple ways, such as triggering various intercellular signaling cascades and by participating in transcellular communication by the transfer of their “cargo” of cytoplasmic components and surface receptors to other cell types. This review describes our current understanding of the potential role of PMVs in mediating hemostasis, inflammation, and angiogenesis and their consequences on the pathogenesis of cardiovascular diseases, such as atherosclerosis, myocardial infarction, and venous thrombosis. Furthermore, new developments of the therapeutic potential of PMVs for the treatment of cardiovascular diseases will be discussed.

Rapid resensitization of purinergic receptor function in human platelets.

Science.gov (United States)

Mundell, S J; Barton, J F; Mayo-Martin, M B; Hardy, A R; Poole, A W

2008-08-01

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), the P2Y(1) and P2Y(12) purinergic receptors. Recently, we demonstrated that both receptors desensitize and internalize in human platelets by differential kinase-dependent mechanisms. To demonstrate whether responses to P2Y(1) and P2Y(12) purinergic receptors resensitize in human platelets and determine the role of receptor traffic in this process. These studies were undertaken either in human platelets or in cells stably expressing epitope-tagged P2Y(1) and P2Y(12) purinergic receptor constructs. In this study we show for the first time that responses to both of these receptors can rapidly resensitize following agonist-dependent desensitization in human platelets. Further, we show that in human platelets or in 1321N1 cells stably expressing receptor constructs, the disruption of receptor internalization, dephosphorylation or subsequent receptor recycling is sufficient to block resensitization of purinergic receptor responses. We also show that, in platelets, internalization of both these receptors is dependent upon dynamin, and that this process is required for resensitization of responses. This study is therefore the first to show that both P2Y(1) and P2Y(12) receptor activities are rapidly and reversibly modulated in human platelets, and it reveals that the underlying mechanism requires receptor trafficking as an essential part of this process.

The effects of platelet lysate patches on the activity of tendon-derived cells.

Science.gov (United States)

Costa-Almeida, Raquel; Franco, Albina R; Pesqueira, Tamagno; Oliveira, Mariana B; Babo, Pedro S; Leonor, Isabel B; Mano, João F; Reis, Rui L; Gomes, Manuela E

2018-03-01

Platelet-derived biomaterials are widely explored as cost-effective sources of therapeutic factors, holding a strong potential for endogenous regenerative medicine. Particularly for tendon repair, treatment approaches that shift the injury environment are explored to accelerate tendon regeneration. Herein, genipin-crosslinked platelet lysate (PL) patches are proposed for the delivery of human-derived therapeutic factors in patch augmentation strategies aiming at tendon repair. Developed PL patches exhibited a controlled release profile of PL proteins, including bFGF and PDGF-BB. Additionally, PL patches exhibited an antibacterial effect by preventing the adhesion, proliferation and biofilm formation by S. aureus, a common pathogen in orthopaedic surgical site infections. Furthermore, these patches supported the activity of human tendon-derived cells (hTDCs). Cells were able to proliferate over time and an up-regulation of tenogenic genes (SCX, COL1A1 and TNC) was observed, suggesting that PL patches may modify the behavior of hTDCs. Accordingly, hTDCs deposited tendon-related extracellular matrix proteins, namely collagen type I and tenascin C. In summary, PL patches can act as a reservoir of biomolecules derived from PL and support the activity of native tendon cells, being proposed as bioinstructive patches for tendon regeneration. Platelet-derived biomaterials hold great interest for the delivery of therapeutic factors for applications in endogenous regenerative medicine. In the particular case of tendon repair, patch augmentation strategies aiming at shifting the injury environment are explored to improve tendon regeneration. In this study, PL patches were developed with remarkable features, including the controlled release of growth factors and antibacterial efficacy. Remarkably, PL patches supported the activity of native tendon cells by up-regulating tenogenic genes and enabling the deposition of ECM proteins. This patch holds great potential towards

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))....

Molecular cloning and functional characterization of the human platelet-derived growth factor alpha receptor gene promoter

NARCIS (Netherlands)

Afink, G. B.; Nistér, M.; Stassen, B. H.; Joosten, P. H.; Rademakers, P. J.; Bongcam-Rudloff, E.; van Zoelen, E. J.; Mosselman, S.

1995-01-01

Expression of the platelet-derived growth factor alpha receptor (PDGF alpha R) is strictly regulated during mammalian development and tumorigenesis. The molecular mechanisms involved in the specific regulation of PDGF alpha R expression are unknown, but transcriptional regulation of the PDGF alpha R

Synthesis and secretion of platelet-derived growth factor by human breast cancer cell lines

International Nuclear Information System (INIS)

Bronzert, D.A.; Pantazis, P.; Antoniades, H.N.; Kasid, A.; Davidson, N.; Dickson, R.B.; Lippman, M.E.

1987-01-01

The authors report that human breast cancer cells secrete a growth factor that is biologically and immunologically similar to platelet-derived growth factor (PDGF). Serum-free medium conditioned by estrogen-independent MDA-MB-231 or estrogen-dependent MCF-7 cells contains a mitogenic or competence activity that is capable of inducing incorporation of [ 3 H] thymidine into quiescent Swiss 3T3 cells in the presence of platelet-poor plasma. Like authentic PDGF, the PDGF-like activity produced by breast cancer cells is stable after acid and heat treatment (95 0 C) and inhibited by reducing agents. The mitogenic activity comigrates with a material of ≅30 kDa on NaDodSO 4 /polyacrylamide gels. Immunoprecipitation with PDGF antiserum of proteins from metabolically labeled cell lysates and conditioned medium followed by analysis on nonreducing NaDodSO 4 /polyacrylamide gels identified proteins of 30 and 34 kDa. Upon reduction, the 30- and 34-kDa bands were converted to 15- and 16-kDa bands suggesting that the immunoprecipitated proteins were made up of two disulfide-linked polypeptides similar to PDGF. Hybridization studies with cDNA probes for the A chain PDGF and the B chain of PDGF/SIS identified transcripts for both PDGF chains in the MCF-7 and MDA-MB-231 cells. The data summarized above provide conclusive evidence for the synthesis and hormonally regulated secretion of a PDGF-like mitogen by breast carcinoma cells. Production of a PDGF-like growth factor by breast cancer cell lines may be important in mediating paracrine stimulation of tumor growth

The influence of platelet- derived products on angiogenesis and tissue repair: a concise update

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Constanza E Martínez

2015-10-01

Full Text Available Platelet degranulation allows the release of a large amount of soluble mediators, is an essential step for wound healing initiation, and stimulates clotting and angiogenesis. The latter process is one of the most critical biological events observed during tissue repair,increasing the growth of blood vessels in the maturing wound. Angiogenesis requires the action of a variety of growth factors that act in an appropriate physiological ratio to assure functional blood vessel restoration. Platelets release main regulators of angiogenesis: Vascular Endothelial Growth Factors (VEGFs, basic fibroblast growth factor (FGF-2, and Platelet derived growth factors (PDGFs, among others. In order to stimulate tissue repair, platelet derived fractions have been used as an autologous source of growth factors and biomolecules, namely Platelet Rich Plasma (PRP, Platelet Poor Plasma (PPP and Platelet Rich Fibrin(PRF. The continuous release of these growth factors has been proposed to promote angiogenesis both in vitro and in vivo. Considering the existence of clinical trials currently evaluating the efficacy of autologous PRP, the present review analyses fundamental questions regarding the putative role of platelet derived fractions as regulators of angiogenesis and evaluates the possible clinical implications of these formulations.

Genome-wide RNA-seq analysis of human and mouse platelet transcriptomes

Science.gov (United States)

Rowley, Jesse W.; Oler, Andrew J.; Tolley, Neal D.; Hunter, Benjamin N.; Low, Elizabeth N.; Nix, David A.; Yost, Christian C.; Zimmerman, Guy A.

2011-01-01

Inbred mice are a useful tool for studying the in vivo functions of platelets. Nonetheless, the mRNA signature of mouse platelets is not known. Here, we use paired-end next-generation RNA sequencing (RNA-seq) to characterize the polyadenylated transcriptomes of human and mouse platelets. We report that RNA-seq provides unprecedented resolution of mRNAs that are expressed across the entire human and mouse genomes. Transcript expression and abundance are often conserved between the 2 species. Several mRNAs, however, are differentially expressed in human and mouse platelets. Moreover, previously described functional disparities between mouse and human platelets are reflected in differences at the transcript level, including protease activated receptor-1, protease activated receptor-3, platelet activating factor receptor, and factor V. This suggests that RNA-seq is a useful tool for predicting differences in platelet function between mice and humans. Our next-generation sequencing analysis provides new insights into the human and murine platelet transcriptomes. The sequencing dataset will be useful in the design of mouse models of hemostasis and a catalyst for discovery of new functions of platelets. Access to the dataset is found in the “Introduction.” PMID:21596849

CRISPR/Cas9-mediated conversion of human platelet alloantigen allotypes.

Science.gov (United States)

Zhang, Nanyan; Zhi, Huiying; Curtis, Brian R; Rao, Sridhar; Jobaliya, Chintan; Poncz, Mortimer; French, Deborah L; Newman, Peter J

2016-02-11

Human platelet alloantigens (HPAs) reside on functionally important platelet membrane glycoproteins and are caused by single nucleotide polymorphisms in the genes that encode them. Antibodies that form against HPAs are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia and posttransfusion purpura. The HPA-1a/HPA-1b alloantigen system, also known as the Pl(A1)/Pl(A2) polymorphism, is the most frequently implicated HPA among whites, and a single Leu33Pro amino acid polymorphism within the integrin β3 subunit is responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes. HPA-1b/b platelets, like those bearing other low-frequency platelet-specific alloantigens, are relatively rare in the population and difficult to obtain for purposes of transfusion therapy and diagnostic testing. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) gene-editing technology to transform Leu33 (+) megakaryocytelike DAMI cells and induced pluripotent stem cells (iPSCs) to the Pro33 allotype. CD41(+) megakaryocyte progenitors derived from these cells expressed the HPA-1b (Pl(A2)) alloantigenic epitope, as reported by diagnostic NciI restriction enzyme digestion, DNA sequencing, and western blot analysis using HPA-1b-specific human maternal alloantisera. Application of CRISPR/Cas9 technology to genetically edit this and other clinically-important HPAs holds great potential for production of designer platelets for diagnostic, investigative, and, ultimately, therapeutic use. © 2016 by The American Society of Hematology.

Scalable Generation of Universal Platelets from Human Induced Pluripotent Stem Cells

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Qiang Feng

2014-11-01

Full Text Available Human induced pluripotent stem cells (iPSCs provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid “surge” capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the β2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.

Isoforms of purified methyltransferase from human blood platelets ...

African Journals Online (AJOL)

... purification from normal human blood platelets have not been investigated, hence, the aim of this study was to purify, characterise the enzyme from human blood platelets and determine its possible role in phospholipid transmethylation. The plasma membranes were purified by velocity and sucrose gradient centrifugation ...

Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

OpenAIRE

Smits, A.; Funa, K.; Vassbotn, F. S.; Beausang-Linder, M.; af Ekenstam, F.; Heldin, C. H.; Westermark, B.; Nistér, M.

1992-01-01

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protei...

Increase in density and accumulation of serotonin by human aging platelets

International Nuclear Information System (INIS)

Mezzano, D.; Aranda, E.; Rodriguez, S.; Foradori, A.; Lira, P.

1984-01-01

51 Cr-labeled autologous platelets were infused into splenectomized subjects and the specific radioactivities of high-density (HD) and low-density (LD) platelet subpopulations were determined sequentially in postinfusion samples. These findings confirm previous observations in eusplenic individuals and support the hypothesis that human LD platelets are, on the average, younger than HD platelets. LD platelets contain 33.8 +/- 13.5 ng serotonin (5HT)/10(8) platelets and HD platelets 76.8 +/- 9.5 ng 5HT/10(8) platelets. Sequential measurements of 5HT in PRP platelets were performed during the recovery phase of thrombocytopenia following splenectomy in patients with idiopathic thrombocytopenic purpura (ITP), a condition associated with aging of platelets in circulation. Presplenectomy platelet 5HT was 17.7 ng/10(8) platelets and on days 1, 6, and 12 after surgery it increased to 18.1, 37.8, and 61.0 ng/10(8) platelets. When three healthy volunteers were given aspirin (500 mg/day) for up to 15 days, no significant change in the 5HT content of circulating platelets was observed. The observation that human HD platelets, enriched with older cells, contain more 5HT than LD platelets taken together with the parallel increase in platelet 5HT and age during the recovery from thrombocytopenia in ITP patients and the lack of effect of aspirin on platelet 5HT content, provides initial evidence that human platelets accumulate 5HT during their life-span in circulation

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

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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.

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)

Bordin, Antonella; Ponti, Donatella; Iudicone, Paola; 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

Human platelet vasopressin receptor identification by direct ultraviolet photoaffinity labeling

International Nuclear Information System (INIS)

Thibonnier, M.

1987-01-01

Tritiated vasopressin ([ 3 H]AVP) was directly crosslinked to its human platelet receptor by using an ultraviolet irradiation procedure. After preincubation with [ 3 H]AVP, the hydrodynamic parameters of the hormone-receptor complexes solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate were derived from Sephacryl S-300 superfine gel filtration and from sucrose density gradient ultracentrifugation experiments. The following values were obtained: Stoke's radius = 5.48 +/- 0.1 nm, apparent sedimentation coefficient = 5.55 +/- 0.1 S, and calculated molecular weight = 132,000. On sodium dodecyl sulfate-8% polyacrylamide slab gel electrophoresis under reducing conditions, [ 3 H]AVP preferentially and specifically labeled a 125,000-dalton protein. The labeling of this protein was suppressed by addition of excess cold vasopressin, whereas angiotensin II did not inhibit incorporation of tritiated vasopressin in this protein. These results suggest that direct UV-photoaffinity labelling with [ 3 H]AVP is a suitable tool for the purification of the human platelet vasopressin receptor

Increased platelet reactivity is associated with circulating platelet-monocyte complexes and macrophages in human atherosclerotic plaques.

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Bert Rutten

Full Text Available Platelet reactivity, platelet binding to monocytes and monocyte infiltration play a detrimental role in atherosclerotic plaque progression. We investigated whether platelet reactivity was associated with levels of circulating platelet-monocyte complexes (PMCs and macrophages in human atherosclerotic carotid plaques.Platelet reactivity was determined by measuring platelet P-selectin expression after platelet stimulation with increasing concentrations of adenosine diphosphate (ADP, in two independent cohorts: the Circulating Cells cohort (n = 244 and the Athero-Express cohort (n = 91. Levels of PMCs were assessed by flow cytometry in blood samples of patients who were scheduled for percutaneous coronary intervention (Circulating Cells cohort. Monocyte infiltration was semi-quantitatively determined by histological examination of atherosclerotic carotid plaques collected during carotid endarterectomy (Athero-Express cohort.We found increased platelet reactivity in patients with high PMCs as compared to patients with low PMCs (median (interquartile range: 4153 (1585-11267 area under the curve (AUC vs. 9633 (3580-21565 AUC, P<0.001. Also, we observed increased platelet reactivity in patients with high macrophage levels in atherosclerotic plaques as compared to patients with low macrophage levels in atherosclerotic plaques (mean ± SD; 8969 ± 3485 AUC vs. 7020 ± 3442 AUC, P = 0.02. All associations remained significant after adjustment for age, sex and use of drugs against platelet activation.Platelet reactivity towards ADP is associated with levels of PMCs and macrophages in human atherosclerotic carotid plaques.

Influence of storage conditions on the release of growth factors in platelet-rich blood derivatives

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Düregger Katharina

2016-09-01

Full Text Available Thrombocytes can be concentrated in blood derivatives and used as autologous transplants e.g. for wound treatment due to the release of growth factors such as platelet derived growth factor (PDGF. Conditions for processing and storage of these platelet-rich blood derivatives influence the release of PDGF from the platelet-bound α-granules into the plasma. In this study Platelet rich plasma (PRP and Platelet concentrate (PC were produced with a fully automated centrifugation system. Storage of PRP and PC for 1 h up to 4 months at temperatures between −20°C and +37°C was applied with the aim of evaluating the influence on the amount of released PDGF. Storage at −20°C resulted in the highest release of PDGF in PRP and a time dependency was determined: prolonged storage up to 1 month in PRP and 10 days in PC increased the release of PDGF. Regardless of the storage conditions, the release of PDGF per platelet was higher in PC than in PRP.

Platelet-Derived Short-Chain Polyphosphates Enhance the Inactivation of Tissue Factor Pathway Inhibitor by Activated Coagulation Factor XI.

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Cristina Puy

Full Text Available Factor (F XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα, in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin.Purified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma.Our data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP.

Aspirin Inhibits Platelet-Derived Sphingosine-1-Phosphate Induced Endothelial Cell Migration.

Science.gov (United States)

Polzin, Amin; Knoop, Betül; Böhm, Andreas; Dannenberg, Lisa; Zurek, Mark; Zeus, Tobias; Kelm, Malte; Levkau, Bodo; Rauch, Bernhard H

2018-01-01

Aspirin plays a crucial role in the prevention of cardiovascular diseases. We previously described that aspirin has effects beyond inhibition of platelet aggregation, as it inhibited thrombin-mediated release of sphingosine-1-phosphate (S1P) from human platelets. S1P is a bioactive lipid with important functions on inflammation and apoptosis. In endothelial cells (EC), S1P is a key regulator of cell migration. In this study, we aimed to analyze the effects of aspirin on platelet-induced EC migration. Human umbilical EC migration was measured by Boyden chamber assay. EC migration was induced by platelet supernatants of thrombin receptor-activating peptide-1 (AP1) stimulated platelets. To investigate the S1P receptor subtype that promotes EC migration, specific inhibitors of S1P receptor subtypes were applied. S1P induced EC migration in a concentration-dependent manner. EC migration induced by AP1-stimulated platelet supernatants was reduced by aspirin. S1P1 receptor inhibition almost completely abolished EC migration induced by activated platelets. The inhibition of S1P2 or S1P3 receptor had no effect. Aspirin inhibits EC migration induced by activated platelets that is in part due to S1P and mediated by the endothelial S1P1 receptor. The clinical significance of this novel mechanism of aspirin action has to be investigated in future studies. © 2017 S. Karger AG, Basel.

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

2016-07-01

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. 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. 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. 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.

Storage of human platelets by freezing

Energy Technology Data Exchange (ETDEWEB)

Kim, B K; Tanoue, K; Baldini, M G

1976-01-01

Prolonged, probably indefinite storage of viable and functional human platelets is now possible by freezing with dimethylsulfoxide (DMSO). The platelets have a nearly normal survival upon reinfusion and are capable of sustained hemostatic effectiveness in thrombocytopenic patients. Adaptation of the freezing technique for large-scale usage has more recently been achieved. The method is mainly based on the following principles: (1) use of plasma for suspension of the platelet concentrate; (2) gradual addition (0.5% every 2 min) of DMSO to a final concentration of 5% and its gradual removal; (3) a slow cooling rate of about 1/sup 0/C per min and rapid thawing (in 1 min); (4) use of a polyolefin plastic bag for freezing; (5) a washing medium of 20% plasma in Hanks' balanced salt solution; (6) final resuspension of the platelets in 50% plasma in Hanks' solution.

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

Science.gov (United States)

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. Copyright© by Royan Institute. All rights reserved.

Generation of Megakaryocytes and Platelets from Human Pluripotent Stem Cells.

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Pick, Marjorie

2016-01-01

Human pluripotent stem cells (hPSC) have the potential to produce any tissue type in the body and thus represent a source of cells for regenerative medicine. Here we have shown that human platelets can be produced from embryonic or induced pluripotent stem cells in a defined culture system. We describe a serum- and feeder-free culture system that enabled the generation of megakaryocyte (Mk) progenitors and functional platelets from hPSCs. After 13 days the differentiated population included precursor cells that formed colonies containing differentiated Mks, and after 20 days these Mks were able to fragment into platelet-like particles that were functional. This protocol represents an important step towards the generation of human platelets for therapeutic use.

Platelets as a Novel Source of Pro-Inflammatory Chemokine CXCL14

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Alexander Witte

2017-03-01

Full Text Available Objective: Platelets are a major source of chemokines. Here, we demonstrate for the first time that platelets express significant amounts of CXCL14 and disclose powerful effects of platelet-derived CXCL14 on monocyte and endothelial migration. Methods: The expression of CXCL14 in platelets and in the supernatant of activated platelets was analysed by immunoblotting, ELISA, and flow cytometry. The effect of platelet-derived CXCL14 on monocyte migration was evaluated using a modified Boyden chamber. The effect of CXCL14 on monocyte phagocytosis was tested by using fluorochrome-labelled E.coli particles. The effect of platelet-derived CXCL14 on endothelial migration was explored by the use of an endothelial scratch assay. Results: Hitherto unrecognized expression of CXCL14 in human and murine platelets was uncovered by immunoblotting. Activation with platelet agonists such as adenosine-di-phosphate (ADP, collagen-related peptide (CRP, or thrombin-receptor activating peptide (TRAP, increased CXCL14 surface expression (flow cytometry and release into the supernatant (immunoblotting, ELISA. Since CXCL14 is known to be chemotactic for CD14+ monocytes, we investigated the chemotactic potential of platelet-derived CXCL14 on human monocytes. Activated platelet supernatant induced monocyte migration, which was counteracted upon neutralization of platelet-derived CXCL14 as compared to IgG control. Blocking of the chemokine receptor CXCR4, but not CXCR7, reduced the number of migratory monocytes towards recombinant CXCL14, suggesting the involvement of CXCR4 in the CXCL14-directed monocyte chemotaxis. Recombinant CXCL14 enhanced the phagocytic uptake of E.coli particles by monocytes. In scratch assays with cultured endothelial cells (HUVECs, platelet-derived CXCL14 counteracted the pro-angiogenic effects of VEGF, supporting its previously recognized angiostatic potential. Conclusions: Platelets are a relevant source of CXCL14. Platelet-derived CXCL14 at the

Platelet-Derived MRP-14 Induces Monocyte Activation in Patients With Symptomatic Peripheral Artery Disease.

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Dann, Rebecca; Hadi, Tarik; Montenont, Emilie; Boytard, Ludovic; Alebrahim, Dornaszadat; Feinstein, Jordyn; Allen, Nicole; Simon, Russell; Barone, Krista; Uryu, Kunihiro; Guo, Yu; Rockman, Caron; Ramkhelawon, Bhama; Berger, Jeffrey S

2018-01-02

Peripheral artery disease (PAD), a diffuse manifestation of atherothrombosis, is a major cardiovascular threat. Although platelets are primary mediators of atherothrombosis, their role in the pathogenesis of PAD remains unclear. The authors sought to investigate the role of platelets in a cohort of symptomatic PAD. The authors profiled platelet activity, mRNA, and effector roles in patients with symptomatic PAD and in healthy controls. Patients with PAD and carotid artery stenosis were recruited into ongoing studies (NCT02106429 and NCT01897103) investigating platelet activity, platelet RNA, and cardiovascular disease. Platelet RNA sequence profiling mapped a robust up-regulation of myeloid-related protein (MRP)-14 mRNA, a potent calcium binding protein heterodimer, in PAD. Circulating activated platelets were enriched with MRP-14 protein, which augmented the expression of the adhesion mediator, P-selectin, thereby promoting monocyte-platelet aggregates. Electron microscopy confirmed the firm interaction of platelets with monocytes in vitro and colocalization of macrophages with MRP-14 confirmed their cross talk in atherosclerotic manifestations of PAD in vivo. Platelet-derived MRP-14 was channeled to monocytes, thereby fueling their expression of key PAD lesional hallmarks and increasing their directed locomotion, which were both suppressed in the presence of antibody-mediated blockade. Circulating MRP-14 was heightened in the setting of PAD, significantly correlated with PAD severity, and was associated with incident limb events. The authors identified a heightened platelet activity profile and unraveled a novel immunomodulatory effector role of platelet-derived MRP-14 in reprograming monocyte activation in symptomatic PAD. (Platelet Activity in Vascular Surgery and Cardiovascular Events [PACE]; NCT02106429; and Platelet Activity in Vascular Surgery for Thrombosis and Bleeding [PIVOTAL]; NCT01897103). Copyright © 2018 American College of Cardiology Foundation

Effect of platelet-derived growth factor-BB on bone formation in calvarial defects: an experimental study in rabbits

DEFF Research Database (Denmark)

Vikjaer, D; Blom, S; Hjørting-Hansen, E

1997-01-01

The effect of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) on bone healing was examined in calvarial defects in rabbits. Bicortical circular (critical size) defects were prepared in the calvarial bone of 16 rabbits. The defects were closed on the dural side and covered externally...

Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets.

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Makhoul, Stephanie; Walter, Elena; Pagel, Oliver; Walter, Ulrich; Sickmann, Albert; Gambaryan, Stepan; Smolenski, Albert; Zahedi, René P; Jurk, Kerstin

2018-06-01

Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca 2+ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Platelet-rich plasma and adipose-derived mesenchymal stem cells for regenerative medicine-associated treatments in bottlenose dolphins (Tursiops truncatus.

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Richard J Griffeth

Full Text Available Dolphins exhibit an extraordinary capacity to heal deep soft tissue injuries. Nevertheless, accelerated wound healing in wild or captive dolphins would minimize infection and other side effects associated with open wounds in marine animals. Here, we propose the use of a biological-based therapy for wound healing in dolphins by the application of platelet-rich plasma (PRP. Blood samples were collected from 9 different dolphins and a specific and simple protocol which concentrates platelets greater than two times that of whole blood was developed. As opposed to a commonly employed human protocol for PRP preparation, a single centrifugation for 3 minutes at 900 rpm resulted in the best condition for the concentration of dolphin platelets. By FACS analysis, dolphin platelets showed reactivity to platelet cell-surface marker CD41. Analysis by electron microscopy revealed that dolphin platelets were larger in size than human platelets. These findings may explain the need to reduce the duration and speed of centrifugation of whole blood from dolphins to obtain a 2-fold increase and maintain proper morphology of the platelets. For the first time, levels of several growth factors from activated dolphin platelets were quantified. Compared to humans, concentrations of PDGF-BB were not different, while TGFβ and VEGF-A were significantly lower in dolphins. Additionally, adipose tissue was obtained from cadaveric dolphins found along the Spanish Mediterranean coast, and adipose-derived mesenchymal stem cells (ASCs were successfully isolated, amplified, and characterized. When dolphin ASCs were treated with 2.5 or 5% dolphin PRP they exhibited significant increased proliferation and improved phagocytotic activity, indicating that in culture, PRP may improve the regenerative capacity of ASCs. Taken together, we show an effective and well-defined protocol for efficient PRP isolation. This protocol alone or in combination with ASCs, may constitute the basis of a

Decrease in platelet activating factor stimulated phosphoinositide turnover during storage of human platelets in plasma

International Nuclear Information System (INIS)

Carter, M.G.; Shukla, S.D.

1987-01-01

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 32 P incorporation into phosphoinositides using platelet rich plasma (PRP). Platelets in PRP were incubated with 1 x 10 -7 M PAF at 37 degree C with gentle shaking and after 5 min their lipids were extracted and analysed by TLC for 32 P-phosphoinositides. The percent stimulation of 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

A bioactive molecule in a complex wound healing process: platelet-derived growth factor.

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Kaltalioglu, Kaan; Coskun-Cevher, Sule

2015-08-01

Wound healing is considered to be particularly important after surgical procedures, and the most important wounds related to surgical procedures are incisional, excisional, and punch wounds. Research is ongoing to identify methods to heal non-closed wounds or to accelerate wound healing; however, wound healing is a complex process that includes many biological and physiological events, and it is affected by various local and systemic factors, including diabetes mellitus, infection, ischemia, and aging. Different cell types (such as platelets, macrophages, and neutrophils) release growth factors during the healing process, and platelet-derived growth factor is a particularly important mediator in most stages of wound healing. This review explores the relationship between platelet-derived growth factor and wound healing. © 2014 The International Society of Dermatology.

Effects of Platelets on Platelet Concentrate Product on the Activation of Human Peripheral Blood Monocyte Cells

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N Sadat Razavi Hoseini

2016-02-01

Full Text Available Introduction: Monocytes can interact with platelets due to their surface molecules such as P-selectin glycoprotein ligand-1 (PSGL-1, and form monocyte-platelet complex. In the present study, the effects of platelets interaction of platelet concentrates (PCs and peripheral blood monocytes were investigated in vitro as a model to predict the probable interactions of these cells and consequently activation of monocytes. Methods: In this experimental study, units of whole blood and PCs were prepared from Tehran Blood Transfusion Center. After isolation of monocytes from the whole blood, these cells were treated with PC- derived platelets. The activation of monocytes was assessed before and after treatment by the analysis of the respiratory burst of monocytes using dihydrorhodamine 123 (DHR-123. The study data were analyzed using the non-parametric test of Wilcoxon. Results: The purity of monocytes was determined as 86.1±2 using NycoPrep method. The respiratory burst of monocytes was increased after exposure with platelets. In fact, the difference was significant when platelets were used on the 5th day of storage (P=0.001. Conclusions: The study findings revealed that platelets have an efficient capacity to stimulate and activate monocytes. The possible involvement of molecules in the interaction of platelet-monocyte demand to be further studied in future.

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.

In vitro effect of sodium nitrite on platelet aggregation in human platelet rich plasma--preliminary report.

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Kadan, M; Doğanci, S; Yildirim, V; Özgür, G; Erol, G; Karabacak, K; Avcu, F

2015-10-01

The role of nitrates and nitric oxide on platelet functions has obtained an increasing attention with respect to their potential effects on cardiovascular disorders. In this study we aimed to analyze the effect of sodium nitrite on platelet functions in human platelets. This in vitro study was designed to show the effect of sodium nitrite on platelet functions in seven healthy volunteers. Blood samples were centrifuged to prepare platelet rich plasma and platelet poor plasma. Platelet rich plasma was diluted with the platelet poor plasma to have a final count of 300,000 ± 25,000 platelets. Platelet rich plasma was incubated with six different increasing doses (from 10 μM to 5 mM) of sodium nitrite for 1 hour at 37°C. Then stimulating agents including collagen (3 μg ml-1), adenosine diphosphate (10 μM), and epinephrine (10 μM) were added to the cuvette. Changes in light transmission were observed for 10 minutes. In addition spontaneous aggregation were performed in control group with all aggregating agents separately. Effect of sodium nitrite on agonist-induced platelet aggregation depends on the concentration of sodium nitrite. Compared with control group, agonist-induced platelet aggregations were significantly suppressed by sodium nitrite at the concentration of 5, 1.0 and 0.5 mM. Our results suggested that sodium nitrite has inhibitory effects in vitro on platelet aggregation in a dose-dependent manner.

Quantification of platelets and platelet derived growth factors from platelet-rich-plasma (PRP) prepared at different centrifugal force (g) and time.

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Arora, Satyam; Doda, Veena; Kotwal, Urvershi; Dogra, Mitu

2016-02-01

Platelet derived biomaterials represent a key source of cytokines and growth factors extensively used for tissue regeneration; wound healing and tissue repair. Our study was to quantify platelets and growth factors released by PRP when prepared at different centrifugal force (g) and time. Our study was approved by the institutional ethical committee. One hundred millilitres of whole blood (WB) was collected in bag with CPDA as the anticoagulant(AC); (14 mL for 100 mL WB ratio). Nine aliquots of 10 mL each were made from the bag and set of three aliquots were made a group. PRP was prepared at varying centrifugal force (group A: -110 g, group B: -208 g & group C: -440 g) & time (1: -5 min, 2: -10 min & 3: -20 min). Contents of each PRP prepared were analysed. Commercial sandwich ELISA kits were used to quantify the concentrations of CD62P (Diaclone SAS; France), Platelet derived growth factors-AB (Qayee-Bio; China), transforming growth factor-β1 (DRG; Germany) and vascular endothelial growth factor (Boster Immuno Leader; USA) released in each PRP prepared. Eight volunteers were enrolled in the study (24-30 years). The baseline blood counts of all the volunteers were comparable (p ≥ 0.05). Mean ± SD of platelet yield of all nine groups ranged from 17.2 ± 4.2% to 78.7 ± 5.7%. Each PRP was activated with calcified thromboplastin to quantify the growth factors released by them. Significantly higher (p < 0.05) transforming growth factor-β1 and vascular endothelial growth factor were released compared to the baseline. Our study highlights the variation in both force (g) and time results in changes at cellular level and growth factor concentrations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Chitosan inhibits platelet-mediated clot retraction, increases platelet-derived growth factor release, and increases residence time and bioactivity of platelet-rich plasma in vivo.

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Deprés-Tremblay, Gabrielle; Chevrier, Anik; Tran-Khanh, Nicolas; Nelea, Monica; Buschmann, Michael D

2017-11-10

Platelet-rich plasma (PRP) has been used to treat different orthopedic conditions, however, the clinical benefits of using PRP remain uncertain. Chitosan (CS)-PRP implants have been shown to improve meniscus, rotator cuff and cartilage repair in pre-clinical models. The purpose of this current study was to investigate in vitro and in vivo mechanisms of action of CS-PRP implants. Freeze-dried formulations containing 1% (w/v) CS (80% degree of deacetylation and number average molar mass 38 kDa), 1% (w/v) trehalose as a lyoprotectant and 42.2 mM calcium chloride as a clot activator were solubilized in PRP. Gravimetric measurements and molecular/cellular imaging studies revealed that clot retraction is inhibited in CS-PRP hybrid clots through physical coating of platelets, blood cells and fibrin strands by chitosan, which interferes with platelet aggregation and platelet-mediated clot retraction. Flow cytometry and ELISA assays revealed that platelets are activated and granules secreted in CS-PRP hybrid clots and that cumulative release of platelet-derived growth factor (PDGF-AB) and epidermal growth factor is higher from CS-PRP hybrid clots compared to PRP clots in vitro. Finally, CS-PRP implants resided for up to 6 weeks in a subcutaneous implantation model and induced cell recruitment and granulation tissue synthesis, confirming greater residency and bioactivity compared to PRP in vivo.

Identification of a second putative receptor of platelet activating factor on human polymorphonuclear leukocytes

International Nuclear Information System (INIS)

Hwang, S.B.

1987-01-01

Due to multiple molecular species of platelet activating factor (PAF) and the existence of high affinity binding sites in a variety of cells and tissues, possible existence of PAF receptor subtypes has been suggested. This report shows differences between specific PAF receptors on human leukocytes and platelets. Human PMN leukocyte membranes showed high affinity binding sites for PAF with an equilibrium dissociation constant (Kd) of 4.7 (+/- 1.4) x 10 -10 M. The maximal number (B/sub max/) of receptor sites was estimated to be 3.13 (+/- 1.4) x 10 -13 mol/mg protein. They compared the relative potencies of several PAF agonists and receptor antagonists between human platelet and human leukocyte membranes. One antagonist (Ono-6240) was found to be 8 times less potent at inhibiting the [ 3 H]PAF specific receptor binding to human leukocytes than to human platelets. Mg 2+ , Ca 2+ and K + ions potentiated the [ 3 H]PAF specific binding in both systems. Na + ions inhibited the [ 3 H]PAF specific binding to human platelets but showed no effects in human leukocytes. K + ions decreased the Mg 2+ -potentiated [ 3 H]PAF binding in human leukocytes but showed no effects in human platelets. These results suggest that the PAF specific receptors in human leukocytes are different structurally and possibly functionally from the receptors identified in human platelets

[Effect of losartan on human platelet activation by thromboxane A2].

Science.gov (United States)

Guerra, J I; Montón, M; Rodríguez-Feo, J A; Farré, J; Jiménez, A M; Núñez, A; Gómez, J; Rico, L; Marcos, P; Castilla, C; Sánchez De Miguel, L; Casado, S; López-Farré, A

2000-04-01

Previous studies have demonstrated that losartan, an AT-1 receptor antagonist of angiotensin II (Ang II) could block the receptor of thromboxane A2 (TXA2) in the vascular wall. The aim of the present study was to assess the effect of losartan on human platelet activation. Platelets were obtained from 15 healthy men between the age 26 and 40. Platelet activation was measured by changes in the light transmission of platelet-rich plasma stimulated by a synthetic TXA2 analogue, U46619 (5 x 10(-6) mol/l). The U46619-stimulated platelet aggregation was significantly inhibited by losartan in a dose-response manner. Only a high dose of EXP 3174 (5 10-5 mol/l), the in vivo active metabolite of losartan, was able to attenuate U46619-induced platelet activation. Captopril, an angiotensin I-converting inhibitor failed to modify U46619-induced platelet aggregation. Despite the platelets expressing AT-1 type receptors, of Ang II exogenous Ang II did not modify platelet aggregation induced by U46619. The binding of U46619 to platelets was competitively inhibited by losartan in dose-dependent manner. However, only a high dose of EXP 3174 reduced the binding of U46619. Captopril failed to modify the binding of U46619 to platelets. Losartan decreased platelet aggregation by a TXA2-dependent mechanism. EXP 3174 showed a lesser potency than losartan to reduce TXA2-platelet activation. Captopril and exogenous angiotensin II had no effect on human platelet activation. These results suggest that losartan reduced TXA2-dependent platelet activation independently of the blockade of AT-1 receptors.

Improved Human Erythropoiesis and Platelet Formation in Humanized NSGW41 Mice

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Susann Rahmig

2016-10-01

Full Text Available Human erythro-megakaryopoiesis does not occur in humanized mouse models, preventing the in vivo analysis of human hematopoietic stem cell (HSC differentiation into these lineages in a surrogate host. Here we show that stably engrafted KIT-deficient NOD/SCID Il2rg−/− KitW41/W41 (NSGW41 mice support much improved human erythropoiesis and platelet formation compared with irradiated NSG recipients. Considerable numbers of human erythroblasts and mature thrombocytes are present in the bone marrow and blood, respectively. Morphology, composition, and enucleation capacity of de novo generated human erythroblasts in NSGW41 mice are comparable with those in human bone marrow. Overexpression of human erythropoietin showed no further improvement in human erythrocyte output, but depletion of macrophages led to the appearance of human erythrocytes in the blood. Human erythropoiesis up to normoblasts and platelet formation is fully supported in NSGW41 mice, allowing the analysis of human HSC differentiation into these lineages, the exploration of certain pathophysiologies, and the evaluation of gene therapeutic approaches.

Xanthohumol, a Prenylated Flavonoid from Hops (Humulus lupulus, Prevents Platelet Activation in Human Platelets

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Ye-Ming Lee

2012-01-01

Full Text Available 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 stimulated by collagen. Xanthohumol inhibited platelet activation accompanied by relative [Ca2+]i mobilization, thromboxane A2 formation, hydroxyl radical (OH● formation, and phospholipase C (PLCγ2, protein kinase C (PKC, mitogen-activated protein kinase (MAPK, and Akt phosphorylation. Neither SQ22536, an inhibitor of adenylate cyclase, nor ODQ, an inhibitor of guanylate cyclase, reversed the xanthohumol-mediated inhibitory effect on platelet aggregation. Furthermore, xanthohumol did not significantly increase nitrate formation in platelets. This study demonstrates for the first time that xanthohumol possesses potent antiplatelet activity which may initially inhibit the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, followed by inhibition of the thromboxane A2 formation, thereby leading to inhibition of [Ca2+]i and finally inhibition of platelet aggregation. Therefore, this novel role of xanthohumol may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.

Women's attitude towards routine human platelet antigen-screening in pregnancy.

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Winkelhorst, Dian; Loeff, Rosanne M; van den Akker-Van Marle, M Elske; de Haas, Masja; Oepkes, Dick

2017-08-01

Fetal and neonatal alloimmune thrombocytopenia is a potentially life-threatening disease with excellent preventative treatment available for subsequent pregnancies. To prevent index cases, the effectiveness of a population-based screening program has been suggested repeatedly. Therefore, we aimed to evaluate women's attitude towards possible future human platelet antigen-screening in pregnancy. We performed a cross-sectional questionnaire study among healthy pregnant women receiving prenatal care in one of seven participating midwifery practices. Attitude was assessed using a questionnaire based on the validated Multidimensional Measurement of Informed Choice model, containing questions assessing knowledge, attitude and intention to participate. A total of 143 of the 220 women (65%) completed and returned the questionnaire. A positive attitude towards human platelet antigen-screening was expressed by 91% of participants, of which 94% was based on sufficient knowledge. Attitude was more likely to be negatively influenced by the opinion that screening can be frightening. Informed choices were made in 87% and occurred significantly less in women from non-European origin, 89% in European women vs. 60% in non-European women (p = 0.03). Pregnant women in the Netherlands expressed a positive attitude towards human platelet antigen-screening in pregnancy. We therefore expect a high rate of informed uptake when human platelet antigen-screening is implemented. In future counseling on human platelet antigen-screening, ethnicity and possible anxiety associated with a screening test need to be specifically addressed. © 2017 Nordic Federation of Societies of Obstetrics and Gynecology.

Myeloperoxidase modulates human platelet aggregation via actin cytoskeleton reorganization and store-operated calcium entry

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Irina V. Gorudko

2013-07-01

Myeloperoxidase (MPO is a heme-containing enzyme released from activated leukocytes into the extracellular space during inflammation. Its main function is the production of hypohalous acids that are potent oxidants. MPO can also modulate cell signaling and inflammatory responses independently of its enzymatic activity. Because MPO is regarded as an important risk factor for cardiovascular diseases associated with increased platelet activity, we studied the effects of MPO on human platelet functional properties. Laser scanning confocal microscopy was used to reveal carbohydrate-independent MPO binding to human platelet membrane. Adding MPO to platelets did not activate their aggregation under basal conditions (without agonist. In contrast, MPO augmented agonist-induced platelet aggregation, which was not prevented by MPO enzymatic activity inhibitors. It was found that exposure of platelets to MPO leads to actin cytoskeleton reorganization and an increase in their elasticity. Furthermore, MPO evoked a rise in cytosolic Ca2+ through enhancement of store-operated Ca2+ entry (SOCE. Together, these findings indicate that MPO is not a direct agonist but rather a mediator that binds to human platelets, induces actin cytoskeleton reorganization and affects the mechanical stiffness of human platelets, resulting in potentiating SOCE and agonist-induced human platelet aggregation. Therefore, an increased activity of platelets in vascular disease can, at least partly, be provided by MPO elevated concentrations.

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

DEFF Research Database (Denmark)

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

2017-01-01

, we tested PLs prepared via three methods from outdated buffy coat-derived platelet concentrates (PCs) to establish an efficient and feasible expansion of ASCs for clinical use. METHODS: PLs were prepared by the freeze-thaw method from freshly drawn platelets or from outdated buffy coat-derived PCs...

Association assessment of platelet derived growth factor B gene ...

African Journals Online (AJOL)

Background: Coronary artery disease (CAD) is the most frequent cause of morbidity and mortality in the world and it is known as a multifactorial disorder which is influenced by both genetic and environmental factors. Based on different assays, the platelet derived growth factor B (PDGF-B) gene is shown to be amongst the ...

Preparation of a viable population of indium-111-labelled human blood platelets

International Nuclear Information System (INIS)

Heyns, A.; Badenhorst, P.N.; Pieters, H.; Loetter, M.G.; Minnaar, P.C.; Duyvene de Wit, L.J.; Reenen, O.R. van; Retief, F.P.; University of the Orange Free State, Bloemfontein; University of the Orange Free State, Bloemfontein; University of the Orange Free State, Bloemfontein

1980-01-01

Factors influencing labelling of human platelets with 111 Indium-8-hydroxyquinoline ([ 111 In]-oxine) in a physiological saline medium were investigated. The efficiency of labelling is influenced by time of incubation, concentration of oxine, and pH of the incubating medium. It was found that a viable platelet population could be labelled under the following conditions: (1) centrifugation of platelet rich plasma in polystyrene conical tubes at 800 g for 15 min; (2) resuspension of the platelet pellet in saline, pH 5.5; (3) incubating for 30 min at 22 0 C with [ 111 In]-oxine at a concentration of 6.25 mg oxine/litre platelet suspension; (4) washing once with platelet poor autologous plasma (PPP); and (5) finally suspending the platelets in PPP. The labelled platelets aggregated normally with collagen and ADP. Electron microscopy, done immediately after labelling, showed internal organelle reorganization characteristic of activated platelets. These ultrastructural features were reversible on incubationin PPP at 37 0 C for 30 min. The 111 In is not released from aggregated platelets and the label does not elute from incubated platelets for at least five hr. We conclude that human platelets thus labelled are suitable for in vivo kinetic studies. (orig.) [de

N-terminal sequence of human leukocyte glycoprotein Mo1: conservation across species and homology to platelet IIb/IIIa.

Science.gov (United States)

Pierce, M W; Remold-O'Donnell, E; Todd, R F; Arnaout, M A

1986-12-12

Mo1 and gp160-gp93 are two surface membrane glycoprotein heterodimers present on granulocytes and monocytes derived from humans and guinea pigs, respectively. We purified both antigens and found that their alpha subunits had identical N-termini which were significantly homologous to the alpha subunit of the human adhesion platelet glycoprotein IIb/IIIa.

Pathogen inactivation efficacy of Mirasol PRT System and Intercept Blood System for non-leucoreduced platelet-rich plasma-derived platelets suspended in plasma.

Science.gov (United States)

Kwon, S Y; Kim, I S; Bae, J E; Kang, J W; Cho, Y J; Cho, N S; Lee, S W

2014-10-01

This study was conducted to evaluate the efficacy of pathogen inactivation (PI) in non-leucoreduced platelet-rich plasma-derived platelets suspended in plasma using the Mirasol PRT System and the Intercept Blood System. Platelets were pooled using the Acrodose PL system and separated into two aliquots for Mirasol and Intercept treatment. Four replicates of each viral strain were used for the evaluation. For bacteria, both low-titre (45-152 CFU/unit) inoculation and high-titre (7·34-10·18 log CFU/unit) inoculation with two replicates for each bacterial strain were used. Platelets with non-detectable bacterial growth and platelets inoculated with a low titre were stored for 5 days, and culture was performed with the BacT/ALERT system. The inactivation efficacy expressed as log reduction for Mirasol and Intercept systems for viruses was as follows: human immunodeficiency virus 1, ≥4·19 vs. ≥4·23; bovine viral diarrhoea virus, 1·83 vs. ≥6·03; pseudorabies virus, 2·73 vs. ≥5·20; hepatitis A virus, 0·62 vs. 0·76; and porcine parvovirus, 0·28 vs. 0·38. The inactivation efficacy for bacteria was as follows: Escherichia coli, 5·45 vs. ≥9·22; Staphylococcus aureus, 4·26 vs. ≥10·11; and Bacillus subtilis, 5·09 vs. ≥7·74. Postinactivation bacterial growth in platelets inoculated with a low titre of S. aureus or B. subtilis was detected only with Mirasol. Pathogen inactivation efficacy of Intercept for enveloped viruses was found to be satisfactory. Mirasol showed satisfactory inactivation efficacy for HIV-1 only. The two selected non-enveloped viruses were not inactivated by both systems. Inactivation efficacy of Intercept was more robust for all bacteria tested at high or low titres. © 2014 International Society of Blood Transfusion.

Comparison of human platelet lysate alternatives using expired and freshly isolated platelet concentrates for adipose-derived stromal cell expansion.

Science.gov (United States)

Dessels, Carla; Durandt, Chrisna; Pepper, Michael S

2018-03-19

Pooled human platelet lysate (pHPL) has been used to expand adipose-derived stromal cells (ASCs) and can be formulated using fresh or expired buffy coats (BCs) which are then resuspended in either plasma or an additive solution. Not much is known about the effects that expired products and additive solutions have on ASC expansion, and the need for quality control and release criteria has been expressed. This pilot study compared proliferation, cell size, morphology and immunophenotype of ASCs expanded in the different pHPL alternatives versus foetal bovine serum (FBS). Quality control criteria were assessed prior to and during the manufacture of the pHPL alternatives. ASCs were then expanded in 1%, 2.5%, 5% or 10% of the different pHPL alternatives or in 10% FBS. Cell size, morphology, cell number and immunophenotype were measured using microscopy and flow cytometry. The majority of the pHPL alternatives were within the recommended ranges for the quality control criteria. ASCs expanded in the pHPL alternatives were smaller in size, displayed a tighter spindle-shaped morphology, increased cell growth and had a similar immunophenotype (with the exception of CD34 and CD36) when compared to ASCs expanded in FBS. Here we report on the effects that expired BC products and additive solutions have on ASC expansion. When taken together, our findings indicate that all of the pHPL alternatives can be considered to be suitable replacements for FBS for ASC expansion, and that expired BC products can be used as an alternative to fresh BC products.

Modified expression of surface glyconjugates in stored human platelets

International Nuclear Information System (INIS)

Dhar, A.; Ganguly, P.

1987-01-01

Platelets are anucleated cells which play an important part in blood coagulation and thrombosis. These cells may be stored in the blood bank for only 4/5 days. In order to improve the storage of platelets, it is essential to first understand the changes in these cells due to storage. In this work, human platelets were stored in autologous plasma at 4 0 or 22 0 and their surface changes were monitored with three lectins - wheat germ afflutinin (WGA), concanavalin A (Con A) and lentil lectin (LL). Blood was drawn from healthy donors and platelet rich plasma (PRP) was collected by slow speed centrifugation. Platelets stored at either temperature for different times showed increased sensitivity to agglutination by WGA after 34-48 hrs. Lectins, Con A and LL, which were not agglutinating to fresh platelets readily caused agglutination after 48-72 hrs. The platelets stored for 25 hrs or longer period were insensitive to thrombin but showed enhanced aggregation with WGA. Labelling of surface glycoconjugates of stored platelets with 3 H-boro-hydride revealed progressive loss of a glycoprotein of Mr 150,000 (GPIb infinity) together with the appearance of components of Mr 69,000; Mr 60,000; Mr 25,000. New high molecular weight glycoproteins were also detected only in stored platelets. The author studies clearly indicate that modification or altered expression of platelets surface glycoproteins may be one factor of storage related dysfunction of platelets

Modified expression of surface glyconjugates in stored human platelets

Energy Technology Data Exchange (ETDEWEB)

Dhar, A.; Ganguly, P.

1987-05-01

Platelets are anucleated cells which play an important part in blood coagulation and thrombosis. These cells may be stored in the blood bank for only 4/5 days. In order to improve the storage of platelets, it is essential to first understand the changes in these cells due to storage. In this work, human platelets were stored in autologous plasma at 4/sup 0/ or 22/sup 0/ and their surface changes were monitored with three lectins - wheat germ afflutinin (WGA), concanavalin A (Con A) and lentil lectin (LL). Blood was drawn from healthy donors and platelet rich plasma (PRP) was collected by slow speed centrifugation. Platelets stored at either temperature for different times showed increased sensitivity to agglutination by WGA after 34-48 hrs. Lectins, Con A and LL, which were not agglutinating to fresh platelets readily caused agglutination after 48-72 hrs. The platelets stored for 25 hrs or longer period were insensitive to thrombin but showed enhanced aggregation with WGA. Labelling of surface glycoconjugates of stored platelets with /sup 3/H-boro-hydride revealed progressive loss of a glycoprotein of Mr 150,000 (GPIb infinity) together with the appearance of components of Mr 69,000; Mr 60,000; Mr 25,000. New high molecular weight glycoproteins were also detected only in stored platelets. The author studies clearly indicate that modification or altered expression of platelets surface glycoproteins may be one factor of storage related dysfunction of platelets.

Global Proteome Analysis Identifies Active Immunoproteasome Subunits in Human Platelets*

Science.gov (United States)

Klockenbusch, Cordula; Walsh, Geraldine M.; Brown, Lyda M.; Hoffman, Michael D.; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

2014-01-01

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. PMID:25146974

Ginsenoside Rg1 and platelet-rich fibrin enhance human breast adipose-derived stem cells function for soft tissue regeneration

Science.gov (United States)

Li, Hong-Mian; Peng, Qi-Liu; Huang, Min-Hong; Li, De-Quan; Liang, Yi-Dan; Chi, Gang-Yi; Li, De-Hui; Yu, Bing-Chao; Huang, Ji-Rong

2016-01-01

Adipose-derived stem cells (ASCs) can be used to repair soft tissue defects, wounds, burns, and scars and to regenerate various damaged tissues. The cell differentiation capacity of ASCs is crucial for engineered adipose tissue regeneration in reconstructive and plastic surgery. We previously reported that ginsenoside Rg1 (G-Rg1 or Rg1) promotes proliferation and differentiation of ASCs in vitro and in vivio. Here we show that both G-Rg1 and platelet-rich fibrin (PRF) improve the proliferation, differentiation, and soft tissue regeneration capacity of human breast adipose-derived stem cells (HBASCs) on collagen type I sponge scaffolds in vitro and in vivo. Three months after transplantation, tissue wet weight, adipocyte number, intracellular lipid, microvessel density, and gene and protein expression of VEGF, HIF-1α, and PPARγ were higher in both G-Rg1- and PRF-treated HBASCs than in control grafts. More extensive new adipose tissue formation was evident after treatment with G-Rg1 or PRF. In summary, G-Rg1 and/or PRF co-administration improves the function of HBASCs for soft tissue regeneration engineering. PMID:27191987

Sequential use of human-derived medium supplements favours cardiovascular tissue engineering

NARCIS (Netherlands)

Riem Vis, P.W.; Sluijter, J.P.G.; Soekhradj - Soechit, R.S.; Herwerden, van L.A.; Kluin, J.; Bouten, C.V.C.

2012-01-01

For clinical application of tissue engineering strategies, the use of animal-derived serum in culture medium is not recommended, because it can evoke immune responses in patients. We previously observed that human platelet-lysate (PL) is favourable for cell expansion, but generates weaker tissue as

Platelet-derived microparticles regulates thrombin generation via phophatidylserine in abdominal sepsis.

Science.gov (United States)

Wang, Yongzhi; Zhang, Su; Luo, Lingtao; Norström, Eva; Braun, Oscar Ö; Mörgelin, Matthias; Thorlacius, Henrik

2018-02-01

Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis. © 2017 Wiley Periodicals, Inc.

Aluminum induces lipid peroxidation and aggregation of human blood platelets

Directory of Open Access Journals (Sweden)

T.J.C. Neiva

1997-05-01

Full Text Available Aluminum (Al3+ intoxication is thought to play a major role in the development of Alzheimer's disease and in certain pathologic manifestations arising from long-term hemodialysis. Although the metal does not present redox capacity, it can stimulate tissue lipid peroxidation in animal models. Furthermore, in vitro studies have revealed that the fluoroaluminate complex induces diacylglycerol formation, 43-kDa protein phosphorylation and aggregation. Based on these observations, we postulated that Al3+-induced blood platelet aggregation was mediated by lipid peroxidation. Using chemiluminescence (CL of luminol as an index of total lipid peroxidation capacity, we established a correlation between lipid peroxidation capacity and platelet aggregation. Al3+ (20-100 µM stimulated CL production by human blood platelets as well as their aggregation. Incubation of the platelets with the antioxidants nor-dihydroguaiaretic acid (NDGA (100 µM and n-propyl gallate (NPG (100 µM, inhibitors of the lipoxygenase pathway, completely prevented CL and platelet aggregation. Acetyl salicylic acid (ASA (100 µM, an inhibitor of the cyclooxygenase pathway, was a weaker inhibitor of both events. These findings suggest that Al3+ stimulates lipid peroxidation and the lipoxygenase pathway in human blood platelets thereby causing their aggregation

Safety assessment of bone marrow derived MSC grown in platelet-rich plasma

Directory of Open Access Journals (Sweden)

Shoji Fukuda

2015-06-01

Full Text Available The injection of endothelial progenitor cells and mononuclear cells derived from bone marrow at the ischemic region of peripheral artery disease patients is reported to be effective for therapeutic angiogenesis; however, these cell therapies require large amounts of bone marrow to obtain sufficient numbers of cells. To solve this problem, we attempted to culture bone-marrow-derived mesenchymal stem cells (BM-MSC, which are supposed to secrete several cytokines that promote angiogenesis. We also focused on using platelet-rich plasma (PRP as a supplement for cell culture instead of fetal bovine serum. Human BM-MSC obtained from healthy volunteers expanded rapidly when cultured with 10% PRP prepared from their own blood. FACS analysis revealed that these cultured human MSC were homogeneous populations, and chromosomal analysis showed a normal karyotype. Moreover, the angiogenetic effect was apparent two weeks after human BM-MSC were injected into the ischemic muscle in SCID mice. Tumor formation was not detected three months after injection into SCID mice either subcutaneously or intramuscularly. To simulate clinical settings, canine BM-MSC were grown with canine PRP and injected into their ischemic muscles. We confirmed that donor cells existed in situ two and six weeks after operation without any side effects. These results suggest that cultured human BM-MSC can be a promising cell source for therapeutic angiogenesis.

Global proteome analysis identifies active immunoproteasome subunits in human platelets.

Science.gov (United States)

Klockenbusch, Cordula; Walsh, Geraldine M; Brown, Lyda M; Hoffman, Michael D; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

2014-12-01

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells.

Science.gov (United States)

Iudicone, Paola; Fioravanti, Daniela; Bonanno, Giuseppina; Miceli, Michelina; Lavorino, Claudio; Totta, Pierangela; Frati, Luigi; Nuti, Marianna; Pierelli, Luca

2014-01-27

Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. Platelet lysate (PL) is a human blood component which may replace animal serum in MSC cultures being rich in various growth factors. Here, we describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC. PL lots were obtained by combining 2 6-unit PLT pools in additive solution (AS) following a transfusional-based procedure including pathogen inactivation (PI) by Intercept technology and 3 cycles of freezing/thawing, followed by membrane removal. Three PI-PL and 3 control PL lots were produced to compare their ability to sustain bone marrow derived MSC selection and expansion. Moreover, two further PL, subjected to PI or not, were also produced starting from the same initial PLT pools to evaluate the impact of PI on growth factor concentration and capacity to sustain cell growth. Additional PI-PL lots were used for comparison with fetal bovine serum (FBS) on MSC expansion. Immunoregulatory properties of PI-PL-generated MSC were documented in vitro by mixed lymphocyte culture (MLC) and peripheral blood mononuclear cells (PBMC) mitogen induced proliferation. PI-PL and PL control lots had similar concentrations of 4 well-described growth factors endowed with MSC stimulating ability. Initial growth and MSC expansion by PI-PL and PL controls were comparable either using different MSC populations or in head to head experiments. Moreover, PI-PL and PL control sustained similar MSC growth of frozen/thawed MSC. Multilineage differentiation of PI-derived and PI-PL-derived MSC were maintained in any MSC cultures as well as their immunoregulatory properties. Finally, no direct impact of PI on growth factor concentration and MSC growth support was observed, whereas the capacity of FBS to sustain MSC expansion in basic

Induced Pluripotent Stem Cell-Derived Red Blood Cells and Platelet Concentrates: From Bench to Bedside.

Science.gov (United States)

Focosi, Daniele; Amabile, Giovanni

2017-12-27

Red blood cells and platelets are anucleate blood components indispensable for oxygen delivery and hemostasis, respectively. Derivation of these blood elements from induced pluripotent stem (iPS) cells has the potential to develop blood donor-independent and genetic manipulation-prone products to complement or replace current transfusion banking, also minimizing the risk of alloimmunization. While the production of erythrocytes from iPS cells has challenges to overcome, such as differentiation into adult-type phenotype that functions properly after transfusion, platelet products are qualitatively and quantitatively approaching a clinically-applicable level owing to advances in expandable megakaryocyte (MK) lines, platelet-producing bioreactors, and novel reagents. Guidelines that assure the quality of iPS cells-derived blood products for clinical application represent a novel challenge for regulatory agencies. Considering the minimal risk of tumorigenicity and the expected significant demand of such products, ex vivo production of iPS-derived blood components can pave the way for iPS translation into the clinic.

Platelet binding and biodistribution of [99mTc]rBitistatin in animal species and humans

International Nuclear Information System (INIS)

Knight, Linda C.; Romano, Jan E.; Bright, Lewis T.; Agelan, Alexis; Kantor, Steven; Maurer, Alan H.

2007-01-01

Introduction: 99m Tc recombinant bitistatin (rBitistatin) is a radioligand for α IIb β 3 (glycoproteins IIb/IIIa) receptor on platelets and is being developed as a diagnostic radiopharmaceutical for in vivo imaging of acute thrombi and emboli. Prior to the first administration of [ 99m Tc]rBitistatin to human subjects, its biodistribution and effects on platelets were evaluated in animals. This paper reports findings in animal studies in comparison with initial findings in normal human subjects. Methods: [ 99m Tc]rBitistatin was administered to mice, guinea pigs and dogs to assess time-dependent organ distribution, urinary excretion and blood disappearance rates. Blood samples were analyzed to determine radioligand binding to circulating platelets and the extent of plasma protein binding. The effect of [ 99m Tc]rBitistatin on circulating platelet count was determined. These factors were also determined in normal human subjects who received [ 99m Tc]rBitistatin as part of a Phase I clinical trial. Results: The main organs that accumulated [ 99m Tc]rBitistatin were kidneys, liver and spleen in all animal species and humans. The main organs seen on human images were the kidneys and spleen. Liver uptake was fainter, and soft-tissue background was low. [ 99m Tc]rBitistatin bound to circulating platelets in blood, with a higher percentage of binding to platelets in guinea pigs and dogs compared to that in humans. Plasma protein binding was low and of little consequence in view of platelet binding. The main route of excretion was through the urine. [ 99m Tc]rBitistatin did not affect platelet counts in humans or dogs. Conclusions: [ 99m Tc]rBitistatin, when administered at low doses for imaging, has no adverse effects on platelets and has the qualitative biodistribution predicted by animal studies. [ 99m Tc]rBitistatin was found to bind to circulating platelets in humans, suggesting that it will be able to bind to activated platelets in vivo in patients with acute

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.

Release of a human platelet specific protein measured by a radioimmunoassay

Energy Technology Data Exchange (ETDEWEB)

Ludlam, C A; Moore, S; Bolton, A E; Pepper, D S; Cash, J D

1975-06-01

Recent studies have demonstrated that it is possible to isolate and characterize a protein released from human platelets during thrombin-induced aggregation. This protein appeared to be unique to platelets and was named ..beta..-thromboglobulin (..beta..-TG). The following communication describes a radioimmunoassay for the measurement of ..beta..-TG and gives an account of preliminary studies to examine the potential application of this assay for the detection of platelet involvement in thromboembolic disorders.

Antiaggregant effects of Arbutus unedo extracts in human platelets.

Science.gov (United States)

El Haouari, Mohammed; López, José J; Mekhfi, Hassane; Rosado, Juan A; Salido, Ginés M

2007-09-05

Platelet hyperaggregability plays a pivotal role in the pathogenesis of cardiovascular diseases. Thrombin evokes aggregation through Ca(2+) mobilization, tyrosine phosphorylation and generation of reactive oxygen species (ROS). We have investigated the antiaggregant properties of Arbutus unedo extracts in human platelets. Changes in cytosolic Ca(2+) concentration and intracellular oxidants production were registered by espectrofluorimetry using fura-2 and dichlorodihydrofluorescein, respectively, platelet aggregation was assessed by aggregometry and protein tyrosine phosphorylation was detected by Western blotting. Platelet treatment with increasing concentrations (0.015-1.5mg/mL) of crude aqueous, ethyl acetate or diethyl ether extracts reduced platelet aggregation evoked by thrombin (0.5 U/mL) and show a potent ROS scavenger activity, preventing thrombin-evoked endogenous generation of ROS. Treatment with Arbutus unedo extracts did not alter thrombin-evoked Ca(2+) release from the intracellular stores but reduced store-operated Ca(2+) entry induced by thrombin or by selective depletion of the two Ca(2+) stores in platelets, the dense tubular system and the acidic stores. In addition, platelet treatment with extracts reduced both basal and thrombin-stimulated protein tyrosine phosphorylation. We conclude that Arbutus unedo extracts show antiaggregant actions due to attenuation of Ca(2+) mobilization, ROS production and protein tyrosine phosphorylation and might be used for the treatment and/or prevention of cardiovascular diseases.

Platelet binding and biodistribution of [{sup 99m}Tc]rBitistatin in animal species and humans

Energy Technology Data Exchange (ETDEWEB)

Knight, Linda C. [Department of Radiology, Temple University School of Medicine, Philadelphia, PA 19140 (United States)], E-mail: lknight@temple.edu; Romano, Jan E. [Department of Radiology, Temple University School of Medicine, Philadelphia, PA 19140 (United States); Bright, Lewis T.; Agelan, Alexis [University Laboratory Animal Resources, Temple University School of Medicine, Philadelphia, PA 19140 (United States); Kantor, Steven; Maurer, Alan H. [Department of Radiology, Temple University School of Medicine, Philadelphia, PA 19140 (United States)

2007-10-15

Introduction: {sup 99m}Tc recombinant bitistatin (rBitistatin) is a radioligand for {alpha}{sub IIb}{beta}{sub 3} (glycoproteins IIb/IIIa) receptor on platelets and is being developed as a diagnostic radiopharmaceutical for in vivo imaging of acute thrombi and emboli. Prior to the first administration of [{sup 99m}Tc]rBitistatin to human subjects, its biodistribution and effects on platelets were evaluated in animals. This paper reports findings in animal studies in comparison with initial findings in normal human subjects. Methods: [{sup 99m}Tc]rBitistatin was administered to mice, guinea pigs and dogs to assess time-dependent organ distribution, urinary excretion and blood disappearance rates. Blood samples were analyzed to determine radioligand binding to circulating platelets and the extent of plasma protein binding. The effect of [{sup 99m}Tc]rBitistatin on circulating platelet count was determined. These factors were also determined in normal human subjects who received [{sup 99m}Tc]rBitistatin as part of a Phase I clinical trial. Results: The main organs that accumulated [{sup 99m}Tc]rBitistatin were kidneys, liver and spleen in all animal species and humans. The main organs seen on human images were the kidneys and spleen. Liver uptake was fainter, and soft-tissue background was low. [{sup 99m}Tc]rBitistatin bound to circulating platelets in blood, with a higher percentage of binding to platelets in guinea pigs and dogs compared to that in humans. Plasma protein binding was low and of little consequence in view of platelet binding. The main route of excretion was through the urine. [{sup 99m}Tc]rBitistatin did not affect platelet counts in humans or dogs. Conclusions: [{sup 99m}Tc]rBitistatin, when administered at low doses for imaging, has no adverse effects on platelets and has the qualitative biodistribution predicted by animal studies. [{sup 99m}Tc]rBitistatin was found to bind to circulating platelets in humans, suggesting that it will be able to bind

Mechanisms of andrographolide-induced platelet apoptosis in human platelets: regulatory roles of the extrinsic apoptotic pathway.

Science.gov (United States)

Lien, Li-Ming; Su, Cheng-Chen; Hsu, Wen-Hsien; Lu, Wan-Jung; Chung, Chi-Li; Yen, Ting-Lin; Chiu, Hou-Chang; Sheu, Joen-Rong; Lin, Kuan-Hung

2013-11-01

Andrographolide, a novel nuclear factor-κB (NF-κB) inhibitor, is isolated from the leaves of Andrographis paniculata. Platelet activation is relevant to a variety of coronary heart diseases. Our recent studies revealed that andrographolide possesses potent antiplatelet activity by inhibition of the p38 MAPK/(●) HO-NF-κB-ERK2 cascade. Although platelets are anucleated cells, apoptotic machinery apparatus recently has been found to regulate platelet activation and limit platelet lifespan. Therefore, we further investigated the regulatory effects of andrographolide on platelet apoptotic events. In this study, apoptotic signaling events for caspase-3, -8, and Bid were time (10-60 min)- and dose (25-100 μΜ)-dependently activated by andrographolide in human platelets. Andrographolide could also disrupt mitrochondrial membrane potential. In addition, caspase-8 inhibitor (z-IETD-fmk, 50 μΜ) was found to reverse andrographolide-induced caspase-8 activation, whereas the antagonistic anti-Fas receptor (ZB4, 500 ng/mL) and anti-tumor necrosis factor-R1 (H398, 10 µg/mL) monoclonal antibodies did not. In conclusion, this study for the first time demonstrated that andrographolide might limit platelet lifespan by initiating the caspase-8-dependent extrinsic apoptotic pathway, in spite of no direct evidence that death receptors are involved in this process proved. Overall, the various medicinal properties of andrographolide suggest its potential value in treating patients with thromboembolic disorders. Copyright © 2012 John Wiley & Sons, Ltd.

Response to platelet-activating factor in human platelets stored and aged in plasma. Decrease in aggregation, phosphoinositide turnover, and receptor affinity

International Nuclear Information System (INIS)

Shukla, S.D.; Morrison, W.J.; Klachko, D.M.

1989-01-01

Human platelet concentrates were stored in polyolefin bags at 22 to 24 degrees C on a horizontal shaker for up to 8 days. At different intervals, aliquots of platelet-rich plasma (PRP) were removed aseptically and five variables, i.e., platelet counts, morphology, platelet-activating factor (PAF)-stimulated aggregation, phosphoinositide turnover, and [3H]PAF binding to platelet receptors, were studied. The number of platelets did not change during the 8 days of storage. Scanning electron microscopy of the platelets revealed a gradual morphologic change from biconcave flat discs to irregular, crenated forms. The PAF-induced aggregation of platelets declined with time of storage. A decrease to 50 percent of the Day 1 aggregatory response to PAF was evident on Day 2, and there was a further decline to about 20 percent by Day 6. Similarly, PAF receptor-coupled phosphoinositide turnover, as monitored by 32P incorporation into individual phosphoinositides, decreased dramatically with storage. After 2 to 3 days of storage, the phosphoinositide turnover was reduced to 50 percent of the original response, and it continued to decline to about 25 percent of original response by Day 5 or 6. The binding of [3H]PAF to washed human platelets indicated subtle changes between Days 2 and 4, which became more noticeable by Day 6. These results have raised the possibility of changes in the number of the receptors and/or their affinity for the ligand during storage. We conclude that although the number of platelets was maintained during storage for 8 days, a general deterioration of their responses to PAF occurred at the levels of cell surface receptor, transmembrane signaling (phosphoinositide turnover), and response (aggregation)

Antimicrobial properties of single-donor-derived, platelet-leukocyte fibrin for fistula occlusion: An in vitro study.

Science.gov (United States)

Wu, Xiuwen; Ren, Jianan; Yuan, Yujie; Luan, Jianfeng; Yao, Genhong; Li, Jieshou

2013-01-01

Fibrin glue is a promising alternative for low-output enterocutaneous fistula closure. Bacterial flora colonizing inside the fistula tract, however, may limit the glue application. Single-donor-derived, platelet-rich materials were hypothesized in this study to have antimicrobial activity against Gram-negative microorganisms. Platelet-leukocyte fibrin (PLF), platelet-rich plasma (PRP), and platelet-poor plasma (PPP) were obtained from healthy volunteers. The amounts of platelet, leukocyte, and complement/antibody were determined. In vitro laboratory susceptibility to PLF and plasmas was determined by the Kirby-Bauer disc-diffusion method. Antimicrobial activity of PLF, PRP, and PPP against three Gram-negative ATCC strains was determined in a bacterial kill assay. Levels of complement and antibody did not significantly differ among PLF, PRP, and PPP (p > 0.05), while platelet and leukocyte counts in platelet-rich biomaterials were significantly higher than those in PPP (p platelets and leukocytes may play an important role in bacterial defense. This is the first study to demonstrate the antibacterial properties of single-unit PLF for fistula closure, presenting a new opportunity for glue sealing.

High-throughput proteomics detection of novel splice isoforms in human platelets.

LENUS (Irish Health Repository)

Power, Karen A

2009-01-01

Alternative splicing (AS) is an intrinsic regulatory mechanism of all metazoans. Recent findings suggest that 100% of multiexonic human genes give rise to splice isoforms. AS can be specific to tissue type, environment or developmentally regulated. Splice variants have also been implicated in various diseases including cancer. Detection of these variants will enhance our understanding of the complexity of the human genome and provide disease-specific and prognostic biomarkers. We adopted a proteomics approach to identify exon skip events - the most common form of AS. We constructed a database harboring the peptide sequences derived from all hypothetical exon skip junctions in the human genome. Searching tandem mass spectrometry (MS\\/MS) data against the database allows the detection of exon skip events, directly at the protein level. Here we describe the application of this approach to human platelets, including the mRNA-based verification of novel splice isoforms of ITGA2, NPEPPS and FH. This methodology is applicable to all new or existing MS\\/MS datasets.

Proteolysis of platelet receptors in humans and other species.

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Qiao, Jian L; Shen, Yang; Gardiner, Elizabeth E; Andrews, Robert K

2010-08-01

In the past 5 years, metalloproteinase-mediated ectodomain shedding of platelet receptors has emerged as a new mechanism for modulating platelet function. By regulating surface expression of the platelet-specific receptors, glycoprotein (GP)VI that binds collagen, and GPIbalpha (the major ligand-binding subunit of the GPIb-IX-V complex) that binds von Willebrand factor (VWF) and other procoagulant and proinflammatory ligands, shedding not only irreversibly downregulates GPVI/GPIbalpha function, but generates proteolytic fragments that might be unique biomarkers or modulators in plasma. This is potentially significant because GPVI and GPIbalpha are involved in initiating thrombotic diseases such as heart attack and stroke, as well as autoimmune diseases where anti-platelet antibodies result in thrombocytopenia. Altered expression levels of GPIbalpha/GPVI are associated with both thrombotic propensity and platelet aging, suggesting an additional role in platelet clearance. Although emerging data are elucidating molecular mechanisms underlying GPIbalpha/GPVI shedding, evidence for the functional consequences of shedding in vivo, either clinically or in animal models, is far more limited. Here we consider recent published evidence for GPVI or GPIbalpha shedding in humans, nonhuman primates and mice, and whether conservation of sheddase cleavage sites across species points to a functional role for metalloproteolytic shedding in vivo.

Ultrastructure and growth factor content of equine platelet-rich fibrin gels.

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Textor, Jamie A; Murphy, Kaitlin C; Leach, J Kent; Tablin, Fern

2014-04-01

To compare fiber diameter, pore area, compressive stiffness, gelation properties, and selected growth factor content of platelet-rich fibrin gels (PRFGs) and conventional fibrin gels (FGs). PRFGs and conventional FGs prepared from the blood of 10 healthy horses. Autologous fibrinogen was used to form conventional FGs. The PRFGs were formed from autologous platelet-rich plasma of various platelet concentrations (100 × 10³ platelets/μL, 250 × 10³ platelets/μL, 500 × 10³ platelets/μL, and 1,000 × 10³ platelets/μL). All gels contained an identical fibrinogen concentration (20 mg/mL). Fiber diameter and pore area were evaluated with scanning electron microscopy. Maximum gelation rate was assessed with spectrophotometry, and gel stiffness was determined by measuring the compressive modulus. Gel weights were measured serially over 14 days as an index of contraction (volume loss). Platelet-derived growth factor-BB and transforming growth factor-β1 concentrations were quantified with ELISAs. Fiber diameters were significantly larger and mean pore areas were significantly smaller in PRFGs than in conventional FGs. Gel weight decreased significantly over time, differed significantly between PRFGs and conventional FGs, and was significantly correlated with platelet concentration. Platelet-derived growth factor-BB and transforming growth factor-β1 concentrations were highest in gels and releasates derived from 1,000 × 10³ platelets/μL. The inclusion of platelets in FGs altered the architecture and increased the growth factor content of the resulting scaffold. Platelets may represent a useful means of modifying these gels for applications in veterinary and human regenerative medicine.

Evaluation of the effects of several zoanthamine-type alkaloids on the aggregation of human platelets.

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Villar, Rosa M; Gil-Longo, José; Daranas, Antonio H; Souto, María L; Fernández, José J; Peixinho, Solange; Barral, Miguel A; Santafé, Gilmar; Rodríguez, Jaime; Jiménez, Carlos

2003-05-15

Ten zoanthamine-type alkaloids from two marine zoanthids belonging to the Zoanthus genus (Zoanthus nymphaeus and Zoanthus sp.) along with one semisynthetic derivative were evaluated for their antiplatelet activities on human platelet aggregation induced by several stimulating agents. 11-Hydroxyzoanthamine (11) and a synthetic derivative of norzoanthamine (16) showed strong inhibition against thrombin-, collagen- and arachidonic acid-induced aggregation, zoanthenol (15) displayed a selective inhibitory activity induced by collagen, while zoanthaminone (10) behaved as a potent aggregant agent. These evaluations allowed us to deduce several structure-activity relationships and suggest some mechanisms of action for this type of compounds.

Pharmacological modulation of human platelet leukotriene C4-synthase.

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Sala, A; Folco, G; Henson, P M; Murphy, R C

1997-03-21

The aim of this study was to test if human platelet leukotriene C4-synthase (LTC4-S) is pharmacologically different from cloned and expressed LTC4-S and, in light of the significant homologies between 5-lipoxygenase activating protein (FLAP) and LTC4-S, if different potencies of leukotriene synthesis inhibitors acting through binding with FLAP (FLAP inhibitors) reflect in different potencies as LTC4-S inhibitors. Leukotriene C4 (LTC4) synthesis by washed human platelets supplemented with synthetic leukotriene A4 (LTA4) was studied in the absence and presence of two different, structurally unrelated FLAP inhibitors (MK-886 and BAY-X1005) as well as a direct 5-lipoxygenase inhibitor (zileuton). LTC4 production was analyzed by RP-HPLC coupled to diode array detection. We report that human platelet LTC4-S was inhibited by MK-886 and BAY-X1005 (IC50 of 4.7 microM and 91.2 microM, respectively), but not by zileuton (inactive up to 300 microM); all 3 compounds were able to inhibit 5-lipoxygenase metabolite biosynthesis in intact human polymorphonuclear leukocytes (IC50 of 0.044 microM, 0.85 microM, and 1.5 microM, respectively). Platelet LTC4-S does not appear pharmacologically different from expression cloned LTC4-S. LTC4-S inhibition by FLAP inhibitors is in agreement with the significant homology reported for expression-cloned LTC4-S with FLAP, Furthermore, functional homology of the binding sites for inhibitors on LTC4-S and FLAP is suggested by the conservation of the relative potencies of MK-886 and BAY-X1005 vs FLAP-dependent 5-lipoxygenase activity and LTC4-S inhibition: MK-886 was 19.3-fold more potent than BAY-X1005 as FLAP inhibitor and 19.6-fold more potent than BAY-X1005 as LTC4-S inhibitor.

Human platelet glycoprotein Ia. One component is only expressed on the surface of activated platelets and may be a granule constituent

International Nuclear Information System (INIS)

Bienz, D.; Clemetson, K.J.

1989-01-01

Glycoprotein Ia (GP Ia) is a relatively minor component of human blood platelets thought to be a receptor involved in collagen-induced platelet activation. However, some difficulties exist with the definition of this glycoprotein. The expression of GP Ia on resting (prostacyclin analogue-treated) and thrombin-activated platelets was compared by surface labeling with 125 I-lactoperoxidase. Intact platelets or platelets solubilized in sodium dodecyl sulfate were labeled with periodate/[ 3 H]NaBH 4 . Analysis on two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed that GP Ia is very poorly labeled in resting platelets. After activation a new spot (GP Ia*) appears with the same relative molecular mass as GP Ia under reducing conditions. GP Ia and Ia* can be clearly separated by two-dimensional nonreduced/reduced gel electrophoresis. Therefore, two glycoproteins which have been termed GP Ia exist in platelets with similar molecular weight and pI under reducing conditions. One of these (GP Ia*) is only surface-labeled when platelets are activated, indicating that it is only exposed on the surface of activated platelets. Supernatant from activated platelets contains this glycoprotein as well as other granule components. This glycoprotein is missing in platelets from two patients with collagen-response defects

Divalent cations and the protein surface co-ordinate the intensity of human platelet adhesion and P-selectin surface expression.

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Whiss, P A; Andersson, R G G

2002-07-01

At sites of blood vessel injury, platelets adhere to exposed vessel components, such as collagen, or immobilized fibrinogen derived from plasma or activated platelets. The divalent cations Mg(2+) and Ca(2+) are essential for platelet adhesion and activation, but Mg(2+) can also inhibit platelet activation. The present study evaluates, by an enzymatic method, the effects of various divalent cations on the adhesion of isolated human platelets to collagen, fibrinogen, albumin or plastic in vitro. By enzyme-linked immunosorbent assay, platelet surface expression of P-selectin was measured to estimate the state of activation on adherence. Mg(2+) increased platelet adhesion exclusively to collagen and fibrinogen at physiologically relevant concentrations. At higher concentrations, the adhesion declined. Ca(2+) induced a weak adhesion only to fibrinogen at physiological doses and a peak of increased adhesion to all protein-coated surfaces at 10 mmol/l. Mn(2+) elicited dose-dependent adhesion only to collagen and fibrinogen. Zn(2+), Ni(2+) and Cu(2+) increased the adhesion of platelets independently of the surface. Ca(2+) dose-dependently inhibited adhesion elicited by Mg(2+) to collagen and fibrinogen. No other combination of divalent cations elicited such an effect. Mg(2+)-dependent platelet adhesion to collagen and Ca(2+)-dependent adhesion to fibrinogen increased P-selectin expression. Thus, the present study shows that the outcome of the platelet adhesion depends on the surface and the access of divalent cations, which co-ordinate the intensity of platelet adhesion and P-selectin surface expression.

Platelet-rich plasma derived growth factors contribute to stem cell differentiation in musculoskeletal regeneration

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Qian, Yun; Han, Qixin; Chen, Wei; Song, Jialin; Zhao, Xiaotian; Ouyang, Yuanming; Yuan, Weien; Fan, Cunyi

2017-10-01

Stem cell treatment and platelet-rich plasma (PRP) therapy are two significant issues in regenerative medicine. Stem cells such as bone marrow mesenchymal stem cells, adipose-derived stem cells and periodontal ligament stem cells can be successfully applied in the field of tissue regeneration. PRP, a natural product isolated from whole blood, can secrete multiple growth factors (GFs) for regulating physiological activities. These GFs can stimulate proliferation and differentiation of different stem cells in injury models. Therefore, combination of both agents receives wide expectations in regenerative medicine, especially in bone, cartilage and tendon repair. In this review, we thoroughly discussed the interaction and underlying mechanisms of platelet-rich plasma derived growth factors with stem cells, and assessed their functions in cell differentiation for musculoskeletal regeneration.

In vitro viability effects on apheresis and buffy-coat derived platelets administered through infusion pumps

Directory of Open Access Journals (Sweden)

Sandgren P

2014-12-01

Full Text Available Per Sandgren,1,2 Veronica Berggren,3 Carl Westling,1,2 Viveka Stiller1 1Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, 2Department of Laboratory Medicine, Karolinska Institutet, 3Department of Neonatology, Karolinska University Hospital, Stockholm, SwedenBackground: Different infusion pump systems as well as gravity infusion have been widely used in neonatal transfusion. However, the limited number of published studies describing the use of infusion pumps on platelets illustrates the necessity for more robust data.Methods: To evaluate the potential in vitro effects on the cellular, metabolic, functional and phenotypic properties of platelets, we set up a four-arm paired study simultaneously comparing the use of different infusion pumps (Alaris® CC/GP with unexposed platelets. The platelet units (n=8 were either produced by the apheresis technique and suspended in 100% plasma or derived from buffy coats to yield platelet units stored in approximately 30% plasma and 70% SSP+. Fresh and 5-day old platelets were tested.Results: Regardless of the production system or storage time used, no significant differences were observed in glucose and lactate concentration, pH, adenosine triphosphate levels, response to extent of shape change, hypotonic shock response reactivity, and CD62P expression. Similarly, no differences were observed in expression of the conformational epitope on glycoprotein IIb/IIIa, determined using procaspase-activating compound 1, or in the expression of CD42b and platelet-endothelial cell adhesion molecule-1 in a comparison between platelets administered through infusion pumps versus unexposed platelets.Conclusion: Using Alaris CC/GP infusion pumps had no influence on the cellular, functional, and phenotypic in vitro properties of platelets. This fact seems not to be affected by different production systems or storage time.Keywords: platelets, neonatal platelet transfusion

Effect of 60Co γ-ray irradiation on human platelets

International Nuclear Information System (INIS)

Wu Guoxin; Zhao Yiming; Ruan Changgeng

1992-01-01

Human platelet-rich plasma was irradiated with various doses (0, 1.25, 2.5, 5, 10, 20, 40 Gy) of 60 Co γ-rays in order to observe the changes of metabolites and the release of internal substance of platelets. At the dose of 5 Gy, alpha-granule membrane protein (GMP-140) molecules expressed on the surface of platelets increased significantly, while glycoproteins (GP) Ib and IIIa did not change apparently; at the dose as low as of 2.5 Gy, thromboxane B 2 production in plasma was remarkably increased; and, at the dose of over 5 Gy, the concentration of von Willebrand factor increased with increasing doses as in the case of GMP-140 molecules. These results indicate that platelets can be activated in vitro when the dose of 60 Co γ-rays exceeds 5 Gy

Effects of 60Co γ-ray irradiation on human platelets

International Nuclear Information System (INIS)

Wu Guoxin; Zhao Yiming; Ruan Changgeng

1991-02-01

Human platelet-rich plasma was irradiated with various doses (0,1.25,2.5,5.0,10,20,40Gy) of 60 Co γ-ray so as to observing the changes of metabolites and releasing substances of platelets. Alpha-granule membrane protein (GMP-140) molecules on the surface of platelet was expressed significantly increasing when the dosage of 60 Co γ-ray was 5 Gy; however, the glycoprotein (GP) I b and III a was not changed significantly; thromboxane B 2 production in plasma was significantly elevated while the γ-ray was only 2.5 Gy; the concentration of von Willebrand factor was increased when the γ-ray was over 5 Gy, this is in accordance with the GMP-140 molecules. These results indicate that platelets could be activated in vitro when the dosage of 60 Co γ was over 5 Gy

cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules

International Nuclear Information System (INIS)

Claesson-Welsh, L.; Eriksson, A.; Moren, A.; Severinsson, L.; Ek, B.; Ostman, A.; Betsholtz, C.; Heldin, C.H.

1988-01-01

The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGFR receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGR receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different /sup 125/I-labeled dimeric forms of PDGF A and B chains showed that the PDGFR receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA

The hemostatic agent ethamsylate enhances P-selectin membrane expression in human platelets and cultured endothelial cells.

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Alvarez-Guerra, Miriam; Hernandez, Maria Rosa; Escolar, Ginés; Chiavaroli, Carlo; Garay, Ricardo P; Hannaert, Patrick

2002-09-15

Ethamsylate possesses antihemorrhagic properties, but whether or not it directly activates blood platelets is unclear. Here we investigated the platelet activation potential of ethamsylate, by measuring membrane P-selectin expression with flow cytometry in human whole blood and also by immunofluorescence imaging of isolated human platelets. Moreover, we measured membrane P-selectin expression in the SV40-transformed aortic rat endothelial cell line (SVAREC) and 14C-ethamsylate membrane binding and/or uptake in platelets and endothelial cells. Whole blood flow cytometry showed a modest, but statistically significant increase by ethamsylate in the percentage of platelets expressing P-selectin (from 2% to 4-5%, p ethamsylate tested (1 microM), with maximal enhancement of P-selectin expression (75-90%) at 10 microM ethamsylate. Similar results were obtained in SVAREC endothelial cells. 14C-ethamsylate specifically bound to platelets and endothelial cell membranes, without significant uptake into the cell interior. In conclusion, ethamsylate enhances membrane P-selectin expression in human platelets and in cultured endothelial cells. Ethamsylate specifically binds to some protein receptor in platelet and endothelial cell membranes, receptor which can signal for membrane P-selectin expression. These results support the view that ethamsylate acts on the first step of hemostasis, by improving platelet adhesiveness and restoring capillary resistance. Copyright 2002 Elsevier Science Ltd.

Secretory products from thrombin-stimulated human platelets exert an inhibitory effect on NK-cytotoxic activity

DEFF Research Database (Denmark)

Skov Madsen, P; Hokland, P; Hokland, M

1987-01-01

We have investigated the interaction between human platelets and the NK-system, with special emphasis on the action of secretory products from platelets in an NK assay with 51Cr-labelled K562 as target cells. Supernatants from thrombin-stimulated platelets added to the NK assay consistently...... decreased the NK-cytotoxicity by 40% +/- 4.3%, indicating the existence of secreted products from platelets as a source of NK-inhibiting substances. In contrast, no direct cytotoxic effect of these secretory products on the target cells (K562) was seen. Thus, normal human platelets, when stimulated...... with thrombin, are capable of secreting different, yet undefined factors, which significantly inhibit NK activity in vitro. The results also suggest that the role of products from contaminating in vitro activated platelets should be borne in mind when performing conventional NK assays. Udgivelsesdato: 1986-Oct...

Post-transcriptional regulation of osteoblastic platelet-derived growth factor receptor-alpha expression by co-cultured primary endothelial cells

DEFF Research Database (Denmark)

Finkenzeller, Günter; Mehlhorn, Alexander T; Schmal, Hagen

2010-01-01

-alpha downregulation is dependent on time and cell number. This effect was specific to endothelial cells and was not observed when hOBs were co-cultured with human primary chondrocytes or fibroblasts. Likewise, HUVEC-mediated suppression of PDGFR-alpha expression was only seen in hOBs and mesenchymal stem cells......Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co......-cultivation of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-alpha expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR...

Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: Evidence for more than one receptor class

International Nuclear Information System (INIS)

Gronwald, R.G.K.; Grant, F.J.; Haldeman, B.A.; Hart, C.E.; O'Hara, P.J.; Hagen, F.S.; Ross, R.; Bowen-Pope, D.F.; Murray, M.J.

1988-01-01

The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A) + RNA from human dermal fibroblasts detects a major and a minor transcript using the cDNA as a probe. Baby hamster kidney cells, transfected with an expression vector containing the receptor cDNA, express an ∼ 190-kDa cell surface protein that is recognized by an anti-human PDGF receptor antibody. The recombinant PDGF receptor is functional in the transfected baby hamster kidney cells as demonstrated by ligand-induced phosphorylation of the receptor. Binding properties of the recombinant PDGF receptor were also assessed with pure preparations of BB and AB isoforms of PDGF. Unlike human dermal fibroblasts, which bind both isoforms with high affinity, the transfected baby hamster kidney cells bind only the BB isoform of PDGF with high affinity. This observation is consistent with the existence of more than one PDGF receptor class

Immunohistochemical examination of effects of kefir, koumiss and commercial probiotic capsules on platelet derived growth factor-c and platelet derived growth factor receptor-alpha expression in mouse liver and kidney.

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Bakir, B; Sari, E K; Aydin, B D; Yildiz, S E

2015-04-01

We investigated using immunohistochemistry the effects of kefir, koumiss and commercial probiotic capsules on the expression of platelet derived growth factor-c (PDGF-C) and platelet derived growth factor receptor-alpha (PDGFR-α) in mouse liver and kidney. Mice were assigned to four groups: group 1 was given commercial probiotic capsules, group 2 was given kefir, group 3 was given koumiss and group 4 was untreated. After oral administration for 15 days, body weights were recorded and liver and kidney tissue samples were obtained. Hematoxylin and eosin staining was used to examine histology. PDGF-C and PDGFR-α in liver and kidney were localized using the streptavidin-biotin peroxidase complex method (ABC). We found that the weights of the mice in the kefir, koumiss and commercial probiotic capsules groups increased compared to the control group. No differences in liver and kidney histology were observed in any of the experimental groups. Kefir, koumiss and the commercial probiotic preparation increased PDGF-C and PDGFR-α expression.

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 (pplatelet 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. 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.

Are the changes in the peripheral brain-derived neurotrophic factor levels due to platelet activation?

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Serra-Millàs, Montserrat

2016-01-01

Brain-derived neurotrophic factor (BDNF) plays an important role in central nervous system development, neurogenesis and neuronal plasticity. BDNF is also expressed in several non-neuronal tissues, and it could play an important role in other processes, such as cancer, angiogenesis, etc. Platelets are the major source of peripheral BDNF. However, platelets also contain high amounts of serotonin; they express specific surface receptors during activation, and a multitude of pro-inflammatory and immunomodulatory bioactive compounds are secreted from the granules. Until recently, there was insufficient knowledge regarding the relationship between BDNF and platelets. Recent studies showed that BDNF is present in two distinct pools in platelets, in α-granules and in the cytoplasm, and only the BDNF in the granules is secreted following stimulation, representing 30% of the total BDNF in platelets. BDNF has an important role in the pathophysiology of depression. Low levels of serum BDNF have been described in patients with major depressive disorder, and BDNF levels increased with chronic antidepressant treatment. Interestingly, there is an association between depression and platelet function. This review analyzed studies that evaluated the relationship between BDNF and platelet activation and the effect of treatments on both parameters. Only a few studies consider this possible confounding factor, and it could be very important in diseases such as depression, which show changes in both parameters. PMID:27014600

Human Platelet-Rich Plasma- and Extracellular Matrix-Derived Peptides Promote Impaired Cutaneous Wound Healing In Vivo

Science.gov (United States)

Demidova-Rice, Tatiana N.; Wolf, Lindsey; Deckenback, Jeffry; Hamblin, Michael R.; Herman, Ira M.

2012-01-01

Previous work in our laboratory has described several pro-angiogenic short peptides derived from endothelial extracellular matrices degraded by bacterial collagenase. Here we tested whether these peptides could stimulate wound healing in vivo. Our experiments demonstrated that a peptide created as combination of fragments of tenascin X and fibrillin 1 (comb1) applied into cranial dermal wounds created in mice treated with cyclophosphamide to impair wound healing, can improve the rate of wound closure. Furthermore, we identify and characterize a novel peptide (UN3) created and modified from two naturally-occurring peptides, which are present in human platelet-rich plasma. In vitro testing of UN3 demonstrates that it causes a 50% increase in endothelial proliferation, 250% increase in angiogenic response and a tripling of epithelial cell migration in response to injury. Results of in vivo experiments where comb1 and UN3 peptides were added together to cranial wounds in cyclophosphamide-treated mice leads to improvement of wound vascularization as shown by an increase of the number of blood vessels present in the wound beds. Application of the peptides markedly promotes cellular responses to injury and essentially restores wound healing dynamics to those of normal, acute wounds in the absence of cyclophosphamide impairment. Our current work is aimed at understanding the mechanisms underlying the stimulatory effects of these peptides as well as identification of the cellular receptors mediating these effects. PMID:22384158

Vertical ridge augmentation using an equine bone and collagen block infused with recombinant human platelet-derived growth factor-BB: a randomized single-masked histologic study in non-human primates.

Science.gov (United States)

Nevins, Myron; Al Hezaimi, Khalid; Schupbach, Peter; Karimbux, Nadeem; Kim, David M

2012-07-01

This study tests the effectiveness of hydroxyapatite and collagen bone blocks of equine origin (eHAC), infused with recombinant human platelet-derived growth factor-BB (rhPDGF-BB), to augment localized posterior mandibular defects in non-human primates (Papio hamadryas). Bilateral critical-sized defects simulating severe atrophy were created at the time of the posterior teeth extraction. Test and control blocks (without growth factor) were randomly grafted into the respective sites in each non-human primate. All sites exhibited vertical ridge augmentation, with physiologic hard- and soft-tissue integration of the blocks when clinical and histologic examinations were done at 4 months after the vertical ridge augmentation procedure. There was a clear, although non-significant, tendency to increased regeneration in the test sites. As in the first two preclinical studies in this series using canines, experimental eHAC blocks infused with rhPDGF-BB proved to be a predictable and technically viable method to predictably regenerate bone and soft tissue in critical-sized defects. This investigation supplies additional evidence that eHAC blocks infused with rhPDGF-BB growth factor is a predictable and technically feasible option for vertical augmentation of severely resorbed ridges.

Platelet antiheparin activity. The isolation and characterisation of platelet factor 4 released from thrombin-aggregated washed human platelets and its dissociation into subunits and the isolation of membrane-bound antiheparin activity.

Science.gov (United States)

Moore, S; Pepper, D S; Cash, J D

1975-02-27

Platelet factor 4 was isolated by gel filtration from the soluble release products of thrombin-aggregated washed human platelets as a proteoglycan-platelet factor 4 complex of molecular weight 358 000, Stokes radius (r-s) of 14.0 nm, sedimentation coefficient (s) of 7.1 S and frictional ratio (f/f-o) of 3.04. The complex was dissociated at high ionic strength (I equals 0.75) and the proteoglycan separated from platelet factor 4 by gel filtration. Platelet factor 4 had a molecular weight of 27 100, r-s of 2.52 nm, s of 2.4 S and f/f-o of 1.26, was insoluble under physiological conditions but readily soluble at pH 3. Under these conditions platelet factor 4 dissociated into four subunits with a molecular weight of 6900, r-s of 1.92 nm, s of 0.8 S, and f/f-o of 1.52. Qualitative N-terminal amino acid analysis showed the presence of glutamic acid or glutamine as the major end group. Platelet factor 4 was compared with protamine sulphate, which has similar biological properties, by electrophoresis at pH 2.2, in which both migrated as single bands but with differing mobility, and by amino acid analysis which showed a more normal distribution of residues than occurred in protamine sulphate. Of the basic amino acids platelet factor 4 (molecular weight 27 100) contained 5.97% arginine, 3.18% histidine, and 12.31% lysine compared to protamine sulphate with 64.2% arginine, 0.6% lysine and no histidine. A partial specific volume (v) of 0.747 was calculated for platelet factor 4 from its amino acid analysis. A membrane fraction with antiheparin activity, an isopycnic density of 1.090-1.110 and r-s of 15-35 nm, was also isolated by sucrose density gradient centrifugation from the ultrasonicated insoluble platelet residue remaining after thrombin-induced aggregation of washed human platelets. Trypsin treatment of the membrane fraction neither solubilised nor destroyed the activity.

Morphine induces expression of platelet-derived growth factor in human brain microvascular endothelial cells: implication for vascular permeability.

Directory of Open Access Journals (Sweden)

Hongxiu Wen

Full Text Available Despite the advent of antiretroviral therapy, complications of HIV-1 infection with concurrent drug abuse are an emerging problem. Morphine, often abused by HIV-infected patients, is known to accelerate neuroinflammation associated with HIV-1 infection. Detailed molecular mechanisms of morphine action however, remain poorly understood. Platelet-derived growth factor (PDGF has been implicated in a number of pathological conditions, primarily due to its potent mitogenic and permeability effects. Whether morphine exposure results in enhanced vascular permeability in brain endothelial cells, likely via induction of PDGF, remains to be established. In the present study, we demonstrated morphine-mediated induction of PDGF-BB in human brain microvascular endothelial cells, an effect that was abrogated by the opioid receptor antagonist-naltrexone. Pharmacological blockade (cell signaling and loss-of-function (Egr-1 approaches demonstrated the role of mitogen-activated protein kinases (MAPKs, PI3K/Akt and the downstream transcription factor Egr-1 respectively, in morphine-mediated induction of PDGF-BB. Functional significance of increased PDGF-BB manifested as increased breach of the endothelial barrier as evidenced by decreased expression of the tight junction protein ZO-1 in an in vitro model system. Understanding the regulation of PDGF expression may provide insights into the development of potential therapeutic targets for intervention of morphine-mediated neuroinflammation.

Sphingosine 1-phosphate (S1P) suppresses the collagen-induced activation of human platelets via S1P4 receptor.

Science.gov (United States)

Onuma, Takashi; Tanabe, Kumiko; Kito, Yuko; Tsujimoto, Masanori; Uematsu, Kodai; Enomoto, Yukiko; Matsushima-Nishiwaki, Rie; Doi, Tomoaki; Nagase, Kiyoshi; Akamatsu, Shigeru; Tokuda, Haruhiko; Ogura, Shinji; Iwama, Toru; Kozawa, Osamu; Iida, Hiroki

2017-08-01

Sphingosine 1-phosphate (S1P) is as an extracellular factor that acts as a potent lipid mediator by binding to specific receptors, S1P receptors (S1PRs). However, the precise role of S1P in human platelets that express S1PRs has not yet been fully clarified. We previously reported that heat shock protein 27 (HSP27) is released from human platelets accompanied by its phosphorylation stimulated by collagen. In the present study, we investigated the effect of S1P on the collagen-induced platelet activation. S1P pretreatment markedly attenuated the collagen-induced aggregation. Co-stimulation with S1P and collagen suppressed collagen-induced platelet activation, but the effect was weaker than that of S1P-pretreatment. The collagen-stimulated secretion of platelet-derived growth factor (PDGF)-AB and the soluble CD40 ligand (sCD40L) release were significantly reduced by S1P. In addition, S1P suppressed the collagen-induced release of HSP27 as well as the phosphorylation of HSP27. S1P significantly suppressed the collagen-induced phosphorylation of p38 mitogen-activated protein kinase. S1P increased the levels of GTP-bound Gαi and GTP-bound Gα13 coupled to S1PPR1 and/or S1PR4. CYM50260, a selective S1PR4 agonist, but not SEW2871, a selective S1PR1 agonist, suppressed the collagen-stimulated platelet aggregation, PDGF-AB secretion and sCD40L release. In addition, CYM50260 reduced the release of phosphorylated-HSP27 by collagen as well as the phosphorylation of HSP27. The selective S1PR4 antagonist CYM50358, which failed to affect collagen-induced HSP27 phosphorylation, reversed the S1P-induced attenuation of HSP27 phosphorylation by collagen. These results strongly suggest that S1P inhibits the collagen-induced human platelet activation through S1PR4 but not S1PR1. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platelet-rich plasma can replace fetal bovine serum in human meniscus cell cultures

NARCIS (Netherlands)

Gonzales, V.K.; Mulder, E.L.W. de; Boer, T. den; Hannink, G.; Tienen, T.G. van; Heerde, W.L. van; Buma, P.

2013-01-01

Concerns over fetal bovine serum (FBS) limit the clinical application of cultured tissue-engineered constructs. Therefore, we investigated if platelet-rich plasma (PRP) can fully replace FBS for meniscus tissue engineering purposes. Human PRP and platelet-poor plasma (PPP) were isolated from three

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.

[Quality of buffy-coat-derived platelet concentrates prepared using automated system terumo automated centrifuge and separator integration (TACSI)].

Science.gov (United States)

Zebrowska, Agnieszka; Lipska, Alina; Rogowska, Anna; Bujno, Magdalena; Nedzi, Marta; Radziwon, Piotr

2011-03-01

Platelet recovery, and viability, and function is strongly dependent on the method of the preparation of platelet concentrate (PC). The glucose consumption, decrease of pH, release of alpha granules during storage in platelet concentrate impair their clinical effectiveness. To compare of the quality of buffy-coat-derieved platelet concentrates prepared using automatic system terumo automated centrifuge and separator integration (TACSI) and stored over 7 days. PCs were prepared from buffy coats using manual method (group I), or automatic system TACSI (group II). Fifteen PCs prepared from the 5 buffy coats each were stored over 7 days in 22-24 degrees C and tested. Samples were taken from the PCs container on days 1 and 7. The following laboratory tests were performed: number of platelets, platelets derived microparticles, CD62P expression, platelet adhesion, pH, glucose, lactate dehydrogenase activity. We have observed higher expression of CD62P in PCs prepared using manual method compared to the PCs produced automatically Platelet recovery was significantly higher in PCs prepared using automatic systems compare to manual method. Compared to manual methods, automatic system for preparation of buffy coats, is more efficient and enable production of platelets concentrates of higher quality.

Effect of cocoa products and flavanols on platelet aggregation in humans: a systematic review.

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Peluso, Ilaria; Palmery, Maura; Serafini, Mauro

2015-07-01

Previous evidence suggested an active role of cocoa products and flavanols in modulating platelet aggregation. However, cocoa flavanols are characterized by a low bioavailability that can deeply affect their presence in biological fluids and raise questions on their biological effect in humans. We performed a systematic search on Medline, Embase, Cochrane and ProQuest databases, until April 2015, on the effect of cocoa products on platelet aggregation in human intervention studies. We identified 13 interventions, of which only five involved repeated administration. Different effects were observed on the basis of the platelet aggregation test used, whereas neither a longer duration of treatment nor a higher dose was associated with a higher inhibition of platelet aggregation. In conclusion, the reviewed results suggest that consumption of cocoa products in bolus administration positively affects platelet aggregation in both healthy subjects and diseased patients. On the other hand, more evidence is required in order to assess the effect of long-term cocoa product ingestion and to identify the bioactive components involved.

Studies on the Biological Effects of Ozone: 10. Release of Factors from Ozonated Human Platelets

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G. Valacchi

1999-01-01

Full Text Available In a previous work we have shown that heparin, in the presence of ozone (O3, promotes a dose-dependent platelet aggregation, while after Ca2+ chelation with citrate, platelet aggregation is almost negligible. These results led us to think that aggregation may enhance the release of platelet components. We have here shown that indeed significantly higher amount of platelet-derived growth factor (PDGF, transforming growth factor β1 (TGF-β1 and interleukin-8(IL-8 are released in a dose-dependent manner after ozonation of heparinised platelet-rich plasma samples. These findings may explain the enhanced healing of torpid ulcers in patients with chronic limbischemia treated with O3 autohaemoteraphy (O3-AHT.

Implementation of buffy-coat-derived pooled platelet concentrates for internal quality control of light transmission aggregometry: a proof of concept study.

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Prüller, F; Rosskopf, K; Mangge, H; Mahla, E; von Lewinski, D; Weiss, E C; Riegler, A; Enko, D

2017-12-01

Essentials In platelet function testing, standardized internal controls (IQC) are not commercially provided. Platelet function testing was performed daily on aliquoted pooled platelet concentrates. Pooled platelet concentrates showed stability for control purposes from Monday to Friday. Pooled platelet concentrates provide the necessary steadiness to serve as IQC material. Background Standardized commercially available control material for internal quality control (IQC) of light transmission aggregometry (LTA) is still lacking. Moreover, the availability of normal blood donors to provide fresh platelets is difficult in small laboratories, where 'volunteers' may be in short supply. Objectives To evaluate the implementation of buffy-coat-derived pooled platelet concentrates (PCs) for IQC material for LTA. Methods We used buffy-coat-derived pooled PCs from the blood bank as IQC material for LTA. On each weekend one PC was prepared (> 200 mL) and aliquoted from the original storage bag on a daily basis in four baby bags (40-50 mL), which were delivered from Monday to Friday to our laboratory. The IQC measurements of at least 85 work-weeks (from Monday to Friday) were evaluated with this new IQC material. LTA was performed on a four-channel Chronolog 700 Aggregometer (Chronolog Corporation, Havertown, PA, USA) (agonists: collagen, adenosine diphosphate [ADP], arachidonic acid [AA] and thrombin receptor activator peptide-6 [TRAP-6]). Results The medians of platelet aggregation from IQC measurements with collagen, ADP and AA from Monday to Friday were 68.0-59.5, 3.0-2.0 and 51.0-50.0%, respectively, and the mean of platelet aggregation with TRAP-6 was 71.2-66.4%. Conclusions Buffy-coat-derived pooled PCs serve as a reliable and robust IQC material for LTA measurements and would be beneficial for the whole laboratory procedure and employees' safety. © 2017 International Society on Thrombosis and Haemostasis.

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

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...

Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

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Smits, A; Funa, K; Vassbotn, F S; Beausang-Linder, M; af Ekenstam, F; Heldin, C H; Westermark, B; Nistér, M

1992-03-01

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protein was found in the malignant tumors, and also in some benign lesions, such as dermatofibroma. In all these cases, benign as well as malignant, the PDGF B-chain mRNA, and less clearly, the PDGF A-chain mRNA, were coexpressed with the beta-receptor. In contrast, high expression of PDGF alpha-receptor mRNA was only found in fully malignant lesions, i.e., malignant fibrous histiocytoma. These data indicate that an autocrine growth stimulation via the PDGF beta-receptor could occur in an early phase of tumorigenesis, and may be a necessary but insufficient event for the progression into fully malignant human connective tissue lesions.

Determination of an unrelated donor pool size for human leukocyte antigen-matched platelets in Brazil

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Carolina Bonet Bub

2016-02-01

Full Text Available ABSTRACT Background: Successful transfusion of platelet refractory patients is a challenge. Many potential donors are needed to sustain human leukocyte antigen matched-platelet transfusion programs because of the different types of antigens and the constant needs of these patients. For a highly mixed population such as the Brazilian population, the pool size required to provide adequate platelet support is unknown. Methods: A mathematical model was created to estimate the appropriate size of an unrelated donor pool to provide human leukocyte antigen-compatible platelet support for a Brazilian population. A group of 154 hematologic human leukocyte antigen-typed patients was used as the potential patient population and a database of 65,500 human leukocyte antigen-typed bone marrow registered donors was used as the donor population. Platelet compatibility was based on the grading system of Duquesnoy. Results: Using the mathematical model, a pool containing 31,940, 1710 and 321 donors would be necessary to match more than 80% of the patients with at least five completely compatible (no cross-reactive group, partial compatible (one cross-reactive group or less compatible (two cross-reactive group donors, respectively. Conclusion: The phenotypic diversity of the Brazilian population has probably made it more difficulty to find completely compatible donors. However, this heterogeneity seems to have facilitated finding donors when cross-reactive groups are accepted as proposed by the grading system of Duquesnoy. The results of this study may help to establish unrelated human leukocyte antigen-compatible platelet transfusions, a procedure not routinely performed in most Brazilian transfusion services.

Enhancement of the repair of dog alveolar cleft by an autologous iliac bone, bone marrow-derived mesenchymal stem cell, and platelet-rich fibrin mixture.

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Yuanzheng, Chen; Yan, Gao; Ting, Li; Yanjie, Fu; Peng, Wu; Nan, Bai

2015-05-01

Autologous bone graft has been regarded as the criterion standard for the repair of alveolar cleft. However, the most prominent issue in alveolar cleft treatment is the high absorption rate of the bone graft. The authors' objective was to investigate the effects of an autologous iliac bone, bone marrow-derived mesenchymal stem cell, and platelet-rich fibrin mixture on the repair of dog alveolar cleft. Twenty beagle dogs with unilateral alveolar clefts created by surgery were divided randomly into four groups: group A underwent repair with an autologous iliac bone, bone marrow-derived mesenchymal stem cell, and platelet-rich fibrin mixture; group B underwent repair with autologous iliac bone and bone marrow-derived mesenchymal stem cells; group C underwent repair with autologous iliac bone and platelet-rich fibrin; and group D underwent repair with autologous iliac bone as the control. One day and 6 months after transplantation, the transplant volumes and bone mineral density were assessed by quantitative computed tomography. All of the transplants were harvested for hematoxylin and eosin staining 6 months later. Bone marrow-derived mesenchymal stem cells and platelet-rich fibrin transplants formed the greatest amounts of new bone among the four groups. The new bone formed an extensive union with the underlying maxilla in groups A, B, and C. Transplants with the bone marrow-derived mesenchymal stem cells, platelet-rich fibrin, and their mixture retained the majority of their initial volume, whereas the transplants in the control group showed the highest absorption rate. Bone mineral density of transplants with the bone marrow-derived mesenchymal stem cells, platelet-rich fibrin, and their mixture 6 months later was significantly higher than in the control group (p platelet-rich fibrin mixed transplants. Hematoxylin and eosin staining showed that the structure of new bones formed the best in group A. Both bone marrow-derived mesenchymal stem cells and platelet

Human Platelet Lysate as a Replacement for Fetal Bovine Serum in Limbal Stem Cell Therapy.

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Suri, Kunal; Gong, Hwee K; Yuan, Ching; Kaufman, Stephen C

2016-10-01

To evaluate the use of human platelet lysate (HPL) as an alternative supplement for limbal explant culture. Culture media were prepared using either 10% pooled HPL (PHPL), single donor HPL, or fetal bovine serum (FBS). Limbal tissues, obtained from the Minnesota Lions Eye Bank, were cultured in each medium on plastic plates or on denuded amniotic membrane (AM). Immunofluorescence staining was performed for ABCG2, tumor protein p63α, and cytokeratin 3 (K3). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate the expression of ABCG2 and p63. Limbal explants grown in each medium were labeled with bromodeoxyuridine (BrdU) to assess the proliferative capacity in each medium. Concentration of growth factors including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and platelet derived growth factor (PDGF) in HPL and PHPL was compared to that in human serum (HS). Immunofluorescence staining on AM showed prominent expression of ABCG2, p63α but sparse expression of K3 in HPL and PHPL supplemented medium. Real time-PCR showed 1.7 fold higher expression of ABCG2 in PHPL supplemented medium (p = 0.03), and similar expression of p63 in HPL and PHPL supplemented medium compared to FBS medium. The proliferation assay showed that LSCs retained their proliferative potential in HPL supplemented medium. Higher concentration of growth factors were found in HPL, compared to HS. Human platelet lysate has higher concentration of grown factors and is effective in maintaining growth and stem cell phenotype of corneal limbal explant cultures.

Comparison of point-of-care methods for preparation of platelet concentrate (platelet-rich plasma).

Science.gov (United States)

Weibrich, Gernot; Kleis, Wilfried K G; Streckbein, Philipp; Moergel, Maximilian; Hitzler, Walter E; Hafner, Gerd

2012-01-01

This study analyzed the concentrations of platelets and growth factors in platelet-rich plasma (PRP), which are likely to depend on the method used for its production. The cellular composition and growth factor content of platelet concentrates (platelet-rich plasma) produced by six different procedures were quantitatively analyzed and compared. Platelet and leukocyte counts were determined on an automatic cell counter, and analysis of growth factors was performed using enzyme-linked immunosorbent assay. The principal differences between the analyzed PRP production methods (blood bank method of intermittent flow centrifuge system/platelet apheresis and by the five point-of-care methods) and the resulting platelet concentrates were evaluated with regard to resulting platelet, leukocyte, and growth factor levels. The platelet counts in both whole blood and PRP were generally higher in women than in men; no differences were observed with regard to age. Statistical analysis of platelet-derived growth factor AB (PDGF-AB) and transforming growth factor β1 (TGF-β1) showed no differences with regard to age or gender. Platelet counts and TGF-β1 concentration correlated closely, as did platelet counts and PDGF-AB levels. There were only rare correlations between leukocyte counts and PDGF-AB levels, but comparison of leukocyte counts and PDGF-AB levels demonstrated certain parallel tendencies. TGF-β1 levels derive in substantial part from platelets and emphasize the role of leukocytes, in addition to that of platelets, as a source of growth factors in PRP. All methods of producing PRP showed high variability in platelet counts and growth factor levels. The highest growth factor levels were found in the PRP prepared using the Platelet Concentrate Collection System manufactured by Biomet 3i.

Human platelet lysate successfully promotes proliferation and subsequent chondrogenic differentiation of adipose-derived stem cells: a comparison with articular chondrocytes.

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Hildner, F; Eder, M J; Hofer, K; Aberl, J; Redl, H; van Griensven, M; Gabriel, C; Peterbauer-Scherb, A

2015-07-01

Fetal calf serum (FCS) bears a potential risk for carrying diseases and eliciting immune reactions. Nevertheless, it still represents the gold standard as medium supplement in cell culture. In the present study, human platelet lysate (PL) was tested as an alternative to FCS for the expansion and subsequent chondrogenic differentiation of human adipose-derived stem cells (ASCs). ASCs were expanded with 10% FCS (group F) or 5% PL (group P). Subsequently, three-dimensional (3D) micromass pellets were created and cultured for 5 weeks in chondrogenic differentiation medium. Additionally, the de- and redifferentiation potential of human articular chondrocytes (HACs) was evaluated and compared to ASCs. Both HACs and ASCs cultured with PL showed strongly enhanced proliferation rates. Redifferentiation of HACs was possible for cells expanded up to 3.3 population doublings (PD). At this stage, PL-expanded HACs demonstrated better redifferentiation potential than FCS-expanded cells. ASCs could also be differentiated following extended passaging. Glycosaminoglycan (GAG) quantification and qRT-PCR of 10 cartilage related markers demonstrated a tendency for increased chondrogenic differentiation of PL-expanded ASCs compared to cells expanded with FCS. Histologically, collagen type II but also collagen type X was mainly present in group P. The present study demonstrates that PL strongly induces proliferation of ASCs, while the chondrogenic differentiation potential is retained. HACs also showed enhanced proliferation and even better redifferentiation when previously expanded with PL. This suggests that PL is superior to FCS as a supplement for the expansion of ASCs and HACs, particularly with regard to chondrogenic (re)differentiation. Copyright © 2013 John Wiley & Sons, Ltd.

Stapled peptides as a new technology to investigate protein-protein interactions in human platelets.

Science.gov (United States)

Iegre, Jessica; Ahmed, Niaz S; Gaynord, Josephine S; Wu, Yuteng; Herlihy, Kara M; Tan, Yaw Sing; Lopes-Pires, Maria E; Jha, Rupam; Lau, Yu Heng; Sore, Hannah F; Verma, Chandra; O' Donovan, Daniel H; Pugh, Nicholas; Spring, David R

2018-05-28

Platelets are blood cells with numerous crucial pathophysiological roles in hemostasis, cardiovascular thrombotic events and cancer metastasis. Platelet activation requires the engagement of intracellular signalling pathways that involve protein-protein interactions (PPIs). A better understanding of these pathways is therefore crucial for the development of selective anti-platelet drugs. New strategies for studying PPIs in human platelets are required to overcome limitations associated with conventional platelet research methods. For example, small molecule inhibitors can lack selectivity and are often difficult to design and synthesise. Additionally, development of transgenic animal models is costly and time-consuming and conventional recombinant techniques are ineffective due to the lack of a nucleus in platelets. Herein, we describe the generation of a library of novel, functionalised stapled peptides and their first application in the investigation of platelet PPIs. Moreover, the use of platelet-permeable stapled Bim BH3 peptides confirms the part of Bim in phosphatidyl-serine (PS) exposure and reveals a role for the Bim protein in platelet activatory processes. Our work demonstrates that functionalised stapled peptides are a complementary alternative to conventional platelet research methods, and could make a significant contribution to the understanding of platelet signalling pathways and hence to the development of anti-platelet drugs.

Radiolabeling of human platelets using four radiopharmaceuticals with Tc-99m

International Nuclear Information System (INIS)

Portillo L, M.C.; Godoy, C.

1991-01-01

The present investigation work has been done in an evaluation of four different radiopharmaceuticals with Tin II (Glucoheptonate, Pyrophosphate, Citrate and DTPA), with the purpose of determining which one of the four would be obtained a higher rate of radiolabeling of platelets with Tc-99m. In order to evaluate the four radiopharmaceuticals it was procede to the separation and labeling of the human platelets. It was worked with samples of blood from five pacients, and the platelets from each one of them were labeled with the radiopharmaceuticals-Tc-99m. Then was observed a significant difference among the four radiopharmaceuticals studied, with a reliable level of 0.05 being the Glucoheptonate-Tc-99m the best to label platelets, obtaining with the same 50.23% of labeling efficiency, while for DTPA, Pyrophosphate and Citrate, It was obtained 33.42%, 29.82% and |2.62% respectively. Also, it was studied the biodistribution of the labeled platelets, using Glucoheptonate-Tc-99m as a radiopharamceutical. The biodistribution was studied in white mice at different times and it was founded that the place of biodistribution of the labeled platelets is given in greater percentage in the liver, spleen, lungs and kydneis. (Author)

Progress in bio-manufacture of platelets for transfusion.

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Heazlewood, Shen Y; Nilsson, Susan K; Cartledge, Kellie; Be, Cheang Ly; Vinson, Andrew; Gel, Murat; Haylock, David N

2017-11-01

Blood transfusion services face an ever-increasing demand for donor platelets to meet clinical needs. Whilst strategies for increasing platelet storage life and improving the efficiency of donor platelet collection are important, in the longer term, platelets generated by bio-manufacturing processes will be required to meet demands. Production of sufficient numbers of in vitro-derived platelets for transfusion represents a significant bioengineering challenge. In this review, we highlight recent progress in this area of research and outline the main technical and biological obstacles that need to be met before this becomes feasible and economic. A critical consideration is assurance of the functional properties of these cells as compared to their fresh, donor collected, counterparts. We contend that platelet-like particles and in vitro-derived platelets that phenotypically resemble fresh platelets must deliver the same functions as these cells upon transfusion. We also note recent progress with immortalized megakaryocyte progenitor cell lines, molecular strategies for reducing expression of HLA Class I to generate universal donor platelets and the move to early clinical studies with in vitro-derived platelets.

An evaluation of platelet-rich plasma without thrombin activation with or without anorganic bone mineral in the treatment of human periodontal intrabony defects.

Science.gov (United States)

Rodrigues, Silvia V; Acharya, Anirudh B; Thakur, Srinath L

2011-01-01

The efficacy of platelet-rich plasma (PRP) in periodontal regeneration is not well understood and the definite clinical viability of blood derived platelets lacks clarity. Also, the use of thrombin for platelet activation is disputed. Hence, the purpose of this study was to evaluate the efficacy of blood derived platelets without thrombin activation, alone or in combination with bovine anorganic bone mineral (ABM), in the treatment of human periodontal intrabony defects. PRP was prepared using a simple tabletop centrifuge and activated using calcium chloride without the addition of thrombin. This PRP was used alone (in Group A) and in combination with bovine ABM (in Group B) in the treatment of human periodontal angular defects. Both the control and the test groups showed definite improvement in clinical parameters. On comparison, however, there was a statistically significant improvement in the probing pocket depths and relative attachment level in Group B over Group A at 3 and 6 months intervals, whereas at the end of 9 months this difference was not statistically significant. There was no statistically significant difference between the groups with respect to the relative defect depth. Within the limitations of this study and the type of PRP used, i.e. without thrombin mediated activation, it can be concluded that both PRP and PRP combined with bovine ABM results in significant clinical improvement. Albeit statistically insignificant, there is a preponderance of better clinical results with the addition of ABM to PRP. Further studies need to be carried out on a larger sample size to confirm the results of the present study.

Platelet-Poor and Platelet-Rich Plasma Stimulate Bone Lineage Differentiation in Periodontal Ligament Stem Cells.

Science.gov (United States)

Martínez, Constanza E; González, Sergio A; Palma, Verónica; Smith, Patricio C

2016-02-01

Plasma-derived fractions have been used as an autologous source of growth factors; however, limited knowledge concerning their biologic effects has hampered their clinical application. In this study, the authors analyze the content and specific effect of both platelet-rich plasma (PRP) and platelet-poor plasma (PPP) on osteoblastic differentiation using primary cultures of human periodontal ligament stem cells (HPLSCs). The authors evaluated the growth factor content of PRP and PPP using a proteome profiler array and enzyme-linked immunosorbent assay. HPLSCs were characterized by flow cytometry and differentiation assays. The effect of PRP and PPP on HPLSC bone differentiation was analyzed by quantifying calcium deposition after 14 and 21 days of treatment. Albeit at different concentrations, the two fractions had similar profiles of growth factors, the most representative being platelet-derived growth factor (PDGF) isoforms (PDGF-AA, -BB, and -AB), insulin-like growth factor binding protein (IGFBP)-2, and IGFBP-6. Both formulations exerted a comparable stimulus on osteoblastic differentiation even at low doses (2.5%), increasing calcium deposits in HPLSCs. PRP and PPP showed a similar protein profile and exerted comparable effects on bone differentiation. Further studies are needed to characterize and compare the effects of PPP and PRP on bone healing in vivo.

Platelet size does not correlate with platelet age.

Science.gov (United States)

Thompson, C B; Love, D G; Quinn, P G; Valeri, C R

1983-08-01

The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense-body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75Se-methionine, (2) in vitro labeling with 51Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51Cr and 14C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation.

Platelet size does not correlate with platelet age

International Nuclear Information System (INIS)

Thompson, C.B.; Love, D.G.; Quinn, P.G.; Valeri, C.R.

1983-01-01

The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense-body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75 Se-methionine, (2) in vitro labeling with 51 Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75 Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51 Cr and 14 C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation

Effects of different concentrations of Platelet-rich Plasma and Platelet-Poor Plasma on vitality and differentiation of autologous Adipose tissue-derived stem cells.

Science.gov (United States)

Felthaus, Oliver; Prantl, Lukas; Skaff-Schwarze, Mona; Klein, Silvan; Anker, Alexandra; Ranieri, Marco; Kuehlmann, Britta

2017-01-01

Autologous fat grafts and adipose-derived stem cells (ASCs) can be used to treat soft tissue defects. However, the results are inconsistent and sometimes comprise tissue resorption and necrosis. This might be due to insufficient vascularization. Platelet-rich plasma (PRP) is a source of concentrated autologous platelets. The growth factors and cytokines released by platelets can facilitate angiogenesis. The simultaneous use of PRP might improve the regeneration potential of fat grafts. The optimal ratio has yet to be elucidated. A byproduct of PRP preparation is platelet-poor plasma (PPP). In this study we investigated the influence of different concentrations of PRP on the vitality and differentiation of ASCs. We processed whole blood with the Arthrex Angel centrifuge and isolated ASCs from the same donor. We tested the effects of different PRP and PPP concentrations on the vitality using resazurin assays and the differentiation of ASCs using oil-red staining. Both cell vitality and adipogenic differentiation increase to a concentration of 10% to 20% PRP. With a PRP concentration of 30% cell vitality and differentiation decrease. Both PRP and PPP can be used to expand ASCs without xenogeneic additives in cell culture. A PRP concentration above 20% has inhibitory effects.

Platelets in Immune Response to Virus and Immunopathology of Viral Infections

Directory of Open Access Journals (Sweden)

Eugenio D. Hottz

2018-04-01

Full Text Available Platelets are essential effector cells in hemostasis. Aside from their role in coagulation, platelets are now recognized as major inflammatory cells with key roles in the innate and adaptive arms of the immune system. Activated platelets have key thromboinflammatory functions linking coagulation to immune responses in various infections, including in response to virus. Recent studies have revealed that platelets exhibit several pattern recognition receptors (PRR including those from the toll-like receptor, NOD-like receptor, and C-type lectin receptor family and are first-line sentinels in detecting and responding to pathogens in the vasculature. Here, we review the main mechanisms of platelets interaction with viruses, including their ability to sustain viral infection and replication, their expression of specialized PRR, and activation of thromboinflammatory responses against viruses. Finally, we discuss the role of platelet-derived mediators and platelet interaction with vascular and immune cells in protective and pathophysiologic responses to dengue, influenza, and human immunodeficiency virus 1 infections.

Culture of human cell lines by a pathogen-inactivated human platelet lysate.

Science.gov (United States)

Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

2016-08-01

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

Further studies on the relationship between platelet buoyant density and platelet age

International Nuclear Information System (INIS)

Boneu, B.; Vigoni, F.; Boneu, A.; Caranobe, C.; Sie, P.

1982-01-01

The relationship between platelet buoyant density and platelet age was investigated in eight human subjects submitted to an autologous chromium labeled platelet survival study. Platelets were isolated after isopycnic centrifugation using eight discontinuous isoosmotic stractan gradients (five subjects), or various continuous and linear isoosmolar gradients (three subjects). A paradoxical radioactivity enrichment of the dense platelets and a premature loss of radioactivity in the light platelets were observed. These results are explained by a shift of the radioactivity distribution curve toward higher densities during the 3-4 days after platelet injection, while the standard deviation of the distribution was conserved throughout the platelet life span. These results suggest that young platelets are heterogeneous and slightly less dense than the total platelet population

Platelet-, leucocyte- and red cell-derived microparticles in stored whole blood, with and without leucofiltration, with and without ionising radiation.

Science.gov (United States)

Saito, Shunnichi; Nollet, Kenneth E; Ngoma, Alain M; Ono, Takako; Ohto, Hitoshi

2018-02-01

Storage lesion, including microparticle formation, has been partially characterised in whole blood, but not in all combinations of pre-storage leucofiltration and/or irradiation. Single-donor whole blood products were processed into four subunits: with and without leucofiltration, with and without X-irradiation (25 Gy). Platelet-, leucocyte-, and erythrocyte-derived microparticles and free haemoglobin were measured periodically throughout 42 days of storage. Pre-storage leucofiltration substantially reduced platelet- and leucocyte-derived microparticle counts throughout storage. Irradiation, in contrast, had no significant effect on microparticle counts. A gate for all microparticles showed a substantial time-dependent increase in unfiltered whole blood. A time-dependent increase in free haemoglobin was greatest in unfiltered, irradiated whole blood. This study indicates that leucofiltration can prevent the formation of leucocyte- and platelet-derived microparticles, and might reduce haemolysis in irradiated whole blood, either by removing factors that provoke haemolysis, or by selective retention of senescent or effete red cells most prone to haemolysis.

Platelet-derived growth factor (PDGF) B-chain gene expression by activated blood monocytes precedes the expression of the PDGF A-chain gene

International Nuclear Information System (INIS)

Martinet, Y.; Jaffe, H.A.; Yamauchi, K.; Betsholtz, C.; Westermark, B.; Heldin, C.H.; Crystal, R.G.

1987-01-01

When activated, normal human blood monocytes are known to express the c-sis proto-oncogene coding for PDGF B-chain. Since normal human platelet PDGF molecules are dimers of A and B chains and platelets and monocytes are derived from the same marrow precursors, activated blood monocytes were simultaneously evaluated for their expression of PDGF A and B chain genes. Human blood monocytes were purified by adherence, cultured with or without activation by lipopolysaccharide and poly(A)+ RNA evaluated using Northern analysis and 32 P-labeled A-chain and B-chain (human c-sis) probes. Unstimulated blood monocytes did not express either A-chain or B-chain genes. In contrast, activated monocytes expressed a 4.2 kb mRNA B-chain transcript at 4 hr, but the B-chain mRNA levels declined significantly over the next 18 hr. In comparison, activated monocytes expressed very little A-chain mRNA at 4 hr, but at 12 hr 1.9, 2.3, and 2.8 kb transcripts were observed and persisted through 24 hr. Thus, activation of blood monocytes is followed by PDGF B-chain gene expression preceding PDGF A-chain gene expression, suggesting a difference in the regulation of the expression of the genes for these two chains by these cells

Metabolism of dehydroepiandrosterone sulfate and estrone-sulfate by human platelets.

Science.gov (United States)

Garrido, A; Munoz, Y; Sierralta, W; Valladares, L

2012-01-01

The aim of the present research was to study the uptake of DHEAS, and to establish the intracrine capacity of human platelets to produce sex steroid hormones. The DHEAS transport was evaluated through the uptake of [(3)H]-DHEAS in the presence or absence of different substrates through the organic anion transporting polypeptide (OATP) family. The activity of sulfatase enzyme was evaluated, and the metabolism of DHEAS was measured by the conversion of [(3)H]-DHEAS to [(3)H]-androstenedione, [(3)H]-testosterone, [(3)H]-estrone and [(3)H]-17beta-estradiol. Results indicated the existence in the plasma membrane of an OATP with high affinity for DHEAS and estrone sulphate (E(1)S). The platelets showed the capacity to convert DHEAS to active DHEA by the steroid-sulfatase activity. The cells resulted to be a potential site for androgens production, since they have the capacity to produce androstenedione and testosterone; in addition, they reduced [(3)H]-estrone to [(3)H]-17beta-estradiol. This is the first demonstration that human platelets are able to import DHEAS and E(1)S using the OATP family and to convert DHEAS to active DHEA, and to transform E(1)S to 17beta-estradiol.

Glycoprotein Ib activation by thrombin stimulates the energy metabolism in human platelets

Science.gov (United States)

Corona de la Peña, Norma; Gutiérrez-Aguilar, Manuel; Hernández-Reséndiz, Ileana; Marín-Hernández, Álvaro

2017-01-01

Thrombin-induced platelet activation requires substantial amounts of ATP. However, the specific contribution of each ATP-generating pathway i.e., oxidative phosphorylation (OxPhos) versus glycolysis and the biochemical mechanisms involved in the thrombin-induced activation of energy metabolism remain unclear. Here we report an integral analysis on the role of both energy pathways in human platelets activated by several agonists, and the signal transducing mechanisms associated with such activation. We found that thrombin, Trap-6, arachidonic acid, collagen, A23187, epinephrine and ADP significantly increased glycolytic flux (3–38 times vs. non-activated platelets) whereas ristocetin was ineffective. OxPhos (33 times) and mitochondrial transmembrane potential (88%) were increased only by thrombin. OxPhos was the main source of ATP in thrombin-activated platelets, whereas in platelets activated by any of the other agonists, glycolysis was the principal ATP supplier. In order to establish the biochemical mechanisms involved in the thrombin-induced OxPhos activation in platelets, several signaling pathways associated with mitochondrial activation were analyzed. Wortmannin and LY294002 (PI3K/Akt pathway inhibitors), ristocetin and heparin (GPIb inhibitors) as well as resveratrol, ATP (calcium-release inhibitors) and PP1 (Tyr-phosphorylation inhibitor) prevented the thrombin-induced platelet activation. These results suggest that thrombin activates OxPhos and glycolysis through GPIb-dependent signaling involving PI3K and Akt activation, calcium mobilization and protein phosphorylation. PMID:28817667

Role of Platelet-derived Microvesicles as Crosstalk Mediators in Atherothrombosis and Future Pharmacology Targets: a Link between Inflammation, Atherosclerosis and Thrombosis

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Lina Badimon

2016-08-01

Full Text Available Reports in the last decade have suggested that the role of platelets in atherosclerosis and its thrombotic complications may be mediated, in part, by local secretion of platelet-derived microvesicles (pMVs, small cell blebs released during the platelet activation process. MVs are the most abundant cell-derived microvesicle subtype in the circulation. High concentrations of circulating MVs have been reported in patients with atherosclerosis, acute vascular syndromes, and/or diabetes mellitus, suggesting a potential correlation between the quantity of microvesicles and the clinical severity of the atherosclerotic disease. pMVs are considered to be biomarkers of disease but new information indicates that pMVs are also involved in signaling functions. pMVs evoke or promote haemostatic and inflammatory responses, neovascularization, cell survival and apoptosis, processes involved in the pathophysiology of cardiovascular disease. This review is focused on the complex cross-talk between platelet-derived microvesicles, inflammatory cells and vascular elements and their relevance in the development of the atherosclerotic disease and its clinical outcomes, providing an updated state-of-the art of pMV involvement in atherothrombosis and pMV potential use as therapeutic agent influencing cardiovascular biomedicine in the future.

86Rb(K) influx and [3H]ouabain binding by human platelets: Evidence for beta-adrenergic stimulation of Na-K ATPase activity

International Nuclear Information System (INIS)

Turaihi, K.; Khokher, M.A.; Barradas, M.A.; Mikhailidis, D.P.; Dandona, P.

1989-01-01

Although active transport of potassium into human platelets has been demonstrated previously, there is hitherto no evidence that human platelets have an ouabain-inhibitable Na-K ATPase in their membrane. The present study demonstrates active rubidium (used as an index of potassium influx), 86 Rb(K), influx into platelets, inhibitable by ouabain, and also demonstrates the presence of specific [ 3 H]ouabain binding by the human platelet. This 86 Rb(K) influx was stimulated by adrenaline, isoprenaline, and salbutamol, but noradrenaline caused a mild inhibition. Active 86 Rb(K) influx by platelets was inhibited markedly by timolol, mildly by atenolol, but not by phentolamine. Therefore, active 86 Rb(K) influx in human platelets is enhanced by stimulation of beta adrenoceptors of the beta 2 subtype. The platelet may therefore replace the leukocyte in future studies of Na-K ATPase activity. This would be a considerable advantage in view of the ease and rapidity of preparation of platelets

Superoxide Dismutase 2 is dispensable for platelet function.

Science.gov (United States)

Fidler, Trevor P; Rowley, Jesse W; Araujo, Claudia; Boudreau, Luc H; Marti, Alex; Souvenir, Rhonda; Dale, Kali; Boilard, Eric; Weyrich, Andrew S; Abel, E Dale

2017-10-05

Increased intracellular reactive oxygen species (ROS) promote platelet activation. The sources of platelet-derived ROS are diverse and whether or not mitochondrial derived ROS, modulates platelet function is incompletely understood. Studies of platelets from patients with sickle cell disease, and diabetes suggest a correlation between mitochondrial ROS and platelet dysfunction. Therefore, we generated mice with a platelet specific knockout of superoxide dismutase 2 (SOD2-KO) to determine if increased mitochondrial ROS increases platelet activation. SOD2-KO platelets demonstrated decreased SOD2 activity and increased mitochondrial ROS, however total platelet ROS was unchanged. Mitochondrial function and content were maintained in non-stimulated platelets. However SOD2-KO platelets demonstrated decreased mitochondrial function following thrombin stimulation. In vitro platelet activation and spreading was normal and in vivo, deletion of SOD2 did not change tail-bleeding or arterial thrombosis indices. In pathophysiological models mediated by platelet-dependent immune mechanisms such as sepsis and autoimmune inflammatory arthritis, SOD2-KO mice were phenotypically identical to wildtype controls. These data demonstrate that increased mitochondrial ROS does not result in platelet dysfunction.

Specific Inflammatory Stimuli Lead to Distinct Platelet Responses in Mice and Humans.

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Lea M Beaulieu

Full Text Available Diverse and multi-factorial processes contribute to the progression of cardiovascular disease. These processes affect cells involved in the development of this disease in varying ways, ultimately leading to atherothrombosis. The goal of our study was to compare the differential effects of specific stimuli--two bacterial infections and a Western diet--on platelet responses in ApoE-/- mice, specifically examining inflammatory function and gene expression. Results from murine studies were verified using platelets from participants of the Framingham Heart Study (FHS; n = 1819 participants.Blood and spleen samples were collected at weeks 1 and 9 from ApoE-/- mice infected with Porphyromonas gingivalis or Chlamydia pneumoniae and from mice fed a Western diet for 9 weeks. Transcripts based on data from a Western diet in ApoE-/- mice were measured in platelet samples from FHS using high throughput qRT-PCR.At week 1, both bacterial infections increased circulating platelet-neutrophil aggregates. At week 9, these cells individually localized to the spleen, while Western diet resulted in increased platelet-neutrophil aggregates in the spleen only. Microarray analysis of platelet RNA from infected or Western diet-fed mice at week 1 and 9 showed differential profiles. Genes, such as Serpina1a, Ttr, Fgg, Rpl21, and Alb, were uniquely affected by infection and diet. Results were reinforced in platelets obtained from participants of the FHS.Using both human studies and animal models, results demonstrate that variable sources of inflammatory stimuli have the ability to influence the platelet phenotype in distinct ways, indicative of the diverse function of platelets in thrombosis, hemostasis, and immunity.

Notch signaling regulates platelet-derived growth factor receptor-β expression in vascular smooth muscle cells

NARCIS (Netherlands)

Jin, S.; Hansson, E.M.; Tikka, S.; Lanner, F.; Sahlgren, C.; Farnebo, F.; Baumann, M.; Kalimo, H.; Lendahl, U.

2008-01-01

Notch signaling is critically important for proper architecture of the vascular system, and mutations in NOTCH3 are associated with CADASIL, a stroke and dementia syndrome with vascular smooth muscle cell (VSMC) dysfunction. In this report, we link Notch signaling to platelet-derived growth factor

Oxidative alterations during human platelet storage

OpenAIRE

Göker, Bahar; Özsavcı, Derya; Şener, Azize; Aksoy, Halil; Bağışgil, Vedat; Yanıkkaya Demirel, Gülderen; Uras, Fikriye

2014-01-01

SUMMARY: During storage of platelet obtained by apheresis several changes occur. The aimof this study was to investigate the effect of storage on activation, apoptosis, protein pattern,lipid peroxidation, and the levels of nitric oxide (NO) and glutathione (GSH) of platelets. In thisstudy, platelets obtained from healty donors (n=7) by apheresis were kept in an agitator fornine days at 20-24°C. The samples were taken on the 1st, 3 rd, 5 th and 9 th days and plateletswere precipitated. Platele...

Study on platelet kinetics

International Nuclear Information System (INIS)

Yui, Tokuo

1981-01-01

Fundamental study: Factors influencing the labeling on human platelets were evaluated and optimal labeling conditions were chosen. Then, platelet survival times were measured and organ distribution of labeled platelets was observed in rat by four different methods. These results were compared with each other. Based on the findings of those studies, the protocol of human platelet labeling with 111 In-oxine for clinical use was established. Clinical study: In normal cases, a platelet survival time and a platelet turnover rate were quite similar to the results from 51 Cr method. In gamma camera imaging, a radioactivity on the heart decreased with the lapse of time, while that on the spleen and liver gradually increased. In patients with idiopathic thrombocytopenic purpura, platelet survival time was markedly shortened and both a platelet turnover rate and an effective production increased. In patient with congestive splenomegaly, a platelet survival time was normal, whereas a platelet pooling on the spleen markedly increased. In patients who were implanted dacron-graft for abdominal aortic aneurysm, a radioactivity accumulated to the graft and a platelet survival time shortened. In a patient with myocardial infarction, the camera imaging clearly showed the thrombus in the left ventricular aneurysm. In three patients with mitral stenosis, thrombi in left atrium were observed at the camera images. Imaging of a platelet distribution and measurement of platelet survival time using 111 In-oxine labeled platelets are considered to be an excellent method for the diagnosis and decision of treatment on various disorders with thrombocytopenia and thrombosis. (J.P.N.)

Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Kawaguchi, Manami [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Kitajima, Kenji [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Kanokoda, Mai [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Suzuki, Hidenori [Division of Morphological and Biomolecular Research, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602 (Japan); Miyashita, Kazuya; Nakajima, Marino [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Nuriya, Hideko [Core Technology and Research Center, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Kasahara, Kohji [Laboratory of Biomembrane, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Hara, Takahiko, E-mail: hara-tk@igakuken.or.jp [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan)

2016-06-03

Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit{sup −}Tie2{sup −}CD41{sup +} Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41{sup +}CD42b{sup +}CD61{sup +} platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future. -- Highlights: •Megakaryocytes are efficiently induced by Gata2 from ESC-derived day 5 HECs. •Gata2-induced ESC-derived megakaryocytes are c-Kit{sup −}Tie2{sup −}CD41{sup +}. •Gata2-induced ESC-derived megakaryocytes produce larger discoid-shaped platelets. •Gata2-induced ESC-derived platelets bind fibrinogen upon thrombin stimulation.

Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

International Nuclear Information System (INIS)

Kawaguchi, Manami; Kitajima, Kenji; Kanokoda, Mai; Suzuki, Hidenori; Miyashita, Kazuya; Nakajima, Marino; Nuriya, Hideko; Kasahara, Kohji; Hara, Takahiko

2016-01-01

Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit − Tie2 − CD41 + Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41 + CD42b + CD61 + platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future. -- Highlights: •Megakaryocytes are efficiently induced by Gata2 from ESC-derived day 5 HECs. •Gata2-induced ESC-derived megakaryocytes are c-Kit − Tie2 − CD41 + . •Gata2-induced ESC-derived megakaryocytes produce larger discoid-shaped platelets. •Gata2-induced ESC-derived platelets bind fibrinogen upon thrombin stimulation.

A modified assay method for determining serotonin uptake in human platelets

International Nuclear Information System (INIS)

Arora, R.C.; Meltzer, H.Y.

1981-01-01

Effects of various experimental conditions on serotonin (5-HT) uptake in human platelets were examined. The experimental design allowed the evaluation of the effect of diffusion and other non-saturable processes on the affinity and maximum activity of the membrane pump for 5-HT uptake. Total 5-HT uptake was determined by incubating platelet-rich plasma (PRP) with increasing concentrations of serotonin at 37 0 C for 4 min. The passive uptake was measured by the addition of various 5-HT concentrations to PRP in buffer at 37 0 C, followed by immediate transfer to an ice-cold water bath. The difference between the total and passive uptake was linear for 6 min. The affinity (Ksub(m)) for active platelet serotonin uptake was 0.45 +- 0.09 μmol/l and maximal rate of uptake (V) was 10.7 +- 2.1 pmol/10 7 platelets/min. The described method provides a convenient and reliable measure of active 5-HT uptake suitable for clinical investigation. The effect of passive diffusion on kinetic parameters is discussed. (Auth.)

Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal stem/stromal cells.

Science.gov (United States)

de Soure, António M; Fernandes-Platzgummer, Ana; Moreira, Francisco; Lilaia, Carla; Liu, Shi-Hwei; Ku, Chen-Peng; Huang, Yi-Feng; Milligan, William; Cabral, Joaquim M S; da Silva, Cláudia L

2017-05-01

Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non-invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate-based culture supplement (UltraGRO TM , AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGRO TM -supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGRO TM -supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13-fold after 5-6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic-free microcarrier-based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Functional alterations of human platelets following indium-111 labelling using different incubation media and labelling agents

International Nuclear Information System (INIS)

Isaka, Yoshinari; Imaizumi, Masatoshi; Kimura, Kazufumi; Matsumoto, Masayasu; Kamada, Takenobu

1991-01-01

Human platelets were labelled in the absence of presence of plasma using 111 In-labelled oxine sulphate, tropolone or 2-mercaptopyridine-N-oxide (MPO). Under in vitro and in vivo conditions, platelet functions were evaluated by measuring their aggregability, survival, recovery and early distribution. High labelling efficiency was achieved in saline labelling, whereas with plasma labelling, it was necessary to concentrate the platelet-rich plasma to 4.8x10 6 platelets/μl. The aggregation of platelets labelled in plasma or saline was compared with that of controls; platelets labelled in saline showed lower aggregability in 2 μM ADP but not in 5 μM ADP nor with collagen. No significant differences in platelet survival and recovery were noted between platelets labelled in plasma and those labelled in saline. Our results indicate that partial loss of ADP aggregability in vitro does not influence the in vivo viability of platelets labelled in saline. Scintigraphic studies showed that platelets labelled in a saline medium were temporarily sequestrated in the liver but not in the spleen or heart. Thus, platelet labelling in saline does not affect platelet function adversely, but platelets labelled in plasma are more desirable for assessing the early distribution of platelets in the reticuloendothelial system. (orig.)

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

Directory of Open Access Journals (Sweden)

Monique Ramos de Oliveira Trugilho

2017-05-01

Full Text Available Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet

The human endogenous circadian system causes greatest platelet activation during the biological morning independent of behaviors.

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Frank A J L Scheer

Full Text Available Platelets are involved in the thromboses that are central to myocardial infarctions and ischemic strokes. Such adverse cardiovascular events have day/night patterns with peaks in the morning (~9 AM, potentially related to endogenous circadian clock control of platelet activation. The objective was to test if the human endogenous circadian system influences (1 platelet function and (2 platelet response to standardized behavioral stressors. We also aimed to compare the magnitude of any effects on platelet function caused by the circadian system with that caused by varied standardized behavioral stressors, including mental arithmetic, passive postural tilt and mild cycling exercise.We studied 12 healthy adults (6 female who lived in individual laboratory suites in dim light for 240 h, with all behaviors scheduled on a 20-h recurring cycle to permit assessment of endogenous circadian function independent from environmental and behavioral effects including the sleep/wake cycle. Circadian phase was assessed from core body temperature. There were highly significant endogenous circadian rhythms in platelet surface activated glycoprotein (GP IIb-IIIa, GPIb and P-selectin (6-17% peak-trough amplitudes; p ≤ 0.01. These circadian peaks occurred at a circadian phase corresponding to 8-9 AM. Platelet count, ATP release, aggregability, and plasma epinephrine also had significant circadian rhythms but with later peaks (corresponding to 3-8 PM. The circadian effects on the platelet activation markers were always larger than that of any of the three behavioral stressors.These data demonstrate robust effects of the endogenous circadian system on platelet activation in humans--independent of the sleep/wake cycle, other behavioral influences and the environment. The 9 AM timing of the circadian peaks of the three platelet surface markers, including platelet surface activated GPIIb-IIIa, the final common pathway of platelet aggregation, suggests that endogenous

Calcium-binding proteins from human platelets

International Nuclear Information System (INIS)

Gogstad, G.O.; Krutnes, M.B.; Solum, N.O.

1983-01-01

Calcium-binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45 Ca 2 + and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45 Ca 2 + . These corresponded to the glycoprotein IIb-IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane-bound and surface-oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb-IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4 Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb-IIIa complex. As another approach, platelet-rich plasma was incubated with 45 Ca 2 + prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was wekly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this antigen was markedly increased when the platelt-rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIB-IIIa precipitate also became apparent. No increased incorporation of calcium occured in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45 Ca 2 + . (orig.)

Comparative Effects of Platelet-Rich Plasma, Platelet Lysate, and Fetal Calf Serum on Mesenchymal Stem Cells.

Science.gov (United States)

Lykov, A P; Bondarenko, N A; Surovtseva, M A; Kim, I I; Poveshchenko, O V; Pokushalov, E A; Konenkov, V I

2017-10-01

We studied the effects of human platelet-rich plasma and platelet lysate on proliferation, migration, and colony-forming properties of rat mesenchymal stem cells. Platelet-rich plasma and platelet lysate stimulated the proliferation, migration, and colony formation of mesenchymal stem cells. A real-time study showed that platelet-rich plasma produces the most potent stimulatory effect, while both platelet-rich plasma and platelet lysate stimulated migration of cells.

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.

Analysis of angiotensin II binding to human platelets: Differences in young and old subjects

International Nuclear Information System (INIS)

Siebers, M.J.; Goodfriend, T.L.; Ball, D.; Elliott, M.E.

1990-01-01

We examined the binding of radiolabeled angiotensin II (AII) to human platelets to characterize the apparent increase in AII receptors observed in older subjects. At 22 degrees C, the amount of radioactivity associated with platelets from older subjects increased continuously for more than 2 hours. The same amount of radioactivity was displaced by addition of unlabeled AII at 30 min and 60 min. In the presence of phenylarsine oxide, in the cold, or when labeled antagonist was the ligand, binding came to equilibrium by 30 min. High pressure liquid chromatography demonstrated that 125 I-AII was the major radioactive compound in the supernatant and platelets after incubation, but the platelets also contained radiolabeled AII fragments. Thus, some degradation accompanied interaction of AII and platelets. Phenylarsine oxide did not prevent degradation of bound AII, suggesting that degradation precedes internalization. On average, maximum binding was greater in older subjects whether platelets were incubated with 125 I-AII alone, with 125 I-AII and phenylarsine oxide to prevent internalization, or when the competitive inhibitor 125 I-sar1,ile8-AII was the radioligand. Variability of binding among subjects also increased with age. Thus, platelets bind, degrade, and internalize AII, and the three processes occur to a greater extent in platelets from some, but not all older subjects

Imipramine binding in subpopulations of normal human blood platelets

International Nuclear Information System (INIS)

Arora, R.C.; Meltzer, H.Y.

1984-01-01

Imipramine binding was studied in platelet membranes isolated with different proportions of heavy (young) and light (old) platelets. The B/sub max/, a measure of the number of binding sites, was greater in the heavier platelets than in the light platelets. However, the dissociation constant K/sub d/ (a reflection of the affinity of imipramine binding) was greater in the lighter platelets compared to the heavy platelets. These results indicate that differences in K/sub d/ and B/sub max/ in particular membrane preparation, could be due to the differences in the relative proportion of heavy and light platelets

Human platelet lysate as a promising growth-stimulating additive for culturing of stem cells and other cell types.

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Shanskii, Ya D; Sergeeva, N S; Sviridova, I K; Kirakozov, M S; Kirsanova, V A; Akhmedova, S A; Antokhin, A I; Chissov, V I

2013-11-01

We compared the composition and biological activity of fetal calf serum and platelet lysate from donor platelet concentrate. In platelet lysate, the concentrations of alkaline phosphatase, lactate dehydrogenase, creatinine, and mineral metabolism parameters were lower, while parameters of lipid and protein metabolism were higher than in fetal calf serum. The concentrations of growth factors (platelet-derived (AA, AB, BB), vascular endothelial, insulin-like, and transforming growth factor β) in platelet lysate 1.7-148.7-fold surpassed the corresponding parameters in fetal calf serum. After replacement of fetal calf serum with platelet lysate in the culture medium (0, 25, 50, 75, and 100%), the count of multipotent mesenchymal stromal cells on day 7 (in comparison with day 1) increased by 154.8, 206.6, 228.2, 367.7, and 396.5%, respectively. Thus, platelet lysate can be an adequate non-xenogenic alternative for fetal calf serum.

Platelet-, monocyte-derived and tissue factor-carrying circulating microparticles are related to acute myocardial infarction severity.

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Gemma Chiva-Blanch

Full Text Available Circulating microparticles (cMPs are phospholipid-rich vesicles released from cells when activated or injured, and contribute to the formation of intracoronary thrombi. Tissue factor (TF, CD142 is the main trigger of fibrin formation and TF-carrying cMPs are considered one of the most procoagulant cMPs. Similar types of atherosclerotic lesions may lead to different types of AMI, although the mechanisms behind are unresolved. Therefore, we aimed to investigate the phenotype of cMPs found in plasma of ACS patients and its relation to AMI severity and thrombotic burden.In a cross-sectional study, two hundred patients aged 75±4 years were included in the study 2-8 weeks after suffering an AMI. Annexin V positive (AV+-cMPs derived from blood and vascular cells were measured by flow cytometry. Plasma procoagulant activity (TF-PCA was measured through a chromogenic assay.STEMI patients (n = 75 showed higher levels of platelet-derived cMPs [CD61+/AV+, CD31+/AV+, CD42b+/AV+ and CD31+/CD42b+/AV+, P = 0.048, 0.038, 0.009 and 0.006, respectively], compared to NSTEMI patients (n = 125. Patients who suffered a heart failure during AMI (n = 17 had increased levels of platelet (CD61+-and monocyte (CD14+-derived cMPs carrying TF (CD142+ (P<0.0001 and 0.004, respectively. Additionally, NYHA class III (n = 23 patients showed higher levels of CD142+/AV+, CD14+/AV+ and CD14+/CD142+/AV+ cMPs than those in class I/II (P = 0.001, 0.015 and 0.014, respectively. The levels of these cMPs positively correlated with TF-PCA (r≥0.166, P≤0.027, all.Platelets and monocytes remain activated in AMI patients treated as per guidelines and release cMPs that discriminate AMI severity. Therefore, TF-MPs, and platelet- and monocyte-MPs may reflect thrombotic burden in AMI patients.

Ligament Tissue Engineering Using a Novel Porous Polycaprolactone Fumarate Scaffold and Adipose Tissue-Derived Mesenchymal Stem Cells Grown in Platelet Lysate.

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Wagner, Eric R; Bravo, Dalibel; Dadsetan, Mahrokh; Riester, Scott M; Chase, Steven; Westendorf, Jennifer J; Dietz, Allan B; van Wijnen, Andre J; Yaszemski, Michael J; Kakar, Sanjeev

2015-11-01

Surgical reconstruction of intra-articular ligament injuries is hampered by the poor regenerative potential of the tissue. We hypothesized that a novel composite polymer "neoligament" seeded with progenitor cells and growth factors would be effective in regenerating native ligamentous tissue. We synthesized a fumarate-derivative of polycaprolactone fumarate (PCLF) to create macro-porous scaffolds to allow cell-cell communication and nutrient flow. Clinical grade human adipose tissue-derived human mesenchymal stem cells (AMSCs) were cultured in 5% human platelet lysate (PL) and seeded on scaffolds using a dynamic bioreactor. Cell growth, viability, and differentiation were examined using metabolic assays and immunostaining for ligament-related markers (e.g., glycosaminoglycans [GAGs], alkaline phosphatase [ALP], collagens, and tenascin-C). AMSCs seeded on three-dimensional (3D) PCLF scaffolds remain viable for at least 2 weeks with proliferating cells filling the pores. AMSC proliferation rates increased in PL compared to fetal bovine serum (FBS) (p ligament and tenogenic growth factor fibroblast growth factor 2 (FGF-2), especially when cultured in the presence of PL (p engineering and ligament regeneration.

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

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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.

Tailor-made purified human platelet lysate concentrated in neurotrophins for treatment of Parkinson's disease.

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Chou, Ming-Li; Wu, Joe-Wei; Gouel, Flore; Jonneaux, Aurélie; Timmerman, Kelly; Renn, Ting-Yi; Laloux, Charlotte; Chang, Hung-Ming; Lin, Liang-Tzung; Devedjian, Jean-Christophe; Devos, David; Burnouf, Thierry

2017-10-01

Human platelet lysates (PLs), which contain multiple neurotrophins, have been proposed for treating neurodegenerative disorders, including Parkinson's disease (PD). However, current PLs suspended in plasma have high protein content and contain fibrinogen/fibrin and, following activation, also proteolytic and thrombogenic enzymes. Upon brain administration, such PLs may saturate the cerebrospinal fluid and exert neurotoxicity. We assessed whether purified PLs, concentrated in neurotrophins, protected dopaminergic neurons in PD models. Platelet concentrates were collected by apheresis and centrifuged to eliminate plasma and recover the platelets. Platelets were lysed by freeze-thaw cycles, and the 10-fold concentrated platelet pellet lysates (PPLs) were heat-treated (at 56 °C for 30 min). The heat-treated PPLs were low in total proteins, depleted in both plasma and platelet fibrinogen, and devoid of thrombogenic and proteolytic activities. They exerted very high neuroprotective activity when non-oncogenic, Lund human mesencephalic (LUHMES) cells that had differentiated into dopaminergic neurons were exposed to the MPP + neurotoxin. Heat treatment improved the neuroprotection and inactivated the neurotoxic blood-borne hepatitis C virus. PPL did not induce inflammation in BV2 microglial cells and inhibited COX-2 expression upon lipopolysaccharide exposure. Intranasal administration in mice revealed (a) diffusion of neurotrophins in the striatum and cortex, and (b) MPTP intoxication neuroprotection in the substantia nigra and striatum and the absence of neuroinflammation. These dedicated heat-treated PPLs can be a safe and valuable candidate for a therapeutic strategy for PD. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characteristics of an autologous leukocyte and platelet-rich fibrin patch intended for the treatment of recalcitrant wounds

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Lundquist, Rasmus; Holmstrøm, Kim; Clausen, Christian

2012-01-01

We have investigated the physical, biochemical, and cellular properties of an autologous leukocyte and platelet-rich fibrin patch. This was generated in an automated device from a sample of a patient's blood at the point of care. Using microscopy, cell counting, enzyme-linked immunosorbent assay...... of chronic wound fluid. By comparison with traditional platelet-rich plasma, differences in immune components were found. The relevance of these findings was assessed by showing a mitogenic and migratory effect on cultured human dermal fibroblasts. Further, we showed that fibrocytes, a cell type important......, antibody arrays, and cell culture assays, we show that the patch is a three-layered membrane comprising a fibrin sheet, a layer of platelets, and a layer of leukocytes. Mean recovery of platelets from the donated blood was 98% (±95%CI 0.8%). Mean levels of platelet-derived growth factor AB, human...

Taurine and platelet aggregation

International Nuclear Information System (INIS)

Nauss-Karol, C.; VanderWende, C.; Gaut, Z.N.

1986-01-01

Taurine is a putative neurotransmitter or neuromodulator. The endogenous taurine concentration in human platelets, determined by amino acid analysis, is 15 μM/g. In spite of this high level, taurine is actively accumulated. Uptake is saturable, Na + and temperature dependent, and suppressed by metabolic inhibitors, structural analogues, and several classes of centrally active substances. High, medium and low affinity transport processes have been characterized, and the platelet may represent a model system for taurine transport in the CNS. When platelets were incubated with 14 C-taurine for 30 minutes, then resuspended in fresh medium and reincubated for one hour, essentially all of the taurine was retained within the cells. Taurine, at concentrations ranging from 10-1000 μM, had no effect on platelet aggregation induced by ADP or epinephrine. However, taurine may have a role in platelet aggregation since 35-39% of the taurine taken up by human platelets appears to be secreted during the release reaction induced by low concentrations of either epinephrine or ADP, respectively. This release phenomenon would imply that part of the taurine taken up is stored directly in the dense bodies of the platelet

Serum platelet-derived growth factor and fibroblast growth factor in patients with benign and malignant ovarian tumors

DEFF Research Database (Denmark)

Madsen, Christine Vestergaard; Steffensen, Karina Dahl; Olsen, Dorte Aalund

2012-01-01

New biological markers with predictive or prognostic value are highly warranted in the treatment of ovarian cancer. The platelet-derived growth factor (PDGF) system and fibroblast growth factor (FGF) system are important components in tumor growth and angiogenesis....

sup 86 Rb(K) influx and ( sup 3 H)ouabain binding by human platelets: Evidence for beta-adrenergic stimulation of Na-K ATPase activity

Energy Technology Data Exchange (ETDEWEB)

Turaihi, K.; Khokher, M.A.; Barradas, M.A.; Mikhailidis, D.P.; Dandona, P. (Royal Free Hospital and School of Medicine, London (England))

1989-08-01

Although active transport of potassium into human platelets has been demonstrated previously, there is hitherto no evidence that human platelets have an ouabain-inhibitable Na-K ATPase in their membrane. The present study demonstrates active rubidium (used as an index of potassium influx), {sup 86}Rb(K), influx into platelets, inhibitable by ouabain, and also demonstrates the presence of specific ({sup 3}H)ouabain binding by the human platelet. This {sup 86}Rb(K) influx was stimulated by adrenaline, isoprenaline, and salbutamol, but noradrenaline caused a mild inhibition. Active {sup 86}Rb(K) influx by platelets was inhibited markedly by timolol, mildly by atenolol, but not by phentolamine. Therefore, active {sup 86}Rb(K) influx in human platelets is enhanced by stimulation of beta adrenoceptors of the beta 2 subtype. The platelet may therefore replace the leukocyte in future studies of Na-K ATPase activity. This would be a considerable advantage in view of the ease and rapidity of preparation of platelets.

VacA, the vacuolating cytotoxin of Helicobacter pylori, binds to multimerin 1 on human platelets

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Satoh, Kaneo; Hirayama, Toshiya; Takano, Katsuhiro; Suzuki-Inoue, Katsue; Sato, Tadashi; Ohta, Masato; Nakagomi, Junko; Ozaki, Yukio

2013-01-01

Platelets were activated under the infection with H. pylori in human and mice. We investigated the role of VacA, an exotoxin released by H. pylori in this context. Acid-activated VacA, but not heated VacA, induced platelet CD62P expression. However, VacA reacted with none of the alleged VacA receptors present on platelet membranes. We therefore analyzed VacA associated proteins obtained through VacA affinity chromatography, using MALDI-TOF-MS. Multimerin1 was detected in two consecutive exper...

The important role of von Willebrand factor in platelet-derived FVIII gene therapy for murine hemophilia A in the presence of inhibitory antibodies.

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Shi, Q; Schroeder, J A; Kuether, E L; Montgomery, R R

2015-07-01

Our previous studies have demonstrated that targeting FVIII expression to platelets results in FVIII storage together with von Willebrand factor (VWF) in platelet α-granules and that platelet-derived FVIII (2bF8) corrects the murine hemophilia A phenotype even in the presence of high-titer anti-FVIII inhibitory antibodies (inhibitors). To explore how VWF has an impact on platelet gene therapy for hemophilia A with inhibitors. 2bF8 transgenic mice in the FVIII(-/-) background (2bF8(tg+/-) F8(-/-) ) with varying VWF phenotypes were used in this study. Animals were analyzed by VWF ELISA, FVIII activity assay, Bethesda assay and tail clip survival test. Only 18% of 2bF8(tg+/-) F8(-/-) VWF(-/-) animals, in which VWF was deficient, survived the tail clip challenge with inhibitor titers of 3-8000 BU mL(-1) . In contrast, 82% of 2bF8(tg+/-) F8(-/-) VWF(+/+) mice, which had normal VWF levels, survived tail clipping with inhibitor titers of 10-50,000 BU mL(-1) . All 2bF8(tg+/-) F8(-/-) VWF(-/-) mice without inhibitors survived tail clipping and no VWF(-/-) F8(-/-) mice survived this challenge. Because VWF is synthesized by endothelial cells and megakaryocytes and is distributed in both plasma and platelets in peripheral blood, we further investigated the effect of each compartment of VWF on platelet-FVIII gene therapy for hemophilia A with inhibitors. In the presence of inhibitors, 42% of animals survived tail clipping in the group with plasma-VWF and 50% survived in the platelet-VWF group. VWF is essential for platelet gene therapy for hemophilia A with inhibitors. Both platelet-VWF and plasma-VWF are required for optimal platelet-derived FVIII gene therapy for hemophilia A in the presence of inhibitors. © 2015 International Society on Thrombosis and Haemostasis.

Development of large-scale manufacturing of adipose-derived stromal cells for clinical applications using bioreactors and human platelet lysate.

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Haack-Sørensen, Mandana; Juhl, Morten; Follin, Bjarke; Harary Søndergaard, Rebekka; Kirchhoff, Maria; Kastrup, Jens; Ekblond, Annette

2018-04-17

In vitro expanded adipose-derived stromal cells (ASCs) are a useful resource for tissue regeneration. Translation of small-scale autologous cell production into a large-scale, allogeneic production process for clinical applications necessitates well-chosen raw materials and cell culture platform. We compare the use of clinical-grade human platelet lysate (hPL) and fetal bovine serum (FBS) as growth supplements for ASC expansion in the automated, closed hollow fibre quantum cell expansion system (bioreactor). Stromal vascular fractions were isolated from human subcutaneous abdominal fat. In average, 95 × 10 6 cells were suspended in 10% FBS or 5% hPL medium, and loaded into a bioreactor coated with cryoprecipitate. ASCs (P0) were harvested, and 30 × 10 6 ASCs were reloaded for continued expansion (P1). Feeding rate and time of harvest was guided by metabolic monitoring. Viability, sterility, purity, differentiation capacity, and genomic stability of ASCs P1 were determined. Cultivation of SVF in hPL medium for in average nine days, yielded 546 × 10 6 ASCs compared to 111 × 10 6 ASCs, after 17 days in FBS medium. ASCs P1 yields were in average 605 × 10 6 ASCs (PD [population doublings]: 4.65) after six days in hPL medium, compared to 119 × 10 6 ASCs (PD: 2.45) in FBS medium, after 21 days. ASCs fulfilled ISCT criteria and demonstrated genomic stability and sterility. The use of hPL as a growth supplement for ASCs expansion in the quantum cell expansion system provides an efficient expansion process compared to the use of FBS, while maintaining cell quality appropriate for clinical use. The described process is an obvious choice for manufacturing of large-scale allogeneic ASC products.

Platelets promote tumor growth and metastasis via direct interaction between Aggrus/podoplanin and CLEC-2.

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Satoshi Takagi

Full Text Available The platelet aggregation-inducing factor Aggrus, also known as podoplanin, is frequently upregulated in several types of tumors and enhances hematogenous metastasis by interacting with and activating the platelet receptor CLEC-2. Thus, Aggrus-CLEC-2 binding could be a therapeutic molecular mechanism for cancer therapy. We generated a new anti-human Aggrus monoclonal antibody, MS-1, that suppressed Aggrus-CLEC-2 binding, Aggrus-induced platelet aggregation, and Aggrus-mediated tumor metastasis. Interestingly, the MS-1 monoclonal antibody attenuated the growth of Aggrus-positive tumors in vivo. Moreover, the humanized chimeric MS-1 antibody, ChMS-1, also exhibited strong antitumor activity against Aggrus-positive lung squamous cell carcinoma xenografted into NOD-SCID mice compromising antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities. Because Aggrus knockdown suppressed platelet-induced proliferation in vitro and tumor growth of the lung squamous cell carcinoma in vivo, Aggrus may be involved in not only tumor metastasis but also tumor growth by promoting platelet-tumor interaction, platelet activation, and secretion of platelet-derived factors in vivo. Our results indicate that molecular target drugs inhibiting specific platelet-tumor interactions can be developed as antitumor drugs that suppress both metastasis and proliferation of tumors such as lung squamous cell carcinoma.

ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

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Kanamarlapudi, Venkateswarlu; Owens, Sian E; Saha, Keya; Pope, Robert J; Mundell, Stuart J

2012-01-01

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1) and P2Y(12) purinoceptors. Recently, we demonstrated that P2Y(1) and P2Y(12) purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y(1) and P2Y(12) purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1) or P2Y(12) purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

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Venkateswarlu Kanamarlapudi

Full Text Available Adenosine diphosphate (ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1 and P2Y(12 purinoceptors. Recently, we demonstrated that P2Y(1 and P2Y(12 purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6 in the internalization and function of P2Y(1 and P2Y(12 purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1 or P2Y(12 purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

In vitro effects of three blood derivatives on human corneal epithelial cells.

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Freire, Vanesa; Andollo, Noelia; Etxebarria, Jaime; Durán, Juan A; Morales, María-Celia

2012-08-15

We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.

[Experimental research on the effects of different activators on the formation of platelet-rich gel and the release of bioactive substances in human platelet-rich plasma].

Science.gov (United States)

Yang, Y; Zhang, W; Cheng, B

2017-01-20

Objective: To explore the effects of calcium gluconate and thrombin on the formation of platelet-rich gel (PRG) and the release of bioactive substances in human platelet-rich plasma (PRP) and the clinical significance. Methods: Six healthy blood donors who met the inclusion criteria were recruited in our unit from May to August in 2016. Platelet samples of each donor were collected for preparation of PRP. (1) PRP in the volume of 10 mL was collected from each donor and divided into thrombin activation group (TA, added with 0.5 mL thrombin solution in dose of 100 U/mL) and calcium gluconate activation group (CGA, added with 0.5 mL calcium gluconate solution in dose of 100 g/L) according to the random number table, with 5 mL PRP in each group. Then the PRP of the two groups was activated in water bath at 37 ℃ for 1 h. The formation time of PRG was recorded, and the formation situation of PRG was observed within 1 hour of activation. After being activated for 1 h, one part of PRG was collected to observe the distribution of fibrous protein with HE staining, and another part of PRG was collected to observe platelet ultrastructure under transmission electron microscope (TEM). After being activated for 1 h, the supernatant was collected to determine the content of transforming growth factor β(1, )platelet-derived growth factor BB (PDGF-BB), vascular endothelial growth factor, basic fibroblast growth factor (bFGF), epidermal growth factor, and insulin-like growth factorⅠby enzyme-linked immunosorbent assay. (2) Another 10 mL PRP from each donor was collected and grouped as above, and the platelet suspension was obtained after two times of centrifugation and resuspension with phosphate buffered saline, respectively. And then they were treated with corresponding activator for 1 h as that in experiment (1). Nanoparticle tracking analyzer was used to detect the concentrations of microvesicles with different diameters and total microvesicles derived from platelet. Data

Sports medicine applications of platelet rich plasma.

Science.gov (United States)

Mishra, Allan; Harmon, Kimberly; Woodall, James; Vieira, Amy

2012-06-01

Platelet rich plasma (PRP) is a powerful new biologic tool in sports medicine. PRP is a fraction of autologous whole blood containing and increased number of platelets and a wide variety of cytokines such as platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and transforming growth factor beta-1 (TGF-B1), fibroblast growth factor (FGF), Insulin-like growth factor-1 (IGF-1) among many others. Worldwide interest in this biologic technology has recently risen sharply. Basic science and preclinical data support the use of PRP for a variety of sports related injuries and disorders. The published, peer reviewed, human data on PRP is limited. Although the scientific evaluation of clinical efficacy is in the early stages, elite and recreational athletes already use PRP in the treatment of sports related injuries. Many questions remain to be answered regarding the use of PRP including optimal formulation, including of leukocytes, dosage and rehabilitation protocols. In this review, a classification for platelet rich plasma is proposed and the in-vitro, preclinical and human investigations of PRP applications in sports medicine will be reviewed as well as a discussion of rehabilitation after a PRP procedure. The regulation of PRP by the World Anti-Doping Agency will also be discussed. PRP is a promising technology in sports medicine; however, it will require more vigorous study in order to better understand how to apply it most effectively.

Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells.

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Michael Lohmann

Full Text Available The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS or other serum supplements such as human platelet lysate (HPL. In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1, but it was significantly higher with HPLs from younger donors (45 years. Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal. HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1 or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3 were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.

Donor Age of Human Platelet Lysate Affects Proliferation and Differentiation of Mesenchymal Stem Cells

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Lohmann, Michael; Walenda, Gudrun; Hemeda, Hatim; Joussen, Sylvia; Drescher, Wolf; Jockenhoevel, Stefan; Hutschenreuter, Gabriele; Zenke, Martin; Wagner, Wolfgang

2012-01-01

The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation. PMID:22662236

Platelets alter gene expression profile in human brain endothelial cells in an in vitro model of cerebral malaria.

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Mathieu Barbier

Full Text Available Platelet adhesion to the brain microvasculature has been associated with cerebral malaria (CM in humans, suggesting that platelets play a role in the pathogenesis of this syndrome. In vitro co-cultures have shown that platelets can act as a bridge between Plasmodium falciparum-infected red blood cells (pRBC and human brain microvascular endothelial cells (HBEC and potentiate HBEC apoptosis. Using cDNA microarray technology, we analyzed transcriptional changes of HBEC in response to platelets in the presence or the absence of tumor necrosis factor (TNF and pRBC, which have been reported to alter gene expression in endothelial cells. Using a rigorous statistical approach with multiple test corrections, we showed a significant effect of platelets on gene expression in HBEC. We also detected a strong effect of TNF, whereas there was no transcriptional change induced specifically by pRBC. Nevertheless, a global ANOVA and a two-way ANOVA suggested that pRBC acted in interaction with platelets and TNF to alter gene expression in HBEC. The expression of selected genes was validated by RT-qPCR. The analysis of gene functional annotation indicated that platelets induce the expression of genes involved in inflammation and apoptosis, such as genes involved in chemokine-, TREM1-, cytokine-, IL10-, TGFβ-, death-receptor-, and apoptosis-signaling. Overall, our results support the hypothesis that platelets play a pathogenic role in CM.

Molecular insight into human platelet antigens: structural and evolutionary conservation analyses offer new perspective to immunogenic disorders

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Landau, Meytal; Rosenberg, Nurit

2011-01-01

BACKGROUND: Human platelet antigens (HPAs) are polymorphisms in platelet membrane glycoproteins (GPs) that can stimulate production of alloantibodies once exposed to foreign platelets (PLTs) with different HPAs. These antibodies can cause neonatal alloimmune thrombocytopenia, posttransfusion purpura, and PLT transfusion refractoriness. Most HPAs are localized on the main PLT receptors: 1) integrin αIIbβ3, known as the fibrinogen receptor; 2) the GPIb-IX-V complex that functions as the recepto...

Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

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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

Platelet-Rich Plasma Derived Growth Factors Contribute to Stem Cell Differentiation in Musculoskeletal Regeneration

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Yun Qian; Yun Qian; Qixin Han; Wei Chen; Wei Chen; Jialin Song; Jialin Song; Xiaotian Zhao; Yuanming Ouyang; Yuanming Ouyang; Weien Yuan; Cunyi Fan

2017-01-01

Stem cell treatment and platelet-rich plasma (PRP) therapy are two significant issues in regenerative medicine. Stem cells such as bone marrow mesenchymal stem cells, adipose-derived stem cells and periodontal ligament stem cells can be successfully applied in the field of tissue regeneration. PRP, a natural product isolated from whole blood, can secrete multiple growth factors (GFs) for regulating physiological activities. These GFs can stimulate proliferation and differentiation of differen...

Platelet-rich plasma and its derivatives as promising bioactive materials for regenerative medicine: basic principles and concepts underlying recent advances.

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Kawase, Tomoyuki

2015-05-01

Over the past decade, platelet-rich plasma (PRP), a platelet-concentrated plasma fraction, has been widely investigated and applied to regenerative medicine. The clinical utility of PRP is supported by evidence that PRP contains high concentrations of platelet-related growth factors and normal concentrations of plasma-derived fibrinogen, both of which contribute synergistically to the regenerative process. Additionally, its superior cost-efficacy versus conventional therapies is attractive to many clinicians. However, current disadvantages of PRP include a relatively complicated preparation procedure and variable operator-dependent efficacy. An additional disadvantage is the use of bovine thrombin, an animal-derived biological, as a coagulant. Many of these disadvantages are overcome by recent advances in preparation procedures and devices; for example, Joseph Choukroun simplified the platelet-rich fibrin preparation procedure and improved handling efficiency without the aid of animal-derived factors. With advancements in cell processing technology, there has been a general shift in cell therapy from autologous to allogeneic treatment; however, autologous PRP therapy will not easily be replaced by allogeneic treatment in the near future. Therefore, to provide more predictable regenerative therapy outcomes using autologous PRP, further investigations should address developing a standardized procedure for PRP preparation to augment its efficacy and potency, independent of donor variability. We would then propose that operators and clinicians prepare PRP according to the standardized protocol and to carefully evaluate the clinical scenario (i.e., recipient factors comprising skeletal defects) to determine which factor(s) should be added to PRP preparations. This careful approach will lead to improved clinical outcomes for patients.

Human platelet lysate improves human cord blood derived ECFC survival and vasculogenesis in three dimensional (3D) collagen matrices.

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Kim, Hyojin; Prasain, Nutan; Vemula, Sasidhar; Ferkowicz, Michael J; Yoshimoto, Momoko; Voytik-Harbin, Sherry L; Yoder, Mervin C

2015-09-01

Human cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Since diminished ECFC survival is known to dampen the vasculogenic response in vivo, we tested how long implanted ECFC survive and generate vessels in three-dimensional (3D) type I collagen matrices in vitro and in vivo. We hypothesized that human platelet lysate (HPL) would promote cell survival and enhance vasculogenesis in the 3D collagen matrices. We report that the percentage of ECFC co-cultured with HPL that were alive was significantly enhanced on days 1 and 3 post-matrix formation, compared to ECFC alone containing matrices. Also, co-culture of ECFC with HPL displayed significantly more vasculogenic activity compared to ECFC alone and expressed significantly more pro-survival molecules (pAkt, p-Bad and Bcl-xL) in the 3D collagen matrices in vitro. Treatment with Akt1 inhibitor (A-674563), Akt2 inhibitor (CCT128930) and Bcl-xL inhibitor (ABT-263/Navitoclax) significantly decreased the cell survival and vasculogenesis of ECFC co-cultured with or without HPL and implicated activation of the Akt1 pathway as the critical mediator of the HPL effect on ECFC in vitro. A significantly greater average vessel number and total vascular area of human CD31(+) vessels were present in implants containing ECFC and HPL, compared to the ECFC alone implants in vivo. We conclude that implantation of ECFC with HPL in vivo promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC. Copyright © 2015 Elsevier Inc. All rights reserved.

Flavonoids purified from parsley inhibit human blood platelet aggregation and adhesion to collagen under flow.

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Gadi, Dounia; Bnouham, Mohamed; Aziz, Mohammed; Ziyyat, Abderrahim; Legssyer, Abdelkhaleq; Bruel, Arlette; Berrabah, Mohamed; Legrand, Chantal; Fauvel-Lafeve, Françoise; Mekhfi, Hassane

2012-08-10

Blood platelets are directly involved in both haemostatic and pathologic thrombotic processes, through their adhesion, secretion and aggregation. In this study, we investigated the effect of genins (aglycone flavonoids without sugar group) isolated from parsley (Petroselinum crispum) leaves in vitro on human platelet aggregation and adhesion to a collagen-coated surface under physiologic flow conditions. The aggregation and adhesion studies were monitored after pre-incubation of platelets with genins. Genins inhibited dose dependently aggregation induced by thrombin, ADP and collagen. The strongest effect was observed in collagen induced aggregation (IC50 = 0.08 ± 0.01 mg/ml). The HPLC identification of genins compounds revealed the presence of keampferol, apigenin and other not identified compounds. The aggregation tests showed that these compounds have anti-aggregating activity. In addition, adhesion of human platelets to collagen was greatly decreased (over 75 %) by genins (0.3 mg/ml). While the mechanism by which genins act is unclear, we suggest that these compounds may interfere with a multiple target step in the haemostasis process. These results show that genins isolated from parsley has a potent antiplatelet activity. It may be an important source of beneficial antiplatelet compounds that decrease thrombosis and cardiovascular diseases.

Platelet Counts in Insoluble Platelet-Rich Fibrin Clots: A Direct Method for Accurate Determination

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Yutaka Kitamura

2018-02-01

Full Text Available Platelet-rich fibrin (PRF clots have been used in regenerative dentistry most often, with the assumption that growth factor levels are concentrated in proportion to the platelet concentration. Platelet counts in PRF are generally determined indirectly by platelet counting in other liquid fractions. This study shows a method for direct estimation of platelet counts in PRF. To validate this method by determination of the recovery rate, whole-blood samples were obtained with an anticoagulant from healthy donors, and platelet-rich plasma (PRP fractions were clotted with CaCl2 by centrifugation and digested with tissue-plasminogen activator. Platelet counts were estimated before clotting and after digestion using an automatic hemocytometer. The method was then tested on PRF clots. The quality of platelets was examined by scanning electron microscopy and flow cytometry. In PRP-derived fibrin matrices, the recovery rate of platelets and white blood cells was 91.6 and 74.6%, respectively, after 24 h of digestion. In PRF clots associated with small and large red thrombi, platelet counts were 92.6 and 67.2% of the respective total platelet counts. These findings suggest that our direct method is sufficient for estimating the number of platelets trapped in an insoluble fibrin matrix and for determining that platelets are distributed in PRF clots and red thrombi roughly in proportion to their individual volumes. Therefore, we propose this direct digestion method for more accurate estimation of platelet counts in most types of platelet-enriched fibrin matrix.

Platelet-derived growth factor (PDGF-BB-mediated induction of monocyte chemoattractant protein 1 in human astrocytes: implications for HIV-associated neuroinflammation

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Bethel-Brown Crystal

2012-12-01

Full Text Available Abstract Chemokine (C-C motif ligand 2, also known as monocyte chemoattractant protein 1 (MCP-1 is an important factor for the pathogenesis of HIV-associated neurocognitive disorders (HAND. The mechanisms of MCP-1-mediated neuropathogenesis, in part, revolve around its neuroinflammatory role and the recruitment of monocytes into the central nervous system (CNS via the disrupted blood-brain barrier (BBB. We have previously demonstrated that HIV-1/HIV-1 Tat upregulate platelet-derived growth factor (PDGF-BB, a known cerebrovascular permeant; subsequently, the present study was aimed at exploring the regulation of MCP-1 by PDGF-BB in astrocytes with implications in HAND. Specifically, the data herein demonstrate that exposure of human astrocytes to HIV-1 LAI elevated PDGF-B and MCP-1 levels. Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels. MCP-1 induction was regulated by activation of extracellular-signal-regulated kinase (ERK1/2, c-Jun N-terminal kinase (JNK and p38 mitogen-activated protein (MAP kinases and phosphatidylinositol 3-kinase (PI3K/Akt pathways and the downstream transcription factor, nuclear factor κB (NFκB. Chromatin immunoprecipitation (ChIP assays demonstrated increased binding of NFκB to the human MCP-1 promoter following PDGF-BB exposure. Conditioned media from PDGF-BB-treated astrocytes increased monocyte transmigration through human brain microvascular endothelial cells (HBMECs, an effect that was blocked by STI-571, a tyrosine kinase inhibitor (PDGF receptor (PDGF-R blocker. PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays. Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND. These results suggest that astrocyte

Characterization of the stimulation of human platelets by stable analogues of PGH2/TXA2

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Morinelli, T.A.

1987-01-01

The specific effects of the TXA 2 /PGH 2 analogues, U46619 (9,11-dideoxy,9α-11α-methanoepoxy-PGF/sub 2α/), and 9,11 aza-PGH 2 , on human platelet shape change, myosin light chain phosphorylation, serotonin release, fibrinogen receptor exposure and platelet aggregation were measured and compared with binding of 3 H-U46619 to platelets. Shape change and myosin light chain phosphorylation were found to saturable and dose dependent. These two effects were competitively inhibited by specific antagonists of TXA 2 /PGH 2 receptors (BM13177 and I-PTA-OH) indicating that they are receptor mediated. Binding of 3 H-U46619 showed two components. Occupancy of high affinity binding sites correlated with platelet shape change and myosin and light chain phosphorylation. A second component with an apparent K/sub d/ of 1.46 +/- 0.47 μM, may represent a second, low-affinity site. Therefore, the platelet release reaction as not directly correlated with occupancy of high affinity receptors but could be related to the second binding component of U46619. Fibrinogen receptor exposure and platelet aggregation caused by U46619 appeared to be events mediated by the release of ADP from platelet dense granules

Senescence and quiescence in adipose-derived stromal cells: Effects of human platelet lysate, fetal bovine serum and hypoxia.

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Søndergaard, Rebekka Harary; Follin, Bjarke; Lund, Lisbeth Drozd; Juhl, Morten; Ekblond, Annette; Kastrup, Jens; Haack-Sørensen, Mandana

2017-01-01

Adipose-derived stromal cells (ASCs) are attractive sources for cell-based therapies. The hypoxic niche of ASCs in vivo implies that cells will benefit from hypoxia during in vitro expansion. Human platelet lysate (hPL) enhances ASC proliferation rates, compared with fetal bovine serum (FBS) at normoxia. However, the low proliferation rates of FBS-expanded ASCs could be signs of senescence or quiescence. We aimed to determine the effects of hypoxia and hPL on the expansion of ASCs and whether FBS-expanded ASCs are senescent or quiescent. ASCs expanded in FBS or hPL at normoxia or hypoxia until passage 7 (P7), or in FBS until P5 followed by culture in hPL until P7, were evaluated by proliferation rates, cell cycle analyses, gene expression and β-galactosidase activity. hPL at normoxia and hypoxia enhanced proliferation rates and expression of cyclins, and decreased G0/G1 fractions and expression of p21 and p27, compared with FBS. The shift from FBS to hPL enhanced cyclin levels, decreased p21 and p27 levels and tended to decrease G0/G1 fractions. Hypoxia does not add to the effect of hPL during ASC expansion with regard to proliferation, cell cycle regulation and expression of cyclins, p21 and p27. hPL rejuvenates FBS-expanded ASCs with regard to cell cycle regulation and expression of cyclins, p21 and p27. This indicates a reversible arrest. Therefore, we conclude that ASCs expanded until P7 are not senescent regardless of culture conditions. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Abnormal platelet-derived growth factor signaling accounting for lung hypoplasia in experimental congenital diaphragmatic hernia.

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Dingemann, Jens; Doi, Takashi; Ruttenstock, Elke; Puri, Prem

2010-10-01

The pathogenesis of pulmonary hypoplasia in congenital diaphragmatic hernia (CDH) is not fully understood. Platelet-derived growth factor A (PDGFA) and platelet-derived growth factor receptor α (PDGFRα) play a crucial role in lung development. It has been reported that PDGF induces H(2)O(2)-production and that oxidative stress may be an important mechanism for the impaired lung development in the nitrofen rat model. We hypothesized that pulmonary expression of PDGFA and PDGFRα is altered in the nitrofen induced CDH model. Pregnant rats received 100 mg nitrofen or vehicle on gestational day 9 (D9) and were sacrificed on D15, D18 or D21. RNA was extracted from fetal left lungs and mRNA levels of PDGFA and PDGFRα were determined using real-time polymerase chain reaction. Immunohistochemistry for protein expression of PDGFA and PDGFRα was performed. Pulmonary H(2)O(2) was measured colorimetrically. mRNA levels of PDGFRα at D15 (4.50 ± 0.87) and PDGFA at D18 (2.90 ± 1.38) were increased in the nitrofen group (P stress during lung development. Copyright © 2010 Elsevier Inc. All rights reserved.

Comparative characterisation of the biofilm-production abilities of Staphylococcus epidermidis isolated from human skin and platelet concentrates.

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Taha, Mariam; Kohnen, Carissa; Mallya, Shruti; Kou, Yuntong; Zapata, Adriana; Ramirez-Arcos, Sandra

2018-02-01

Staphylococcus epidermidis is the predominant contaminant of platelet concentrates (PCs), a blood product used to treat patients with platelet deficiencies. This microorganism is able to form surface-attached aggregates (biofilms) in human skin. Herein, the abundance of S. epidermidis biofilm-producers in contaminated PCs compared to skin isolates was explored. Furthermore, the potential positive selection of S. epidermidis biofilm-producers during the blood donation process and PC manufacturing was investigated. Twenty-four S. epidermidis isolates obtained from contaminated PCs and 48 S. epidermidis isolates obtained from the venipuncture area of human volunteers were compared for their ability to form biofilms in laboratory media and in PCs using a semi quantitative crystal violet assay. Also, the presence of the biofilm-associated icaA and icaD genes was assessed by PCR-amplification.Results/Key findings.Biofilm production in laboratory media showed a higher number of S. epidermidis biofilm-producers in the skin-derived group (43.7 %) compared to the PC-derived isolates (25 %). However, all skin and PC isolates formed biofilms in PCs. The prevalence of ica-positive biofilm-producer isolates was similar in PC and skin isolates (16.6 and 18.8 %, respectively). In contrast, the abundance of ica-negative biofilm-producers was lower in PC isolates compared to skin isolates (8.3 vs 25 %, respectively). Positive selection of S. epidermidis biofilm-producers during blood donation and PC manufacturing was not observed. Interestingly, ica-negative biofilm-producers seem to be negatively affected by skin disinfection, blood processing and PC storage. Furthermore, this study shows that S. epidermidis adopts a biofilm-forming phenotype in PCs regardless of its genetic background or origin.

Human plasma fibrinogen adsorption and platelet adhesion to polystyrene.

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Tsai, W B; Grunkemeier, J M; Horbett, T A

1999-02-01

The purpose of this study was to further investigate the role of fibrinogen adsorbed from plasma in mediating platelet adhesion to polymeric biomaterials. Polystyrene was used as a model hydrophobic polymer; i.e., we expected that the role of fibrinogen in platelet adhesion to polystyrene would be representative of other hydrophobic polymers. Platelet adhesion was compared to both the amount and conformation of adsorbed fibrinogen. The strategy was to compare platelet adhesion to surfaces preadsorbed with normal, afibrinogenemic, and fibrinogen-replenished afibrinogenemic plasmas. Platelet adhesion was determined by the lactate dehydrogenase (LDH) method, which was found to be closely correlated with adhesion of 111In-labeled platelets. Fibrinogen adsorption from afibrinogenemic plasma to polystyrene (Immulon I(R)) was low and polystyrene preadsorbed with fibrinogen-replenished afibrinogenemic plasma. Addition of even small, subnormal concentrations of fibrinogen to afibrinogenemic plasma greatly increased platelet adhesion. In addition, surface-bound fibrinogen's ability to mediate platelet adhesion was different, depending on the plasma concentration from which fibrinogen was adsorbed. These differences correlated with changes in the binding of a monoclonal antibody that binds to the Aalpha chain RGDS (572-575), suggesting alteration in the conformation or orientation of the adsorbed fibrinogen. Platelet adhesion to polystyrene preadsorbed with blood plasma thus appears to be a strongly bivariate function of adsorbed fibrinogen, responsive to both low amounts and altered states of the adsorbed molecule. Copyright 1999 John Wiley & Sons, Inc.

Effect of activated autologous platelet-rich plasma on proliferation and osteogenic differentiation of human adipose-derived stem cells in vitro

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Xu, Fang-Tian; Li, Hong-Mian; Yin, Qing-Shui; Liang, Zhi-Jie; Huang, Min-Hong; Chi, Guang-Yi; Huang, Lu; Liu, Da-Lie; Nan, Hua

2015-01-01

To investigate whether activated autologous platelet-rich plasma (PRP) can promote proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) in vitro. hASCs were isolated from lipo-aspirates, and characterized by specific cell markers and multilineage differentiation capacity after culturing to the 3rd passage. PRP was collected and activated from human peripheral blood of the same patient. Cultured hASCs were treated with normal osteogenic inductive media alone (group A, control) or osteogenic inductive media plus 5%, 10%, 20%, 40%PRP (group B, C, D, E, respectively). Cell proliferation was assessed by CCK-8 assay. mRNA expression of osteogenic marker genes including alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN) and core binding factor alpha 1 (Cbfa1) were determined by Real-Time Quantitative PCR Analysis (qPCR). Data revealed that different concentrations of activated autologous PRP significantly promoted hASCs growth in the proliferation phase compared to the without PRP group and resulted in a dose-response relationship. At 7-d and 14-d time point of the osteogenic induced stage, ALP activity in PRP groups gradually increased with the increasing of concentrations of PRP and showed that dose-response relationship. At 21-d time point of the osteogenic induced stage, PRP groups make much more mineralization and mRNA relative expression of ALP, OPN, OCN and Cbfa1 than that without PRP groups and show that dose-response relationship. This study indicated that different concentrations of activated autologous PRP can promote cell proliferation at earlier stage and promote osteogenic differentiation at later stage of hASCs in vitro. Moreover, it displayed a dose-dependent effect of activated autologous PRP on cell proliferation and osteogenic differentiation of hASCs in vitro. PMID:25901195

Mechanisms of Nifedipine-Downregulated CD40L/sCD40L Signaling in Collagen Stimulated Human Platelets.

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Tso-Hsiao Chen

Full Text Available The platelet-derived soluble CD40L (sCD40L release plays a critical role in the development of atherosclerosis. Nifedipine, a dihydropyridine-based L-type calcium channel blocker (CCB, has been reported to have an anti-atherosclerotic effect beyond its blood pressure-lowering effect, but the molecular mechanisms remain unclear. The present study was designed to investigate whether nifedipine affects sCD40L release from collagen-stimulated human platelets and to determine the potential role of peroxisome proliferator-activated receptor-β/-γ (PPAR-β/-γ. We found that treatment with nifedipine significantly inhibited the platelet surface CD40L expression and sCD40L release in response to collagen, while the inhibition was markedly reversed by blocking PPAR-β/-γ activity with specific antagonist such as GSK0660 and GW9662. Meanwhile, nifedipine also enhanced nitric oxide (NO and cyclic GMP formation in a PPAR-β/-γ-dependent manner. When the NO/cyclic GMP pathway was suppressed, nifedipine-mediated inhibition of sCD40L release was abolished significantly. Collagen-induced phosphorylation of p38MAPK, ERK1/2 and HSP27, matrix metalloproteinase-2 (MMP-2 expression/activity and reactive oxygen species (ROS formation were significantly inhibited by nifedipine, whereas these alterations were all attenuated by co-treatment with PPAR-β/-γ antagonists. Collectively, these results demonstrate that PPAR-β/-γ-dependent pathways contribute to nifedipine-mediated downregulation of CD40L/sCD40L signaling in activated platelets through regulation of NO/ p38MAPK/ERK1/2/HSP27/MMP-2 signalings and provide a novel mechanism regarding the anti-atherosclerotic effect of nifedipine.

Identification of Aspergillus fumigatus Surface Components That Mediate Interaction of Conidia and Hyphae With Human Platelets.

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Rambach, Günter; Blum, Gerhard; Latgé, Jean-Paul; Fontaine, Thierry; Heinekamp, Thorsten; Hagleitner, Magdalena; Jeckström, Hanna; Weigel, Günter; Würtinger, Philipp; Pfaller, Kristian; Krappmann, Sven; Löffler, Jürgen; Lass-Flörl, Cornelia; Speth, Cornelia

2015-10-01

Platelets were recently identified as a part of innate immunity. They are activated by contact with Aspergillus fumigatus; putative consequences include antifungal defense but also thrombosis, excessive inflammation, and thrombocytopenia. We aimed to identify those fungal surface structures that mediate interaction with platelets. Human platelets were incubated with Aspergillus conidia and hyphae, isolated wall components, or fungal surface mutants. Interaction was visualized microscopically; activation was quantified by flow cytometry of specific markers. The capacity of A. fumigatus conidia to activate platelets is at least partly due to melanin, because this effect can be mimicked with "melanin ghosts"; a mutant lacking melanin showed reduced platelet stimulating potency. In contrast, conidial hydrophobin masks relevant structures, because an A. fumigatus mutant lacking the hydrophobin protein induced stronger platelet activation than wild-type conidia. A. fumigatus hyphae also contain surface structures that interact with platelets. Wall proteins, galactomannan, chitin, and β-glucan are not the relevant hyphal components; instead, the recently identified fungal polysaccharide galactosaminogalactan potently triggered platelet activation. Conidial melanin and hydrophobin as well as hyphal galactosaminogalactan represent important pathogenicity factors that modulate platelet activity and thus might influence immune responses, inflammation, and thrombosis in infected patients. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Relative turnover of [3H]arachidonic acid and [14C]eicosapentaenoic acid in stimulated human platelets

International Nuclear Information System (INIS)

Weaver, B.J.; Holub, B.J.

1986-01-01

The relative release of arachidonic acid (AA) versus eicosapentaenoic acid (EPA) from platelet phospholipids may be important in accounting for the potential of dietary fish oil containing EPA to alter platelet reactivity. Human platelets enriched in EPA and prelabelled with [ 3 H]AA and [ 14 C]EPA were used to examine the relative losses of these fatty acids from platelet phospholipids upon stimulation. Washed dual-labelled platelets were incubated with and without thrombin in the presence of BW755C and in the presence and absence of trifluoperazine. The platelet lipids were extracted and the individual phospholipids as well as diacylglycerol (DG), phosphatidic acid (PA), etc. were separated by thin-layer chromatography and the radioactivity in each fraction determined. The [ 3 H]AA/[ 14 C]EPA dpm ratio for the loss in radioactivity from PC upon thrombin stimulation was similar to that for the PC in resting platelets. This suggests no marked selectivity in the degradation of AA versus EPA species of PC during platelet activation. The [ 3 H]/[ 14 C] ratios for the increased radioactivity in DG and PA upon thrombin stimulation were slightly higher than the ratio in PI from resting platelets suggesting only a minor preference for 1-acyl 2-arachidonoyl PI over 1-acyl 2-eicosapentaenoyl PI in the pathway from PI to DG to PA

The Antimicrobial Peptide Human Beta-Defensin-3 Is Induced by Platelet-Released Growth Factors in Primary Keratinocytes

OpenAIRE

Andreas Bayer; Justus Lammel; Mersedeh Tohidnezhad; Sebastian Lippross; Peter Behrendt; Tim Klüter; Thomas Pufe; Jochen Cremer; Holger Jahr; Franziska Rademacher; Regine Gläser; Jürgen Harder

2017-01-01

Platelet-released growth factors (PRGF) and its related clinically used formulations (e.g., Vivostat Platelet-Rich Fibrin (PRF?)) contain a variety of chemokines, cytokines, and growth factors and are therefore used to support healing of chronic, hard-to-heal, or infected wounds. Human beta-defensin-3 (hBD-3) is an antimicrobial peptide inducibly expressed in human keratinocytes especially upon wounding. The potent antimicrobial activity of hBD-3 together with its wound closure-promoting acti...

Human platelet lysate supports the formation of robust human periodontal ligament cell sheets.

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Tian, Bei-Min; Wu, Rui-Xin; Bi, Chun-Sheng; He, Xiao-Tao; Yin, Yuan; Chen, Fa-Ming

2018-04-01

The use of stem cell-derived sheets has become increasingly common in a wide variety of biomedical applications. Although substantial evidence has demonstrated that human platelet lysate (PL) can be used for therapeutic cell expansion, either as a substitute for or as a supplement to xenogeneic fetal bovine serum (FBS), its impact on cell sheet production remains largely unexplored. In this study, we manufactured periodontal ligament stem cell (PDLSC) sheets in vitro by incubating PDLSCs in sheet-induction media supplemented with various ratios of PL and FBS, i.e. 10% PL without FBS, 7.5% PL + 2.5% FBS, 5% PL + 5% FBS, 2.5% PL + 7.5% FBS or 10% FBS without PL. Cultures with the addition of all the designed supplements led to successful cell sheet production. In addition, all the resultant cellular materials exhibited similar expression profiles of matrix-related genes and proteins, such as collagen I, fibronectin and integrin β1. Interestingly, the cell components within sheets generated by media containing both PL and FBS exhibited improved osteogenic potential. Following in vivo transplantation, all sheets supported significant new bone formation. Our data suggest that robust PDLSC sheets can be produced by applying PL as either an alternative or an adjuvant to FBS. Further examination of the relevant influences of human PL that benefit cell behaviour and matrix production will pave the way towards optimized and standardized conditions for cell sheet production. Copyright © 2017 John Wiley & Sons, Ltd.

The content of bone morphogenetic proteins in platelets varies greatly between different platelet donors

International Nuclear Information System (INIS)

Kalen, Anders; Wahlstroem, Ola; Linder, Cecilia Halling; Magnusson, Per

2008-01-01

Platelet derivates and platelet rich plasma have been used to stimulate bone formation and wound healing because of the rich content of potent growth factors. However, not all reports have been conclusive since some have not been able to demonstrate a positive effect. We investigated the interindividual variation of bone morphogenetic proteins (BMPs) in platelets from healthy donors, and the pH-dependent effect on the release of BMPs in preparations of lysed platelets in buffer (LPB). Platelet concentrates from 31 healthy donors were prepared in pH 4.3 and pH 7.4 buffers and investigated with respect to BMP-2, -4, -6, and -7. BMP-2 and BMP-4 were significantly more common in acidic LPBs in comparison with neutral preparations. We also observed a considerable variation among platelet donors with respect to the release of BMPs at pH 4.3 and 7.4. In conclusion, a considerable variation was found among platelet donors, which may be of importance considering the ambiguous results previously reported on osteoblast proliferation and differentiation

Platelet-Rich Plasma Influences Expansion and Paracrine Function of Adipose-Derived Stromal Cells in a Dose-Dependent Fashion

NARCIS (Netherlands)

Willemsen, Joep C. N.; Spiekman, Maroesjka; Stevens, H. P. Jeroen; van der Lei, Berend; Harmsen, Martin C.

Background: Lipofilling is a treatment modality to restore tissue volume. Both platelet-rich plasma and adipose-derived stromal cells have been reported to augment the efficacy of lipofilling, yet results are not conclusive. The authors hypothesized that the variation reported in literature is

Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells.

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Naskou, Maria C; Sumner, Scarlett M; Chocallo, Anna; Kemelmakher, Hannah; Thoresen, Merrilee; Copland, Ian; Galipeau, Jacques; Peroni, John F

2018-03-22

Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed

Bacteria-induced release of white cell--and platelet-derived vascular endothelial growth factor in vitro

DEFF Research Database (Denmark)

Nielsen, Hans Jørgen; Werther, K; Mynster, T

2001-01-01

BACKGROUND AND OBJECTIVES: Poor prognosis after resection of primary colorectal cancer may be related to the combination of perioperative blood transfusion and subsequent development of infectious complications. White blood cell--and platelet-derived cancer growth substances, including vascular...... endothelial growth factor (VEGF), may be involved in this process. Therefore, we studied the in vitro release of VEGF from white blood cells and platelets stimulated by bacterial antigens and supernatants from stored red cell components. MATERIALS AND METHODS: Eight units of whole blood (WB) and eight units...... of buffy-coat-depleted red cell (SAGM) blood were donated by healthy blood donors. Subsequently, half of every unit was leucocyte depleted by filtration, and all 32 half-units were stored under standard conditions for 35 days. Just after storage, and on days 7, 21 and 35 during storage, aliquots...

Evaluation of platelet aggregation in platelet concentrates: storage implications

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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.

Mitochondrial respiration in human viable platelets-Methodology and influence of gender, age and storage

DEFF Research Database (Denmark)

Sjövall, Fredrik; Ehinger, Johannes K H; Marelsson, Sigurður E

2013-01-01

Studying whole cell preparations with intact mitochondria and respiratory complexes has a clear benefit compared to isolated or disrupted mitochondria due to the dynamic interplay between mitochondria and other cellular compartments. Platelet mitochondria have a potential to serve as a source...... of human viable mitochondria when studying mitochondrial physiology and pathogenic mechanisms, as well as for the diagnostics of mitochondrial diseases. The objective of the present study was to perform a detailed evaluation of platelet mitochondrial respiration using high-resolution respirometry. Further...

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.

Morphofunctional reaction of bacteria treated with antimicrobial peptides derived from farm animal platelets.

Science.gov (United States)

Vasilchenko, Alexey S; Dymova, Veronica V; Kartashova, Olga L; Sycheva, Maria V

2015-03-01

Classical microbiological approach and atomic force microscopy were used to evaluate the mechanisms of biological activity of antimicrobial peptides (AMPs) derived from platelets of farm animals. It is established that AMPs inhibit both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) microorganisms. Differences revealed in the biological activity of AMP preparations obtained from the organisms of various species can be reduced to quantitative differences. While qualitative changes of bacterial cells were substantially similar, changes in the integrity of cell walls resulted in disintegration of the bacterial outer and/or cytoplasmic membranes.

Portable dynamic light scattering instrument and method for the measurement of blood platelet suspensions

International Nuclear Information System (INIS)

Maurer-Spurej, Elisabeth; Brown, Keddie; Labrie, Audrey; Marziali, Andre; Glatter, Otto

2006-01-01

No routine test exists to determine the quality of blood platelet transfusions although every year millions of patients require platelet transfusions to survive cancer chemotherapy, surgery or trauma. A new, portable dynamic light scattering instrument is described that is suitable for the measurement of turbid solutions of large particles under temperature-controlled conditions. The challenges of small sample size, short light path through the sample and accurate temperature control have been solved with a specially designed temperature-controlled sample holder for small diameter, disposable capillaries. Efficient heating and cooling is achieved with Peltier elements in direct contact with the sample capillary. Focusing optical fibres are used for light delivery and collection of scattered light. The practical use of this new technique was shown by the reproducible measurement of latex microspheres and the temperature-induced morphological changes of human blood platelets. The measured parameters for platelet transfusions are platelet size, number of platelet-derived microparticles and the response of platelets to temperature changes. This three-dimensional analysis provides a high degree of confidence for the determination of platelet quality. The experimental data are compared to a matrix and facilitate automated, unbiased quality testing

Serotonin binding in vitro by releasable proteins from human blood platelets

International Nuclear Information System (INIS)

Heemstra, V.L.

1983-11-01

Among the substances released from human blood platelets are serotonin and various proteins. It was hypothesized that one of these proteins binds serotonin and that serotonin might be important to the protein's function or that the protein might be important to serotonin's function. Two platelet-specific proteins, platelet factor 4 (PF4) and β-thromboglobulin (βTG) were found to bind serotonin in vitro. Endogenous PF4 was isolated by serotonin-affinity chromatography and was identified by radioimmunoassay. Purified [ 125 I] -PF4 and native PF4 bound to and eluted from a serotonin-affinity column similarly. Ultrafiltration of the homologous protein, βTG, with [ 14 C]-serotonin demonstrated binding of about 8 moles serotonin per mole tetrameric βTG with a dissociation constant of about 4 X 10(sup-8) M. Equilibrium dialysis of PF4 with radiolabelled serotonin was attempted, but no binding constant values were obtained because serotonin apparently bound to the dialysis membrane. Since EDTA was one of the two agents that eluted PF4 from the serotonin-affinity gel, calcium binding by PF4 was investigated by equilibrium dialysis. Evidence was obtained for positively cooperative binding of calcium ions by PF4. It is concluded that PF4 and βTG bind serotonin in vitro, that they may also bind in vivo when platelets undergo release, and that the functions of serotonin, PF4 and βTG may be mediated in part by serotonin-protein associations

Generation of Platelet Microparticles after Cryopreservation of Apheresis Platelet Concentrates Contributes to Hemostatic Activity

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İbrahim Eker

2017-03-01

Full Text Available Objective: In the last decade, substantial evidence has accumulated about the use of cryopreserved platelet concentrates, especially in trauma. However, little reference has been made in these studies to the morphological and functional changes of platelets. Recently platelets have been shown to be activated by cryopreservation processes and to undergo procoagulant membrane changes resulting in the generation of platelet-derived microparticles (PMPs, platelet degranulation, and release of platelet-derived growth factors (PDGFs. We assessed the viabilities and the PMP and PDGF levels of cryopreserved platelets, and their relation with thrombin generation. Materials and Methods: Apheresis platelet concentrates (APCs from 20 donors were stored for 1 day and cryopreserved with 6% dimethyl sulfoxide. Cryopreserved APCs were kept at -80 °C for 1 day. Thawed APCs (100 mL were diluted with 20 mL of autologous plasma and specimens were analyzed for viabilities and PMPs by flow cytometry, for thrombin generation by calibrated automated thrombogram, and for PDGFs by enzyme-linked immunosorbent assay testing. Results: The mean PMP and PDGF levels in freeze-thawed APCs were significantly higher (2763±399.4/μL vs. 319.9±80.5/μL, p<0.001 and 550.9±73.6 pg/mL vs. 96.5±49 pg/mL, p<0.001, respectively, but the viability rates were significantly lower (68.2±13.7% vs. 94±7.5%, p<0.001 than those of fresh APCs. The mean endogenous thrombin potential (ETP of freeze-thawed APCs was significantly higher than that of the fresh APCs (3406.1±430.4 nM.min vs. 2757.6±485.7 nM.min, p<0.001. Moreover, there was a significant positive poor correlation between ETP levels and PMP levels (r=0.192, p=0.014. Conclusion: Our results showed that, after cryopreservation, while levels of PMPs were increasing, significantly higher and earlier thrombin formation was occurring in the samples analyzed despite the significant decrease in viability. Considering the damage caused

Structure activity relationships of quinoxalin-2-one derivatives as platelet-derived growth factor-beta receptor (PDGFbeta R) inhibitors, derived from molecular modeling.

Science.gov (United States)

Mori, Yoshikazu; Hirokawa, Takatsugu; Aoki, Katsuyuki; Satomi, Hisanori; Takeda, Shuichi; Aburada, Masaki; Miyamoto, Ken-ichi

2008-05-01

We previously reported a quinoxalin-2-one compound (Compound 1) that had inhibitory activity equivalent to existing platelet-derived growth factor-beta receptor (PDGFbeta R) inhibitors. Lead optimization of Compound 1 to increase its activity and selectivity, using structural information regarding PDGFbeta R-ligand interactions, is urgently needed. Here we present models of the PDGFbeta R kinase domain complexed with quinoxalin-2-one derivatives. The models were constructed using comparative modeling, molecular dynamics (MD) and ligand docking. In particular, conformations derived from MD, and ligand binding site information presented by alpha-spheres in the pre-docking processing, allowed us to identify optimal protein structures for docking of target ligands. By carrying out molecular modeling and MD of PDGFbeta R in its inactive state, we obtained two structural models having good Compound 1 binding potentials. In order to distinguish the optimal candidate, we evaluated the structural activity relationships (SAR) between the ligand-binding free energies and inhibitory activity values (IC50 values) for available quinoxalin-2-one derivatives. Consequently, a final model with a high SAR was identified. This model included a molecular interaction between the hydrophobic pocket behind the ATP binding site and the substitution region of the quinoxalin-2-one derivatives. These findings should prove useful in lead optimization of quinoxalin-2-one derivatives as PDGFb R inhibitors.

Platelet Rich Plasma- mechanism of action and clinical applications

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Cristina N. Cozma

2016-11-01

Full Text Available Platelet-rich plasma (PRP is a blood-derived fraction containing high level of platelets, a high concentration of leukocytes and growth factors. PRP therapy has been growing as a viable treatment alternative for a number of clinical applications and has a potential benefit for use in wound healing. Nowadays platelet rich plasma is used in stimulating wound healing in skin and soft tissue ulcerations, accelerating wound healing in diabetic patients and facilitating bone proliferation in orthopedic and trauma surgery. It has also applications in maxillofacial surgery, spinal surgery, plastic and esthetic surgery, heart surgery and burns. This review of the literature shows a limited number of studies realized on humans that sustain PRP applications in orthopedic and plastic surgery. As the use of PRP increases, more properly structured clinical studies are necessary to confirm the results and to establish clearly the techniques of preparing, the conditions and the clinical indications of applying this therapy.

Matrix metalloproteinase-2 of human carotid atherosclerotic plaques promotes platelet activation. Correlation with ischaemic events.

Science.gov (United States)

Lenti, Massimo; Falcinelli, Emanuela; Pompili, Marcella; de Rango, Paola; Conti, Valentina; Guglielmini, Giuseppe; Momi, Stefania; Corazzi, Teresa; Giordano, Giuseppe; Gresele, Paolo

2014-06-01

Purified active matrix metalloproteinase-2 (MMP-2) is able to promote platelet aggregation. We aimed to assess the role of MMP-2 expressed in atherosclerotic plaques in the platelet-activating potential of human carotid plaques and its correlation with ischaemic events. Carotid plaques from 81 patients undergoing endarterectomy were tested for pro-MMP-2 and TIMP-2 content by zymography and ELISA. Plaque extracts were incubated with gel-filtered platelets from healthy volunteers for 2 minutes before the addition of a subthreshold concentration of thrombin receptor activating peptide-6 (TRAP-6) and aggregation was assessed. Moreover, platelet deposition on plaque extracts immobilised on plastic coverslips under high shear-rate flow conditions was measured. Forty-three plaque extracts (53%) potentiated platelet aggregation (+233 ± 26.8%), an effect prevented by three different specific MMP-2 inhibitors (inhibitor II, TIMP-2, moAb anti-MMP-2). The pro-MMP-2/TIMP-2 ratio of plaques potentiating platelet aggregation was significantly higher than that of plaques not potentiating it (3.67 ± 1.21 vs 1.01 ± 0.43, p<0.05). Moreover, the platelet aggregation-potentiating effect, the active-MMP-2 content and the active MMP-2/pro-MMP-2 ratio of plaque extracts were significantly higher in plaques from patients who developed a subsequent major cardiovascular event. In conclusion, atherosclerotic plaques exert a prothrombotic effect by potentiating platelet activation due to their content of MMP-2; an elevated MMP-2 activity in plaques is associated with a higher rate of subsequent ischaemic cerebrovascular events.

Hippophae rhamnoides L. Fruits Reduce the Oxidative Stress in Human Blood Platelets and Plasma

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Beata Olas

2016-01-01

Full Text Available Effects of the phenolic fraction from Hippophae rhamnoides fruits on the production of thiobarbituric acid reactive substances (TBARS, a marker of lipid peroxidation and the generation of superoxide anion (O2-∙ in human blood platelets (resting platelets and platelets stimulated by a strong physiological agonist, thrombin were studied in vitro. We also examined antioxidant properties of this fraction against human plasma lipid peroxidation and protein carbonylation induced by a strong biological oxidant, hydrogen peroxide (H2O2 or H2O2/Fe (a donor of hydroxyl radicals. The tested fraction of H. rhamnoides (0.5– 50 µg/mL; the incubation time: 15 and 60 min inhibited lipid peroxidation induced by H2O2 or H2O2/Fe. The H. rhamnoides phenolic fraction inhibited not only plasma lipid peroxidation, but also plasma protein carbonylation stimulated by H2O2 or H2O2/Fe. Moreover, the level of O2-∙ in platelets significantly decreased. In comparative experiments, the H. rhamnoides fraction was a more effective antioxidant than aronia extract or grape seed extract (at the highest tested concentration, 50 µg/mL. The obtained results suggest that H. rhamnoides fruits may be a new, promising source of natural compounds with antioxidant and antiplatelet activity beneficial not only for healthy people, but also for those with oxidative stress-associated diseases.

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

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. 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. 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. PRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the presence of platelet-derived

Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma.

Directory of Open Access Journals (Sweden)

Andrew L Frelinger

Full Text Available 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.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.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.PRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the presence of platelet-derived

Beta 3 and PDI proteins isolated from human platelets bind with ECwt rotavirus in vitro

International Nuclear Information System (INIS)

Mayorga, Diana; Rubio, Linda; Guerrero-Fonseca, Carlos A; Acosta-Losada, Orlando

2010-01-01

Commercial integrin Beta 3 is currently not available and commercial PDI is too expensive, which is making access difficult to these proteins needed for conducting experiments aimed at the establishment of possible interactions between integrin Beta 3 and PDI and wild type rotavirus strains. Objective. To explore a methodology allowing isolation of proteins Beta 3 and PDI from human platelets to be used as antigens in the generation of rabbit polyclonal antibodies useful in the assessment of interactions between these proteins and rotavirus ECwt. Materials and methods. Proteins Beta 3 and PDI from human platelet lysates were separated using preparative electrophoresis under reducing conditions and then eluted. Interactions of these proteins with rotavirus ECwt were analyzed using co-immunoprecipitation, Western blotting and capture ELISA. Results. Proteins from human platelet lysates were separated by preparative electrophoresis under reducing conditions. The identification of proteins Beta 3 and PDI present in a gel slice was performed through their reaction with commercial antibodies in a Western blotting analysis. Protein purity was established after electro elution from a gel slice. Polyclonal antibodies against protein Beta 3 were generated in rabbit. Incubation of eluted proteins Beta 3 and PDI with rotavirus ECwt showed in co-immunoprecipitation and ELISA assays that these proteins bound virus in vitro. The same binding was showed to occur when rotavirus was incubated with isolated small intestinal villi from suckling mice. Conclusions. Relatively high amounts of proteins Beta 3 and PDI were partially purified from human platelets by preparative electrophoresis. The isolation of these proteins allowed the generation of polyclonal antibodies against Beta 3 in addition to the establishment of the in vitro interaction of proteins Beta 3 and PDI with rotavirus ECwt. This interaction was also demonstrated in vivo after incubating the virus with isolated small

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

International Nuclear Information System (INIS)

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

1984-01-01

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 [ 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

Pluripotent stem cells reveal the developmental biology of human megakaryocytes and provide a source of platelets for clinical application.

Science.gov (United States)

Takayama, Naoya; Eto, Koji

2012-10-01

Human pluripotent stem cells [PSCs; including human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)] can infinitely proliferate in vitro and are easily accessible for gene manipulation. Megakaryocytes (MKs) and platelets can be created from human ESCs and iPSCs in vitro and represent a potential source of blood cells for transfusion and a promising tool for studying the human thrombopoiesis. Moreover, disease-specific iPSCs are a powerful tool for elucidating the pathogenesis of hematological diseases and for drug screening. In that context, we and other groups have developed in vitro MK and platelet differentiation systems from human pluripotent stem cells (PSCs). Combining this co-culture system with a drug-inducible gene expression system enabled us to clarify the novel role played by c-MYC during human thrombopoiesis. In the next decade, technical advances (e.g., high-throughput genomic sequencing) will likely enable the identification of numerous gene mutations associated with abnormal thrombopoiesis. Combined with such technology, an in vitro system for differentiating human PSCs into MKs and platelets could provide a novel platform for studying human gene function associated with thrombopoiesis.

Platelet kinetics with indium-111 platelets: comparison with chromium-51 platelets

International Nuclear Information System (INIS)

Peters, A.M.; Lavender, J.P.

1983-01-01

The application of 111In-oxine to platelet labeling has contributed to the understanding of platelet kinetics along three lines: 1. It allows the measurement of new parameters of splenic function, such as the intrasplenic platelet transit time, which has shed new light on the physiology of splenic blood cell handling. 2. It facilitates the measurement of platelet life span in conditions, such as ITP, in which 51Cr may undergo undesirable elution from the platelet as a result of platelet-antibody interaction. 3. It allows the determination of the fate of platelets, that is, the site of platelet destruction in conditions in which reduced platelet life span is associated with abnormal platelet consumption, as a result of either premature destruction of ''abnormal'' platelets by the RE system, or the consumption (or destruction) of normal platelets after their interaction with an abnormal vasculature. Future research using 111In platelets may yield further valuable information on the control as well as the significance of intrasplenic platelet pooling, on the role of platelets in the development of chronic vascular lesions, and on the sites of platelet destruction in ITP. With regard to the latter, methods will have to be developed for harvesting sufficient platelets representative of the total circulating platelet population from severely thrombocytopenic patients for autologous platelet labeling. This would avoid the use of homologous platelets, which is likely to be responsible for some of the contradictory data relating to the use of radiolabeled platelet studies for the prediction of the response of patients with ITP to splenectomy

Electrospun silk fibroin fibers for storage and controlled release of human platelet lysate.

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Pignatelli, Cataldo; Perotto, Giovanni; Nardini, Marta; Cancedda, Ranieri; Mastrogiacomo, Maddalena; Athanassiou, Athanassia

2018-04-17

Human platelet lysate (hPL) is a pool of growth factors and cytokines able to induce regeneration of different tissues. Despite its good potentiality as therapeutic tool for regenerative medicine applications, hPL has been only moderately exploited in this field. A more widespread adoption has been limited because of its rapid degradation at room temperature that decreases its functionality. Another limiting factor for its extensive use is the difficulty of handling the hPL gels. In this work, silk fibroin-based patches were developed to address several points: improving the handling of hPL, enabling their delivery in a controlled manner and facilitating their storage by creating a device ready to use with expanded shelf life. Patches of fibroin loaded with hPL were synthesized by electrospinning to take advantage of the fibrous morphology. The release kinetics of the material was characterized and tuned through the control of fibroin crystallinity. Cell viability assays, performed with primary human dermal fibroblasts, demonstrated that fibroin is able to preserve the hPL biological activity and prolong its shelf-life. The strategy of storing and preserving small active molecules within a naturally-derived, protein-based fibrous scaffold was successfully implemented, leading to the design of a biocompatible device, which can potentially simplify the storage and the application of the hPL on a human patient, undergoing medical procedures such as surgery and wound care. Human platelets lysate (hPL) is a mixture of growth factors and cytokines able to induce the regeneration of damaged tissues. This study aims at enclosing hPL in a silk fibroin electrospun matrix to expand its utilization. Silk fibroin showed the ability to preserve the hPL activity at temperature up to 60 °C and the manipulation of fibroin's crystallinity provided a tool to modulate the hPL release kinetic. This entails the possibility to fabricate the hPL silk fibroin patches in advance and

Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures.

Science.gov (United States)

Mojica-Henshaw, Mariluz P; Jacobson, Pam; Morris, Julie; Kelley, Linda; Pierce, Jan; Boyer, Michael; Reems, Jo-Anna

2013-12-01

Fetal bovine serum (FBS) is commonly used as a serum supplement for culturing human mesenchymal stromal cells (hMSCs). However, human cells grown in FBS, especially for extended periods, risk potential exposure to bovine immunogenic proteins and infectious agents. To address this issue, we investigated the ability of a novel human platelet serum supplement to substitute for FBS in hMSC cultures. Platelet lysate-serum (PL-serum) was converted from platelet lysate-plasma (PL-plasma) that was manufactured from pooled platelet-rich plasma (PRP) apheresis units. Growth factor levels and the number of residual intact platelets in PL-serum and PL-plasma were compared with enzyme-linked immunosorbent assays and flow cytometry, respectively. Proliferation responses of hMSCs cultured in PL-serum, PL-plasma, or FBS were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the immunophenotype of harvested hMSCs was evaluated by flow cytometry and tri-lineage differentiation potential was evaluated by assessing adipogenic, osteogenic and chondrogenic development. Selected growth factor levels in PL-serum were not significantly different from PL-plasma (P > 0.05). hMSC cultures supplemented with PL-serum had comparable growth kinetics to PL-plasma, and hMSC yields were consistently greater than with FBS. hMSCs harvested from cultures supplemented with PL-serum, PL-plasma or FBS had similar cell surface phenotypes and maintained tri-lineage differentiation potential. PL-serum, similar to PL-plasma, can substitute for FBS in hMSC cultures. Use of PL-serum, in contrast to PL-plasma, has an added advantage of not requiring addition of a xenogeneic source of heparin, providing a completely xeno-free culture medium. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Human cord blood-derived platelet lysate enhances the therapeutic activity of adipose-derived mesenchymal stromal cells isolated from Crohn's disease patients in a mouse model of colitis.

Science.gov (United States)

Forte, Dorian; Ciciarello, Marilena; Valerii, Maria Chiara; De Fazio, Luigia; Cavazza, Elena; Giordano, Rosaria; Parazzi, Valentina; Lazzari, Lorenza; Laureti, Silvio; Rizzello, Fernando; Cavo, Michele; Curti, Antonio; Lemoli, Roberto M; Spisni, Enzo; Catani, Lucia

2015-09-09

Due to their immunomodulatory properties, mesenchymal stromal cells (MSCs) have been used for auto-immune disease treatment. Crohn disease (CD) and ulcerative colitis are two major inflammatory bowel diseases (IBDs), resulting from pathological immune responses to environmental or microbial antigens. Preclinical and clinical studies have suggested that MSC-based cellular therapy hold promising potential for IBD treatment. However, open issues include the selection of the proper cell dose, the source and the optimal route of administration of MSCs for more effective results. Platelet lysate has gained clinical interest due to its efficacy in accelerating wound healing. Thus, we propose to combine the administration of MSCs with a human umbilical cord blood-derived platelet lysate (hCBPL) as a novel strategy to improve MSC-based therapy for IBD resolution. Colitis was induced in 8-week-old C57BL/6J mice by daily oral administration of dextran sulphate sodium (DSS) (1.5 % w/v in tap water) for 9 days. MSCs were isolated from adipose tissue of CD patients (adCD-MSCs), expanded in proliferation medium, resuspended in hCBPL or PBS and administrated via enema for three times (1 × 10(6) cells/mouse/time) every other day starting on day +7 from DSS induction. The colitis evolution was evaluated by daily monitoring of body weight, stool consistency and bleeding. Histopathological analysis was performed. Inflammatory cytokine plasma levels were determined. adCD-MSCs stained with lipophilic membrane dye Nile Red, were injected in DSS mice as described above. Colon section of mice sacrificed 24 hours after last cell administration, were analyzed by confocal microscopy. We found that adCD-MSCs could be easily isolated and expanded from CD patients. Upon injection, adCD-MSCs exerted a therapeutic effect on DSS-induced colitis. Moreover, hCBPL increased adCD-MSCs efficacy by significantly reducing colitis scores, extension of the colon inflamed area and plasma levels of

Platelet-Rich Plasma Derived Growth Factors Contribute to Stem Cell Differentiation in Musculoskeletal Regeneration

Directory of Open Access Journals (Sweden)

Yun Qian

2017-10-01

Full Text Available Stem cell treatment and platelet-rich plasma (PRP therapy are two significant issues in regenerative medicine. Stem cells such as bone marrow mesenchymal stem cells, adipose-derived stem cells and periodontal ligament stem cells can be successfully applied in the field of tissue regeneration. PRP, a natural product isolated from whole blood, can secrete multiple growth factors (GFs for regulating physiological activities. These GFs can stimulate proliferation and differentiation of different stem cells in injury models. Therefore, combination of both agents receives wide expectations in regenerative medicine, especially in bone, cartilage and tendon repair. In this review, we thoroughly discussed the interaction and underlying mechanisms of PRP derived GFs with stem cells, and assessed their functions in cell differentiation for musculoskeletal regeneration.

Platelet-rich plasma differs according to preparation method and human variability.

Science.gov (United States)

Mazzocca, Augustus D; McCarthy, Mary Beth R; Chowaniec, David M; Cote, Mark P; Romeo, Anthony A; Bradley, James P; Arciero, Robert A; Beitzel, Knut

2012-02-15

Varying concentrations of blood components in platelet-rich plasma preparations may contribute to the variable results seen in recently published clinical studies. The purposes of this investigation were (1) to quantify the level of platelets, growth factors, red blood cells, and white blood cells in so-called one-step (clinically used commercial devices) and two-step separation systems and (2) to determine the influence of three separate blood draws on the resulting components of platelet-rich plasma. Three different platelet-rich plasma (PRP) separation methods (on blood samples from eight subjects with a mean age [and standard deviation] of 31.6 ± 10.9 years) were used: two single-spin processes (PRPLP and PRPHP) and a double-spin process (PRPDS) were evaluated for concentrations of platelets, red and white blood cells, and growth factors. Additionally, the effect of three repetitive blood draws on platelet-rich plasma components was evaluated. The content and concentrations of platelets, white blood cells, and growth factors for each method of separation differed significantly. All separation techniques resulted in a significant increase in platelet concentration compared with native blood. Platelet and white blood-cell concentrations of the PRPHP procedure were significantly higher than platelet and white blood-cell concentrations produced by the so-called single-step PRPLP and the so-called two-step PRPDS procedures, although significant differences between PRPLP and PRPDS were not observed. Comparing the results of the three blood draws with regard to the reliability of platelet number and cell counts, wide variations of intra-individual numbers were observed. Single-step procedures are capable of producing sufficient amounts of platelets for clinical usage. Within the evaluated procedures, platelet numbers and numbers of white blood cells differ significantly. The intra-individual results of platelet-rich plasma separations showed wide variations in

Platelet lysate obtained via plateletpheresis performed in standing and awake equine donors.

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Sumner, Scarlett M; Naskou, Maria C; Thoresen, Merrilee; Copland, Ian; Peroni, John F

2017-07-01

Platelet preparations containing growth factors, attachment factors, and enzymes are appealing to enhance healing of injured tissues and as an alternative to xenogenic serum in cell culture media. Plateletpheresis is commonly used to collect platelets in human medicine but has not been validated in horses. Plateletpheresis to collect platelet concentrate was performed on six female, mixed breed, chemically restrained horses using commercially available apheresis equipment. Before and immediately after plateletpheresis, we performed physical examinations and collected blood for chemistry and coagulation panels and then again at 8, 16, 24, and 48 hours after the procedure. To produce platelet lysate, the platelet concentrate underwent two freeze-thaw cycles followed by centrifugation and filtration processing. The platelet lysate was then analyzed for cellular debris, fibrinogen, and growth factors. The collected platelet concentration contained a mean platelet yield of 390 × 10 3 /μL. Donor platelet count decreased from a mean of 193 × 10 3 /μL to 138 × 10 3 /μL after plateletpheresis, but no individual was at risk for hemorrhage. Pooled platelet lysate had minimal cellular residue and contained growth factor concentrations at 6.1 ng/mL for transforming growth factor-β1, at 3.5 ng/mL for platelet-derived growth factor-BB, and at 13.8 ng/mL for vascular endothelial growth factor-A. Plateletpheresis using commercially available apheresis equipment is a feasible option for collecting platelet concentrate from equine donors. The lysate generated from the apheresis product contains growth factors and has potential to be used as a fetal bovine serum substitute for cell culture. © 2017 AABB.

Human platelet antigens in Burmese, Karen and north-eastern Thais.

Science.gov (United States)

Phuangtham, R; Romphruk, A; Puapairoj, C; Leelayuwat, C; Romphruk, A V

2017-02-01

A comparative study of allele frequencies at HPA-1 to -6 and HPA-15 in Burmese and Karen populations as well as at HPA-15 in north-eastern Thais (NET) is presented. Human platelet antigens (HPAs) are clinically important in several immune platelet disorders, including foetal and neonatal alloimmune thrombocytopenia (FNAIT), post-transfusion purpura (PTP) and platelet transfusion refractoriness (PTR). The knowledge of antigen frequencies in a population is essential for the evaluation of patients suffering from immune-mediated platelet disorders. A total of 285 unrelated, healthy Burmese, 242 Karen and 300 NET were recruited to this study. Genotype and allele frequencies of HPA-1 to -6 and HPA-15 were defined using polymerase chain reaction sequence-specific primers (PCR-SSP) RESULTS: No individuals homozygous for HPA-1bb, -2bb, -4bb, -5bb and -6bb were detected. HPA-1a, -2a, -4a, -5a and -6a were present in all samples of Burmese and Karen origin. HPA-1b, -2b, -4b, -5b and -6b were rare in these populations. The frequencies of HPA-3a/-3b were 60·4/39·6% in Burmese and 55·8/44·2% in Karen, respectively. Frequencies of HPA-15a/-15b were 57·2/42·8% in Burmese, 52·5/47·5% in Karen and 49·8/50·2% in NET. The frequencies of HPA genotypes in our study indicates that HPA-1a, -2a, -4a, -5a and -6a are unlikely involved in FNAIT, PTP and PTR in Burmese and Karen populations. However, HPA-1b, -2b, -3a, -3b, -4b, -5b, -6b, -15a and -15b may likely stimulate alloantibodies in these populations. © 2016 British Blood Transfusion Society.

Association of coatomer proteins with the beta-receptor for platelet-derived growth factor

DEFF Research Database (Denmark)

Hansen, Klaus; Rönnstrand, L; Rorsman, C

1997-01-01

The nonreceptor tyrosine kinase Src binds to and is activated by the beta-receptor for platelet-derived growth factor (PDGF). The interaction leads to Src phosphorylation of Tyr934 in the kinase domain of the receptor. In the course of the functional characterization of this phosphorylation, we...... of intracellular vesicle transport. In order to explore the functional significance of the interaction between alpha- and beta'-COP and the PDGF receptor, a receptor mutant was made in which the conserved histidine residue 928 was mutated to an alanine residue. The mutant receptor, which was unable to bind alpha...

Thrombocytopenia impairs host defense in gram-negative pneumonia-derived sepsis in mice

NARCIS (Netherlands)

de Stoppelaar, Sacha F.; van 't Veer, Cornelis; Claushuis, Theodora A. M.; Albersen, Bregje J. A.; Roelofs, Joris J. T. H.; van der Poll, Tom

2014-01-01

Thrombocytopenia is a common finding in sepsis and associated with a worse outcome. We used a mouse model of pneumonia-derived sepsis caused by the human pathogen Klebsiella pneumoniae to study the role of platelets in host response to sepsis. Platelet counts (PCs) were reduced to less than a median

High-resolution molecular validation of self-renewal and spontaneous differentiation in adipose-tissue derived human mesenchymal stem cells cultured in human platelet lysate

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Dudakovic, Amel Dudakovic; Camilleri, Emily; Riester, Scott M.; Lewallen, Eric A.; Kvasha, Sergiy; Chen, Xiaoyue; Radel, Darcie J.; Anderson, Jarett M.; Nair, Asha A.; Evans, Jared M.; Krych, Aaron J.; Smith, Jay; Deyle, David R.; Stein, Janet L.; Stein, Gary S.; Im, Hee-Jeong; Cool, Simon M.; Westendorf, Jennifer J.; Kakar, Sanjeev; Dietz, Allan B.; van Wijnen, Andre J.

2014-01-01

Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1 and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10 fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while up-regulating WNT-related genes (WISP2, SFRP2 and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility. PMID:24905804

The pro-inflammatory effects of platelet contamination in plasma and mitigation strategies for avoidance

Science.gov (United States)

Bercovitz, R. S.; Kelher, M. R.; Khan, S. Y.; Land, K. J.; Berry, T. H.; Silliman, C. C.

2013-01-01

Background and Objectives Plasma and platelet concentrates are disproportionately implicated in transfusion-related acute lung injury (TRALI). Platelet-derived pro-inflammatory mediators, including soluble CD40 ligand (sCD40L), accumulate during storage. We hypothesized that platelet contamination induces sCD40L generation that causes neutrophil [polymorphonuclear leucocyte (PMN)] priming and PMN-mediated cytotoxicity. Materials and Methods Plasma was untreated, centrifuged (12 500 g) or separated from leucoreduced whole blood (WBLR) prior to freezing. Platelet counts and sCD40L concentrations were measured 1–5 days post-thaw. The plasma was assayed for PMN priming activity and was used in a two-event in vitro model of PMN-mediated human pulmonary microvascular endothelial cell (HMVEC) cytotoxicity. Results Untreated plasma contained 42 ± 4.2 × 103/μl platelets, which generated sCD40L accumulation (1.6-eight-fold vs. controls). Priming activity and HMVEC cytotoxicity were directly proportional to sCD40L concentration. WBLR and centrifugation reduced platelet and sCD40L contamination, abrogating the pro-inflammatory potential. Conclusion Platelet contamination causes sCD40L accumulation in stored plasma that may contribute to TRALI. Platelet reduction is potentially the first TRALI mitigation effort in plasma manufacturing. PMID:22092073

Indium-111 platelet imaging for detection of platelet deposition in abdominal aneurysms and prosthetic arterial grafts

International Nuclear Information System (INIS)

Ritchie, J.L.; Stratton, J.R.; Thiele, B.; Haminton, G.W.; Warrick, L.N.; Huang, T.W.; Harker, L.A.

1981-01-01

Thirty-four platelet imaging studies were performed in 23 patients to determine whether platelet deposition could be detected in patients with vascular aneurysms (18 patients) or in patients in whom Dacron prosthetic grafts had been placed (5 patients). In patients in whom abnormal platelet deposition was detected, the effect of administration of platelet-active drugs on platelet deposition was examined. Of the 18 patients with an aneurysm, 12 had equivocally positive studies on initial imaging and 2 had equivocally positive images. Of five patients with Dacron arterial grafts in place, four had diffuse platelet deposition in the grafts; the fifth patient had a platelet deposition only in a pseudoaneurysm. Eight patients with an abdominal aneurysm and positive or equivocally positive baseline images were restudied during platelet-active drug therapy either with aspirin plus dipyridamole (seven patients) or with sulfinpyrazone (four patients). No patient studied during treatment with aspirin plus dipyridamole had detectably decreased platelet deposition compared with baseline determinations. In contrast, two of four patients studied while receiving sulfinpyrazone showed decreased platelet deposition. Thus, platelet imaging may be of value for studying platelet physiology in vivo and for assessing platelet-active drugs and the thrombogenicity of prosthetic graft materials in human beings

Namibia's transition from whole blood-derived pooled platelets to single-donor apheresis platelet collections

NARCIS (Netherlands)

Pitman, John P.; Basavaraju, Sridhar V.; Shiraishi, Ray W.; Wilkinson, Robert; von Finckenstein, Bjorn; Lowrance, David W.; Marfin, Anthony A.; Postma, Maarten; Mataranyika, Mary; Smit Sibinga, Cees Th.

BACKGROUNDFew African countries separate blood donations into components; however, demand for platelets (PLTs) is increasing as regional capacity to treat causes of thrombocytopenia, including chemotherapy, increases. Namibia introduced single-donor apheresis PLT collections in 2007 to increase PLT

[Single-donor (apheresis) platelets and pooled whole-blood-derived platelets--significance and assessment of both blood products].

Science.gov (United States)

Hitzler, Walter E

2014-01-01

The transfusion efficacy of ATK, which contain fully functional platelets, is beyond all doubt. The equivalence of ATK and PTK has been subject of many studies. Some of those studies show the superiority of ATK's, while others do not, but there have been no studies that demonstrated a superiority of PTK's. The superiority of platelets stored in plasma and in third generation additive solution was demonstrated in clinical studies; therefore, it cannot be said that all the platelet concentrates on the German market are equivalent in efficacy. Of decisive importance, above all, is the risk of transfusion-transmitted infections with known pathogens, or those not yet discovered. This risk is different for ATK compared to PTK. Taking this difference in risk and the difference in donor exposure of transfused patients into account, it can definitely be said that ATK and PTK are not equivalent. In 2012, the Robert-Koch-Institute (RKI) published a mathematical risk model for different platelet concentrates and assessed the risk of transmitting known pathogens such as HIV, HCV, and HBV. The risk was higher for PTK compared to ATK. The relative risks for PTK derived from 4BCs were 2.2 (95%--CI: 2.1-2.4) for HIV, 2.7 (95%--CI: 2.5-3.0) for HCV, and 2.2 (95%--CI: 2.8-3.7) for HBV. At the present time, these are the relative risks of transfusion-transmitted infections with the traditional pathogens for PTK compared to ATK. In addition to the RKI assessed risks, there is the theoretical risk of a new, unknown agent, transmitted through blood exposure. The magnitude of this risk is hardly predictable for PTK. The experience gathered so far, especially in the last three decades, with the emergence of HIV, prions, and West Nil virus, shows that the biological nature of a next transfusion-transmissible infectious agent cannot be predictable. This agent, if we think at a conventional sexually transmissible agent with nucleic acid and long latent period, would spread first in areas with

Human Platelet Lysate versus Fetal Calf Serum: These Supplements Do Not Select for Different Mesenchymal Stromal Cells.

Science.gov (United States)

Fernandez-Rebollo, Eduardo; Mentrup, Birgit; Ebert, Regina; Franzen, Julia; Abagnale, Giulio; Sieben, Torsten; Ostrowska, Alina; Hoffmann, Per; Roux, Pierre-François; Rath, Björn; Goodhardt, Michele; Lemaitre, Jean-Marc; Bischof, Oliver; Jakob, Franz; Wagner, Wolfgang

2017-07-11

Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements - it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types.

Imaging platelet deposition on Dacron bifurcation grafts in man: Quantification by a dual-tracer method using 111In-labeled platelets and sup(99m)Tc-labeled human serum albumin

International Nuclear Information System (INIS)

Isaka, Yoshinari; Kimura, Kazufumi; Etani, Hideki; Uehara, Akira; Yoneda, Shotaro; Kamada, Takenobu; Kusunoki, Masahito; Kim, Teak Dong; Ohshiro, Takeshi

1986-01-01

A dual tracer technique using 111 In-labeled platelets and sup(99m)Tc-labeled human serum albumin was applied to evaluate the thrombogenicity of Dacron, bifurcation arterial grafts. The level of platelet accumulation over the whole of the graft was estimated from the ratio of 111 In-platelet radioactivity deposited on the vascular wall to these radioactivity circulating in the blood pool, i.e., the platelet-accumulation index (PAI). Furthermore, the PAI value was calculated for each pixel in digitized images and the PAI distribution image (PAI image) was reconstructed. Eighteen patients with DeBakey knitted Dacron bifurcation grafts and 11 normal volunteers were studied. Of the 18 patients, 11 had no graft occlusion (group I) and the remaining 7 (group II) had occlusion. The mean PAI value (+-SD) over the whole of the graft in group I was 32.6%+-33.7% as compared to -8.8%+-4.5% in the control group (P 2 =10.55; P<0.005). The method used for platelet imaging in the present study may be useful in the study of platelet reactions on Dacron arterial prostheses. (orig.)

Thermo-mechanical stress analysis in platelet reinforced composites with bonded and debonded platelet end

International Nuclear Information System (INIS)

Fattahi, A. M.; Moaddab, E.; Bibishahrbanoei, N.

2015-01-01

An analytical model has been performed to analyze the stress transfer in platelet reinforced composite subjected to both tensile loading and residual thermal stresses. Two sets of the matrix/platelet displacement solutions, which were called respectively as the far-field solution and the transient solution, were exactly derived based on the theory of elasticity. These two sets of the displacement solutions were then superposed to obtain simplified analytical expressions for the matrix/platelet stress field components. The main difference with the previous works here were that the thermal residual stresses were considered in this article. The analytical results obtained here are then validated by the FEM modeling. Interestingly, good agreements are found between the analytical and numerical predictions. Another superiority of the proposed analytical model was in its capability of solving problems with platelet/matrix debonding defect.

Interaction between the Staphylococcus aureus extracellular adherence protein Eap and its subdomains with platelets.

Science.gov (United States)

Palankar, Raghavendra; Binsker, Ulrike; Haracska, Bianca; Wesche, Jan; Greinacher, Andreas; Hammerschmidt, Sven

2018-04-18

S. aureus associated bacteremia can lead to severe infections with high risk of mortality (e.g. sepsis, infective endocarditis). Many virulence factors and adhesins of S. aureus are known to directly interact with platelets. Extracellular adherence protein, Eap, one of the most important virulence factors in S. aureus mediated infections is a multi-tandem domain protein and has been shown to interact with almost all cell types in the human circulatory system. By using amine reactive fluorescent N-hydroxysuccinimidyl (NHS)-ester dyes and by direct detection with primary fluorescently conjugated anti-histidine (His-tag) antibodies against detect N-terminal His6, we show Eap subdomain Eap D 3 D 4 specifically interacts and rapidly activates human platelets. Furthermore, we validate our finding by using site directed directional immobilization of Eap D 3 D 4 through N-terminal His 6 on nickel (II)-nitrilotriacetic acid (Ni-NTA) functionalized bacteriomimetic microbead arrays to visualize real-time platelet activation through calcium release assay. These methods offer an easily adoptable protocols for screening of S.aureus derived virulence factors and adhesins with platelets. Copyright © 2018 Elsevier GmbH. All rights reserved.

Deciphering of ADP-induced, phosphotyrosine-dependent signaling networks in human platelets by Src-homology 2 region (SH2)-profiling.

Science.gov (United States)

Schweigel, Hardy; Geiger, Jörg; Beck, Florian; Buhs, Sophia; Gerull, Helwe; Walter, Ulrich; Sickmann, Albert; Nollau, Peter

2013-03-01

Tyrosine phosphorylation plays a central role in signal transduction controlling many important biological processes. In platelets, the activity of several signaling proteins is controlled by tyrosine phosphorylation ensuring proper platelet activation and aggregation essential for regulation of the delicate balance between bleeding and hemostasis. Here, we applied Src-homology 2 region (SH2)-profiling for deciphering of the phosphotyrosine state of human platelets activated by adenosine diphosphate (ADP). Applying a panel of 31 SH2-domains, rapid and complex regulation of the phosphotyrosine state of platelets was observed after ADP stimulation. Specific inhibition of platelet P2Y receptors by synthetic drugs revealed a major role for the P2Y1 receptor in tyrosine phosphorylation. Concomitant activation of protein kinase A (PKA) abolished ADP-induced tyrosine phosphorylation in a time and concentration-dependent manner. Given the fact that PKA activity is negatively regulated by the P2Y12 receptor, our data provide evidence for a novel link of synergistic control of the state of tyrosine phosphorylation by both P2Y receptors. By SH2 domain pull down and MS/MS analysis, we identified distinct tyrosine phosphorylation sites in cell adhesion molecules, intracellular adapter proteins and phosphatases suggesting a major, functional role of tyrosine phosphorylation of theses candidate proteins in ADP-dependent signaling in human platelets. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Are genetic variants in the platelet-derived growth factor [beta] gene associated with chronic pancreatitis?

Science.gov (United States)

Muddana, Venkata; Park, James; Lamb, Janette; Yadav, Dhiraj; Papachristou, Georgios I; Hawes, Robert H; Brand, Randall; Slivka, Adam; Whitcomb, David C

2010-11-01

Platelet-derived growth factor [beta] (PDGF-[beta]) is a major signal in proliferation and matrix synthesis through activated pancreatic stellate cells, leading to fibrosis of the pancreas. Recurrent acute pancreatitis (RAP) seems to predispose to chronic pancreatitis (CP) in some patients but not others. We tested the hypothesis that 2 known PDGF-[beta] polymorphisms are associated with progression from RAP to CP. We also tested the hypothesis that PDGF-[beta] polymorphisms in combination with environmental risk factors such as alcohol and smoking are associated with CP. Three hundred eighty-two patients with CP (n = 176) and RAP (n = 206) and 251 controls were evaluated. Platelet-derived growth factor [beta] polymorphisms +286 A/G (rs#1800818) seen in 5'-UTR and +1135 A/C (rs#1800817) in first intron were genotyped using single-nucleotide polymorphism polymerase chain reaction approach and confirmed by DNA sequencing. The genotypic frequencies for PDGF-[beta] polymorphisms in positions +286 and +1135 were found to be similar in controls and patients with RAP and CP. There was no difference in genotypic frequencies among RAP, CP, and controls in subjects in the alcohol and smoking subgroups. Known variations in the PDGF-[beta] gene do not have a significant effect on promoting or preventing fibrogenesis in pancreatitis. Further evaluation of this important pathway is warranted.

The role of platelets during reproduction.

Science.gov (United States)

Isermann, Berend; Nawroth, Peter P

2006-01-01

The availability of mice with defined defects within the hemostatic system enabled researchers to identify a role the coagulation system for embryonic and placental development. However, the role of platelets during development has only recently been experimentally addressed, giving some insight into potential functions of platelets during development. Thus, a quantitative embryonic platelet defect (severe thrombopenia secondary to NF-E2 deficiency) is associated with an embryonic growth retardation and reduced vascularisation of the placenta. Maternal platelet deficiency is associated with placental hemorrhage, which, however, does not impair embryonic or maternal survival. In vitro studies established that platelets or platelet conditioned medium regulate the invasive properties of human extravillous trophoblast cells and induce a phenotypical switch of trophoblast cells. These data imply that platelets are of relevance during placentation. Conversely, platelets and the formation of platelet-fibrin aggregates are dispensable for the development of the embryo proper, establishing that the lethal phenotypes observed in some embryo slacking coagulation regulators does not result from an inability to form platelet-fibrin aggregates, but likely reflects altered protease dependent signaling during vascular development.

Preparing Platelet-Rich Plasma with Whole Blood Harvested Intraoperatively During Spinal Fusion.

Science.gov (United States)

Shen, Bin; Zhang, Zheng; Zhou, Ning-Feng; Huang, Yu-Feng; Bao, Yu-Jie; Wu, De-Sheng; Zhang, Ya-Dong

2017-07-22

BACKGROUND Platelet-rich plasma (PRP) has gained growing popularity in use in spinal fusion procedures in the last decade. Substantial intraoperative blood loss is frequently accompanied with spinal fusion, and it is unknown whether blood harvested intraoperatively qualifies for PRP preparation. MATERIAL AND METHODS Whole blood was harvested intraoperatively and venous blood was collected by venipuncture. Then, we investigated the platelet concentrations in whole blood and PRP, the concentration of growth factors in PRP, and the effects of PRP on the proliferation and viability of human bone marrow-derived mesenchymal stem cells (HBMSCs). RESULTS Our results revealed that intraoperatively harvested whole blood and whole blood collected by venipuncture were similar in platelet concentration. In addition, PRP formulations prepared from both kinds of whole blood were similar in concentration of platelet and growth factors. Additional analysis showed that the similar concentrations of growth factors resulted from the similar platelet concentrations of whole blood and PRP between the two groups. Moreover, these two kinds of PRP formulations had similar effects on promoting cell proliferation and enhancing cell viability. CONCLUSIONS Therefore, intraoperatively harvested whole blood may be a potential option for preparing PRP spinal fusion.

Current trends in platelet transfusions practice: The role of ABO-RhD and human leukocyte antigen incompatibility

Directory of Open Access Journals (Sweden)

Serena Valsami

2015-01-01

Full Text Available Platelet transfusions have contributed to the revolutionary modern treatment of hypoproliferative thrombocytopenia. Despite the long-term application of platelet transfusion in therapeutics, all aspects of their optimal use (i.e., in cases of ABO and/or Rh (D incompatibility have not been definitively determined yet. We reviewed the available data on transfusion practices and outcome in ABO and RhD incompatibility and platelet refractoriness due to anti-human leukocyte antigen (HLA antibodies. Transfusion of platelets with major ABO-incompatibility is related to reduced posttransfusion platelet (PLT count increments, compared to ABO-identical and minor, but still are equally effective in preventing clinical bleeding. ABO-minor incompatible transfusions pose the risk of an acute hemolytic reaction of the recipient that is not always related to high anti-A, B donor titers. ABO-identical PLT transfusion seems to be the most effective and safest therapeutic strategy. Exclusive ABO-identical platelet transfusion policy could be feasible, but alternative approaches could facilitate platelet inventory management. Transfusion of platelets from RhD positive donors to RhD negative patients is considered to be effective and safe though is associated with low rate of anti-D alloimmunization due to contaminating red blood cells. The prevention of D alloimmunization is recommended only for women of childbearing age. HLA alloimmunization is a major cause of platelet refractoriness. Managing patients with refractoriness with cross-matched or HLA-matched platelets is the current practice although data are still lacking for the efficacy of this practice in terms of clinical outcome. Leukoreduction contributes to the reduction of both HLA and anti-D alloimmunization.

Human platelet as an independent unit for sulfate conjugation

International Nuclear Information System (INIS)

Khoo, B.Y.; Sit, K.H.; Wong, K.P.

1988-01-01

The human platelets possess a full complement of enzymes capable of synthesizing N-acetyldopamine (NADA) 35 sulfate from ATP, Mg ++ and sodium 35 sulfate. The pH optimum for this three-step overall sulfate conjugation (comprising of the ATP sulfurylase, APS kinase and phenolsulfotransferase reactions) is 8.6 and the reactions proceeded progressively for several hours. Both ATP and Mg ++ ions, above their respective optimal concentrations of 5 and 7 mM, inhibited the sulfate conjugation of NADA. The apparent Km values for NADA as determined by the phenolsulfotransferase (PST) and overall reactions were similar in magnitude being 2.6 and 4.8 μM, respectively, while that for sodium 35 sulfate was 202 μM. A comparison of these two activities in 62 platelet preparations of normal subjects showed that the rate of the PST reaction was generally higher than the overall reaction even though the PST assay was carried out at suboptimal concentration of PAPS. There was a positive correlation (r=0.82) between the two sets of data, suggesting that the PST reaction probably has some control over the rate of overall sulfate conjugation

Demonstration of a specific C3a receptor on guinea pig platelets

International Nuclear Information System (INIS)

Fukuoka, Y.; Hugli, T.E.

1988-01-01

Guinea pig platelets reportedly contain receptors specific for the anaphylatoxin C3a based on both ligand-binding studies and functional responses. A portion of the human 125I-C3a that binds to guinea pig platelets is competitively displaced by excess unlabeled C3a; however, the majority of ligand uptake was nonspecific. Uptake of 125I-C3a by guinea pig platelets is maximal in 1 min, and stimulation of guinea pig platelets by thrombin, ADP, or the Ca2+ ionophore A23187 showed little influence on binding of the ligand. Scatchard analysis indicated that approximately 1200 binding sites for C3a exist per cell with an estimated Kd of 8 x 10(-10) M. Human C3a des Arg also binds to guinea pig platelets, but Scatchard analysis indicated that no specific binding occurred. Because the ligand-binding studies were complicated by high levels of nonspecific uptake, we attempted to chemically cross-link the C3a molecule to a specific component on the platelet surface. Cross-linkage of 125I-C3a to guinea pig platelets with bis(sulfosuccinimidyl)suberate revealed radioactive complexes at 105,000 and 115,000 m.w. on SDS-PAGE gels by autoradiographic analysis. In the presence of excess unlabeled C3a, complex formation was inhibited. No cross-linkage could be demonstrated between the inactive 125I-C3a des Arg and the putative C3a-R on guinea pig platelets. Human C3a, but not C3a des Arg induces serotonin release and aggregation of the guinea pig platelets. Human C3a was unable to induce either serotonin release or promote aggregation of human platelets. Uptake of human 125I-C3a by human platelets was not saturable, and Scatchard analysis was inconclusive. Attempts to cross-link 125I-C3a to components on the surface of human platelets also failed to reveal a ligand-receptor complex. Therefore, we conclude that guinea pig platelets have specific surface receptors to C3a and that human platelets appear devoid of receptors to the anaphylatoxin

Human Platelet Senescence Study.

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1980-03-01

ability to measure certain enzymes to their oxidation-reduc other enzymes which can be measured by o phosphatase , acid phosphatase , chymotryp...alkaline sin, trypsin, esterases (17)); M use of n A or wheat germ agglutinin in the second etect specific carbohydrate constituents. We have...Von Willebrand factor. Nurden and Caen also demonstrated that GPI was rich in sialic acid (5) and probably responsible for the platelets’ surface

Equid herpesvirus type 1 activates platelets.

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Tracy Stokol

Full Text Available Equid herpesvirus type 1 (EHV-1 causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression and platelet microvesiculation (increased small events double positive for CD41 and Annexin V. Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM. A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis

Assessment of quality of platelets preserved in plasma and platelet additive solution: A Malaysian experience

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Munirah Binti Mokhtar

2016-01-01

Full Text Available Background: A use of platelet additives solution (PAS improves storage conditions so as to give increased shelf life to platelets and to maintain hemostatic function. Objective: The present study was aimed to compare in vitro quality of platelet rich plasma (PRP-derived platelet concentrate (PC during extended period of storage in plasma and in additive solution (Composol PS and Fresenius. Study Design: Randomized 19 PCs each were used in the study for plasma and PAS as the storage medium. The measurement parameters, including pH, total white blood cell (WBC count, total platelet count, and platelet activation rate, were studied on day 1, day 5, and day 8 of the storage period. The sterility test was carried out on the eighth day of storage. Results: pH of PC suspended in PAS was significantly lower as compared to that in plasma (P < 0.001 for all the three days of sampling. The WBC count, both in plasma and in PAS, showed an acceptable values of being <0.2 Χ 10 9 /unit during the storage period. Platelet count in PAS was higher as compared to that in plasma, though it was not statistically significant. While both the groups showed increased platelet activation rate during the storage, the PCs suspended in PAS showed significantly higher platelet activation rate (p0.001. Results from sterility test showed no bacterial growth in the PCs in both the groups. Conclusion: Most parameters studied on platelet storage in suspending medium of native plasma and PAS remained well within the acceptable limits. However, the pH values and platelet activation rate significantly differed in PAS as compared with plasma.

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

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Laduca, F.M.; Bell, W.R.; Bettigole, R.E.

1987-01-01

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 [ 3 H]serotonin, or alter the dose-responsive binding of 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

Effects of delayed laboratory processing on platelet serotonin levels.

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Sanner, Jennifer E; Frazier, Lorraine; Udtha, Malini

2013-01-01

Despite the availability of established guidelines for measuring platelet serotonin, these guidelines may be difficult to follow in a hospital setting where time to processing may vary from sample to sample. The purpose of this study was to evaluate the effect of the time to processing of human blood samples on the stability of the enzyme-linked immunosorbent assay (ELISA) for the determination of platelet serotonin levels in human plasma. Human blood samples collected from a convenience sample of eight healthy volunteers were analyzed to determine platelet serotonin levels from plasma collected in ethylene diamine tetra acetic acid (EDTA) tubes and stored at 4°C for 3 hr, 5 hr, 8 hr, and 12 hr. Refrigeration storage at 4°C for 3 hr, 5 hr, 8 hr, and 12 hr altered the platelet serotonin measurement when compared to immediate processing. The bias for the samples stored at 4°C for 3 hr was 102.3 (±217.39 ng/10(9) platelets), for 5 hr was 200.1 (±132.76 ng/10(9) platelets), for 8 hr was 146.9 (±221.41 ng/10(9) platelets), and for 12 hr was -67.6 (±349.60 ng/10(9) platelets). Results from this study show that accurate measurement of platelet serotonin levels is dependent on time to processing. Researchers should therefore follow a standardized laboratory guideline for obtaining immediate platelet serotonin levels after blood sample collection.

Platelet function in brown bear (Ursus arctos compared to man

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Särndahl Eva

2010-06-01

Full Text Available Abstract Background Information on hemostasis and platelet function in brown bear (Ursus arctos is of importance for understanding the physiological, protective changes during hibernation. Objective The study objective was to document platelet activity values in brown bears shortly after leaving the den and compare them to platelet function in healthy humans. Methods Blood was drawn from immobilized wild brown bears 7-10 days after leaving the den in mid April. Blood samples from healthy human adults before and after clopidogrel and acetylsalicylic acid administration served as control. We analyzed blood samples by standard blood testing and platelet aggregation was quantified after stimulation with various agonists using multiple electrode aggregometry within 3 hours of sampling. Results Blood samples were collected from 6 bears (3 females between 1 and 16 years old and from 10 healthy humans. Results of adenosine diphosphate, aspirin, and thrombin receptor activating peptide tests in bears were all half or less of those in humans. Platelet and white blood cell counts did not differ between species but brown bears had more and smaller red blood cells compared with humans. Conclusion Using three different tests, we conclude that platelet function is lower in brown bears compared to humans. Our findings represent the first descriptive study on platelet function in brown bears and may contribute to explain how bears can endure denning without obvious thrombus building. However, the possibility that our findings reflect test-dependent and not true biological variations in platelet reactivity needs further studies.

Cultivation and irradiation of human fibroblasts in a medium enriched with platelet lysate for obtaining feeder layer in epidermal cell culture

International Nuclear Information System (INIS)

Yoshito, Daniele

2011-01-01

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)

Platelet Immunology in China: Research and Clinical Applications.

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Wu, Guoguang; Zhou, Yan; Li, Lilan; Zhong, Zhoulin; Li, Hengchong; Li, Haiyan; Yu, Mei; Shen, Weidong; Ni, Heyu

2017-04-01

Immunization against human platelet alloantigens (HPAs) is associated with a number of clinical complications. The detection and identification of clinically relevant platelet antibodies are important for the diagnosis and management of patients affected with immune-mediated thrombocytopenias. Human platelet alloantigen frequencies and the characteristics of antiplatelet antibodies vary widely between ethnic groups. Since 2008, the importance of platelet immunology in the field of transfusion medicine has gained greater recognition by clinical laboratories in China. Laboratories in China have established and improved methods for platelet antibody detection and HPA genotyping techniques, which are used for the diagnosis of alloimmune platelet disorders in clinic and research environments. Research has revealed the frequencies of HPA alleles in different Chinese ethnic groups and compared the differences in HPA gene frequencies between the Chinese Han and other ethnic groups of the world. Production of anti-CD36 isoantibodies is an important risk factor for immune-mediated thrombocytopenia in the Chinese population. Advances in research and clinical application of platelet immunology have significantly improved the clinical diagnosis, treatment including transfusion support, and prevention of alloimmune platelet disorders in the Chinese population. Copyright © 2017. Published by Elsevier Inc.

Next Generation Sequencing Analysis of Human Platelet PolyA+ mRNAs and rRNA-Depleted Total RNA

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Kissopoulou, Antheia; Jonasson, Jon; Lindahl, Tomas L.; Osman, Abdimajid

2013-01-01

Background Platelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA. Materials and Methods We used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT) isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts. Results Consistent with previous findings our data suggests that mitochondrially expressed genes comprise a substantial fraction of the platelet transcriptome. We also identified high transcript levels for protein coding genes related to the cytoskeleton function, chemokine signaling, cell adhesion, aggregation, as well as receptor interaction between cells. Certain transcripts were particularly abundant in platelets compared with other cell and tissue types represented by RNA-Seq data from the Illumina Human Body Map 2.0 project. Irrespective of the different library preparation and sequencing protocols, there was good agreement between samples from the 4 individuals. Eighteen differentially expressed genes were identified in the two sexes at 10% false discovery rate using DESeq. Conclusion The present data suggests that platelets may have a unique transcriptome profile characterized by a relative over-expression of mitochondrially encoded genes and also of genomic transcripts related to the cytoskeleton function, chemokine signaling and surface components compared with other cell and

Next generation sequencing analysis of human platelet PolyA+ mRNAs and rRNA-depleted total RNA.

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Antheia Kissopoulou

Full Text Available BACKGROUND: Platelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA. MATERIALS AND METHODS: We used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts. RESULTS: Consistent with previous findings our data suggests that mitochondrially expressed genes comprise a substantial fraction of the platelet transcriptome. We also identified high transcript levels for protein coding genes related to the cytoskeleton function, chemokine signaling, cell adhesion, aggregation, as well as receptor interaction between cells. Certain transcripts were particularly abundant in platelets compared with other cell and tissue types represented by RNA-Seq data from the Illumina Human Body Map 2.0 project. Irrespective of the different library preparation and sequencing protocols, there was good agreement between samples from the 4 individuals. Eighteen differentially expressed genes were identified in the two sexes at 10% false discovery rate using DESeq. CONCLUSION: The present data suggests that platelets may have a unique transcriptome profile characterized by a relative over-expression of mitochondrially encoded genes and also of genomic transcripts related to the cytoskeleton function, chemokine signaling and surface components

The phase diagram for the ternary system propylene glycol-sodium chloride-water and their application to platelet cryopreservation.

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Liu, Jing-Han; Gao, Dayong; He, Li-Qun; Moey, Lip Kean; Hua, Kegang; Liu, Zuo-Bin

2003-02-01

In order to acquire freezing model of the cryopretective solution (NaCl-propylene glycol-water ternary system) for platelet, the melting points (T(f)) of this cryopretective solutions with different solute concentration and different ratio of PG mass to NaCl mass were measured by using a differential scanning calorimeter (DSC), and these experimental data were fitting by computer. An empirical equation was derived which characterized the Tf as a function of the solute concentration and the ratio of PG mass to NaCl mass inside this solution. It was concluded that the equilibrium freezing model for human platelets in this system could be used to instruct platelet cryopreserving techniques.

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

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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.

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.

A novel β-lactam derivative, albactam from the flowers of Albizia lebbeck with platelets anti-aggregatory activity in vitro.

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El-Gamal, Ali Ali; Abd-El-Halim, Mohamed Farag; Kalil, Ashraf Taha; Basudan, Omer Ahmed; Al-Rehaily, Adnan Jathlan; Ahmad, Mohamed Shamim; El-Tahir, Kamal Hussin; Al-Massarani, Shaza Mohamed; Abdel-Mageed, Wael Moustafa

2015-03-01

A novel β-lactam derivative, albactam, was isolated from the alcoholic extract of the flowers of Albizia lebbeck. It showed a significant anti-aggregatory activity against adenosine diphosphate and arachidonic acid induced guinea-pigs' platelets aggregation in vitro. Six more known compounds were also isolated and fully characterized by measuring 1D and 2D NMR, two of them are the triterpenes β-amyrin and 11α, 12α-oxidotaraxerol, two ceramide derivatives and two flavonoids, kampferol 3-O-rutinoside and rutin.

DMSO inhibits human platelet activation through cyclooxygenase-1 inhibition. A novel agent for drug eluting stents?

International Nuclear Information System (INIS)

Asmis, Lars; Tanner, Felix C.; Sudano, Isabella; Luescher, Thomas F.; Camici, Giovanni G.

2010-01-01

Background: DMSO is routinely infused together with hematopoietic cells in patients undergoing myeloablative therapy and was recently found to inhibit smooth muscle cells proliferation and arterial thrombus formation in the mouse by preventing tissue factor (TF), a key activator of the coagulation cascade. This study was designed to investigate whether DMSO prevents platelet activation and thus, whether it may represent an interesting agent to be used on drug eluting stents. Methods and results: Human venous blood from healthy volunteers was collected in citrated tubes and platelet activation was studied by cone and platelet analyzer (CPA) and rapid-platelet-function-assay (RPFA). CPA analysis showed that DMSO-treated platelets exhibit a lower adherence in response to shear stress (-15.54 ± 0.9427%, n = 5, P < 0.0001 versus control). Additionally, aggregometry studies revealed that DMSO-treated, arachidonate-stimulated platelets had an increased lag phase (18.0% ± 4.031, n = 9, P = 0.0004 versus control) as well as a decreased maximal aggregation (-6.388 ± 2.212%, n = 6, P = 0.0162 versus control). Inhibitory action of DMSO could be rescued by exogenous thromboxane A2 and was mediated, at least in part, by COX-1 inhibition. Conclusions: Clinically relevant concentrations of DMSO impair platelet activation by a thromboxane A2-dependent, COX-1-mediated effect. This finding may be crucial for the previously reported anti-thrombotic property displayed by DMSO. Our findings support a role for DMSO as a novel drug to prevent not only proliferation, but also thrombotic complications of drug eluting stents.

Comparison between human cord blood serum and platelet-rich plasma supplementation for Human Wharton's Jelly Stem Cells and dermal fibroblasts culture

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Hashemi SS

2016-08-01

Full Text Available We carried out a side-by-side comparison of the effects of Human cord blood serum (HcbS versus embryonic PRP on Human Wharton's Jelly Stem Cells(hWMSCand dermal fibroblasts proliferation. Human umbilical cord blood was collected to prepare activated serum (HCS and platelet-rich plasma (CPRP.Wharton's Jelly Stem Cells and dermal fibroblasts were cultured in complete medium with10% CPRP, 10%HCSor 10% fetal bovine serumand control (serum-free media.The efficiency of the protocols was evaluated in terms of the number of adherent cells and their expansion and Cell proliferation. We showed that proliferation of fibroblasts and mesenchymal stem cells in the presence of cord blood serum and platelet-rich plasma significantly more than the control group (p≤0/05. As an alternative to FBS, cord blood serum has been proved as an effective component in cell tissue culture applications and embraced a vast future in clinical applications of regenerative medicine. However, there is still a need to explore the potential of HCS and its safe applications in humanized cell therapy or tissue engineering.

In vivo quantitation of platelet deposition on human peripheral arterial bypass grafts using indium-111-labeled platelets. Effect of dipyridamole and aspirin

International Nuclear Information System (INIS)

Pumphrey, C.W.; Chesebro, J.H.; Dewanjee, M.K.; Wahner, H.W.; Hollier, L.H.; Pairolero, P.C.; Fuster, V.

1983-01-01

Indium-111-labeled autologous platelets, injected 48 hours after operation, were used to evaluate the thrombogenicity of prosthetic material and the effect of platelet inhibitor therapy in vivo. Dacron double-velour (Microvel) aortofemoral artery bifurcation grafts were placed in 16 patients and unilateral polytetrafluoroethylene femoropopliteal grafts were placed in 10 patients. Half the patients in each group received platelet inhibitors before operation (dipyridamole, 100 mg 4 times a day) and after operation (dipyridamole, 75 mg, and acetylsalicylic acid, 325 mg 3 times a day); the rest of the patients served as control subjects. Five-minute scintigrams of the graft region were taken with a gamma camera interfaced with a computer 48, 72, and 96 hours after injection of the labeled platelets. Platelet deposition was estimated from the radioactivities of the grafts and expressed as counts per 100 pixels per microcurie injected. Dipyridamole and aspirin therapy significantly reduced the number of platelets deposited on Dacron grafts and prevented platelet accumulation over 3 days. With the small amount of platelet deposition on polytetrafluoroethylene femoropopliteal artery grafts even in control patients, platelet inhibitor therapy had no demonstrable effect on platelet deposition on these grafts. It is concluded that (1) platelet deposition on vascular grafts in vivo can be quantitated by noninvasive methods, and (2) dipyridamole and aspirin therapy reduced platelet deposition on Dacron aortofemoral artery grafts

Platelet serotonin level and impulsivity in human self-destructive behavior: A biological and psychological study

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S Era Dutta

2017-01-01

Full Text Available Context: Suicide is a disease and a global public health problem. Suicidology has come to become a topic of study for intervention and research. The serotonin (5-hydroxytryptamine [5HT] system has remained a prime area of investigation. The neurons and platelets display structural and functional similarities. Ninety-nine percent of 5HT is contained in platelets, which shares similar 5HT uptake and release mechanisms with 5HT neurons. Aims: This study aims to study human self-destructive behavior (HSDB. Objectives: Exploring the biological (serotonin levels in platelets and psychological aspects (impulsivity of attempted suicide or HSDB. Settings and Design: Thirty-one patients, above the age of 18 years, with a recent history of HSDB, were studied and given an International Classification of Diseases-10 diagnosis, after a detailed interview. Subjects and Methods: For the platelet 5HT estimation, blood samples were collected, and enzyme immunometric assay carried out. Detailed assessment of the impulsivity was done by the 25-item structured diagnostic interview for borderlines by Zanarini et al. Statistical Analysis Used: We obtained both categorical and continuous data. Chi-square test, Fisher's test, Student's t-test, and Pearson's product moment correlation were used. Results: Female subjects outnumbered males by 2:1. Major depression, adjustment disorder, personality disorder were predominant diagnoses. The mean platelet serotonin concentration for males = 57.3 ng/ml, that of females = 56.05 ng/ml (P > 0.05. Platelet 5HT levels were found to be negatively correlated with impulsivity scores (P < 0.05. Conclusions: Platelet serotonin levels in our study sample were quite low when compared with those reported in published literature. Low serotonin levels were inversely related to impulsivity, but only in males.

Dose- and time-related platelet response with apheresis platelet concentrates and pooled platelets

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Mohammad Mizanur Rahman

2017-02-01

Full Text Available This study was carried out to compare the post-transfusion platelet increment between the apheresis platelet concentrate (n=74 and pooled platelets (n=54. Pre- and post-transfusion platelet count of the recipient were carried out by automated hematology analyzer. In apheresis platelet concentrate group, the mean 24 hours post-transfusion platelet increment was 47 x 109/L which was statistically significant (p<0.001. On the other hand, in pooled platelets group, the mean 24 hours post–transfusions platelet count increment was 11.0 x 109/L which was also statistically significant (p<0.001. This study concluded that the transfusion of apheresis platelet concentrate was more useful than the transfusion of pooled platelets in terms of platelet count increment and requirement of donor.

The role of platelets in hemostasis and the effects of snake venom toxins on platelet function.

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de Queiroz, Mayara Ribeiro; de Sousa, Bruna Barbosa; da Cunha Pereira, Déborah Fernanda; Mamede, Carla Cristine Neves; Matias, Mariana Santos; de Morais, Nadia Cristina Gomes; de Oliveira Costa, Júnia; de Oliveira, Fábio

2017-07-01

The human body has a set of physiological processes, known as hemostasis, which keeps the blood fluid and free of clots in normal vessels; in the case of vascular injury, this process induces the local formation of a hemostatic plug, preventing hemorrhage. The hemostatic system in humans presents complex physiological interactions that involve platelets, plasma proteins, endothelial and subendothelial structures. Disequilibrium in the regulatory mechanisms that control the growth and the size of the thrombus is one of the factors that favors the development of diseases related to vascular disorders such as myocardial infarction and stroke, which are among the leading causes of death in the western world. Interfering with platelet function is a strategy for the treatment of thrombotic diseases. Antiplatelet drugs are used mainly in cases related to arterial thrombosis and interfere in the formation of the platelet plug by different mechanisms. Aspirin (acetylsalicylic acid) is the oldest and most widely used antithrombotic drug. Although highly effective in most cases, aspirin has limitations compared to other drugs used in the treatment of homeostatic disorders. For this reason, research related to molecules that interfere with platelet aggregation are of great relevance. In this regard, snake venoms are known to contain a number of molecules that interfere with hemostasis, including platelet function. The mechanisms by which snake venom components inhibit or activate platelet aggregation are varied and can be used as tools for the diagnosis and the treatment of several hemostatic disorders. The aim of this review is to present the role of platelets in hemostasis and the mechanisms by which snake venom toxins interfere with platelet function. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of platelet thromboxane radioimmunoassay method to measure platelet life-span: Comparison with /sup 111/indium-platelet method

International Nuclear Information System (INIS)

Vallabhajosula, S.; Machac, J.; Badimon, L.; Lipszyc, H.; Goldsmith, S.J.; Fuster, V.

1985-01-01

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 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

Expression of a splice variant of the platelet-activating factor receptor transcript 2 in various human cancer cell lines

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Ibtissam Youlyouz

2002-01-01

Full Text Available Platelet-activating factor receptor (PAF-R transcripts were analysed by reverse transcriptase-polymerase chain reaction in five human cancer cell lines derived from the breast (BT20, SKBR3 and T47D cells, the pancreas (Miapaca cells and the bladder (5637 cells in order to confirm the existence of a splice variant of the PAF-R transcript 2. After cloning and sequencing, we confirmed its existence in all cell lines. It consisted of the PAF-R transcript 2 lengthening with 82 nucleotides from the 3' end of exon 1 of the PAF-R gene. The role of this elongated form of the tissue-type PAF-R transcript in cell physiology remains to be elucidated.

Physiopathology of blood platelets and development of platelet substitutes. Progress report, August 1, 1974--July 31, 1975

International Nuclear Information System (INIS)

Baldini, M.G.

1975-01-01

Progress is reported on investigations of methods for the storage of human blood platelets. Good results were obtained when platelets were frozen using 5 percent dimethyl sulfoxide (DMSO) as a cryoprotective agent. There was no evidence of toxic effects of trace amounts of DMSO in experimental animals. Blood platelet storage at 22 0 C with nucleotide additives in the storage medium was also investigated. The effects of x irradiation at doses varying from 100 to 1000 R on the aggregation of blood platelets following exposure in vitro and the effect of Vitamin E as an antiaggregating agent were studied. (U.S.)

Human Platelets Exhibit Chemotaxis using Functional N-Formyl Peptide Receptors

National Research Council Canada - National Science Library

Czapiga, Meggan; Gao, Ji-Liang; Kirk, Allen; Lekstrom-Himes, Julie

2005-01-01

Activated platelets participate in inflammatory and microbicidal processes by upregulation of surface selectins, shedding of CD40 ligand, and release of platelet microbicidal proteins and microparticles...

21 CFR 864.6675 - Platelet aggregometer.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Platelet aggregometer. 864.6675 Section 864.6675 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6675 Platelet...

Platelet adhesion studies on dipyridamole coated polyurethane surfaces

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Aldenhoff Y. B.J.

2003-06-01

Full Text Available Surface modification of polyurethanes (PUs by covalent attachment of dipyridamole (Persantinregistered is known to reduce adherence of blood platelets upon exposure to human platelet rich plasma (PRP. This effect was investigated in further detail. First platelet adhesion under static conditions was studied with four different biomaterial surfaces: untreated PU, PU immobilised with conjugate molecule 1, PU immobilised with conjugate molecule 2, and PU immobilised with conjugate molecule 3. In PU immobilised with 1 dipyridamole is directly linked to the surface, in PU immobilised with 2 there is a short hydrophilic spacer chain in between the surface and the dipyridamole, while conjugate molecule 3 is merely the spacer chain. Scanning electron microscopy (SEM was used to characterise platelet adhesion from human PRP under static conditions, and fluorescence imaging microscopy was used to study platelet adhesion from whole blood under flow. SEM experiments encompassed both density measurements and analysis of the morphology of adherent platelets. In the static experiments the surface immobilised with 2 showed the lowest platelet adherence. No difference between the three modified surfaces emerged from the flow experiments. The surfaces were also incubated with washed blood platelets and labeled with Oregon-Green Annexin V. No capture of Oregon-Green Annexin V was seen, implying that the adhered platelets did not expose any phosphatidyl serine at their exteriour surface.

Defining the identity of human adipose-derived mesenchymal stem cells.

Science.gov (United States)

Montelatici, Elisa; Baluce, Barbara; Ragni, Enrico; Lavazza, Cristiana; Parazzi, Valentina; Mazzola, Riccardo; Cantarella, Giovanna; Brambilla, Massimiliano; Giordano, Rosaria; Lazzari, Lorenza

2015-02-01

Adipose-derived mesenchymal stem cells (ADMSCs) are an ideal population for regenerative medical application. Both the isolation procedure and the culturing conditions are crucial steps, since low yield can limit further cell therapies, especially when minimal adipose tissue harvests are available for cell expansion. To date, a standardized procedure encompassing both isolation sites and expansion methods is missing, thus making the choice of the most appropriate conditions for the preparation of ADMSCs controversial, especially in view of the different applications needed. In this study, we compared the effects of three different commercial media (DMEM, aMEM, and EGM2), routinely used for ADMSCs expansion, and two supplements, FBS and human platelet lysate, recently proven to be an effective alternative to prevent xenogeneic antibody transfer and immune alloresponse in the host. Notably, all the conditions resulted in being safe for ADMSCs isolation and expansion with platelet lysate supplementation giving the highest isolation and proliferation rates, together with a commitment for osteogenic lineage. Then, we proved that the high ADMSC hematopoietic supportive potential is performed through a constant and abundant secretion of both GCSF and SCF. In conclusion, this study further expands the knowledge on ADMSCs, defining their identity definition and offers potential options for in vitro protocols for clinical production, especially related to HSC expansion without use of exogenous cytokines or genetic modifications.

Blood platelet kinetics and platelet transfusion.

Science.gov (United States)

Aster, Richard H

2013-11-01

The discovery of citrate anticoagulant in the 1920s and the development of plastic packs for blood collection in the 1960s laid the groundwork for platelet transfusion therapy on a scale not previously possible. A major limitation, however, was the finding that platelet concentrates prepared from blood anticoagulated with citrate were unsuitable for transfusion because of platelet clumping. We found that this could be prevented by simply reducing the pH of platelet-rich plasma to about 6.5 prior to centrifugation. We used this approach to characterize platelet kinetics and sites of platelet sequestration in normal and pathologic states and to define the influence of variables such as anticoagulant and ABO incompatibility on post-transfusion platelet recovery. The "acidification" approach enabled much wider use of platelet transfusion therapy until alternative means of producing concentrates suitable for transfusion became available.

Ligament Tissue Engineering Using a Novel Porous Polycaprolactone Fumarate Scaffold and Adipose Tissue-Derived Mesenchymal Stem Cells Grown in Platelet Lysate

Science.gov (United States)

Wagner, Eric R.; Bravo, Dalibel; Dadsetan, Mahrokh; Riester, Scott M.; Chase, Steven; Westendorf, Jennifer J.; Dietz, Allan B.; van Wijnen, Andre J.; Yaszemski, Michael J.

2015-01-01

Purpose: Surgical reconstruction of intra-articular ligament injuries is hampered by the poor regenerative potential of the tissue. We hypothesized that a novel composite polymer “neoligament” seeded with progenitor cells and growth factors would be effective in regenerating native ligamentous tissue. Methods: We synthesized a fumarate-derivative of polycaprolactone fumarate (PCLF) to create macro-porous scaffolds to allow cell–cell communication and nutrient flow. Clinical grade human adipose tissue-derived human mesenchymal stem cells (AMSCs) were cultured in 5% human platelet lysate (PL) and seeded on scaffolds using a dynamic bioreactor. Cell growth, viability, and differentiation were examined using metabolic assays and immunostaining for ligament-related markers (e.g., glycosaminoglycans [GAGs], alkaline phosphatase [ALP], collagens, and tenascin-C). Results: AMSCs seeded on three-dimensional (3D) PCLF scaffolds remain viable for at least 2 weeks with proliferating cells filling the pores. AMSC proliferation rates increased in PL compared to fetal bovine serum (FBS) (p < 0.05). Cells had a low baseline expression of ALP and GAG, but increased expression of total collagen when induced by the ligament and tenogenic growth factor fibroblast growth factor 2 (FGF-2), especially when cultured in the presence of PL (p < 0.01) instead of FBS (p < 0.05). FGF-2 and PL also significantly increased immunostaining of tenascin-C and collagen at 2 and 4 weeks compared with human fibroblasts. Summary: Our results demonstrate that AMSCs proliferate and eventually produce a collagen-rich extracellular matrix on porous PCLF scaffolds. This novel scaffold has potential in stem cell engineering and ligament regeneration. PMID:26413793

Metabolism of 5,6-epoxyeicosatrienoic acid by the human platelet. Formation of novel thromboxane analogs.

Science.gov (United States)

Balazy, M

1991-12-15

Radiolabeled cis-(+-)-5,6-epoxyeicosatrienoic acid (5(6)-EpETrE) was incubated with a suspension of isolated human platelets in order to study its metabolic fate. The epoxide slowly disappeared from the suspension and was completely metabolized within 30 min. After extraction and analysis by reverse-phase high performance liquid chromatography, seven metabolites were found. Addition of either indomethacin (0.01 mM, cyclooxygenase inhibitor) or BW755C (0.1 mM, cyclooxygenase/lipoxygenase inhibitor) to the incubations blocked the formation of four and six metabolites, respectively, 1,2-Epoxy-3,3,3-trichloropropane (inhibitor of microsomal epoxide hydrolase) failed to inhibit the formation of 5,6-dihydroxyeicosatrienoic acid (5,6-DiHETrE), a hydrolysis product of the precursor 5(6)-EpETrE. The metabolites were characterized by UV spectroscopy, negative ion chemical ionization liquid chromatography/mass spectrometry, gas chromatography/mass spectrometry and, in one instance, coelution with synthetic standard. Three primary platelet metabolites were structurally determined to be 5,6-epoxy-12-hydroxyeicosatrienoic acid, 5,6-epoxy-12-hydroxyheptadecadienoic acid, and a unique bicyclic metabolite, 5-hydroxy-6,9-epoxy-thromboxane B1, which originated from intramolecular hydrolysis of 5,6-epoxythromboxane-B1. This thromboxane analog was partially separated into stereoisomers and coeluted with the racemic synthetic standard in gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Three other metabolites were characterized as 5,6,12-trihydroxyeicosatrienoic acid, 5,6,12-trihydroxyheptadecadienoic acid, and 5,6-dihydroxythromboxane-B1, and resulted from the hydrolysis of the corresponding epoxides rather than from the metabolism of 5,6-DiHETrE. The latter was not metabolized by platelet cyclooxygenase or lipoxygenase. The biosynthesis of two cyclooxygenase metabolites indicated the formation of unstable 5,6-epoxythromboxane-A1 as an intermediate

Inflammatory role and prognostic value of platelet chemokines in acute coronary syndrome

NARCIS (Netherlands)

Blanchet, X.; Cesarek, K.; Brandt, J.; Herwald, H.; Teupser, D.; Küchenhoff, H.; Karshovska, E.; Mause, S. F.; Siess, W.; Wasmuth, H.; Soehnlein, O.; Koenen, R. R.; Weber, C.; von Hundelshausen, P.

2014-01-01

Activated platelets and neutrophils exacerbate atherosclerosis. Platelets release the chemokines CXCL4, CXCL4L1 and CCL5, whereas myeloperoxidase (MPO) and azurocidin are neutrophil-derived. We investigated whether plasma levels of these platelet and neutrophil mediators are affected by the acute

Platelet size and age determine platelet function independently

International Nuclear Information System (INIS)

Thompson, C.B.; Jakubowski, J.A.; Quinn, P.G.; Deykin, D.; Valeri, C.R.

1984-01-01

A study was undertaken to examine the interaction of platelet size and age in determining in vitro platelet function. Baboon megakaryocytes were labeled in vivo by the injection of 75Se-methionine. Blood was collected when the label was predominantly associated with younger platelets (day 2) and with older platelets (day 9). Size-dependent platelet subpopulations were prepared on both days by counterflow centrifugation. The reactivity of each platelet subpopulation was determined on both days by measuring thrombin-induced aggregation. Platelets were fixed after partial aggregation had occurred by the addition of EDTA/formalin. After removal of the aggregated platelets by differential centrifugation, the supernatant medium was assayed for remaining platelets and 75Se radioactivity. Comparing day 2 and day 9, no significant difference was seen in the rate of aggregation of a given subpopulation. However, aggregation was more rapid in the larger platelet fractions than in the smaller ones on both days. A greater percentage of the 75Se radioactivity appeared in the platelet aggregates on day 2 than on day 9. This effect was independent of platelet size, as it occurred to a similar extent in the unfractionated platelets and in each of the size-dependent platelet subpopulations. The data indicate that young platelets are more active than older platelets. This study demonstrates that size and age are both determinants of platelet function, but by independent mechanisms

Platelet lysate: a replacement for fetal bovine serum in animal cell culture?

OpenAIRE

Johansson, Liselott; Klinth, Jeanna; Holmqvist, Olov; Ohlson, Sten

2003-01-01

A new cell culture supplement, platelet lysate, was evaluated with reference to fetal bovine serum (FBS), an established industrial medium for animal cell culture. Chemical and bacteriological profiles were conducted including the presence of platelet-derived growth factor (PDGF). PDGF was detected in the platelet lysate but it was not present in FBS. The platelet lysate medium demonstrated lack of microorganisms, mycoplasma and endotoxins. The platelet lysate was investigated in culture stud...

DioxolaneA3-phosphatidylethanolamines are generated by human platelets and stimulate neutrophil integrin expression

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Maceler Aldrovandi

2017-04-01

Full Text Available Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA3. Herein, we demonstrate that significant amounts of DXA3 are rapidly attached to phosphatidylethanolamine (PE forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA3-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA3 generated by platelets, are formed in ng amounts (24.3±6.1 ng/2×108 and remain membrane bound. Pharmacological studies revealed DXA3-PE formation involves cyclooxygenase-1 (COX, protease-activated receptors (PAR 1 and 4, cytosolic phospholipase A2 (cPLA2, phospholipase C and intracellular calcium. They are generated primarily via esterification of newly formed DXA3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA3-PEs were detected in human clots. Purified platelet DXA3-PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells.

Increasing platelet concentrations in leukocyte-reduced platelet-rich plasma decrease collagen gene synthesis in tendons.

Science.gov (United States)

Boswell, Stacie G; Schnabel, Lauren V; Mohammed, Hussni O; Sundman, Emily A; Minas, Tom; Fortier, Lisa A

2014-01-01

Platelet-rich plasma (PRP) is used for the treatment of tendinopathy. There are numerous PRP preparations, and the optimal combination of platelets and leukocytes is not known. Within leukocyte-reduced PRP (lrPRP), there is a plateau effect of platelet concentration, with increasing platelet concentrations being detrimental to extracellular matrix synthesis. Controlled laboratory study. Different formulations of lrPRP with respect to the platelet:leukocyte ratio were generated from venous blood of 8 horses. Explants of the superficial digital flexor tendon were cultured in lrPRP products for 96 hours. Platelet-derived growth factor-BB (PDGF-BB), tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), and interleukin-1β (IL-1β) concentrations were determined in the media by enzyme-linked immunosorbent assay. Gene expression in tendon tissue for collagen type I and III (COL1A1 and COL3A1, respectively), matrix metalloproteinase-3 and -13 (MMP-3 and MMP-13, respectively), cartilage oligomeric matrix protein (COMP), and IL-1β was determined. Data were divided into 3 groups of lrPRP based on the ratio of platelets:leukocytes and evaluated to determine the effect of platelet concentration. Complete blood counts verified leukocyte reduction and platelet enrichment in all PRP preparations. In the lrPRP preparation, the anabolic growth factors PDGF-BB and TGF-β1 were increased with increasing platelet concentrations, and the catabolic cytokine IL-1β was decreased with increasing platelet concentrations. Increasing the platelet concentration resulted in a significant reduction in COL1A1 and COL3A1 synthesis in tendons. Increasing the platelet concentration within lrPRP preparations results in the delivery of more anabolic growth factors and less proinflammatory cytokines, but the biological effect on tendons is diminished metabolism as indicated by a decrease in the synthesis of both COL1A1 and COL3A1. Together, this information suggests that

USE OF VITAL STAINING IN STORED HUMAN PLATELETS MORPHOFUNCTIONAL ANALYSIS

Directory of Open Access Journals (Sweden)

M. S. Makarov

2014-01-01

Full Text Available Apheresis and pooled platelet concentrates, stored at 22°C during 5 days, were studied with morho-functional platelet rate analysis, based on vital cell staining and registration with fluorescent microscope. It was revealed that apheresis and pooled PC had, on the average, normal values of morphological and functional parameters. On the other hand, both PC kept MFPR of cells only for 2 days storage. Longer PC storage caused the significant decay of morphological and functional platelet parameters.

Platelet transfusion therapy: from 1973 to 2005.

NARCIS (Netherlands)

Brand, A.; Novotny, V.M.J.; Tomson, B.

2006-01-01

Platelet transfusions are indispensable for supportive care of patients with hematological diseases. We describe the developments in platelet products for transfusion since the 1970s, when, in particular, support for patients with allo-antibodies against human leukocyte antigens was a laborious

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

Science.gov (United States)

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.

Platelets and Multi-Organ Failure in Sepsis

Directory of Open Access Journals (Sweden)

Elisabetta Greco

2017-10-01

Full Text Available Platelets have received increasing attention for their role in the pathophysiology of infectious disease, inflammation, and immunity. In sepsis, a low platelet count is a well-known biomarker for disease severity and more recently authors have focused their attention on the active role of platelets in the pathogenesis of multi-organ failure. Septic shock is characterised by a dysregulated inflammatory response, which can impair the microcirculation and lead to organ injury. Being at the crossroads between the immune system, clotting cascade, and endothelial cells, platelets seem to be an appealing central mediator and possible therapeutic target in sepsis. This review focuses on the pathogenic role of platelets in septic organ dysfunction in humans and animal models.

Platelets and Multi-Organ Failure in Sepsis.

Science.gov (United States)

Greco, Elisabetta; Lupia, Enrico; Bosco, Ornella; Vizio, Barbara; Montrucchio, Giuseppe

2017-10-20

Platelets have received increasing attention for their role in the pathophysiology of infectious disease, inflammation, and immunity. In sepsis, a low platelet count is a well-known biomarker for disease severity and more recently authors have focused their attention on the active role of platelets in the pathogenesis of multi-organ failure. Septic shock is characterised by a dysregulated inflammatory response, which can impair the microcirculation and lead to organ injury. Being at the crossroads between the immune system, clotting cascade, and endothelial cells, platelets seem to be an appealing central mediator and possible therapeutic target in sepsis. This review focuses on the pathogenic role of platelets in septic organ dysfunction in humans and animal models.

Platelet-Rich Plasma Increases Pigmentation.

Science.gov (United States)

Uysal, Cagri A; Ertas, Nilgun Markal

2017-11-01

Platelet-rich plasma (PRP) is an autologous solution of plasma containing 4 to 7 times the baseline concentration of human platelets. Platelet-rich plasma has been widely popular in facial rejuvenation to attenuate wrinkles and has been practically used. The authors have been encountering various patients of increased hiperpigmentation following PRP applications that were performed to attenuate the postinflammatory hiperpigmentation especially after laser treatment. The authors have been using PRP for facial rejuvenation in selected patients and in 1 patient the authors have encountered increased pigmentation over the pigmented skin lesions that were present before the application. The authors recommend that the PRP might increase pigmentation especially in the face region and precautions might be taken before and after the application. Platelet-rich plasma should not be used for the treatment of post inflammatory hiperpigmentation.

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...... published parallel data set obtained for apheresis-derived PLTs. STUDY DESIGN AND METHODS: During storage we measured more than 150 variables in 8 PLT units, prepared by the buffy coat method. Samples were collected at seven different time points resulting in a data set containing more than 8000...... after their collection. The transition was evident in PLT produced by both production methods. Apheresis-derived PLTs showed a clearer phenotype of PLT activation during early days of storage. The activated phenotype of apheresis PLTs was accompanied by a higher metabolic activity, especially related...

Storage of platelets: effects associated with high platelet content in platelet storage containers.

Science.gov (United States)

Gulliksson, Hans; Sandgren, Per; Sjödin, Agneta; Hultenby, Kjell

2012-04-01

A major problem associated with platelet storage containers is that some platelet units show a dramatic fall in pH, especially above certain platelet contents. The aim of this study was a detailed investigation of the different in vitro effects occurring when the maximum storage capacity of a platelet container is exceeded as compared to normal storage. Buffy coats were combined in large-volume containers to create primary pools to be split into two equal aliquots for the preparation of platelets (450-520×10(9) platelets/unit) in SSP+ for 7-day storage in two containers (test and reference) with different platelet storage capacity (n=8). Exceeding the maximum storage capacity of the test platelet storage container resulted in immediate negative effects on platelet metabolism and energy supply, but also delayed effects on platelet function, activation and disintegration. Our study gives a very clear indication of the effects in different phases associated with exceeding the maximum storage capacity of platelet containers but throw little additional light on the mechanism initiating those negative effects. The problem appears to be complex and further studies in different media using different storage containers will be needed to understand the mechanisms involved.

Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4

Science.gov (United States)

Semeraro, Fabrizio; Ammollo, Concetta T.; Morrissey, James H.; Dale, George L.; Friese, Paul; Esmon, Naomi L.

2011-01-01

The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process. PMID:21673343

Human platelet glycoprotein IX: An adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures

International Nuclear Information System (INIS)

Hickey, M.J.; Williams, S.A.; Roth, G.J.

1989-01-01

The glycoprotein (GP) Ib-IX complex on the surface of human platelets functions as the von Willebrand factor receptor and mediates von Willebrand factor-dependent platelet adhesion to blood vessels. GPIX is a relatively small (M r , 17,000) protein that may provide for membrane insertion and orientation of the larger component of the complex. GPIb (M r , 165,000). Using antibody screening, the authors cloned a cDNA encoding GPIX from a human erythroleukemia cell cDNA library constructed in phage λgt11. Lacking a 5' untranslated region and start codon, the cDNA sequence includes 604 nucleotides, beginning with 495 bases at the 5' end coding for 165 amino acids, followed by a stop codon and 106 noncoding bases at the 3' end. By Northern blot analysis, the GPIX cDNA hybridizes with a single 1.0-kilobase species of platelet poly(A) + RNA. Translation of the cDNA sequence gives a predicted protein sequence beginning with a truncated putative signal sequence of 5 amino acids followed by a sequence of 17 amino acids matching that determined directly by Edman degradation of intact GPIX. GPIX contains a leucine-rich glycoprotein (LRG) sequence of 24 amino acids similar to conserved LRG sequences in GPIb and other proteins from humans, Drosophila, and yeast. The role of the flank-LRG center-flank structure in the evolution and function of the LRG proteins remains to be defined

Polyphosphate nanoparticles on the platelet surface trigger contact system activation

NARCIS (Netherlands)

Verhoef, Johan J F; Barendrecht, Arjan D; Nickel, Katrin F; Dijkxhoorn, Kim; Kenne, Ellinor; Labberton, Linda; McCarty, Owen J T; Schiffelers, Raymond; Heijnen, Harry F G; Hendrickx, Antoni P A; Schellekens, Huub; Fens, Marcel H; de Maat, Steven; Renné, Thomas; Maas, Coen

2017-01-01

Polyphosphate is an inorganic polymer that can potentiate several interactions in the blood coagulation system. Blood platelets contain polyphosphate, and the secretion of platelet-derived polyphosphate has been associated with increased thrombus formation and activation of coagulation factor XII.

Senescence and quiescence in adipose-derived stromal cells

DEFF Research Database (Denmark)

Søndergaard, Rebekka Harary; Follin, Bjarke; Lund, Lisbeth Drozd

2017-01-01

Background aims. Adipose-derived stromal cells (ASCs) are attractive sources for cell-based therapies. The hypoxic niche of ASCs in vivo implies that cells will benefit from hypoxia during in vitro expansion. Human platelet lysate (hPL) enhances ASC proliferation rates, compared with fetal bovine...

Production and characterization of monoclonal antibodies against rat platelet GPIIb/IIIa

International Nuclear Information System (INIS)

Miyazaki, H.; Tamura, S.; Sudo, T.; Suzuki, T.

1990-01-01

Four murine monoclonal antibodies against rat platelets were produced by fusion of spleen cells from mice intravenously immunized with whole rat platelets. All four antibodies immunoprecipitated two major platelet membrane proteins with apparent molecular weights of 130,000 and 82,000 (nonreduced) and of 120,000 and 98,000 (reduced), which were structurally analogous to human glycoprotein (GP) IIb/IIIa, i.e. rat GPIIb/IIIa. Two of four antibodies, named P9 and P55, strongly inhibited adenosine diphosphate (ADP)-induced aggregation of washed rat platelets and caused approximately 50% inhibition of human fibrinogen binding to ADP-stimulated rat platelets, suggesting that rat GPIIb/IIIa serves as a fibrinogen receptor in ADP-induced aggregation. In contrast, two other antibodies, named P14 and P34, themselves caused aggregation of rat platelets in platelet-rich plasma (PRP) and the secretion of 14C-serotonin from 14C-serotonin-labeled PRP. These results indicate that rat GPIIb/IIIa plays an important role in platelet aggregation

Platelet-Derived Growth Factor BB Influences Muscle Regeneration in Duchenne Muscle Dystrophy.

Science.gov (United States)

Piñol-Jurado, Patricia; Gallardo, Eduard; de Luna, Noemi; Suárez-Calvet, Xavier; Sánchez-Riera, Carles; Fernández-Simón, Esther; Gomis, Clara; Illa, Isabel; Díaz-Manera, Jordi

2017-08-01

Duchenne muscular dystrophy (DMD) is characterized by a progressive loss of muscle fibers, and their substitution by fibrotic and adipose tissue. Many factors contribute to this process, but the molecular pathways related to regeneration and degeneration of muscle are not completely known. Platelet-derived growth factor (PDGF)-BB belongs to a family of growth factors that regulate proliferation, migration, and differentiation of mesenchymal cells. The role of PDGF-BB in muscle regeneration in humans has not been studied. We analyzed the expression of PDGF-BB in muscle biopsy samples from controls and patients with DMD. We performed in vitro experiments to understand the effects of PDGF-BB on myoblasts involved in the pathophysiology of muscular dystrophies and confirmed our results in vivo by treating the mdx murine model of DMD with repeated i.m. injections of PDGF-BB. We observed that regenerating and necrotic muscle fibers in muscle biopsy samples from DMD patients expressed PDGF-BB. In vitro, PDGF-BB attracted myoblasts and activated their proliferation. Analysis of muscles from the animals treated with PDGF-BB showed an increased population of satellite cells and an increase in the number of regenerative fibers, with a reduction in inflammatory infiltrates, compared with those in vehicle-treated mice. Based on our results, PDGF-BB may play a protective role in muscular dystrophies by enhancing muscle regeneration through activation of satellite cell proliferation and migration. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Platelet Rich Plasma- mechanism of action and clinical applications

OpenAIRE

Cristina N. Cozma; Laura Raducu; Cristian R. Jecan

2016-01-01

Platelet-rich plasma (PRP) is a blood-derived fraction containing high level of platelets, a high concentration of leukocytes and growth factors. PRP therapy has been growing as a viable treatment alternative for a number of clinical applications and has a potential benefit for use in wound healing. Nowadays platelet rich plasma is used in stimulating wound healing in skin and soft tissue ulcerations, accelerating wound healing in diabetic patients and facilitating bone proliferation in ortho...

Human platelet ( sup 125 I)R-DOI binding sites. Characterization by in vitro autoradiography

Energy Technology Data Exchange (ETDEWEB)

Himeno, A.; Saavedra, J.M. (National Institute of Mental Health, Bethesda, MD (USA))

1990-02-01

We quantified binding sites for 2,5-dimethoxy-4-iodo-phenylisopropylamine (DOI), a 5-HT2 agonist and hallucinogen, in human platelets. We incubated sections from human platelet pellets with ({sup 125}I)R-DOI with or without 1 mumol/L ketanserin, followed by autoradiography and computerized microdensitometry. We corrected the values of binding density by the protein content of each section with a densitometric protein assay. The present method revealed a single class of high affinity binding sites for ({sup 125}I)R-DOI, with a Kd of 6.4 +/- 0.7 nmol/L and a Bmax of 100 +/- 10 fmol/mg protein. Kd and Bmax for ({sup 125}I)R-DOI determined by the classical membrane binding assay, were 2.7 +/- 0.4 nmol/L and 100 +/- 10 fmol/mg protein, respectively. The present method is precise, very sensitive, and allows the characterization of ({sup 125}I)R-DOI binding in sections obtained from as little as 3 ml of blood. Standardization is possible after correction by the protein content of each individual section.

Identification and characterization of a putative human platelet thromboxane A2/prostaglandin H2 receptor

International Nuclear Information System (INIS)

Saussy, D.L. Jr.

1986-01-01

The thromboxane A 2 (TXA 2 ) analog, 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15αβ-omega-tetranor TXA 2 (I-PTA-OH) was characterized as a competitive antagonist of TXA 2 mimetic-induced platelet aggregation, with a K/sub d/ of 190 nM in platelet rich plasma. This antagonism was specific for the putative thromboxane A 2 /prostaglandin H 2 (TXA 2 /PGH 2 ) receptor, since I-PTA-OH had no inhibitory effects on platelet aggregation stimulated by agonists which act independently of TXA 2 /PGH 2 , and did not inhibit platelet TXA 2 synthesis. [ 125 I]-PTA-OH binding to a particulate fraction from human platelets was saturable, displaceable, and linear with protein concentration. Scatchard analysis of equilibrium binding revealed a single class of high affinity binding sites, with a K/sub d/ of 30 +/- 4 nM and a B/sub max/ of 1.8 +/- 0.3 pmol/mg protein. Kinetic analysis yielded a k 1 of 1.35 x 10 6 M -1 x min -1 and a k√ 1 of 0.032 min -1 , K/sub d/ = k√ 1 /k 1 = 24 nM. The subcellular localization of the putative TXA 2 /PGH 2 receptor was determined using [ 125 I]-PTA-OH binding as a marker for the receptor. [ 125 I]-PTA-OH binding as a marker for the receptor. [ 125 I]-PTA-OH binding, was coenriched with markers for plasma membranes and dense tubular system; but not with markers for cytoplasmic constituents, mitochondria, or granules

Potential fluid mechanic pathways of platelet activation.

Science.gov (United States)

Shadden, Shawn C; Hendabadi, Sahar

2013-06-01

Platelet activation is a precursor for blood clotting, which plays leading roles in many vascular complications and causes of death. Platelets can be activated by chemical or mechanical stimuli. Mechanically, platelet activation has been shown to be a function of elevated shear stress and exposure time. These contributions can be combined by considering the cumulative stress or strain on a platelet as it is transported. Here, we develop a framework for computing a hemodynamic-based activation potential that is derived from a Lagrangian integral of strain rate magnitude. We demonstrate that such a measure is generally maximized along, and near to, distinguished material surfaces in the flow. The connections between activation potential and these structures are illustrated through stenotic flow computations. We uncover two distinct structures that may explain observed thrombus formation at the apex and downstream of stenoses. More broadly, these findings suggest fundamental relationships may exist between potential fluid mechanic pathways for mechanical platelet activation and the mechanisms governing their transport.

Biologic effects of platelet-derived growth factor receptor α blockade in uterine cancer.

Science.gov (United States)

Roh, Ju-Won; Huang, Jie; Hu, Wei; Yang, XiaoYun; Jennings, Nicholas B; Sehgal, Vasudha; Sohn, Bo Hwa; Han, Hee Dong; Lee, Sun Joo; Thanapprapasr, Duangmani; Bottsford-Miller, Justin; Zand, Behrouz; Dalton, Heather J; Previs, Rebecca A; Davis, Ashley N; Matsuo, Koji; Lee, Ju-Seog; Ram, Prahlad; Coleman, Robert L; Sood, Anil K

2014-05-15

Platelet-derived growth factor receptor α (PDGFRα) expression is frequently observed in many kinds of cancer and is a candidate for therapeutic targeting. This preclinical study evaluated the biologic significance of PDGFRα and PDGFRα blockade (using a fully humanized monoclonal antibody, 3G3) in uterine cancer. Expression of PDGFRα was examined in uterine cancer clinical samples and cell lines, and biologic effects of PDGFRα inhibition were evaluated using in vitro (cell viability, apoptosis, and invasion) and in vivo (orthotopic) models of uterine cancer. PDGFRα was highly expressed and activated in uterine cancer samples and cell lines. Treatment with 3G3 resulted in substantial inhibition of PDGFRα phosphorylation and of downstream signaling molecules AKT and mitogen-activated protein kinase (MAPK). Cell viability and invasive potential of uterine cancer cells were also inhibited by 3G3 treatment. In orthotopic mouse models of uterine cancer, 3G3 monotherapy had significant antitumor effects in the PDGFRα-positive models (Hec-1A, Ishikawa, Spec-2) but not in the PDGFRα-negative model (OVCA432). Greater therapeutic effects were observed for 3G3 in combination with chemotherapy than for either drug alone in the PDGFRα-positive models. The antitumor effects of therapy were related to increased apoptosis and decreased proliferation and angiogenesis. These findings identify PDGFRα as an attractive target for therapeutic development in uterine cancer. ©2014 American Association for Cancer Research.

Peptide-Mediated Platelet Capture at Gold Micropore Arrays.

Science.gov (United States)

Adamson, Kellie; Spain, Elaine; Prendergast, Una; Moran, Niamh; Forster, Robert J; Keyes, Tia E

2016-11-30

Ordered spherical cap gold cavity arrays with 5.4, 1.6, and 0.98 μm diameter apertures were explored as capture surfaces for human blood platelets to investigate the impact of surface geometry and chemical modification on platelet capture efficiency and their potential as platforms for surface enhanced Raman spectroscopy of single platelets. The substrates were chemically modified with single-constituent self-assembled monolayers (SAM) or mixed SAMs comprised of thiol-functionalized arginine-glycine-aspartic acid (RGD, a platelet integrin target) with or without 1-octanethiol (adhesion inhibitor). As expected, platelet adhesion was promoted and inhibited at RGD and alkanethiol modified surfaces, respectively. Platelet adhesion was reversible, and binding efficiency at the peptide modified substrates correlated inversely with pore diameter. Captured platelets underwent morphological change on capture, the extent of which depended on the topology of the underlying substrate. Regioselective capture of the platelets enabled study for the first time of the surface enhanced Raman spectroscopy of single blood platelets, yielding high quality Raman spectroscopy of individual platelets at 1.6 μm diameter pore arrays. Given the medical importance of blood platelets across a range of diseases from cancer to psychiatric illness, such approaches to platelet capture may provide a useful route to Raman spectroscopy for platelet related diagnostics.

Quantitation of thrombogenicity of hemodialyzer with technetium-99m and indium-111 labeled platelets

International Nuclear Information System (INIS)

Dewanjee, M.K.; Kapadvanjwala, Mansoor; Ruzius, Kees; Serafini, A.N.; Zilleruelo, G.E.; Sfakianakis, G.N.

1993-01-01

The platelet thromobogenicity of a hemodialyzer was quantified with 99m Tc- and 111 In-labeled platelets. The platelets collected from blood of Beagle dogs, Yorkshire pigs and human volunteers were labeled with 111 in-tropolone (detergent-free) and 99m Tc-HMPAO. Hemodialysis was performed with a hollow-fiber dialyzer (HFD) in a flow-loop, the temperature of which was maintained at 37 o C, with flow-rates of 7, 150 and 270 mL/min; after dialysis, the HFD radioactivity was measured with an ionization chamber and imaged with a γ-camera. The radioactivity of samples of hollow-fibers taken from the top, middle and bottom of the dialyzer was determined with a γ-counter. The mean values of hemodialyzer-adherent platelet radioactivity were calculated for both radionuclides. The canine platelets were found to be more thrombogenic than porcine and human platelets. The adhesivity of porcine platelets to the biomaterial (cellulose-acetate) of the dialyzer approximated that of human platelets. The 99m Tc label underestimated the thrombus formation (P 111 In- and 99m Tc-labeled platelets suggests that both radionuclides could be used for measurement of device-induced thrombogenicity and may provide an estimation of prosthesis-induced thrombogenicity of human platelets from animal studies. (Author)

Extending The Shelf Life Of Blood Platelets

Science.gov (United States)

Surgenor, Douglas M.

1988-01-01

New method of storing human blood platelets extends vitality for transfusions. Packaged as suspension in sterile liquid in plastic blood bags. Each bag placed between pair of plastic grids, and rubberbands placed around sandwich thus formed to hold together. Stored upright in open air or in container through which air pumped at rate of at least 45 L/min. Ensures that platelets receive ample oxygen and expiratory carbon dioxide form platelets removed before pH drops to harmful levels.

Platelet concentration in platelet-rich plasma affects tenocyte behavior in vitro.

Science.gov (United States)

Giusti, Ilaria; D'Ascenzo, Sandra; Mancò, Annalisa; Di Stefano, Gabriella; Di Francesco, Marianna; Rughetti, Anna; Dal Mas, Antonella; Properzi, Gianfranco; Calvisi, Vittorio; Dolo, Vincenza

2014-01-01

Since tendon injuries and tendinopathy are a growing problem, sometimes requiring surgery, new strategies that improve conservative therapies are needed. Platelet-rich plasma (PRP) seems to be a good candidate by virtue of its high content of growth factors, most of which are involved in tendon healing. This study aimed to evaluate if different concentrations of platelets in PRP have different effects on the biological features of normal human tenocytes that are usually required during tendon healing. The different platelet concentrations tested (up to 5 × 10(6) plt/µL) stimulated differently tenocytes behavior; intermediate concentrations (0.5 × 10(6), 1 × 10(6) plt/µL) strongly induced all tested processes (proliferation, migration, collagen, and MMPs production) if compared to untreated cells; on the contrary, the highest concentration had inhibitory effects on proliferation and strongly reduced migration abilities and overall collagen production but, at the same time, induced increasing MMP production, which could be counterproductive because excessive proteolysis could impair tendon mechanical stability. Thus, these in vitro data strongly suggest the need for a compromise between extremely high and low platelet concentrations to obtain an optimal global effect when inducing in vivo tendon healing.

Indium-111 labelled platelets: experimental and clinical studies

International Nuclear Information System (INIS)

Gjerloeff Schmidt, K.

1985-10-01

The object of the present study became to develop a method of effective and gentle isolation and 111-In labelling of human platelets, as well as to employ these platelets in human clinical studies with the object of elucidating a number of physiological and pathophysiological mechanisms and processes in which platelets take part. 111-In-oxine presents obvious advantages over 51-Cr-sodium chromate; a high labelling efficiency, and more advantageous physical properties (a half life of 68 hours (against the half life of 28 days for 51-Cr) and considerably more effective gamma emission), making external registration by means of a gamma camera possible. Considering the role played by platelets in the development of atherosclerosis and its thromboembolic complications, in the early phases of deep venous thrombosis, and in graft rejection, it is natural that attempts have been made to use 111-In-labelled platelets for scintigraphic and kinetic evaluation of thromboembolic processes. Accumulation of 111-In-labelled platelets at sites of vessel wall injury, on pulmonary emboli (presumably on deep vein thrombi as well), and on catheter material has been demonstrated. Beyond this, the number of publications concerning the use of 111-In-labelled platelets for visualization of atherosclerosis, venous thromboembolism, arterial grafts, intracardiac thrombi, aortic aneurysms, renal allograft rejection, and other situations in which platelet thromboembolism takes place, provides evidence that a tool has finally been found for the study of their nature and response to therapeutic intervention. (eg)

Evaluation of the antioxidant properties of N-acetylcysteine in human platelets: prerequisite for bioconversion to glutathione for antioxidant and antiplatelet activity.

Science.gov (United States)

Gibson, Kyle R; Neilson, Ilene L; Barrett, Fiona; Winterburn, Tim J; Sharma, Sushma; MacRury, Sandra M; Megson, Ian L

2009-10-01

N-Acetylcysteine (NAC) is a frequently used "antioxidant" in vitro, but the concentrations applied rarely correlate with those encountered with oral dosing in vivo. Here, we investigated the in vitro antioxidant and antiplatelet properties of NAC at concentrations (10-100 microM) that are achievable in plasma with tolerable oral dosing. The impact of NAC pretreatment (2 hours) on aggregation of platelets from healthy volunteers in response to thrombin and adenosine diphosphate and on platelet-derived nitric oxide (NO) was examined. NAC was found to be a weak reducing agent and a poor antioxidant compared with glutathione (reduced form) (GSH). However, platelets treated with NAC showed enhanced antioxidant activity and depression of reactive oxygen species generation associated with increases in intraplatelet GSH levels. An approximately 2-fold increase in NO synthase-derived nitrite was observed with 10 microM NAC treatment, but the effect was not concentration dependent. Finally, NAC significantly reduced both thrombin-induced and adenosine diphosphate-induced platelet aggregation. NAC should be considered a weak antioxidant that requires prior conversion to GSH to convey antioxidant and antithrombotic benefit at therapeutically relevant concentrations. Our results suggest that NAC might be an effective antiplatelet agent in conditions where increased oxidative stress contributes to heightened risk of thrombosis but only if the intraplatelet machinery to convert it to GSH is functional.

Platelet-derived growth factor (PDGF)-signaling mediates radiation-induced apoptosis in human prostate cancer cells with loss of p53 function

International Nuclear Information System (INIS)

Kim, Harold E.; Han, Sue J.; Kasza, Thomas; Han, Richard; Choi, Hyeong-Seon; Palmer, Kenneth C.; Kim, Hyeong-Reh C.

1997-01-01

Platelet-derived growth factor (PDGF) signals a diversity of cellular responses in vitro, including cell proliferation, survival, transformation, and chemotaxis. PDGF functions as a 'competence factor' to induce a set of early response genes expressed in G 1 including p21 WAF1/CIP1 , a functional mediator of the tumor suppressor gene p53 in G 1 /S checkpoint. For PDGF-stimulated cells to progress beyond G 1 and transit the cell cycle completely, progression factors in serum such as insulin and IGF-1 are required. We have recently shown a novel role of PDGF in inducing apoptosis in growth-arrested murine fibroblasts. The PDGF-induced apoptosis is rescued by insulin, suggesting that G 1 /S checkpoint is a critical determinant for PDGF-induced apoptosis. Because recent studies suggest that radiation-induced signal transduction pathways interact with growth factor-mediated signaling pathways, we have investigated whether activation of the PDGF-signaling facilitates the radiation-induced apoptosis in the absence of functional p53. For this study we have used the 125-IL cell line, a mutant p53-containing, highly metastatic, and hormone-unresponsive human prostate carcinoma cell line. PDGF signaling is constitutively activated by transfection with a p28 v-sis expression vector, which was previously shown to activate PDGF α- and β- receptors. Although the basal level of p21 WAF1/CIP1 expression and radiation-induced apoptosis were not detectable in control 125-IL cells as would be predicted in mutant p53-containing cells, activation of PDGF-signaling induced expression of p21 WAF1/CIP1 and radiation-induced apoptosis. Our study suggests that the level of 'competence' growth factors including PDGF may be one of the critical determinants for radiation-induced apoptosis, especially in cells with loss of p53 function at the site of radiotherapy in vivo

Manufacture of pooled platelets in additive solution and storage in an ELX container after an overnight warm temperature hold of platelet-rich plasma.

Science.gov (United States)

Alhumaidan, Hiba; Cheves, Tracey; Holme, Stein; Sweeney, Joseph D

2011-10-01

The processing of whole blood-derived platelet-rich plasma (PRP) to a platelet concentrate and platelet-poor plasma is currently performed within 8 hours to comply with the requirements to manufacture fresh frozen plasma. Maintaining PRP at room temperature for a longer period can have the advantage of shifting the completion of component manufacture onto day shifts. Pairs of ABO-identical prepooled platelets were manufactured by the PRP method, using the current approach with platelet storage in a CLX HP container (Pall Medical, Covina, CA) and plasma, or a novel approach with an 18- to a 24-hour room temperature hold of the PRP and the manufacture of pooled platelets in a glucose-containing additive solution (AS) and storage in a new ELX container (Pall Medical). Standard in vitro assays were performed on days 2, 5, and 7. The results showed that the AS platelets in ELX have in vitro characteristics that are equivalent or superior to those of the standard product.

The Efficacy and Safety of Platelet-Rich Plasma and Adipose-Derived Stem Cells: An Update

Directory of Open Access Journals (Sweden)

Jaehoon Choi

2012-11-01

Full Text Available During the past decade, many studies using platelet-rich plasma (PRP or adipose-derivedstem cells (ASCs have been conducted in various medical fields, from cardiovascular researchto applications for corneal diseases. Nonetheless, there are several limitations of practicalapplications of PRP and ASCs. Most reports of PRP are anecdotal and few include controlsto determine the specific role of PRP. There is little consensus regarding PRP production andcharacterization. Some have reported the development of an antibody to bovine thrombin,which was the initiator of platelet activation. In the case of ASCs, good manufacturing practicesare needed for the production of clinical-grade human stem cells, and in vitro expansion ofASCs requires approval of the Korea Food and Drug Administration, such that considerableexpense and time are required. Additionally, some have reported that ASCs could have apotential risk of transformation to malignant cells. Therefore, the authors tried to investigatethe latest research on the efficacy and safety of PRP and ASCs and report on the current stateand regulation of these stem cell-based therapies.

Measurement of platelet aggregation, independently of patient platelet count

DEFF Research Database (Denmark)

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

2017-01-01

with collagen-related peptide). Platelet aggregation had a negative predictive value of 100% for a bleeding tendency among patients. Conclusion The established platelet aggregation assay was applicable for thrombocytopenic patients, and improved the identification of bleeding risk.......Essentials •Platelet function may influence bleeding risk in thrombocytopenia, but useful tests are needed. •A flow cytometric platelet aggregation test independent of the patient platelet count was made. •Platelet aggregation was reduced in thrombocytopenic patients with hematological cancer....... •High 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...

21 CFR 864.6650 - Platelet adhesion test.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Platelet adhesion test. 864.6650 Section 864.6650 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6650 Platelet adhesion...

Radiolabeled platelets

International Nuclear Information System (INIS)

Datz, F.L.; Taylor, A.T.

1986-01-01

Initial interest in developing techniques to radiolabel platelets was spurred by the lack of an accurate method for measuring platelet life span in both normals and in thrombocytopenic patients. Early investigators could obtain only rough estimates of platelet life spans by monitoring the platelet counts of thrombocytopenic patients undergoing platelet transfusions. Labels were also sought that would allow imaging of platelets in vivo in order to better understand the pathophysiology of atherosclerosis, thrombophlebitis, and clotting disorders, and to improve the clinical diagnosis of these diseases. Two types of platelet labels were investigated: cohort (pulse) labels and random labels. Cohort labels are taken up by megakaryocytes in the bone marrow and incorporated in the DNA and other components of the forming platelet. In theory, only freshly released platelets of a uniform age are labeled. Random labels, on the other hand, tag platelets in the peripheral blood, labeling platelets of all ages

Elemental composition of platelets. Part I. Sampling and sample preparation of platelets for trace-element analysis

International Nuclear Information System (INIS)

Iyengar, G.V.; Borberg, H.; Kasperek, K.; Kiem, J.; Siegers, M.; Feinendegen, L.E.; Gross, R.

1979-01-01

Sampling of platelets for trace-element analysis poses special problems: obtaining adequate sample materials, achieving a sufficient cell purity, preserving viability (integrity), correcting for trapped plasma, and controlling contamination. We used a blood-cell separator for the primary isolation of platelets from blood, and differential centrifugation in natural plasma to further isolate them. The pyrimidopyrimidine RA233 was used as a stabilizer to maintain viability. 131 I-labeled human serum albumin was used to estimate trapped plasma. Contamination was controlled by using five-times-distilled water to simulate donor's blood in the system and by comparing three fractions: the serum, the first portion of the platelet-rich plasma, and the supernatant plasma after the final centrifugation. Neutron activation analysis was used for the elemental analysis. A single differential centrifugation of the platelet-rich plasma from the blood-cell separator at 400 x g for 8 min was optimum (mean mass fractions: erythrocytes/platelets < 5 mg/g and leukocytes/platelets < 20 mg/g). The trapped plasma in the wet platelet samples amounted to about 0.40 g/g. No appreciable contamination from the sampling system was found for the elements Ag, Cd, Co, Cr, Cs, Cu, Fe, Mo, Rb, Sb, Se, and Zn. 2 figures, 3 tables

Thermophoresis of Nano Platelets in Confined Space

Science.gov (United States)

Hardt, Steffen

2007-11-01

For several decades it has been known that a rigid body immersed in a gas of nonuniform temperature experiences a thermophoretic force aligned with the temperature gradient. If however, a body is placed in a narrow gap between two solid walls of uniform, but different temperature, a thermophoretic force perpendicular to the temperature gradient can be created. In this work an analytical expression is derived for the force experienced by a nano platelet between two walls of different temperature. It is assumed that the in-plane extension of the platelet is larger than the gap width and that the gas dynamics occurs in the free-molecular flow regime. For the case that the gas molecules are diffusively reflected from the walls, it can be shown that the wall collision rate is constant over the whole computational domain. Based on this insight, expressions for the force and the torque on a nano platelet in a narrow gap are derived. It is shown that the torque vanishes, whereas there is a net force perpendicular to the temperature gradient. When considering thermal fluctuations on top of the average force it becomes apparent that in such a way a thermophoretic motion of a platelet can be induced.

Three-dimensional structure and cytokine distribution of platelet-rich fibrin.

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Bai, Meng-Yi; Wang, Ching-Wei; Wang, Jyun-Yi; Lin, Ming-Fang; Chan, Wing P

2017-02-01

Previous reports have revealed that several cytokines (including platelet-derived growth factor-BB, transforming growth factors-β1 and insulin-like growth factor-1) can enhance the rate of bone formation and synthesis of extracellular matrix in orthopaedics or periodontology. This study aimed to determine the concentration of cytokines within platelet-rich fibrin microstructures and investigate whether there are differences in the different portions of platelet-rich fibrin, which has implications for proper clinical use of platelet-rich fibrin gel. Whole blood was obtained from six New Zealand rabbits (male, 7 to 39 weeks old, weight 2.7-4 kg); it was then centrifuged for preparation of platelet-rich fibrin gels and harvest of plasma. The resultant platelet-rich fibrin gels were used for cytokine determination, histological analyses and scanning electron microscopy. All plasmas obtained were subject to the same cytokine determination assays for the purpose of comparison. Cytokines platelet-derived growth factor-BB and transforming growth factor-β1 formed concentration gradients from high at the red blood cell end of the platelet-rich fibrin gel (p=1.88×10-5) to low at the plasma end (p=0.19). Insulin-like growth factor-1 concentrations were similar at the red blood cell and plasma ends. The porosities of the platelet-rich fibrin samples taken in sequence from the red blood cell end to the plasma end were 6.5% ± 4.9%, 24.8% ± 7.5%, 30.3% ± 8.5%, 41.4% ± 12.3%, and 40.3% ± 11.7%, respectively, showing a gradual decrease in the compactness of the platelet-rich fibrin network. Cytokine concentrations are positively associated with platelet-rich fibrin microstructure and portion in a rabbit model. As platelet-rich fibrin is the main entity currently used in regenerative medicine, assessing cytokine concentration and the most valuable portion of PRF gels is essential and recommended to all physicians.

Three-dimensional structure and cytokine distribution of platelet-rich fibrin

Directory of Open Access Journals (Sweden)

Meng-Yi Bai

Full Text Available OBJECTIVES: Previous reports have revealed that several cytokines (including platelet-derived growth factor-BB, transforming growth factors-β1 and insulin-like growth factor-1 can enhance the rate of bone formation and synthesis of extracellular matrix in orthopaedics or periodontology. This study aimed to determine the concentration of cytokines within platelet-rich fibrin microstructures and investigate whether there are differences in the different portions of platelet-rich fibrin, which has implications for proper clinical use of platelet-rich fibrin gel. METHODS: Whole blood was obtained from six New Zealand rabbits (male, 7 to 39 weeks old, weight 2.7-4 kg; it was then centrifuged for preparation of platelet-rich fibrin gels and harvest of plasma. The resultant platelet-rich fibrin gels were used for cytokine determination, histological analyses and scanning electron microscopy. All plasmas obtained were subject to the same cytokine determination assays for the purpose of comparison. RESULTS: Cytokines platelet-derived growth factor-BB and transforming growth factor-β1 formed concentration gradients from high at the red blood cell end of the platelet-rich fibrin gel (p=1.88×10-5 to low at the plasma end (p=0.19. Insulin-like growth factor-1 concentrations were similar at the red blood cell and plasma ends. The porosities of the platelet-rich fibrin samples taken in sequence from the red blood cell end to the plasma end were 6.5% ± 4.9%, 24.8% ± 7.5%, 30.3% ± 8.5%, 41.4% ± 12.3%, and 40.3% ± 11.7%, respectively, showing a gradual decrease in the compactness of the platelet-rich fibrin network. CONCLUSION: Cytokine concentrations are positively associated with platelet-rich fibrin microstructure and portion in a rabbit model. As platelet-rich fibrin is the main entity currently used in regenerative medicine, assessing cytokine concentration and the most valuable portion of PRF gels is essential and recommended to all physicians.

Visualisation of lectin binding sites on the surface of human platelets using lectins adsorbed to gold granules.

Science.gov (United States)

Nurden, A T; Horisberger, M; Savariau, E; Caen, J P

1980-10-15

Washed human platelets have been incubated with the lectins WGA, ConA and RCA1, adsorbed to different-sized gold particles. Plasma membrane receptors for each lectin were then located by scanning and transmission electron microscopy.

Pathogen reduction by ultraviolet C light effectively inactivates human white blood cells in platelet products.

Science.gov (United States)

Pohler, Petra; Müller, Meike; Winkler, Carla; Schaudien, Dirk; Sewald, Katherina; Müller, Thomas H; Seltsam, Axel

2015-02-01

Residual white blood cells (WBCs) in cellular blood components induce a variety of adverse immune events, including nonhemolytic febrile transfusion reactions, alloimmunization to HLA antigens, and transfusion-associated graft-versus-host disease (TA-GVHD). Pathogen reduction (PR) methods such as the ultraviolet C (UVC) light-based THERAFLEX UV-Platelets system were developed to reduce the risk of transfusion-transmitted infection. As UVC light targets nucleic acids, it interferes with the replication of both pathogens and WBCs. This preclinical study aimed to evaluate the ability of UVC light to inactivate contaminating WBCs in platelet concentrates (PCs). The in vitro and in vivo function of WBCs from UVC-treated PCs was compared to that of WBCs from gamma-irradiated and untreated PCs by measuring cell viability, proliferation, cytokine secretion, antigen presentation in vitro, and xenogeneic GVHD responses in a humanized mouse model. UVC light was at least as effective as gamma irradiation in preventing GVHD in the mouse model. It was more effective in suppressing T-cell proliferation (>5-log reduction in the limiting dilution assay), cytokine secretion, and antigen presentation than gamma irradiation. The THERAFLEX UV-Platelets (MacoPharma) PR system can substitute gamma irradiation for TA-GVHD prophylaxis in platelet (PLT) transfusion. Moreover, UVC treatment achieves suppression of antigen presentation and inhibition of cytokine accumulation during storage of PCs, which has potential benefits for transfusion recipients. © 2014 AABB.

Low level of procoagulant platelet microparticles is associated with impaired coagulation and transfusion requirements in trauma patients

DEFF Research Database (Denmark)

Windeløv, Nis Agerlin; Johansson, Pär Ingemar; Sørensen, Anne Marie

2014-01-01

BACKGROUND: Following activation, platelets release small vesicles called platelet-derived microparticles (PMPs). PMPs accelerate thrombin generation and thus clot formation at sites of injury by exposing the procoagulant membrane phospholipid phosphatidylserine (PS). The role of PMPs in coagulop......BACKGROUND: Following activation, platelets release small vesicles called platelet-derived microparticles (PMPs). PMPs accelerate thrombin generation and thus clot formation at sites of injury by exposing the procoagulant membrane phospholipid phosphatidylserine (PS). The role of PMPs...

Platelet-derived growth factor-C and -D in the cardiovascular system and diseases.

Science.gov (United States)

Lee, Chunsik; Li, Xuri

2018-08-01

The cardiovascular system is among the first organs formed during development and is pivotal for the formation and function of the rest of the organs and tissues. Therefore, the function and homeostasis of the cardiovascular system are finely regulated by many important molecules. Extensive studies have shown that platelet-derived growth factors (PDGFs) and their receptors are critical regulators of the cardiovascular system. Even though PDGF-C and PDGF-D are relatively new members of the PDGF family, their critical roles in the cardiovascular system as angiogenic and survival factors have been amply demonstrated. Understanding the functions of PDGF-C and PDGF-D and the signaling pathways involved may provide novel insights into both basic biomedical research and new therapeutic possibilities for the treatment of cardiovascular diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Palmitic acid-labeled lipids selectively incorporated into platelet cytoskeleton during aggregation

International Nuclear Information System (INIS)

Packham, M.A.; Guccione, M.A.; Bryant, N.L.; Livne, A.

1990-01-01

Previous experiments showed that during the early stages (20-30 seconds) of aggregation induced by adenosine diphosphate (ADP, 2 microM) or thrombin (0.1 U/mL) of rabbit or human platelets prelabeled with [3H]palmitic acid, labeled lipid became associated with the cytoskeleton isolated after lysis with 1% Triton X-100, 5 mM EGTA [ethylene glycol-bis-(beta-aminoethyl ether)]-N,N,N',N'-tetra-acetic acid. The association appeared to be related to the number of sites of contact and was independent of the release of granule contents. We have now investigated the nature of the labeled lipids by thin-layer and column chromatography and found differences between the distribution of the label in intact platelets (both stimulated and unstimulated) and the isolated cytoskeletons. In both species, and with either ADP or thrombin as aggregating agent, 70-85% of the label in both intact platelets and in the cytoskeletons was in phospholipids. The distribution of label among the phospholipids in the cytoskeletons was similar to that in intact platelets except that the percentage of label in phosphatidylcholine was significantly higher in the cytoskeletons of human platelets than in the intact platelets, and the percentage of label in phosphatidylserine/phosphatidylinositol was significantly lower in the cytoskeletons of rabbit platelets and thrombin-aggregated human platelets than in intact platelets. The cytoskeletons contained a lower percentage of label in triacylglycerol, diacylglycerol, and cholesterol ester than the intact platelets. Contrary to a report in the literature, we found no evidence for the incorporation of diacylglycerol and palmitic acid into the cytoskeleton

Concentration of platelets and growth factors in platelet-rich plasma from Goettingen minipigs.

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Jungbluth, Pascal; Grassmann, Jan-Peter; Thelen, Simon; Wild, Michael; Sager, Martin; Windolf, Joachim; Hakimi, Mohssen

2014-01-01

In minipigs little is known about the concentration of growth factors in plasma, despite their major role in several patho-physiological processes such as healing of fractures. This prompted us to study the concentration of platelets and selected growth factors in plasma and platelet-rich plasma (PRP) preparation of sixteen Goettingen minipigs. Platelet concentrations increased significantly in PRP in comparison to native blood plasma. Generally, significant increase in the concentration of all growth factors tested was observed in the PRP in comparison to the corresponding plasma or serum. Five of the plasma samples examined contained detectable levels of bone morphogenic protein 2 (BMP-2) whereas eleven of the plasma or serum samples contained minimal amounts of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF-bb) respectively. On the other hand variable concentrations of bone morphogenic protein 7 (BMP-7) and transforming growth factor β1 (TGF-β1) were measured in all plasma samples. In contrast, all PRP samples contained significantly increased amounts of growth factors. The level of BMP-2, BMP-7, TGF-β1, VEGF and PDGF-bb increased by 17.6, 1.5, 7.1, 7.2 and 103.3 fold, in comparison to the corresponding non-enriched preparations. Moreover significant positive correlations were found between platelet count and the concentrations of BMP-2 (r=0.62, pplatelet-rich plasma of minipigs which might thus serve as a source of autologous growth factors.

Exposure to acrolein by inhalation causes platelet activation

International Nuclear Information System (INIS)

Sithu, Srinivas D.; Srivastava, Sanjay; Siddiqui, Maqsood A.; Vladykovskaya, Elena; Riggs, Daniel W.; Conklin, Daniel J.; Haberzettl, Petra; O'Toole, Timothy E.; Bhatnagar, Aruni; D'Souza, Stanley E.

2010-01-01

Acrolein is a common air pollutant that is present in high concentrations in wood, cotton, and tobacco smoke, automobile exhaust and industrial waste and emissions. Exposure to acrolein containing environmental pollutants such as tobacco smoke and automobile exhaust has been linked to the activation of the coagulation and hemostasis pathways and thereby to the predisposition of thrombotic events in human. To examine the effects of acrolein on platelets, adult male C57Bl/6 mice were subjected acute (5 ppm for 6 h) or sub-chronic (1 ppm, 6 h/day for 4 days) acrolein inhalation exposures. The acute exposure to acrolein did not cause pulmonary inflammation and oxidative stress, dyslipidemia or induce liver damage or muscle injury. Platelet GSH levels in acrolein-exposed mice were comparable to controls, but acrolein-exposure increased the abundance of protein-acrolein adducts in platelets. Platelets isolated from mice, exposed to both acute and sub-chronic acrolein levels, showed increased ADP-induced platelet aggregation. Exposure to acrolein also led to an increase in the indices of platelet activation such as the formation of platelet-leukocyte aggregates in the blood, plasma PF4 levels, and increased platelet-fibrinogen binding. The bleeding time was decreased in acrolein exposed mice. Plasma levels of PF4 were also increased in mice exposed to environmental tobacco smoke. Similar to inhalation exposure, acrolein feeding to mice also increased platelet activation and established a pro-thrombotic state in mice. Together, our data suggest that acrolein is an important contributing factor to the pro-thrombotic risk in human exposure to pollutants such as tobacco smoke or automobile exhaust, or through dietary consumption.

Exposure to acrolein by inhalation causes platelet activation

Energy Technology Data Exchange (ETDEWEB)

Sithu, Srinivas D [Department of Physiology and Biophysics, University of Louisville, Louisville, KY 40202 (United States); Diabetes and Obesity Center, University of Louisville, Louisville, KY 40202 (United States); Srivastava, Sanjay; Siddiqui, Maqsood A; Vladykovskaya, Elena; Riggs, Daniel W; Conklin, Daniel J; Haberzettl, Petra; O' Toole, Timothy E; Bhatnagar, Aruni [Diabetes and Obesity Center, University of Louisville, Louisville, KY 40202 (United States); D' Souza, Stanley E., E-mail: sedsou01@louisville.ed [Department of Physiology and Biophysics, University of Louisville, Louisville, KY 40202 (United States)

2010-10-15

Acrolein is a common air pollutant that is present in high concentrations in wood, cotton, and tobacco smoke, automobile exhaust and industrial waste and emissions. Exposure to acrolein containing environmental pollutants such as tobacco smoke and automobile exhaust has been linked to the activation of the coagulation and hemostasis pathways and thereby to the predisposition of thrombotic events in human. To examine the effects of acrolein on platelets, adult male C57Bl/6 mice were subjected acute (5 ppm for 6 h) or sub-chronic (1 ppm, 6 h/day for 4 days) acrolein inhalation exposures. The acute exposure to acrolein did not cause pulmonary inflammation and oxidative stress, dyslipidemia or induce liver damage or muscle injury. Platelet GSH levels in acrolein-exposed mice were comparable to controls, but acrolein-exposure increased the abundance of protein-acrolein adducts in platelets. Platelets isolated from mice, exposed to both acute and sub-chronic acrolein levels, showed increased ADP-induced platelet aggregation. Exposure to acrolein also led to an increase in the indices of platelet activation such as the formation of platelet-leukocyte aggregates in the blood, plasma PF4 levels, and increased platelet-fibrinogen binding. The bleeding time was decreased in acrolein exposed mice. Plasma levels of PF4 were also increased in mice exposed to environmental tobacco smoke. Similar to inhalation exposure, acrolein feeding to mice also increased platelet activation and established a pro-thrombotic state in mice. Together, our data suggest that acrolein is an important contributing factor to the pro-thrombotic risk in human exposure to pollutants such as tobacco smoke or automobile exhaust, or through dietary consumption.

Platelet fibrinogen binding in Basset Hound Hereditary Thrombopathy

International Nuclear Information System (INIS)

Patterson, W.; Estry, D.; Schwartz, K.; Bell, T.

1986-01-01

Platelets from dogs with Basset Hound Hereditary Thrombopathy (BHT) display a thrombasthenia-like aggregation defect but have been shown to have normal amounts of platelet membrane glycoproteins IIb and IIIa (GP IIb-IIIa). In order to investigate the possibility of a functionally abnormal GPIIb-IIIa complex, which might be unable to bind fibrinogen after stimulation, fibrinogen binding in BHT was evaluated. Two canine fibrinogen preparations were used, one from BHT dogs and one from normal control dogs, as well as a human fibrinogen preparation. Platelets from BHT and normal dogs were activated with 1 x 10 -5 M ADP in the presence of 125 I-labeled fibrinogen and the surface bound radioactivity quantitated. For all fibrinogen preparations, the amount of fibrinogen bound by BHT platelets was not significantly different than that bound by normal dog platelets. BHT platelets bound 23,972 +/- 3612 and normal dog platelets bound 23,033 +/- 3971 molecules of fibrinogen per platelet. The BHT platelet aggregation defect does not seem to be caused by a functionally abnormal GP IIb-IIIa complex, since BHT platelets bind normal amounts of fibrinogen. The results suggest that fibrinogen binding is not sufficient for platelet aggregation, and other factors, perhaps receptor mobility and membrane phospholipid content should be investigated in BHT

Blood platelet kinetics and platelet transfusion

OpenAIRE

Aster, Richard H.

2013-01-01

The discovery of citrate anticoagulant in the 1920s and the development of plastic packs for blood collection in the 1960s laid the groundwork for platelet transfusion therapy on a scale not previously possible. A major limitation, however, was the finding that platelet concentrates prepared from blood anticoagulated with citrate were unsuitable for transfusion because of platelet clumping. We found that this could be prevented by simply reducing the pH of platelet-rich plasma to about 6.5 pr...

Determination of human platelet antigen typing by molecular methods: Importance in diagnosis and early treatment of neonatal alloimmune thrombocytopenia.

Science.gov (United States)

Arinsburg, Suzanne A; Shaz, Beth H; Westhoff, Connie; Cushing, Melissa M

2012-05-01

Neonatal alloimmune thrombocytopenia (NAIT) is the most common cause of severe thrombocytopenia and intracranial hemorrhage in the perinatal period. While the gold standard for making a diagnosis of NAIT is detection of a human platelet antigen (HPA)-specific antibody in maternal serum, together with identifying an incompatibility between the parents for the cognate HPA antigen, platelet genotyping is the gold standard method for HPA typing. Platelet genotyping is critical in screening at-risk fetuses for the presence ofthe HPA corresponding to the maternal antibody. In addition, platelet genotyping may play a role in population screening to identify women at risk for sensitization, and thus, fetuses at risk for NAIT. The most commonly used methods of platelet genotyping are sequence-specific primer-polymerase chain reaction (PCR-SSP), restriction fragment length polymorphism-PCR (PCR-RFLP), and TaqMan real-time PCR. PCR-SSP and PCR-RFLP are relatively inexpensive and technically simple methods, but they are not easily automated and require expertise for reliable interpretation of results. Newer methods that allow for multiplexing, automation, and easily interpretable results, such as bead arrays, are currently in development and available for research purposes. Copyright © 2012 Wiley Periodicals, Inc.

An aptamer-based biosensing platform for highly sensitive detection of platelet-derived growth factor via enzyme-mediated direct electrochemistry

International Nuclear Information System (INIS)

Deng Kun; Xiang Yang; Zhang Liqun; Chen Qinghai; Fu Weiling

2013-01-01

Highlights: ► Direct electrochemistry of glucose oxidase used for signal generation in aptasensor. ► Using novel nanocomposite for immobilization and signal amplification. ► Sensitive electrochemical detection of platelet-derived growth factor. - Abstract: In this work, a new label-free electrochemical aptamer-based sensor (aptasensor) was constructed for detection of platelet-derived growth factor (PDGF) based on the direct electrochemistry of glucose oxidase (GOD). For this proposed aptasensor, poly(diallyldimethylammonium chloride) (PDDA)-protected graphene-gold nanoparticles (P-Gra-GNPs) composite was firstly coated on electrode surface to form the interface with biocompatibility and huge surface area for the adsorption of GOD layer. Subsequently, gold nanoclusters (GNCs) were deposited on the surface of GOD to capture PDGF binding aptamer (PBA). Finally, GOD as a blocking reagent was employed to block the remaining active sites of the GNCs and avoid the nonspecific adsorption. With the direct electron transfer of double layer GOD membranes, the aptasensor showed excellent electrochemical response and the peak current decreased linearly with increasing logarithm of PDGF concentration from 0.005 nM to 60 nM with a relatively low limit of detection of 1.7 pM. The proposed aptasensor exhibited high specificity, good reproducibility and long-term stability, which provided a new promising technique for aptamer-based protein detection.

Functionalized Magnetic Resonance Contrast Agent Selectively Binds to Glycoprotein IIb/IIIa on Activated Human Platelets under Flow Conditions and Is Detectable at Clinically Relevant Field Strengths

Directory of Open Access Journals (Sweden)

Constantin von zur Mühlen

2008-03-01

Full Text Available Recent progress in molecular magnetic resonance imaging (MRI provides the opportunity to image cells and cellular receptors using microparticles of iron oxide (MPIOs. However, imaging targets on vessel walls remains challenging owing to the quantity of contrast agents delivered to areas of interest under shear stress conditions. We evaluated ex vivo binding characteristics of a functional MRI contrast agent to ligand-induced binding sites (LIBSs on activated glycoprotein IIb/IIIa receptors of human platelets, which were lining rupture-prone atherosclerotic plaques and could therefore facilitate detection of platelet-mediated pathology in atherothrombotic disease. MPIOs were conjugated to anti-LIBS single-chain antibodies (LIBS-MPIO or control antibodies (control MPIO. Ex vivo binding to human platelet-rich clots in a dose-dependent manner was confirmed on a 3 T clinical MRI scanner and by histology (p < .05 for LIBS-MPIO vs control MPIO. By using a flow chamber setup, significant binding of LIBS-MPIO to a platelet matrix was observed under venous and arterial flow conditions, but not for control MPIO (p < .001. A newly generated MRI contrast agent detects activated human platelets at clinically relevant magnetic field strengths and binds to platelets under venous and arterial flow conditions, conveying high payloads of contrast to specific molecular targets. This may provide the opportunity to identify vulnerable, rupture-prone atherosclerotic plaques via noninvasive MRI.

Platelet lysate induces chondrogenic differentiation of umbilical cord-derived mesenchymal stem cells.

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Hassan, Ghmkin; Bahjat, Mohammad; Kasem, Issam; Soukkarieh, Chadi; Aljamali, Majd

2018-01-01

Articular cartilage has a poor capacity for self-repair, and thus still presents a major challenge in orthopedics. Mesenchymal stem cells (MSCs) are multipotent stem cells with the potential to differentiate into chondrocytes in the presence of transforming growth factor beta (TGF-β). Platelet lysate (PL) contains a relatively large number of growth factors, including TGF-β, and has been shown to ameliorate cartilage repair. Here, we investigated the ability of PL to direct chondrogenic differentiation of MSCs along with other standard differentiation components in a pellet culture system. We isolated and expanded MSCs from human umbilical cords using a PL-supplemented medium and characterized the cells based on immunophenotype and potential for differentiation to adipocytes and osteocytes. We further cultured MSCs as pellets in a chondrogenic-differentiation medium supplemented with PL. After 21 days, the pellets were processed for histological analysis and stained with alician blue and acridine orange. The expression of SOX9 was investigated using RT-PCR. MSCs maintained their stemness characteristics in the PL-supplemented medium. However, the distribution of cells in the pellets cultured in the PL-supplemented chondrogenic differentiation medium had a greater similarity to cartilage tissue-derived chondrocytes than to the negative control. The intense alician blue staining indicated an increased production of mucopolysaccharides in the differentiated pellets, which also showed elevated expression of SOX9 . Our data suggest that MSCs could be differentiated to chondrocytes in the presence of PL and absence of exogenous TGF-β. Further research needs to be conducted to understand the exact role and potential of PL in chondrogenic differentiation and chondrocyte regeneration.

Forced expression of platelet-derived growth factor B in the mouse cerebellar primordium changes cell migration during midline fusion and causes cerebellar ectopia

NARCIS (Netherlands)

Andrae, Johanna; Afink, Gijs; Zhang, Xiao-Qun; Wurst, Wolfgang; Nistér, Monica

2004-01-01

The platelet-derived growth factor (PDGF) and receptors are expressed in the developing central nervous system and in brain tumors. To investigate the role of PDGF during normal cerebellar development, we created transgenic mice where PDGF-B was introduced into the endogenous Engrailed1 locus (En1).

Basic study of platelet labeling with 111In-oxine

International Nuclear Information System (INIS)

Yui, Tokuo; Uchida, Tatsumi; Matsuda, Shin; Muroi, Shuichi; Tanaka, Tetsugoro

1981-01-01

Indium-111-oxine has recently been suggested as a new isotopic labeling agent of platelets. In this paper, the results on the investigation of in vitro labeling of human platelets with In-111-oxine and those of platelet kinetics in rats are presented. Based on the findings of those studies, the protocol of human platelet labeling with In-111-oxine for clinical use was established. All operations should be carried out with sterile techniques at 20 - 25 0 C. 1) Forty four ml venous blood is drawn into a 50 ml polystyrene syringe containing 6 ml ACD-A. 2) The blood is transferred to a 50 ml tube and centrifuged at 300 g for 15 min. 3) Supernatant platelet rich plasma (PRP) is transferred to other 50 ml tube. Then, the pH is adjusted to 6.5 by addition of 1 ml ACD-A per 20 ml PRP. 4) Platelets are sedimented by centrifuging at 1,500 g for 15 min and resuspended in 3 ml ACD-A-saline solution (pH 6.5). 5) Three hundreds μCi of In-111-oxine is added to the platelet suspension. The mixture is incubated for 20 min at room temperature. 6) About 15 ml of the platelet poor autologous plasma (PPP) is added into the incubated mixture, followed by the sedimentation of labeled platelets (1,500 g, 15 min). 7) The labeled platelets are suspended in 10 ml PPP and the contaminating red cells are sedimented by centrifuging at 200 g for 5 min. 8) One hundred and fifty μCi of labeled platelet suspension is injected to the patient intravenously. The labeling efficiency in this method was 62 +- 5% (mean +- 1S.D., n = 6). (author)

Platelet cyclooxygenase expression in normal dogs.

Science.gov (United States)

Thomason, J; Lunsford, K; Mullins, K; Stokes, J; Pinchuk, L; Wills, R; McLaughlin, R; Langston, C; Pruett, S; Mackin, A

2011-01-01

Human platelets express both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Variation in COX-2 expression could be a mechanism for variable response to aspirin. The hypotheses were that circulating canine platelets express COX-1 and COX-2, and that aspirin alters COX expression. The objective was to identify changes in platelet COX expression and in platelet function caused by aspirin administration to dogs. Eight female, intact hounds. A single population, repeated measures design was used to evaluate platelet COX-1 and COX-2 expression by flow cytometry before and after aspirin (10 mg/kg Q12h for 10 days). Platelet function was analyzed via PFA-100(®) (collagen/epinephrine), and urine 11-dehydro-thromboxane B(2) (11-dTXB(2)) was measured and normalized to urinary creatinine. Differences in COX expression, PFA-100(®) closure times, and urine 11-dTXB(2 ): creatinine ratio were analyzed before and after aspirin administration. Both COX-1 and COX-2 were expressed in canine platelets. COX-1 mean fluorescent intensity (MFI) increased in all dogs, by 250% (range 63-476%), while COX-2 expression did not change significantly (P = 0.124) after aspirin exposure, with large interindividual variation. PFA-100(®) closure times were prolonged and urine 11-dTXB(2) concentration decreased in all dogs after aspirin administration. Canine platelets express both COX isoforms. After aspirin exposure, COX-1 expression increased despite impairment of platelet function, while COX-2 expression varied markedly among dogs. Variability in platelet COX-2 expression should be explored as a potential mechanism for, or marker of, variable aspirin responsiveness. Copyright © 2011 by the American College of Veterinary Internal Medicine.

The mitochondrial membrane potential in human platelets: a sensitive parameter for platelet quality

NARCIS (Netherlands)

Verhoeven, Arthur J.; Verhaar, Robin; Gouwerok, Eric G. W.; de Korte, Dirk

2005-01-01

BACKGROUND: Deterioration of platelet (PLT) quality during storage is accompanied by an increase in lactate production, indicating a decrease in mitochondrial function. In this study, the optimal conditions under which the fluorescent dye JC-1 can be used to detect changes in mitochondrial function

Anti-platelet activity of water dispersible curcuminoids in rat platelets.

Science.gov (United States)

Maheswaraiah, Anikisetty; Rao, Lingamallu Jaganmohan; Naidu, Kamatham Akhilender

2015-03-01

Curcuminoids are active principle of turmeric with plethora of health beneficial properties. In this study, we have evaluated for the first time the effect of water dispersible curcuminoids on rat platelet aggregation. Curcuminoids (10-30 µg/mL) significantly inhibited platelet aggregation induced by agonists viz., collagen, ADP and arachidonic acid. Curcuminoids were found to be two-fold more potent than curcumin in inhibiting platelet aggregation. Intracellular curcuminoid concentration was relatively higher than curcumin in rat platelets. Curcuminoids significantly attenuated thromboxane A2 , serotonin levels in rat platelets which play an important role in platelet aggregation. Curcuminoid treatment increased nitric oxide (NO) levels in platelets treated with agonists. Curcuminoids inhibited free radicals such as superoxide anion released from activated platelets, which ultimately inhibits platelet aggregation. Further, curcuminoids inhibited 12-lipoxygenase activity and formation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE) in activated rat platelets which regulates platelet aggregation. The results suggest that curcuminoids have remarkable anti-platelet activity by modulating multiple mechanisms involved in platelet aggregation. Thus curcuminoids may have a therapeutic potential to prevent platelet activation related disorders. Copyright © 2015 John Wiley & Sons, Ltd.

Stimulus-response coupling in platelets

International Nuclear Information System (INIS)

Huang, E.M.

1986-01-01

To understand the mechanism of stimulus-response coupling in platelets, the potentiating effect of succinate and lithium on platelet activation was examined. The action of succinate was immediate; preincubation with succinate did not lead to desensitization. Succinate was comparable to ADP in lowering cAMP levels previously elevated by PGl 2 . Since inhibition of cAMP is not a prerequisite for platelet activation, the mechanism of potentiation of succinate remains undefined. Lithium has also been shown to inhibit adenylate cyclase in PGl 2 -pretreated platelets. Lithium, however, can also inhibit inositol phosphate (InsP) phosphatase and lead to an accumulation of InsP. In human platelets, lithium also enhanced the thrombin-induced accumulation of [ 3 H]inositol-labelled inositol trisphosphate (InsP 3 ), and inositol bisphosphate (InsP 2 ). One hour after thrombin addition, all 3 inositol phosphates returned to near basal levels. In the presence of lithium, while labelled InsP 2 and InsP 3 returned to their respective basal levels, the InsP level remained elevated, consistent with the known inhibitory effect of lithium on InsP phosphatase. In thrombin-stimulated platelets prelabeled with [ 32 P]phosphate, lithium led to a decrease in labelled phosphatidylinositol 4-phosphate (PtdIns4P) as well as an enhanced production of labelled lysophosphatidylinositol, suggesting multiple effects of lithium on platelet phosphoinositide metabolism. These observed effects, however, occurred too slowly to be the mechanism by which lithium potentiated agonist-induced platelet activation. To study the agonist-receptor interaction, the effect of the specific, high affinity thrombin inhibitor, hirudin, on thrombin-induced accumulation of [ 3 H]inositol-labelled inositol phosphates was studied

Radioimmunoassay for platelet activation specific protein GMP-140 on the platelet surface and in plasma

International Nuclear Information System (INIS)

Wu Guoxin; Li Jianyong; Ruan Changgeng

1991-08-01

Using monoclonal antibody (McAb) SZ-51 which is specific for an alpha-granule membrane protein (GMP-140) on the surface of human activated platelets, the platelet GMP-140 expression in fixed whole blood was measured by direct radioimmunoassay and GMP-140 microparticles in plasma was measured by sandwich method. The GMP-140 molecules per platelet or milliliter (mL) were calculated for the following subjects; acute myocardial infarction; cerebro thrombosis; diabetic mellitus; asthma attack; epidemic hemorrhagic fever etc.. By comparing with the concentration of thromboxane B 2 (TXB 2 ) and von Willebrand factor (vWF) in plasma, it is confirmed that the measurement of GMP-140 molecules is better than that of TXB 2 and vWF. It is a sensitive and specific method for evaluating the platelet activation degree in vivo. The establishment of this method will be useful to diagnosing the thrombotic disorders and studying the pathogenesis of some other diseases

A new method for high yield purification of type beta transforming growth factor from human platelets

NARCIS (Netherlands)

Eijnden-van Raaij, A.J.M. van den; Koornneef, I.; Zoelen, E.J.J. van

1988-01-01

A new method was developed for the purification of type beta transforming growth factor from human platelets. This method is a three-step procedure including gel filtration, weak cation exchange HPLC and reverse phase HPLC. All steps are carried out at low pH using exclusively volatile acidic buffer

Glycoprotein Ibα receptor instability is associated with loss of quality in platelets produced in culture.

Science.gov (United States)

Robert, Amélie; Boyer, Lucie; Pineault, Nicolas

2011-03-01

The development of culture processes for hematopoietic progenitors could lead to the development of a complementary source of platelets for therapeutic purposes. However, functional characterization of culture-derived platelets remains limited, which raises some uncertainties about the quality of platelets produced in vitro. The aim of this study was to define the proportion of functional platelets produced in cord blood CD34+ cell cultures. Toward this, the morphological and functional properties of culture-derived platelet-like particles (PLPs) were critically compared to that of blood platelets. Flow cytometry combined with transmission electron microscopy analyses revealed that PLPs formed a more heterogeneous population of platelets at a different stage of maturation than blood platelets. The majority of PLPs harbored the fibrinogen receptor αIIbβ3, but a significant proportion failed to maintain glycoprotein (GP)Ibα surface expression, a component of the vWF receptor essential for platelet functions. Importantly, GPIbα extracellular expression correlated closely with platelet function, as the GPIIb+ GPIbα+ PLP subfraction responded normally to agonist stimulation as evidenced by α-granule release, adhesion, spreading, and aggregation. In contrast, the GPIIb+ GPIbα⁻ subfraction was unresponsive in most functional assays and appeared to be metabolically inactive. The present study confirms that functional platelets can be generated in cord blood CD34+ cell cultures, though these are highly susceptible to ectodomain shedding of receptors associated with loss of function. Optimization of culture conditions to prevent these deleterious effects and to homogenize PLPs is necessary to improve the quality and yields of culture-derived platelets before they can be recognized as a suitable complementary source for therapeutic purposes.

Functional alterations of human platelets following 111In labeling with different ligands and incubation media

International Nuclear Information System (INIS)

Mieno, Masayuki; Isaka, Yoshinori; Kimura, Kazutumi

1990-01-01

We studied the effects of various 111 In-water soluble chelates and incubation media on labeling efficiency of platelets and in vitro platelet aggregability. High labeling efficiency of platelets in ACD-saline was achieved with 111 In-oxine sulfate, 111 In-tropolone and 111 In-MPO (2-mercaptopyridine-N-oxide). In the condition with 4.8 x 10 6 /mm 3 platelets in ACD-plasma, 111 In-oxine-sulfate had low labeling efficiency and inconsistent labeling, while 111 In-tropolone and 111 In-MPO had high labeling efficiency. In vitro platelet aggregability (ADP 2μM) was reduced when platelets were labeled in the absence of plasma. However, there was no significant difference in platelet aggregability among 111 In-platelets labeled by three different chelates. In conclusion, to maintain aggregation activity of the platelets with relatively high labeling efficiency, the best result was obtained by using MPO or tropolone chelate in plasma at 4.8 x 10 6 /μl platelet concentration. (author)

Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches.

Science.gov (United States)

Saury, Charlotte; Lardenois, Aurélie; Schleder, Cindy; Leroux, Isabelle; Lieubeau, Blandine; David, Laurent; Charrier, Marine; Guével, Laëtitia; Viau, Sabrina; Delorme, Bruno; Rouger, Karl

2018-05-02

Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL). hMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3'-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL. HS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives. Our

Effects of platelet-poor plasma, platelet-rich plasma, and platelet-rich fibrin on healing of extraction sockets with buccal dehiscence in dogs.

Science.gov (United States)

Hatakeyama, Ichiro; Marukawa, Eriko; Takahashi, Yukinobu; Omura, Ken

2014-02-01

Alveolar bone resorption generally occurs during healing after tooth extraction. This study aimed to evaluate the effects of platelet-poor plasma (PPP), platelet-rich plasma (PRP), and platelet-rich fibrin (PRF) on healing in a ridge-augmentation model of the canine socket with dehiscence of the buccal wall. The third mandibular premolars of 12 beagle dogs were extracted and a 3 mm buccal dehiscence from the alveolar crest to the buccal wall of the extraction socket was created. These sockets were then divided into four groups on the basis of the material used to fill the sockets: PPP, PRP, PRF, and control (no graft material) groups. Results were evaluated at 4 and 8 weeks after surgery. The ultrastructural morphology and constructs of each blood product were studied by a scanning electron microscope (SEM) or calculating concentrations of platelets, fibrinogen, platelet-derived growth factor, and transforming growth factor-β. A total of five microcomputed tomography images of specimens were selected for measurement, and the area occupied by the newly formed bone as well as the horizontal bone width were measured. Moreover, decalcified tissue specimens from each defect were analyzed histologically. The median area of new bone at 4 and 8 weeks and median horizontal bone width at 8 weeks were the highest in the PPP group. However, bone maturation in the PRF and the PRP groups was more progressed than that in the PPP and control groups. By SEM findings, the PRF group showed a more highly condensed fibrin fiber network that was regularly arranged when compared with the PPP and PRP groups. The growth factors released from platelets in PRP indicated higher concentrations than that in PRF. Under more severe conditions for bone formation, as in this experiment, the growth factors released from platelets had a negative effect on bone formation. This study showed that PPP is an effective material for the preservation of sockets with buccal dehiscence.

Glu- and Lys-forms of plasminogen differentially affect phosphatidylserine exposure on the platelet surface

Directory of Open Access Journals (Sweden)

D. D. Zhernossekov

2017-04-01

Full Text Available Plasminogen/plasmin system is known for its ability to support hemostatic balance of blood. However, plasminogen may be considered as an adhesive ligand and in this way could affect the functioning of blood cells. We showed that exogenous Lys-plasminogen, but not its Glu-form, inhibited platelet aggregation and suppressed platelet α-granule secretion. The aim of this work was to investigate the influence of Glu- and Lys-form of plasminogen on the formation of platelet procoagulant surface using phosphatidylserine exposure as a marker. Human platelets were obtained from human platelet-rich plasma (donors were healthy volunteers, men aged 30-40 years by gel-filtration on Sepharose 2B. Phosphatidylserine exposure on the platelet surface was evaluated by flow cytometry with FITC-conjugated annexin A5. Glu- and Lys-plasminogen have different impact on the platelet functioning. Exogenous Lys-plasminogen has no significant effect on phosphatidylserine exposure, while Glu-plasminogen increases phosphatidylserine exposure on the surface of thrombin- and collagen-activated human platelets. Glu-plasminogen can be considered as a co-stimulator of agonist-induced platelet secretion and procoagulant surface formation. Meanwhile effects of Lys-plasminogen are probably directed at platelet-platelet interactions and not related to agonist-stimulated pro-apoptotic changes. The observed different effects of Glu- and Lys-plasminogen on phosphatidylserine exposure can be explained by their structural peculiarities.

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.

The Oligo Fucoidan Inhibits Platelet-Derived Growth Factor-Stimulated Proliferation of Airway Smooth Muscle Cells

Directory of Open Access Journals (Sweden)

Chao-Huei Yang

2016-01-01

Full Text Available In the pathogenesis of asthma, the proliferation of airway smooth muscle cells (ASMCs is a key factor in airway remodeling and causes airway narrowing. In addition, ASMCs are also the effector cells of airway inflammation. Fucoidan extracted from marine brown algae polysaccharides has antiviral, antioxidant, antimicrobial, anticlotting, and anticancer properties; however, its effectiveness for asthma has not been elucidated thus far. Platelet-derived growth factor (PDGF-treated primary ASMCs were cultured with or without oligo-fucoidan (100, 500, or 1000 µg/mL to evaluate its effects on cell proliferation, cell cycle, apoptosis, and Akt, ERK1/2 signaling pathway. We found that PDGF (40 ng/mL increased the proliferation of ASMCs by 2.5-fold after 48 h (p < 0.05. Oligo-fucoidan reduced the proliferation of PDGF-stimulated ASMCs by 75%–99% after 48 h (p < 0.05 and induced G1/G0 cell cycle arrest, but did not induce apoptosis. Further, oligo-fucoidan supplementation reduced PDGF-stimulated extracellular signal-regulated kinase (ERK1/2, Akt, and nuclear factor (NF-κB phosphorylation. Taken together, oligo-fucoidan supplementation might reduce proliferation of PDGF-treated ASMCs through the suppression of ERK1/2 and Akt phosphorylation and NF-κB activation. The results provide basis for future animal experiments and human trials.

Difluorothromboxane A2 and stereoisomers: Stable derivatives of thromboxane A2 with differential effects on platelets and blood vessels

International Nuclear Information System (INIS)

Morinelli, T.A.; Okwu, A.K.; Mais, D.E.; Halushka, P.V.; John, V.; Chen, Chienkuang; Fried, J.

1989-01-01

The present study reports on the selective effects on human platelets and canine saphenous veins of four stable difluorinated analogues and thromboxane A 2 (TXA 2 ), in which the characteristic 2,6-dioxa[3.1.1]bicycloheptane structure of TXA 2 has been retained. The four compounds differ in their stereochemistry of the 5,6 double bond and/or the 15-hydroxyl group. Only 10,10-difluoro-TXA 2 (compound I) with the natural stereochemistry of TXA 2 was an agonist in both platelets and canine saphenous veins. (15R)-10,10-Difluoro-TXA 2 (compound II), (5E)-10,10-difluoro-TXA 2 (compound III), and (5E,15R)-10,10-difluoro-TXA 2 (compound IV) were antagonists of platelet aggregation stimulated by compound I. However, compounds II, III, and IV stimulated contraction of canine saphenous veins. All four compounds could displace the TXA 2 /prostaglandin H 2 antagonist 9,11-dimethylmethano-11,12-methano-16-(3- 125 I-4-hydroxyphenyl)-13,14-dihydro-13-aza-15αβ-ω-tetranor-TXA 2 from its platelet receptor. These results support the existence of two subtypes of TXA 2 /prostaglandin H 2 receptors and emphasize the importance of the stereochemical requirements of these TXA 2 analogues for interaction with these receptors. These stable fluorinated TXA 2 analogues should prove useful tools for the further characterization of these and other TXA 2 /prostaglandin H 2 receptors

Ristocetin induces phosphorylated-HSP27 (HSPB1) release from the platelets of type 2 DM patients: Anti-platelet agent-effect on the release.

Science.gov (United States)

Tokuda, Haruhiko; Kuroyanagi, Gen; Onuma, Takashi; Enomoto, Yukiko; Doi, Tomoaki; Iida, Hiroki; Otsuka, Takanobu; Ogura, Shinji; Iwama, Toru; Kojima, Kumi; Kozawa, Osamu

2018-04-01

It has been previously reported that HSP27 is released from platelets in type 2 diabetes mellitus (DM) patients according to phosphorylation. In the present study, we investigated the effect of ristocetin, a glycoprotein (GP)Ib/IX/V activator, on the release of HSP27 and the effect of anti-platelet agents, such as acetylsalicylic acid (ASA), on this release. Forty-six patients with type 2 DM were recruited, and classified into two groups depending on administration of anti-platelet agents, resulting in 31 patients without these agents (control group) and 15 patients with them (anti-platelet group). Ristocetin potently induced the aggregation of platelets in the two groups. Ristocetin-induced changes of the area under the curve of light transmittance and the ratio of the size of platelet aggregates in the anti-platelet group were slightly different from those in the control group. On the other hand, the levels of phosphorylated-HSP27 induced by ristocetin in the platelets from a patient of the anti-platelet group taking ASA were significantly lower than those from a patient of the control group. The levels of HSP27 release from the ristocetin-stimulated platelets were significantly correlated with the levels of phosphorylated-HSP27 in the platelets from patients in the two groups. The levels of HSP27 release and those of platelet-derived growth factor-AB (PDGF-AB) secretion stimulated by ristocetin in the anti-platelet group were lower than those in the control group. In addition, the levels of HSP27 release and those of PDGF-AB secretion stimulated by ADP in the anti-platelet group were lower than those in the control group. These results strongly suggest that anti-platelet agents inhibit the HSP27 release from platelets stimulated by ristocetin but not the aggregation in type 2 DM patients.

The Antimicrobial Peptide Human Beta-Defensin-3 Is Induced by Platelet-Released Growth Factors in Primary Keratinocytes

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Andreas Bayer

2017-01-01

Full Text Available Platelet-released growth factors (PRGF and its related clinically used formulations (e.g., Vivostat Platelet-Rich Fibrin (PRF® contain a variety of chemokines, cytokines, and growth factors and are therefore used to support healing of chronic, hard-to-heal, or infected wounds. Human beta-defensin-3 (hBD-3 is an antimicrobial peptide inducibly expressed in human keratinocytes especially upon wounding. The potent antimicrobial activity of hBD-3 together with its wound closure-promoting activities suggests that hBD-3 may play a crucial role in wound healing. Therefore, we analyzed the influence of PRGF on hBD-3 expression in human primary keratinocytes in vitro. In addition, we investigated the influence of Vivostat PRF on hBD-3 expression in artificially generated human skin wounds in vivo. PRGF treatment of primary keratinocytes induced a significant, concentration- and time-dependent increase in hBD-3 gene expression which was partially mediated by the epidermal growth factor receptor (EGFR. In line with these cell culture data, in vivo experiments revealed an enhanced hBD-3 expression in experimentally produced human wounds after the treatment with Vivostat PRF. Thus, the induction of hBD-3 may contribute to the beneficial effects of thrombocyte concentrate lysates in the treatment of chronic or infected wounds.

The Antimicrobial Peptide Human Beta-Defensin-3 Is Induced by Platelet-Released Growth Factors in Primary Keratinocytes

Science.gov (United States)

Lammel, Justus; Tohidnezhad, Mersedeh; Lippross, Sebastian; Behrendt, Peter; Klüter, Tim; Pufe, Thomas; Cremer, Jochen; Jahr, Holger; Rademacher, Franziska; Gläser, Regine; Harder, Jürgen

2017-01-01

Platelet-released growth factors (PRGF) and its related clinically used formulations (e.g., Vivostat Platelet-Rich Fibrin (PRF®)) contain a variety of chemokines, cytokines, and growth factors and are therefore used to support healing of chronic, hard-to-heal, or infected wounds. Human beta-defensin-3 (hBD-3) is an antimicrobial peptide inducibly expressed in human keratinocytes especially upon wounding. The potent antimicrobial activity of hBD-3 together with its wound closure-promoting activities suggests that hBD-3 may play a crucial role in wound healing. Therefore, we analyzed the influence of PRGF on hBD-3 expression in human primary keratinocytes in vitro. In addition, we investigated the influence of Vivostat PRF on hBD-3 expression in artificially generated human skin wounds in vivo. PRGF treatment of primary keratinocytes induced a significant, concentration- and time-dependent increase in hBD-3 gene expression which was partially mediated by the epidermal growth factor receptor (EGFR). In line with these cell culture data, in vivo experiments revealed an enhanced hBD-3 expression in experimentally produced human wounds after the treatment with Vivostat PRF. Thus, the induction of hBD-3 may contribute to the beneficial effects of thrombocyte concentrate lysates in the treatment of chronic or infected wounds. PMID:28811680

Inhibition of platelet-derived growth factor signaling prevents muscle fiber growth during skeletal muscle hypertrophy.

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Sugg, Kristoffer B; Korn, Michael A; Sarver, Dylan C; Markworth, James F; Mendias, Christopher L

2017-03-01

The platelet-derived growth factor receptors alpha and beta (PDGFRα and PDGFRβ) mark fibroadipogenic progenitor cells/fibroblasts and pericytes in skeletal muscle, respectively. While the role that these cells play in muscle growth and development has been evaluated, it was not known whether the PDGF receptors activate signaling pathways that control transcriptional and functional changes during skeletal muscle hypertrophy. To evaluate this, we inhibited PDGFR signaling in mice subjected to a synergist ablation muscle growth procedure, and performed analyses 3 and 10 days after induction of hypertrophy. The results from this study indicate that PDGF signaling is required for fiber hypertrophy, extracellular matrix production, and angiogenesis that occur during muscle growth. © 2017 Federation of European Biochemical Societies.

21 CFR 864.7695 - Platelet factor 4 radioimmunoassay.

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2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Platelet factor 4 radioimmunoassay. 864.7695 Section 864.7695 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7695 Platelet...

A novel approach to optimal blood platelets logistics

NARCIS (Netherlands)

Smit Sibinga, C.; Dijk, van N.M.; Haijema, R.; Wal, van der J.

2006-01-01

The production and inventory management of blood platelets at blood establishments and hospital blood banks is a service problem of general human interest. Shortages or inadequate supplies may pur lives at risk and thus have to be managed and kept to a minimum. However, platelets have a limited

Release kinetics of platelet-derived and plasma-derived growth factors from autologous plasma rich in growth factors.

Science.gov (United States)

Anitua, Eduardo; Zalduendo, Mari Mar; Alkhraisat, Mohammad Hamdan; Orive, Gorka

2013-10-01

Many studies have evaluated the biological effects of platelet rich plasma reporting the final outcomes on cell and tissues. However, few studies have dealt with the kinetics of growth factor delivery by plasma rich in growth factors. Venous blood was obtained from three healthy volunteers and processed with PRGF-Endoret technology to prepare autologous plasma rich in growth factors. The gel-like fibrin scaffolds were then incubated in triplicate, in a cell culture medium to monitor the release of PDGF-AB, VEGF, HGF and IGF-I during 8 days of incubation. A leukocyte-platelet rich plasma was prepared employing the same technology and the concentrations of growth factors and interleukin-1β were determined after 24h of incubation. After each period, the medium was collected, fibrin clot was destroyed and the supernatants were stored at -80°C until analysis. The growth factor delivery is diffusion controlled with a rapid initial release by 30% of the bioactive content after 1h of incubation and a steady state release when almost 70% of the growth factor content has been delivered. Autologous fibrin matrix retained almost 30% of the amount of the growth factors after 8 days of incubation. The addition of leukocytes to the formula of platelet rich plasma did not increase the concentration of the growth factors, while it drastically increased the presence of pro-inflammatory IL-1β. Further studies employing an in vitro inflammatory model would be interesting to study the difference in growth factors and pro-inflammatory cytokines between leukocyte-free and leukocyte-rich platelet rich plasma. Copyright © 2013 Elsevier GmbH. All rights reserved.

Platelet lysate supports the in vitro expansion of human periodontal ligament stem cells for cytotherapeutic use.

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Wu, Rui-Xin; Yu, Yang; Yin, Yuan; Zhang, Xi-Yu; Gao, Li-Na; Chen, Fa-Ming

2017-08-01

Human platelet lysate (PL) produced under optimal conditions of standardization and safety has been increasingly suggested as the future 'gold standard' supplement to replace fetal bovine serum (FBS) for the ex vivo propagation of mesenchymal stem cells for translational medicine and cell therapy applications. However, the multifaceted effects of PL on tissue-specific stem cells remain largely unexplored. In the present study, we investigated the stem cell behaviours of human periodontal ligament stem cells (PDLSCs) in media with or without PL. Our data indicate that human PL, either as an adjuvant for culture media or as a substitute for FBS, supports the proliferation and expansion of human PDLSCs derived from either 'young' or 'old' donors to the same extent as FBS, without interfering with their immunomodulatory capacities. Although PL appears to inhibit the in vitro differentiation of 'young' or 'old' PDLSCs, their decreased osteogenic potential may be restored to similar or higher levels compared with FBS-expanded cells. PL- and FBS-expanded PDLSCs exhibited a similar potential to form mineralized nodules and expressed similar levels of osteogenic genes. Our data indicate that large clinically relevant quantities of PDLSCs may be yielded by the use of human PL; however, further analysis of its precise composition and function will pave the way for determining optimized, defined culture conditions. In addition to the potential increase in patient safety, our findings highlight the need for further research to develop the potential of PL-expanded PDLSCs for clinical use. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Platelet microvesicles in health and disease.

Science.gov (United States)

Melki, Imene; Tessandier, Nicolas; Zufferey, Anne; Boilard, Eric

2017-05-01

Interest in cell-derived extracellular vesicles and their physiological and pathological implications is constantly growing. Microvesicles, also known as microparticles, are small extracellular vesicles released by cells in response to activation or apoptosis. Among the different microvesicles present in the blood of healthy individuals, platelet-derived microvesicles (PMVs) are the most abundant. Their characterization has revealed a heterogeneous cargo that includes a set of adhesion molecules. Similarly to platelets, PMVs are also involved in thrombosis through support of the coagulation cascade. The levels of circulatory PMVs are altered during several disease manifestations such as coagulation disorders, rheumatoid arthritis, systemic lupus erythematosus, cancers, cardiovascular diseases, and infections, pointing to their potential contribution to disease and their development as a biomarker. This review highlights recent findings in the field of PMV research and addresses their contribution to both healthy and diseased states.

A sensitive new method of ex vivo platelet deposition

International Nuclear Information System (INIS)

Badimon, L.; Fuster, V.; Dewanjee, M.K.; Romero, J.C.

1982-01-01

In 1978, an in vivo quantitative method of platelet aggregation based on the increment of weight of a rabbit tendon when superfused with flowing blood (3 ml/min) derived from a carotid artery of a cat and reentering the contralateral jugular vein was reported. TO increase the sensitivity of the method, researches labeled platelets with indium-111 and reinjected them after two hours; then, with a gamma counter, researches quantitated the 111 In-labeled platelets deposited on the superfused rabbit tendon. Results of the radioactivity method and of the weight method were compared. Researchers found that the 111 In-labeling of platelets was more precise and reproducible method, rendering possible the use of a small amount of blood without need for reentry into the venous system

Generation and characterization of human smooth muscle cell lines derived from atherosclerotic plaque.

Science.gov (United States)

Bonin, L R; Madden, K; Shera, K; Ihle, J; Matthews, C; Aziz, S; Perez-Reyes, N; McDougall, J K; Conroy, S C

1999-03-01

The study of atherogenesis in humans has been restricted by the limited availability and brief in vitro life span of plaque smooth muscle cells (SMCs). We describe plaque SMC lines with extended life spans generated by the expression of the human papillomavirus (HPV)-16 E6 and E7 genes, which has been shown to extend the life span of normal adult human aortic SMCs. Resulting cell lines (pdSMC1A and 2) demonstrated at least 10-fold increases in life span; pdSMC1A became immortal. The SMC identity of both pdSMC lines was confirmed by SM22 mRNA expression. pdSMC2 were generally diploid but with various structural and numerical alterations; pdSMC1A demonstrated several chromosomal abnormalities, most commonly -Y, +7, -13, anomalies previously reported in both primary pdSMCs and atherosclerotic tissue. Confluent pdSMC2 appeared grossly similar to HPV-16 E6/E7-expressing normal adult aortic SMCs (AASMCs), exhibiting typical SMC morphology/growth patterns; pdSMC1A displayed irregular cell shape/organization with numerous mitotic figures. Dedifferentiation to a synthetic/proliferative phenotype has been hypothesized as a critical step in atherogenesis, because rat neonatal SMCs and adult intimal SMCs exhibit similar gene expression patterns. To confirm that our pdSMC lines likewise express this apparent plaque phenotype, osteopontin, platelet-derived growth factor B, and elastin mRNA levels were determined in pdSMC1A, pdSMC2, and AASMCs. However, no significant increases in osteopontin or platelet-derived growth factor B expression levels were observed in either pdSMC compared with AASMCs. pdSMC2 alone expressed high levels of elastin mRNA. Lower levels of SM22 mRNA in pdSMC1A suggested greater dedifferentiation and/or additional population doublings in pdSMC1A relative to pdSMC2. Both pdSMC lines (particularly 1A) demonstrated high message levels for matrix Gla protein, previously reported to be highly expressed by human neointimal SMCs in vitro. These results describe 2

Quantitation of thrombogenicity of hemodialyzer with technetium-99m and indium-111 labeled platelets

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Dewanjee, M.K.; Kapadvanjwala, Mansoor; Ruzius, Kees; Serafini, A.N.; Zilleruelo, G.E.; Sfakianakis, G.N. (Miami Univ., FL (United States). School of Medicine Althin CD-Medical Inc., Miami Lakes, FL (United States))

1993-07-01

The platelet thromobogenicity of a hemodialyzer was quantified with [sup 99m]Tc- and [sup 111]In-labeled platelets. The platelets collected from blood of Beagle dogs, Yorkshire pigs and human volunteers were labeled with [sup 111]in-tropolone (detergent-free) and [sup 99m]Tc-HMPAO. Hemodialysis was performed with a hollow-fiber dialyzer (HFD) in a flow-loop, the temperature of which was maintained at 37[sup o]C, with flow-rates of 7, 150 and 270 mL/min; after dialysis, the HFD radioactivity was measured with an ionization chamber and imaged with a [gamma]-camera. The radioactivity of samples of hollow-fibers taken from the top, middle and bottom of the dialyzer was determined with a [gamma]-counter. The mean values of hemodialyzer-adherent platelet radioactivity were calculated for both radionuclides. The canine platelets were found to be more thrombogenic than porcine and human platelets. The adhesivity of porcine platelets to the biomaterial (cellulose-acetate) of the dialyzer approximated that of human platelets. The [sup 99m]Tc label underestimated the thrombus formation (P < 0.01 ). The dynamic processes of thrombosis and embolization from the hemodialyzer resulted in the large standard deviations around the mean values of the adherent thrombus. In spite of this limitation of the dynamic pathology, the quantitation of comparative throbogenicity with [sup 111]In- and [sup 99m]Tc-labeled platelets suggests that both radionuclides could be used for measurement of device-induced thrombogenicity and may provide an estimation of prosthesis-induced thrombogenicity of human platelets from animal studies. (Author).