Atividade da enzima acetil-hidrolase do fator ativador de plaquetas (PAF-AH em pacientes com diabete melito tipo 1 Platelet-activating factor acetylhydrolase (PAF-AH activity in patients with type 1 diabetes mellitus

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Simone Henriques de Castro

2007-02-01

Full Text Available OBJETIVO: Avaliar a atividade da acetil-hidrolase do fator ativador de plaquetas (PAF-AH e sua relação com variáveis clinicodemográficas, com o controle metabólico, os níveis de apolipoproteínas A e B e a suscetibilidade da lipoproteína de baixa densidade (LDL à oxidação in vitro em pacientes com DM tipo 1 (DM 1. MÉTODOS: Foram avaliados 42 pacientes com DM1 (27 mulheres e 48 não-diabéticos (16 mulheres, pareados por sexo, idade e índice de massa corporal (IMC. Os exames realizados foram: glicemia de jejum (GJ e pós-prandial (GPP, lipidograma, ácido úrico (AU, hemoglobina glicosilada (HbA1c e coeficiente de oxidação da lipoproteína de baixa densidade (LDL por espectrofotometria. A análise da atividade da PAF-AH foi realizada por espectrofotometria (Cayman Chemical. RESULTADOS: A análise da atividade da PAF-AH mostrou haver maior atividade enzimática nos pacientes com DM 1 do que nos não-diabéticos (0,0150 ± 0,0051 versus 0,0116 ± 0,0041; p OBJECTIVE: To evaluate platelet-activating factor acetylhydrolase (PAF-AH activity and its relationship with clinical and demographic variables, metabolic control, apolipoprotein A and B levels and the susceptibility of low-density lipoprotein (LDL to in vitro oxidation in patients with type 1 diabetes mellitus (DM 1. METHODS: Forty two patients with DM 1 (27 females and 48 control subjects (16 females matched for gender, age and body mass index (BMI were evaluated. The following tests were performed: fast plasma glucose (FG and postprandial plasma glucose (PPG, lipid profile, uric acid (UA, glycosylated hemoglobin (HbA1c, and low-density lipoprotein (LDL oxidation rate using colorimetric assay. The PAF-AH activity was analyzed using colorimetric assay (Cayman Chemical. RESULTS: The analysis of PAF-AH activity showed a higher enzyme activity in patients with DM 1 than in control subjects (0.0150 ± 0.0051 vs. 0.0116 ± 0.0041; p < 0.001. In patients with DM 1, a direct correlation

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.

Platelet factor 4 impairs the anticoagulant activity of activated protein C.

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Preston, Roger J S

2012-02-01

Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

Platelet factor 4 impairs the anticoagulant activity of activated protein C.

LENUS (Irish Health Repository)

Preston, Roger J S

2009-02-27

Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

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

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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.

Platelet activating factor activity in the phospholipids of bovine spermatozoa

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Parks, J.E.; Hough, S.; Elrod, C. (Cornell Univ., Ithaca, NY (USA))

1990-11-01

Platelet activating factor (PAF) has been detected in sperm from several mammalian species and can affect sperm motility and fertilization. Because bovine sperm contain a high percentage of ether-linked phospholipid precursors required for PAF synthesis, a study was undertaken to determine the PAF activity of bovine sperm phospholipids. Total lipids of washed, ejaculated bull sperm were extracted, and phospholipids were fractionated by thin-layer chromatography. Individual phospholipid fractions were assayed for PAF activity on the basis of (3H)serotonin release from equine platelets. PAF activity was detected in the PAF fraction (1.84 pmol/mumol total phospholipid) and in serine/inositol (PS/PI), choline (CP), and ethanolamine phosphoglyceride (EP) and cardiolipin (CA) fractions. Activity was highest in the CP fraction (8.05 pmol/mumol total phospholipid). Incomplete resolution of PAF and neutral lipids may have contributed to the activity in the PS/PI and CA fractions, respectively. Phospholipids from nonsperm sources did not stimulate serotonin release. Platelet activation by purified PAF and by sperm phospholipid fractions was inhibited by the receptor antagonist SRI 63-675. These results indicate that bovine sperm contain PAF and that other sperm phospholipids, especially CP and EP, which are high in glycerylether components, are capable of receptor-mediated platelet activation.

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

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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

Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha-receptor.

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Vassbotn, F S; Havnen, O K; Heldin, C H; Holmsen, H

1994-05-13

Human platelets contain platelet-derived growth factor (PDGF) in their alpha-granules which is released during platelet exocytosis. We show by immunoprecipitation and 125I-PDGF binding experiments that human platelets have functionally active PDGF alpha-receptors, but not beta-receptors. The PDGF alpha-receptor (PDGFR-alpha) was identified as a 170-kDa glycosylated protein-tyrosine kinase as found in other cell types. Stimulation of platelets with 0.1 unit/ml thrombin resulted in a significant increase (2-5-fold) of the tyrosine phosphorylation of the PDGFR-alpha, as determined by immunoprecipitation with phosphotyrosine antiserum as well as with PDGFR-alpha antiserum. The observed thrombin-induced autophosphorylation of the PDGFR-alpha was inhibited by the addition of a neutralizing monoclonal PDGF antibody. Thus, our results suggest that the platelet PDGFR-alpha is stimulated in an autocrine manner by PDGF secreted during platelet activation. Preincubation of platelets with PDGF inhibited thrombin-induced platelet aggregation and secretion of ATP + ADP and beta-hexosaminidase. Thrombin-induced platelet aggregation was also reversed when PDGF was added 30 s after thrombin stimulation. Inhibition of the autocrine PDGF pathway during platelet activation by the PDGF antibody led to a potentiation of thrombin-induced beta-hexosaminidase secretion. Thus, the PDGFR-alpha takes part in a negative feedback regulation during platelet activation. Our demonstration of PDGF alpha-receptors on human platelets and its inhibitory function during platelet activation identifies a new possible role of PDGF in the regulation of thrombosis.

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

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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.

Staurosporine potentiates platelet activating factor stimulated phospholipase C activity in rabbit platelets but does not block desensitization by platelet activating factor

International Nuclear Information System (INIS)

Morrison, W.J.; Dhar, A.; Shukla, S.D.

1989-01-01

The possible involvement of protein kinase C activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets. PAF stimulated incorporation of 32 P into proteins and caused [ 3 H]InsP 3 levels to increase about 260% of control. These responses were compared after platelets were pretreated with either PAF, phorbol 12-myristate 13-acetate (PMA) or staurosporine and also after pretreatments with staurosporine followed by PAF or PMA. Pretreating platelets with staurosporine potentiated PAF-stimulated [ 3 H]InsP 3 levels by 54% and blocked protein phosphorylation. Pretreatments with PAF and PMA caused PAF-stimulated [ 3 H]InsP 3 levels to decrease to 115 and 136%, respectively. Staurosporine pretreatment blocked the decrease caused by the PMA pretreatment but not that by PAF. This study demonstrates that PAF-stimulated PLC activity is negatively affected by protein kinase C (PKC) activation and that inhibition of PKC activity did not prevent desensitization of PLC by PAF

Effect of montelukast on platelet activating factor- and tachykinin induced mucus secretion in the rat

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Groneberg David A

2008-02-01

Full Text Available Abstract Background Platelet activating factor and tachykinins (substance P, neurokinin A, neurokinin B are important mediators contributing to increased airway secretion in the context of different types of respiratory diseases including acute and chronic asthma. Leukotriene receptor antagonists are recommended as add-on therapy for this disease. The cys-leukotriene-1 receptor antagonist montelukast has been used in clinical asthma therapy during the last years. Besides its inhibitory action on bronchoconstriction, only little is known about its effects on airway secretions. Therefore, the aim of this study was to evaluate the effects of montelukast on platelet activating factor- and tachykinin induced tracheal secretory activity. Methods The effects of montelukast on platelet activating factor- and tachykinin induced tracheal secretory activity in the rat were assessed by quantification of secreted 35SO4 labelled mucus macromolecules using the modified Ussing chamber technique. Results Platelet activating factor potently stimulated airway secretion, which was completely inhibited by the platelet activating factor receptor antagonist WEB 2086 and montelukast. In contrast, montelukast had no effect on tachykinin induced tracheal secretory activity. Conclusion Cys-leukotriene-1 receptor antagonism by montelukast reverses the secretagogue properties of platelet activating factor to the same degree as the specific platelet activating factor antagonist WEB 2086 but has no influence on treacheal secretion elicited by tachykinins. These results suggest a role of montelukast in the signal transduction pathway of platelet activating factor induced secretory activity of the airways and may further explain the beneficial properties of cys-leukotriene-1 receptor antagonists.

Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

International Nuclear Information System (INIS)

Morrison, W.J.

1988-01-01

The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. [ 3 H]PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 μM. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRPγS and GDPβS, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA)

Platelet-activating factor podoplanin: from discovery to drug development.

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Takemoto, Ai; Miyata, Kenichi; Fujita, Naoya

2017-06-01

Tumor cell-induced platelet aggregation facilitates hematogenous metastasis by promoting tumor embolization, preventing immunological assaults and shear stress, and the platelet-releasing growth factors support tumor growth and invasion. Podoplanin, also known as Aggrus, is a type I transmembrane mucin-like glycoprotein and is expressed on wide range of tumor cells. Podoplanin has a role in platelet aggregation and metastasis formation through the binding to its platelet receptor, C-type lectin-like receptor 2 (CLEC-2). The podoplanin research was originally started from the cloning of highly metastatic NL-17 subclone from mouse colon 26 cancer cell line and from the establishment of 8F11 monoclonal antibody (mAb) that could neutralize NL-17-induced platelet aggregation and hematogenous metastasis. Later on, podoplanin was identified as the antigen of 8F11 mAb, and its ectopic expression brought to cells the platelet-aggregating abilities and hematogenous metastasis phenotypes. From the 8F11 mAb recognition epitopes, podoplanin is found to contain tandemly repeated, highly conserved motifs, designated platelet aggregation-stimulating (PLAG) domains. Series of analyses using the cells expressing the mutants and the established neutralizing anti-podoplanin mAbs uncovered that both PLAG3 and PLAG4 domains are associated with the CLEC-2 binding. The neutralizing mAbs targeting PLAG3 or PLAG4 could suppress podoplanin-induced platelet aggregation and hematogenous metastasis through inhibiting the podoplanin-CLEC-2 binding. Therefore, these domains are certainly functional in podoplanin-mediated metastasis through its platelet-aggregating activity. This review summarizes the platelet functions in metastasis formation, the role of platelet aggregation-inducing factor podoplanin in pathological and physiological situations, and the possibility to develop podoplanin-targeting drugs in the future.

Platelet-activating factor increases platelet-dependent glycoconjugate secretion from tracheal submucosal gland

International Nuclear Information System (INIS)

Sasaki, T.; Shimura, S.; Ikeda, K.; Sasaki, H.; Takishima, T.

1989-01-01

Using isolated glands from feline trachea, we examined the effect of platelet-activating factor (PAF) on radiolabeled glycoconjugate release and glandular contraction by measuring induced tension in the absence or presence of platelets. PAF alone did not produce any significant glandular contraction nor any significant change in glycoconjugate release from isolated glands. In the presence of purified platelets containing no plasma, PAF (10(-8) to 10(-5) M) produced significant glycoconjugate secretion in a dose-dependent fashion, but it produced no significant glandular contraction. PAF-evoked glycoconjugate secretion was time dependent, reaching a peak response of 277% of control 15-30 min after the exposure of isolated glands to 10(-5) M PAF in the presence of platelets and returning to 135% of controls at 2 h. Platelets alone did not produce any significant stimulation in glycoconjugate release. CV-3988, a known PAF antagonist, inhibited the secretory response to PAF. Methysergide, a known antagonist to receptors for 5-hydroxytryptamine, did not alter PAF-evoked glycoconjugate secretion. Both indomethacin and SQ 29,548, a thromboxane receptor antagonist, abolished the PAF-evoked glycoconjugate secretion from isolated submucosal glands. Epithiomethanothromboxane A2, a stable thromboxane A2 analogue, produced a significant increase in glycoconjugate secretion in a dose-dependent fashion. These findings indicate that PAF increases glycoconjugate release in the presence of platelets and that the increase is dependent on some aspect of platelet function, namely thromboxane generation

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.

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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.

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

Exercise and the platelet activator calcium chloride both influence the growth factor content of platelet-rich plasma (PRP): overlooked biochemical factors that could influence PRP treatment

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Hamilton, Bruce; Tol, Johannes L.; Knez, Wade; Chalabi, Hakim

2015-01-01

There is strong evidence that exercise affects platelet haemostasis factors, but this potential effect on growth factor concentrations in platelet-rich plasma (PRP) has never been studied. In addition, there is a paucity of studies focusing on the effects of activating agents used in conjunction

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

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

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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...

Thrombin generation by activated factor VII on platelet activated by different agonists. Extending the cell-based model of hemostasis

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Herrera Maria

2006-04-01

Full Text Available Abstract Background Platelet activation is crucial in normal hemostasis. Using a clotting system free of external tissue factor, we investigated whether activated Factor VII in combination with platelet agonists increased thrombin generation (TG in vitro. Methods and results TG was quantified by time parameters: lag time (LT and time to peak (TTP, and by amount of TG: peak of TG (PTG and area under thrombin formation curve after 35 minutes (AUC→35min in plasma from 29 healthy volunteers using the calibrated automated thrombography (CAT technique. TG parameters were measured at basal conditions and after platelet stimulation by sodium arachidonate (AA, ADP, and collagen (Col. In addition, the effects of recombinant activated FVII (rFVIIa alone or combined with the other platelet agonists on TG parameters were investigated. We found that LT and TTP were significantly decreased (p 35min were significantly increased (p 35min (but not PTG when compared to platelet rich plasma activated with agonists in the absence of rFVIIa. Conclusion Platelets activated by AA, ADP, Col or rFVIIa triggered TG. This effect was increased by combining rFVIIa with other agonists. Our intrinsic coagulation system produced a burst in TG independent of external tissue factor activity an apparent hemostatic effect with little thrombotic capacity. Thus we suggest a modification in the cell-based model of hemostasis.

Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the crosslinking of alpha 2-antiplasmin to fibrin

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Reed, G. L.; Matsueda, G. R.; Haber, E.

1992-01-01

Platelet clots resist fibrinolysis by plasminogen activators. We hypothesized that platelet factor XIII may enhance the fibrinolytic resistance of platelet-rich clots by catalyzing the crosslinking of alpha 2-antiplasmin (alpha 2AP) to fibrin. Analysis of plasma clot structure by polyacrylamide gel electrophoresis and immunoblotting revealed accelerated alpha 2AP-fibrin crosslinking in platelet-rich compared with platelet-depleted plasma clots. A similar study of clots formed with purified fibrinogen (depleted of factor XIII activity), isolated platelets, and specific factor XIII inhibitors indicated that this accelerated crosslinking was due to the catalytic activity of platelet factor XIII. Moreover, when washed platelets were aggregated by thrombin, there was evidence of platelet factor XIII-mediated crosslinking between platelet alpha 2AP and platelet fibrin(ogen). Specific inhibition (by a monoclonal antibody) of the alpha 2AP associated with washed platelet aggregates accelerated the fibrinolysis of the platelet aggregate. Thus in platelet-rich plasma clots, and in thrombin-induced platelet aggregates, platelet factor XIII actively formed alpha 2AP-fibrin crosslinks, which appeared to enhance the resistance of platelet-rich clots to fibrinolysis.

The glycoprotein Ib-IX-V complex contributes to tissue factor-independent thrombin generation by recombinant factor VIIa on the activated platelet surface

NARCIS (Netherlands)

Weeterings, Cees; de Groot, Philip G.; Adelmeijer, Jelle; Lisman, Ton

2008-01-01

Several lines of evidence suggest that recombinant factor VIIa (rFVIIa) is able to activate factor X on an activated platelet, in a tissue factor-independent manner. We hypothesized that, besides the anionic surface, a receptor on the activated platelet surface is involved in this process. Here, we

Platelet-activating factor (PAF) receptor-binding antagonist activity of Malaysian medicinal plants.

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Jantan, I; Rafi, I A A; Jalil, J

2005-01-01

Forty-nine methanol extracts of 37 species of Malaysian medicinal plants were investigated for their inhibitory effects on platelet-activating factor (PAF) binding to rabbit platelets, using 3H-PAF as a ligand. Among them, the extracts of six Zingiberaceae species (Alpinia galanga Swartz., Boesenbergia pandurata Roxb., Curcuma ochorrhiza Val., C. aeruginosa Roxb., Zingiber officinale Rosc. and Z. zerumbet Koenig.), two Cinnamomum species (C. altissimum Kosterm. and C. pubescens Kochummen.), Goniothalamus malayanus Hook. f. Momordica charantia Linn. and Piper aduncum L. are potential sources of new PAF antagonists, as they showed significant inhibitory effects with IC50 values ranging from 1.2 to 18.4 microg ml(-1).

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.

Modulation of protein C activation by histones, platelet factor 4, and heparinoids: new insights into activated protein C formation.

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Kowalska, M Anna; Zhao, Guohua; Zhai, Li; David, George; Marcus, Stephen; Krishnaswamy, Sriram; Poncz, Mortimer

2014-01-01

Histones are detrimental in late sepsis. Both activated protein C (aPC) and heparin can reverse their effect. Here, we investigated whether histones can modulate aPC generation in a manner similar to another positively charged molecule, platelet factor 4, and how heparinoids (unfractionated heparin or oxygen-desulfated unfractionated heparin with marked decrease anticoagulant activity) may modulate this effect. We measured in vitro and in vivo effects of histones, platelet factor 4, and heparinoids on aPC formation, activated partial thromboplastin time, and murine survival. In vitro, histones and platelet factor 4 both affect thrombin/thrombomodulin aPC generation following a bell-shaped curve, with a peak of >5-fold enhancement. Heparinoids shift these curves rightward. Murine aPC generation studies after infusions of histones, platelet factor 4, and heparinoids supported the in vitro data. Importantly, although unfractionated heparin and 2-O, 3-O desulfated heparin both reversed the lethality of high-dose histone infusions, only mice treated with 2-O, 3-O desulfated heparin demonstrated corrected activated partial thromboplastin times and had significant levels of aPC. Our data provide a new contextual model of how histones affect aPC generation, and how heparinoid therapy may be beneficial in sepsis. These studies provide new insights into the complex interactions controlling aPC formation and suggest a novel therapeutic interventional strategy.

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

Effect of cigarette smoke on monocyte procoagulant activity: Focus on platelet-derived brain-derived neurotrophic factor (BDNF).

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Amadio, Patrizia; Baldassarre, Damiano; Sandrini, Leonardo; Weksler, Babette B; Tremoli, Elena; Barbieri, Silvia S

2017-01-01

Cigarette smoke (CS) activates platelets, promotes vascular dysfunction, and enhances Tissue Factor (TF) expression in blood monocytes favoring pro-thrombotic states. Brain-derived neurotrophic factor (BDNF), a member of the family of neurotrophins involved in survival, growth, and maturation of neurons, is released by activated platelets (APLTs) and plays a role in the cardiovascular system. The effect of CS on circulating levels of BDNF is controversial and the function of circulating BDNF in atherothrombosis is not fully understood. Here, we have shown that human platelets, treated with an aqueous extract of CS (CSE), released BDNF in a dose-dependent manner. In addition, incubation of human monocytes with BDNF or with the supernatant of platelets activated with CSE increased TF activity by a Tropomyosin receptor kinase B (TrkB)-dependent mechanism. Finally, comparing serum and plasma samples of 12 male never smokers (NS) and 29 male active smokers (AS) we observed a significant increase in microparticle-associated TF activity (MP-TF) as well as BDNF in AS, while in serum, BDNF behaved oppositely. Taken together these findings suggest that platelet-derived BDNF is involved in the regulation of TF activity and that CS plays a role in this pathway by favoring a pro-atherothrombotic state.

Novel direct factor Xa inhibitory compounds from Tenebrio molitor with anti-platelet aggregation activity.

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Lee, Wonhwa; Kim, Mi-Ae; Park, InWha; Hwang, Jae Sam; Na, MinKyun; Bae, Jong-Sup

2017-11-01

Tenebrio molitor is an edible insect that has antimicrobial, anticancer, and antihypertensive effects. The aim of this study was to identify the unreported bioactive compounds from T. molitor larvae with inhibitory activities against factor Xa (FXa) and platelet aggregation. Isolated compounds were evaluated for their anti-FXa and anti-platelet aggregation properties by monitoring clotting time, platelet aggregation, FXa activity, and thrombus formation. A diketopiperazine (1, cyclo( L -Pro- L -Tyr)) and a phenylethanoid (2, N-acetyltyramine) were isolated and inhibited the catalytic activity of FXa in a mixed inhibition model and inhibited platelet aggregation induced by adenosine diphosphate (ADP) and U46619. They inhibited ADP- and U46619-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) and the expression of P-selectin and PAC-1 in platelets. They also improved the production of nitric oxide and inhibited the oversecretion of endothelin-1 compared to that of the ADP- or U46619-treated group. In an animal model of arterial and pulmonary thrombosis, the isolated compounds showed enhanced antithrombotic effects. They also elicited anticoagulant effects in mice. Compounds 1-2 inhibited ADP-, collagen-, or U46619-induced platelet aggregation and showed similar anti-thrombotic efficacy to rivaroxaban, a positive control. Therefore, 1-2 could serve as candidates and provide scaffolds for the development of new anti-FXa and anti-platelet drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of lecithin-cholesterol acyltransferase in the metabolism of oxidized phospholipids in plasma: studies with platelet-activating factor-acetyl hydrolase-deficient plasma.

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Subramanian, V S; Goyal, J; Miwa, M; Sugatami, J; Akiyama, M; Liu, M; Subbaiah, P V

1999-07-09

To determine the relative importance of platelet-activating factor-acetylhydrolase (PAF-AH) and lecithin-cholesterol acyltransferase (LCAT) in the hydrolysis of oxidized phosphatidylcholines (OXPCs) to lyso-phosphatidylcholine (lyso-PC), we studied the formation and metabolism of OXPCs in the plasma of normal and PAF-AH-deficient subjects. Whereas the loss of PC following oxidation was similar in the deficient and normal plasmas, the formation of lyso-PC was significantly lower, and the accumulation of OXPC was higher in the deficient plasma. Isolated LDL from the PAF-AH-deficient subjects was more susceptible to oxidation, and stimulated adhesion molecule synthesis in endothelial cells, more than the normal LDL. Oxidation of 16:0-[1-14C]-18:2 PC, equilibrated with plasma PC, resulted in the accumulation of labeled short- and long-chain OXPCs, in addition to the labeled aqueous products. The formation of the aqueous products decreased by 80%, and the accumulation of short-chain OXPC increased by 110% in the deficient plasma, compared to the normal plasma, showing that PAF-AH is predominantly involved in the hydrolysis of the truncated OXPCs. Labeled sn-2-acyl group from the long-chain OXPC was not only hydrolyzed to free fatty acid, but was preferentially transferred to diacylglycerol, in both the normal and deficient plasmas. In contrast, the acyl group from unoxidized PC was transferred only to cholesterol, showing that the specificity of LCAT is altered by OXPC. It is concluded that, while PAF-AH carries out the hydrolysis of mainly truncated OXPCs, LCAT hydrolyzes and transesterifies the long-chain OXPCs.

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

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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.

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.

Involvement of nuclear factor κB in platelet CD40 signaling

International Nuclear Information System (INIS)

Hachem, Ahmed; Yacoub, Daniel; Zaid, Younes; Mourad, Walid; Merhi, Yahye

2012-01-01

Highlights: ► sCD40L induces TRAF2 association to CD40 and NF-κB activation in platelets. ► IκBα phosphorylation downstream of CD40L/CD40 signaling is independent of p38 MAPK phosphorylation. ► IκBα is required for sCD40L-induced platelet activation and potentiation of aggregation. -- Abstract: CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-κB). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of IκBα, which are abolished by CD40L blockade. Inhibition of IκBα phosphorylation reverses sCD40L-induced IκBα phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of IκBα phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-κB activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo-inflammatory disorders.

Oxalic acid biosynthesis and oxalacetate acetylhydrolase activity in Streptomyces cattleya

International Nuclear Information System (INIS)

Houck, D.R.; Inamine, E.

1987-01-01

In addition to producing the antibiotic thienamycin, Streptomyces cattleya accumulates large amounts of oxalic acid during the course of a fermentation. Washed cell suspensions were utilized to determine the specific incorporation of carbon-14 into oxalate from a number of labeled organic and amino acids. L-[U- 14 C]aspartate proved to be the best precursor, whereas only a small percentage of label from [1,5- 14 C]citrate was found in oxalate. Cell-free extracts catalyzed the formation of [ 14 C]oxalate and [ 14 C]acetate from L-[U- 14 C]aspartate. When L-[4- 14 C]aspartate was the substrate only [ 14 C]acetate was formed. The cell-free extracts were found to contain oxalacetate acetylhydrolase, the enzyme that catalyzes the hydrolysis of oxalacetate to oxalate and acetate. The enzyme is constitutive and is analogous to enzymes in fungi that produce oxalate from oxalacetate. Properties of the crude enzyme were examined

Of von Willebrand factor and platelets.

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Bryckaert, Marijke; Rosa, Jean-Philippe; Denis, Cécile V; Lenting, Peter J

2015-01-01

Hemostasis and pathological thrombus formation are dynamic processes that require multiple adhesive receptor-ligand interactions, with blood platelets at the heart of such events. Many studies have contributed to shed light on the importance of von Willebrand factor (VWF) interaction with its platelet receptors, glycoprotein (GP) Ib-IX-V and αIIbβ3 integrin, in promoting primary platelet adhesion and aggregation following vessel injury. This review will recapitulate our current knowledge on the subject from the rheological aspect to the spatio-temporal development of thrombus formation. We will also discuss the signaling events generated by VWF/GPIb-IX-V interaction, leading to platelet activation. Additionally, we will review the growing body of evidence gathered from the recent development of pathological mouse models suggesting that VWF binding to GPIb-IX-V is a promising target in arterial and venous pathological thrombosis. Finally, the pathological aspects of VWF and its impact on platelets will be addressed.

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

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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

Platelet-activating factor synthesis and receptor-mediated signaling are downregulated in ovine newborn lungs: relevance in postnatal pulmonary adaptation and persistent pulmonary hypertension of the newborn.

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Renteria, L S; Cruz, E; Ibe, B O

2013-12-01

Platelet-activating factor (PAF) is a phospholipid with a wide range of biological activities. We studied PAF metabolism and PAF receptor (PAFR) signaling in perinatal ovine lungs to understand PAF's role in transition of the perinatal pulmonary hemodynamics and pathophysiology of persistent pulmonary hypertension of the newborn. We hypothesized that downregulation of PAF synthesis with upregulation of PAF catabolism by acetylhydrolase (PAF-Ah) in the newborn lung is needed for fetus-to-newborn pulmonary adaptation. Studies were conducted on fetal and newborn lamb pulmonary arteries (PA), veins (PV) and smooth muscle cells (SMC). PAF metabolism, PAFR binding and cell proliferation were studied by cell culture; gene expression was studied by qPCR. Fetal lungs synthesized 60% more PAF than newborn lungs. Compared with the fetal PVs and SMCs, PAF-Ah activity in newborn was 40-60% greater. PAF-Ah mRNA expression in newborn vessels was different from the expression by fetal PA. PAF-Ah gene clone activity confirmed deletion of hypoxia-sensitive site. PAFR mRNA expression by the PVs and SMC-PV of the fetus and newborn was greater than by corresponding PAs and SMC-PA. Q-PCR study of PAFR expression by the SMC-PV of both groups was greater than SMC-PA. Fetal SMCs bound more PAF than the newborn SMCs. PAFR antagonist, CV-3988, inhibited PAFR binding and DNA synthesis by the fetal SMCs, but augmented binding and DNA synthesis by newborn cells. We show different PAF-PAFR mediated effects in perinatal lungs, suggesting both transcriptional and translational regulation of PAF-Ah and PAFR expression in the perinatal lamb lungs. These indicate that the downregulation of PAF-mediated effects postnatally protects against persistent pulmonary hypertension of the newborn.

Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions.

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Biasetti, Jacopo; Sampath, Kaushik; Cortez, Angel; Azhir, Alaleh; Gilad, Assaf A; Kickler, Thomas S; Obser, Tobias; Ruggeri, Zaverio M; Katz, Joseph

2017-01-01

Tracking cells and proteins' phenotypic changes in deep suspensions is critical for the direct imaging of blood-related phenomena in in vitro replica of cardiovascular systems and blood-handling devices. This paper introduces fluorescence imaging techniques for space and time resolved detection of platelet activation, von Willebrand factor (VWF) conformational changes, and VWF-platelet interaction in deep suspensions. Labeled VWF, platelets, and VWF-platelet strands are suspended in deep cuvettes, illuminated, and imaged with a high-sensitivity EM-CCD camera, allowing detection using an exposure time of 1 ms. In-house postprocessing algorithms identify and track the moving signals. Recombinant VWF-eGFP (rVWF-eGFP) and VWF labeled with an FITC-conjugated polyclonal antibody are employed. Anti-P-Selectin FITC-conjugated antibodies and the calcium-sensitive probe Indo-1 are used to detect activated platelets. A positive correlation between the mean number of platelets detected per image and the percentage of activated platelets determined through flow cytometry is obtained, validating the technique. An increase in the number of rVWF-eGFP signals upon exposure to shear stress demonstrates the technique's ability to detect breakup of self-aggregates. VWF globular and unfolded conformations and self-aggregation are also observed. The ability to track the size and shape of VWF-platelet strands in space and time provides means to detect pro- and antithrombotic processes.

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)

In vitro effects of recombinant activated factor VII on thrombin generation and coagulation following inhibition of platelet procoagulant activity by prasugrel.

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Mazzeffi, Michael; Szlam, Fania; Jakubowski, Joseph A; Tanaka, Kenichi A; Sugidachi, Atsuhiro; Levy, Jerrold H

2013-07-01

Prasugrel is a thienopyridyl P2Y12 antagonist with potent antiplatelet effects. At present, little is known about its effects on thrombin generation or what strategies may emergently reverse its anticoagulant effects. In the current study we evaluated whether recombinant activated factor VII may reverse prasugrel induced effects and increase thrombin generation in an in vitro model. The effect of prasugrel active metabolite, PAM (R-138727), was evaluated on platelet aggregation, thrombin generation, and rotational thromboelastometry parameters using blood from 20 healthy volunteers. Additionally, we evaluated the effects of adenosine diphosphate (ADP) and recombinant activated factor VII on restoring these parameters towards baseline values. PAM reduced maximum platelet aggregation and led to platelet disaggregation. It also decreased peak thrombin, increased lag time, and increased time to peak thrombin. Treatment with recombinant activated factor VII restored all three parameters of thrombin generation towards baseline. ADP decreased lag time and time to peak thrombin, but had no effect on peak thrombin. When recombinant activated factor VII and ADP were combined they had a greater effect on thrombin parameters than either drug alone. PAM also increased thromboelastometric clotting time and clot formation time, but had no effect on maximum clot firmness. Treatment with either recombinant activated factor VII or ADP restored these values towards baseline. Recombinant activated factor VII restores thrombin generation in the presence of PAM. In patients taking prasugrel with life-threatening refractory bleeding it has the potential to be a useful therapeutic approach. Additional clinical studies are needed to validate our findings. Copyright © 2013 Elsevier Ltd. All rights reserved.

Involvement of nuclear factor {kappa}B in platelet CD40 signaling

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Hachem, Ahmed [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Yacoub, Daniel [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Centre Hospitalier Universite de Montreal, 264 boul. Rene-Levesque est, Montreal, Quebec, Canada H2X 1P1 (Canada); Zaid, Younes [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Mourad, Walid [Universite de Montreal, Department of Medicine, 2900 boul. Edouard-Montpetit, Montreal, Quebec, Canada H3T 1J4 (Canada); Centre Hospitalier Universite de Montreal, 264 boul. Rene-Levesque est, Montreal, Quebec, Canada H2X 1P1 (Canada); Merhi, Yahye, E-mail: yahye.merhi@icm-mhi.org [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Universite de Montreal, Department of Medicine, 2900 boul. Edouard-Montpetit, Montreal, Quebec, Canada H3T 1J4 (Canada)

2012-08-17

Highlights: Black-Right-Pointing-Pointer sCD40L induces TRAF2 association to CD40 and NF-{kappa}B activation in platelets. Black-Right-Pointing-Pointer I{kappa}B{alpha} phosphorylation downstream of CD40L/CD40 signaling is independent of p38 MAPK phosphorylation. Black-Right-Pointing-Pointer I{kappa}B{alpha} is required for sCD40L-induced platelet activation and potentiation of aggregation. -- Abstract: CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-{kappa}B). Given that platelets contain NF-{kappa}B, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of I{kappa}B{alpha}, which are abolished by CD40L blockade. Inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced I{kappa}B{alpha} phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on I{kappa}B{alpha} phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-{kappa}B activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo

Thrombopoietin contributes to enhanced platelet activation in cigarette smokers.

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Lupia, Enrico; Bosco, Ornella; Goffi, Alberto; Poletto, Cesare; Locatelli, Stefania; Spatola, Tiziana; Cuccurullo, Alessandra; Montrucchio, Giuseppe

2010-05-01

Thrombopoietin (TPO) is a humoral growth factor that primes platelet activation in response to several agonists. We recently showed that TPO enhances platelet activation in unstable angina and sepsis. Aim of this study was to investigate the role of TPO in platelet function abnormalities described in cigarette smokers. In a case-control study we enrolled 20 healthy cigarette smokers and 20 nonsmokers, and measured TPO and C-reactive protein (CRP), as well as platelet-leukocyte binding and P-selectin expression. In vitro we evaluated the priming activity of smoker or control plasma on platelet activation, and the role of TPO in this effect. We then studied the effects of acute smoking and smoking cessation on TPO levels and platelet activation indices. Chronic cigarette smokers had higher circulating TPO levels than nonsmoking controls, as well as increased platelet-leukocyte binding, P-selectin expression, and CRP levels. Serum cotinine concentrations correlated with TPO concentrations, platelet-monocyte aggregates and P-selectin expression. In addition, TPO levels significantly correlated with ex vivo platelet-monocyte aggregation and P-selectin expression. In vitro, the plasma from cigarette smokers, but not from nonsmoking controls, primed platelet-monocyte binding, which was reduced when an inhibitor of TPO was used. We also found that acute smoking slightly increased TPO levels, but did not affect platelet-leukocyte binding, whereas smoking cessation induced a significant decrease in both circulating TPO and platelet-leukocyte aggregation. Elevated TPO contributes to enhance platelet activation and platelet-monocyte cross-talk in cigarette smokers. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

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

The measurement of platelet activation by radioimmunoassay in asthma

International Nuclear Information System (INIS)

Wu Guoxin; Sun Jian; Li Jianyong; Ruan Changgeng

1992-02-01

Radioimmunoassay with specific monoclonal antibody was used to evaluate the platelet activation in 14 cases of acute bronchial asthma. The result showed that the number of molecules of alpha-granule membrane protein (GMP-140) which was exposed on the surface of platelet following secretion significantly increased on the surface of platelet and in plasma, while the number of molecules of glycoprotein (GP) I b and GPIII a did not change significantly; the concentration of thromboxane B 2 in plasma was raised, while the concentration of 6-keto-PGF 1a was within the normal limits; the concentrations of β-thromboglobulin (β-TG) and platelet factor 4(PF 4 ) in plasma increased significantly; the number of platelets decreased. These results strongly confirmed that the degree of platelet activation was enhanced during acute asthmatic attack. The significance of platelet activation in the pathogenesis of asthma should be further investigated

Platelet-rich preparations to improve healing. Part II: platelet activation and enrichment, leukocyte inclusion, and other selection criteria.

Science.gov (United States)

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

2014-08-01

Multiple platelet-rich preparations have been reported to improve wound and bone healing, such as platelet-rich plasma (PRP) and platelet rich fibrin (PRF). The different methods employed during their preparation are important, as they influence the quality of the product applied to a wound or surgical site. Besides the general protocol for preparing the platelet-rich product (discussed in Part 1 of this review), multiple choices need to be considered during its preparation. For example, activation of the platelets is required for the release and enmeshment of growth factors, but the method of activation may influence the resulting matrix, growth factor availability, and healing. Additionally, some methods enrich leukocytes as well as platelets, but others are designed to be leukocyte-poor. Leukocytes have many important roles in healing and their inclusion in PRP results in increased platelet concentrations. Platelet and growth factor enrichment reported for the different types of platelet-rich preparations are also compared. Generally, TGF-β1 and PDGF levels were higher in preparations that contain leukocytes compared to leukocyte-poor PRP. However, platelet concentration may be the most reliable criterion for comparing different preparations. These and other criteria are described to help guide dental and medical professionals, in large and small practices, in selecting the best procedures for their patients. The healing benefits of platelet-rich preparations along with the low risk and availability of simple preparation procedures should encourage more clinicians to incorporate platelet-rich products in their practice to accelerate healing, reduce adverse events, and improve patient outcomes.

Platelet activation and inflammation markers as emerging risk ...

African Journals Online (AJOL)

Platelet activation and inflammation markers were assessed by measuring plasma levels of sP-selectin, platelet factor 4 (PF4), IL-6 and tumor necrosis factor alpha (TNF-α). Results: HIV infected patients had higher levels of sP- selectin, PF4 and IL-6 than uninfected controls (p<0.001). ART naïve subjects had higher levels ...

Platelet-released growth factors can accelerate tenocyte proliferation and activate the anti-oxidant response element.

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Tohidnezhad, M; Varoga, D; Wruck, C J; Brandenburg, L O; Seekamp, A; Shakibaei, M; Sönmez, T T; Pufe, Thomas; Lippross, S

2011-05-01

Little is know about the pathophysiology of acute and degenerative tendon injuries. Although most lesions are uncomplicated, treatment is long and unsatisfactory in a considerable number of cases. Besides the common growth factors that were shown to be relevant for tendon integrity more recently protection against oxidative stress was shown to promote tendon healing. To improve tendon regeneration, many have advocated the use of platelet-rich plasma (PRP), a thrombocyte concentrate that can serve as an autologous source of growth factors. In this study, we investigated the effect of platelet-released growth factors (PRGF) on tenocytes. Tenocytes were isolated from the Achilles tendon of postnatal rats. Tenocyte cell cultures were stimulated with PRGF. We used a CyQuant assay and WST assay to analyse tendon cell growth and viability in different concentrations of PRGF. Migration and proliferation of cells grown in PRGF were assessed by a scratch test. A dual-luciferase assay was used to demonstrate the activation of the anti-oxidant response element (ARE) in tenocytes. A positive effect of PRGF could be shown on tendon cell growth and migratory capacity. PRGF activated the Nrf2-ARE pathway in a dose-dependent manner. Here, we provide evidence of a biological effect of PRGF on tenocytes by the promotion of tenocyte growth and activation of the Nrf2-ARE pathway. This is a novel aspect of the action of platelet concentrates on tendon growth.

Purification of human platelet-derived growth factor

International Nuclear Information System (INIS)

Raines, E.W.; Ross, R.

1985-01-01

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

Platelets and the innate immune system: mechanisms of bacterial-induced platelet activation.

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Cox, D; Kerrigan, S W; Watson, S P

2011-06-01

It has become clear that platelets are not simply cell fragments that plug the leak in a damaged blood vessel; they are, in fact, also key components in the innate immune system, which is supported by the presence of Toll-like receptors (TLRs) on platelets. As the cells that respond first to a site of injury, they are well placed to direct the immune response to deal with any resulting exposure to pathogens. The response is triggered by bacteria binding to platelets, which usually triggers platelet activation and the secretion of antimicrobial peptides. The main platelet receptors that mediate these interactions are glycoprotein (GP)IIb-IIIa, GPIbα, FcγRIIa, complement receptors, and TLRs. This process may involve direct interactions between bacterial proteins and the receptors, or can be mediated by plasma proteins such as fibrinogen, von Willebrand factor, complement, and IgG. Here, we review the variety of interactions between platelets and bacteria, and look at the potential for inhibiting these interactions in diseases such as infective endocarditis and sepsis. © 2011 International Society on Thrombosis and Haemostasis.

Do methodological differences account for the current controversy on tissue factor expression in platelets?

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Brambilla, Marta; Rossetti, Laura; Zara, Chiara; Canzano, Paola; Giesen, Peter L A; Tremoli, Elena; Camera, Marina

2018-06-01

Tissue factor (TF), the key activator of the blood coagulation cascade and of thrombus formation, is also expressed by circulating human platelets. Despite the documented in-depth characterization of platelet TF carried out in the past 15 years, some authors still fail to identify TF in platelets, especially when assessment in platelet-rich plasma (PRP) or washed platelets is carried out. This study aims to extend the characterization of the subset of TF-positive platelets in PRP from healthy subjects and to verify how different centrifugation forces, used to prepare the PRP, could affect the analysis of TF-positive platelets. Data indicate that large-size platelets express significantly higher amount of TF compared to small-size cells, in terms of both TF protein and TF mRNA. Upon stimulation, large platelets readily expose on the cell membrane TF, which is functionally active, i.e., able to generate factor Xa (FXa) as well as thrombin. By contrast, TF activity in small platelets is almost completely quenched by tissue factor pathway inhibitor (TFPI), becoming indeed detectable only after treatment with an anti-TFPI antibody. Our data highlight that particular attention must be paid to the preparation and collection of the PRP since such preanalytical variables may influence the platelet recovery and in turn affect subsequent analysis, whether it is flow cytometry, functional activity tests, proteome, or transcriptome analysis. Indeed, the TF-positive subset of large platelets can easily be lost if centrifugation protocols are not optimized, thus erroneously leading to a false-negative result.

Effect of Atorvastatin intensive therapy on the serum inflammatory factors, platelet activity and fibrinolytic activity in patients with acute coronary syndrome

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Xiao-Li Zhu

2016-05-01

Full Text Available Objective: To observe the effect of Atorvastatin intensive therapy on the serum inflammatory factors, platelet activity and fibrinolytic activity in patients with acute coronary syndrome (ACS. Methods: A total of 92 patients with ACS were randomly divided into observation group (47 cases and control group (45 cases. The control group was given Atorvastatin (10mg/d based on the conventional therapy, while the observation group was given Atorvastatin at an intensive dose (40 mg/d based on the conventional therapy. Half a month later, the changes of IL-6, IL-8, hs-CRP, TNF-α, TXB2, GMP-140, PAI-1 and t-PA were observed and compared between the two groups. Results: After treatment, the inflammatory factors (IL-6, IL-8, hs-CRP and TNF-α and the indicators of platelet activity (TXB2, GMP-140 and PAI-1 were obviously decreased, while the indicator of fibrinolytic activity (t-PA was apparently increased in the two groups. Besides, the amplitudes of change referring to these indicators in the observation group were bigger than those in the control group after treatment, and the differences were statistically significant. Conclusion: The intensive therapy with the administration of Atorvastatin at a dose of 40 mg/d was better than the conventional therapy (Atorvastatin: 10 mg/d in aspects of reducing inflammatory factors, inhibiting platelet activity and correcting the high coagulation state of fibrinolytic system.

Deletion of Crry and DAF on murine platelets stimulates thrombopoiesis and increases factor H-dependent resistance of peripheral platelets to complement attack.

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Barata, Lidia; Miwa, Takashi; Sato, Sayaka; Kim, David; Mohammed, Imran; Song, Wen-Chao

2013-03-15

Complement receptor 1-related gene/protein y (Crry) and decay-accelerating factor (DAF) are two murine membrane C3 complement regulators with overlapping functions. Crry deletion is embryonically lethal whereas DAF-deficient mice are generally healthy. Crry(-/-)DAF(-/-) mice were viable on a C3(-/-) background, but platelets from such mice were rapidly destroyed when transfused into C3-sufficient mice. In this study, we used the cre-lox system to delete platelet Crry in DAF(-/-) mice and studied Crry/DAF-deficient platelet development in vivo. Rather than displaying thrombocytopenia, Pf4-Cre(+)-Crry(flox/flox) mice had normal platelet counts and their peripheral platelets were resistant to complement attack. However, chimera mice generated with Pf4-Cre(+)-Crry(flox/flox) bone marrows showed platelets from C3(-/-) but not C3(+/+) recipients to be sensitive to complement activation, suggesting that circulating platelets in Pf4-Cre(+)-Crry(flox/flox) mice were naturally selected in a complement-sufficient environment. Notably, Pf4-Cre(+)-Crry(flox/flox) mouse platelets became complement susceptible when factor H function was blocked. Examination of Pf4-Cre(+)-Crry(flox/flox) mouse bone marrows revealed exceedingly active thrombopoiesis. Thus, under in vivo conditions, Crry/DAF deficiency on platelets led to abnormal platelet turnover, but peripheral platelet count was compensated for by increased thrombopoiesis. Selective survival of Crry/DAF-deficient platelets aided by factor H protection and compensatory thrombopoiesis demonstrates the cooperation between membrane and fluid phase complement inhibitors and the body's ability to adaptively respond to complement regulator deficiencies.

Characterization of cutaneous vascular permeability induced by platelet-activating factor in guinea pigs and rats and its inhibition by a platelet-activating factor receptor antagonist

International Nuclear Information System (INIS)

Hwang, S.B.; Li, C.L.; Lam, M.H.; Shen, T.Y.

1985-01-01

Mechanisms of platelet-activating factor (PAF)-induced increases of cutaneous vascular permeability in guinea pigs and in rats were further explored. PAF so far is the most potent vasoactive mediator, being more than 1000-fold more potent than histamine and bradykinin in both species. In guinea pigs, there is a time delay of 5 to 10 minutes before PAF action, whereas, in the rat, the increased vasopermeability occurs immediately following the intradermal PAF injection. Relative vasoactive potencies of PAF and several structure-related analogues in both species correlate very well with their relative inhibition of the binding of 3 H-PAF to specific receptor sites on isolated rabbit platelet plasma membranes and their aggregatory abilities of rabbit platelets. Furthermore, the PAF-induced cutaneous vascular permeability is inhibitable by a competitive specific PAF receptor antagonist, kadsurenone, suggesting that binding of PAF to its specific receptor site is the first step to initiate its action of increased cutaneous vascular permeability. Several pure cyclooxygenase inhibitors, including indomethacin, diflunisal, and flurbiprofen, and the dual cyclooxygenase/lipoxygenase inhibitor, BW755C, but not the histamine antagonists, inhibit the PAF-induced vasopermeability in guinea pigs. The inhibition by indomethacin or BW755C can be fully reversed by coinjection intradermally with PAF and prostaglandin E1 but not leukotriene B4. Also, prostaglandin E1 but not leukotriene B4 enhances the guinea pig in vivo response to PAF in this model. However, in rats, none of the cyclooxygenase inhibitors, histamine antagonists, or BW755C inhibit the PAF effect of cutaneous phenomena

LDL oxidation by platelets propagates platelet activation via an oxidative stress-mediated mechanism.

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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.

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.

Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: Studies with genistein and monoclonal antibody to phosphotyrosine

International Nuclear Information System (INIS)

Dhar, A.; Paul, A.K.; Shukla, S.D.

1990-01-01

Platelet-activating factor (PAF) is a proinflammatory lipid that has platelet-stimulating property. PAF receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) and phosphorylation of several proteins has already been established in our laboratory. To investigate further the molecular mechanism and relationship between activation of PLC and protein phosphorylation, we have used Genistein (a putative inhibitor of tyrosine-specific protein kinases), phosphotyrosine antibody, and phosphoamino acid analysis to probe the involvement of tyrosine kinase in this process. Washed rabbit platelets were loaded with myo-[2-3H]inositol and challenged with PAF (100 nM) after pretreatment with Genistein. PLC-mediated production of radioactive inositol monophosphate, inositol diphosphate, and inositol triphosphate was monitored. PAF alone caused stimulation of PLC activity [( 3H]inositol triphosphate production), whereas pretreatment with Genistein (0.5 mM) diminished PAF-stimulated PLC activity to basal level. Genistein also blocked PAF-stimulated platelet aggregation at this dose. In contrast to Genistein, staurosporine which inhibits protein kinase C, potentiated PAF-stimulated [3H]inositol triphosphate production. Genistein substantially inhibited the combined effects of staurosporine and PAF on inositol triphosphate production. Genistein also reduced PAF-induced phosphorylation of Mr 20,000 and 50,000 proteins. Phorbol 12-myristate 13-acetate-induced Mr 40,000 protein phosphorylation was also affected by Genistein. The above results suggested that Genistein inhibited tyrosine kinase at an early stage of signal transduction by inhibiting PLC. This, in turn, decreased the activation of protein kinase C and, therefore, caused a reduction in Mr 40,000 protein phosphorylation

Thrombopoietin contributes to enhanced platelet activation in patients with unstable angina.

Science.gov (United States)

Lupia, Enrico; Bosco, Ornella; Bergerone, Serena; Dondi, Anna Erna; Goffi, Alberto; Oliaro, Elena; Cordero, Marco; Del Sorbo, Lorenzo; Trevi, Giampaolo; Montrucchio, Giuseppe

2006-12-05

We sought to investigate the potential role of elevated levels of thrombopoietin (TPO) in platelet activation during unstable angina (UA). Thrombopoietin is a humoral growth factor that does not induce platelet aggregation per se, but primes platelet activation in response to several agonists. No data concerning its contribution to platelet function abnormalities described in patients with UA are available. We studied 15 patients with UA and, as controls, 15 patients with stable angina (SA) and 15 healthy subjects. We measured TPO and C-reactive protein (CRP), as well as monocyte-platelet binding and the platelet expression of P-selectin and of the TPO receptor, c-Mpl. The priming activity of patient or control plasma on platelet aggregation and monocyte-platelet binding and the role of TPO in this effect also were studied. Patients with UA showed higher circulating TPO levels, as well as increased monocyte-platelet binding, platelet P-selectin expression, and CRP levels, than those with SA and healthy control subjects. The UA patients also showed reduced platelet expression of the TPO receptor, c-Mpl. In vitro, the plasma from UA patients, but not from SA patients or healthy controls, primed platelet aggregation and monocyte-platelet binding, which were both reduced when an inhibitor of TPO was used. Thrombopoietin may enhance platelet activation in the early phases of UA, potentially participating in the pathogenesis of acute coronary syndromes.

Platelet-activating factor and group I metabotropic glutamate receptors interact for full development and maintenance of long-term potentiation in the rat medial vestibular nuclei.

Science.gov (United States)

Grassi, S; Francescangeli, E; Goracci, G; Pettorossi, V E

1999-01-01

In rat brainstem slices, we investigated the interaction between platelet-activating factor and group I metabotropic glutamate receptors in mediating long-term potentiation within the medial vestibular nuclei. We analysed the N1 field potential wave evoked in the ventral portion of the medial vestibular nuclei by primary vestibular afferent stimulation. The group I metabotropic glutamate receptor antagonist, (R,S)-1-aminoindan-1,5-dicarboxylic acid, prevented long-term potentiation induced by a platelet-activating factor analogue [1-O-hexadecyl-2-O-(methylcarbamyl)-sn-glycero-3-phosphocholine], as well as the full development of potentiation, induced by high-frequency stimulation under the blocking agent for synaptosomal platelet-activating factor receptors (ginkolide B), at drug washout. However, potentiation directly induced by the group I glutamate metabotropic receptor agonist, (R,S)-3,5-dihydroxyphenylglycine, was reduced by ginkolide B. These findings suggest that platelet-activating factor, whether exogenous or released following potentiation induction, exerts its effect through presynaptic group I metabotropic glutamate receptors, mediating the increase of glutamate release. In addition, we found that this mechanism, which led to full potentiation through presynaptic group I metabotropic glutamate receptor activation, was inactivated soon after application of potentiation-inducing stimulus. In fact, the long-lasting block of the platelet-activating factor and metabotropic glutamate receptors prevented the full potentiation development and the induced potentiation progressively declined to null. Moreover, ginkolide B, given when high-frequency-dependent potentiation was established, only reduced it within 5 min after potentiation induction. We conclude that to fully develop vestibular long-term potentiation requires presynaptic events. Platelet-activating factor, released after the activation of postsynaptic mechanisms which induce potentiation, is necessary

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.

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

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

Platelet activation in outpatients undergoing esophagogastroduodenoscopy

International Nuclear Information System (INIS)

Sagripanti, A.; Polloni, A.; Materazzi, F.; Ferdeghini, M.; Pinori, E.; Bianchi, R.

1989-01-01

To evaluate the influence of emotional stress on platelet function mesured by radioimmunoassay in plasma two platelet factor 4, in a series of outpatients undergoing esophagogastroduodenoscopy for upper digestive complaints has been measured. The plasma levels of β-thromboglobulin and platelet factor 4, determined just before the instrumental examination, were significantly more elevated as compared to basal values, checked a week later. These results provide evidence of enhanced in vivo platelet release reaction during emotional stress

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.

Glucose Transporter 3 Potentiates Degranulation and Is Required for Platelet Activation.

Science.gov (United States)

Fidler, Trevor P; Middleton, Elizabeth A; Rowley, Jesse W; Boudreau, Luc H; Campbell, Robert A; Souvenir, Rhonda; Funari, Trevor; Tessandier, Nicolas; Boilard, Eric; Weyrich, Andrew S; Abel, E Dale

2017-09-01

On activation, platelets increase glucose uptake, glycolysis, and glucose oxidation and consume stored glycogen. This correlation between glucose metabolism and platelet function is not well understood and even less is known about the role of glucose metabolism on platelet function in vivo. For glucose to enter a cell, it must be transported through glucose transporters. Here we evaluate the contribution of GLUT3 (glucose transporter 3) to platelet function to better understand glucose metabolism in platelets. Platelet-specific knockout of GLUT3 was generated by crossing mice harboring GLUT3 floxed allele to a PF4 (platelet factor 4)-driven Cre recombinase. In platelets, GLUT3 is localized primarily on α-granule membranes and under basal conditions facilitates glucose uptake into α-granules to be used for glycolysis. After activation, platelets degranulate and GLUT3 translocates to the plasma membrane, which is responsible for activation-mediated increased glucose uptake. In vivo, loss of GLUT3 in platelets increased survival in a collagen/epinephrine model of pulmonary embolism, and in a K/BxN model of autoimmune inflammatory disease, platelet-specific GLUT3 knockout mice display decreased disease progression. Mechanistically, loss of GLUT3 decreased platelet degranulation, spreading, and clot retraction. Decreased α-granule degranulation is due in part to an impaired ability of GLUT3 to potentiate exocytosis. GLUT3-mediated glucose utilization and glycogenolysis in platelets promotes α-granule release, platelet activation, and postactivation functions. © 2017 American Heart Association, Inc.

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.

Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation.

Science.gov (United States)

Binsker, Ulrike; Palankar, Raghavendra; Wesche, Jan; Kohler, Thomas P; Prucha, Josephine; Burchhardt, Gerhard; Rohde, Manfred; Schmidt, Frank; Bröker, Barbara M; Mamat, Uwe; Pané-Farré, Jan; Graf, Anica; Ebner, Patrick; Greinacher, Andreas; Hammerschmidt, Sven

2018-04-01

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection. Schattauer GmbH Stuttgart.

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

Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors.

Science.gov (United States)

Amable, Paola Romina; Carias, Rosana Bizon Vieira; Teixeira, Marcus Vinicius Telles; da Cruz Pacheco, Italo; Corrêa do Amaral, Ronaldo José Farias; Granjeiro, José Mauro; Borojevic, Radovan

2013-06-07

Platelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed. PRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure. Our method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 10(6) to 1.9 × 10(6) platelets/μl). Platelets were highly purified, because only platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of several growth factors, including platelet-derived growth factor and TGF. Our study resulted in a consistent PRP preparation method that yielded a cytokine and growth factor pool

Influence of cardiopulmonary bypass on the interaction of recombinant factor VIIa with activated platelets

DEFF Research Database (Denmark)

Kjalke, M.; Runge, M.; Rojkjaer, R.

2009-01-01

Recombinant factor VIIa (rFVIIa) interacts preferentially with coated platelets characterized by a high exposure of phosphatidyl serine (PS), FV, FVIII, FIX, and FX binding, and fibrinogen. Cardiopulmonary bypass (CPB) is known to impair platelet function. In this study, the influence of CPB...

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors

Science.gov (United States)

2013-01-01

Introduction Platelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed. Methods PRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure. Results Our method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 106 to 1.9 × 106 platelets/μl). Platelets were highly purified, because only blood cells and leukocytes was present in the final PRP preparation. We also quantified growth factors, cytokines and chemokines secreted by the concentrated platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of

Potential fluid mechanic pathways of platelet activation

OpenAIRE

Shadden, Shawn C.; Hendabadi, Sahar

2012-01-01

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 ...

Observations on human smooth muscle cell cultures from hyperplastic lesions of prosthetic bypass grafts: Production of a platelet-derived growth factor-like mitogen and expression of a gene for a platelet-derived growth factor receptor--a preliminary study

International Nuclear Information System (INIS)

Birinyi, L.K.; Warner, S.J.; Salomon, R.N.; Callow, A.D.; Libby, P.

1989-01-01

Prosthetic bypass grafts placed to the distal lower extremity often fail because of an occlusive tissue response in the perianastomotic region. The origin of the cells that comprise this occlusive lesion and the causes of the cellular proliferation are not known. To increase our understanding of this process we cultured cells from hyperplastic lesions obtained from patients at the time of reexploration for lower extremity graft failure, and we studied their identity and growth factor production in tissue culture. These cultures contain cells that express muscle-specific actin isoforms, shown by immunohistochemical staining, consistent with vascular smooth muscle origin. These cultures also released material that stimulated smooth muscle cell growth. A portion of this activity was similar to platelet-derived growth factor, since preincubation with antibody-to-human platelet-derived growth factor partially blocked the mitogenic effect of medium conditioned by human anastomotic hyperplastic cells. These conditioned media also contained material that competed with platelet-derived growth factor for its receptor, as measured in a radioreceptor assay. Northern blot analysis showed that these cells contain messenger RNA that encodes the A chain but not the B chain of platelet-derived growth factor. In addition, these cells contain messenger RNA that encodes a platelet-derived growth factor receptor. We conclude that cultured smooth muscle cells from human anastomotic hyperplastic lesions express genes for platelet-derived growth factor A chain and a platelet-derived growth factor receptor and secrete biologically active molecules similar to platelet-derived growth factor

Changes of platelet GMP-140 in diabetic nephropathy and its multi-factor regression analysis

International Nuclear Information System (INIS)

Wang Zizheng; Du Tongxin; Wang Shukui

2001-01-01

The relation of platelet GMP-140 and its related factors with diabetic nephropathy was studied. 144 patients of diabetic mellitus without nephropathy (group without DN, mean suffering duration of 25.5 +- 18.6 months); 80 with diabetic nephropathy (group DN, mean suffering duration of 58.7 +- 31.6 months) and 50 normal controls were chosen in the research. Platelet GMP-140, plasma α 1 -MG, β 2 -MG, and 24 hour urine albumin (ALB), IgG, α 1 -MG, β 2 -MG were detected by RIA, while HBA 1 C via chromatographic separation and FBG, PBG, Ch, TG, HDL, FG via biochemical methods. All the data had been processed with software on computer with t-test and linear regression, and multi-factor analysis were done also. The levels of platelet GMP-140, FG, DBP, TG, HBA 1 C and PBG in group DN were significantly higher than those of group without DN and normal control (P 0.05), while they were higher than those of normal controls. Multi-factor analysis of platelet GMP-140 with TG, DBP and HBA 1 C were performed in 80 patients with DN (P 1 C are the independent factors enhancing the activation of platelets. The disturbance of lipid metabolism in type II diabetic mellitus may also enhance the activation of platelets. Elevation of blood pressure may accelerate the initiation and deterioration of DN in which change of platelet GMP-140 is an independent factor. Elevation of HBA 1 C and blood glucose are related closely to the diabetic nephropathy

Platelet-activating factor receptor agonists mediate xeroderma pigmentosum A photosensitivity.

Science.gov (United States)

Yao, Yongxue; Harrison, Kathleen A; Al-Hassani, Mohammed; Murphy, Robert C; Rezania, Samin; Konger, Raymond L; Travers, Jeffrey B

2012-03-16

To date, oxidized glycerophosphocholines (Ox-GPCs) with platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPCs play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein xeroderma pigmentosum type A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor ultraviolet B (UVB) radiation resulted in increased reactive oxygen species and PAF receptor (PAF-R) agonistic activity in comparison with gene-corrected cells. The UVB irradiation-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison with control mice. The increased UVB irradiation-mediated skin inflammation and TNF-α production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R-stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chain Ox-GPCs along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPCs in the pathophysiology of XPA photosensitivity.

Platelet activation in pregnancy-induced hypertension.

Science.gov (United States)

Karalis, Ioannis; Nadar, Sunil K; Al Yemeni, Eman; Blann, Andrew D; Lip, Gregory Y H

2005-01-01

Although excess platelet activation, as indicated by increased plasma beta thromboglobulin (beta-TG), has been shown in pregnancy-induced hypertension (PIH), platelet adhesion, platelet morphology and a comparison of platelet and soluble (plasma) levels of the adhesion molecules P-selectin (pPsel and sPsel, respectively) have not been studied. We conducted a cross-sectional study of 35 consecutive women with PIH (age 31+/-6 years), 31 consecutive women with normotensive pregnancies (age 29+/-5 years) and 30 normotensive non pregnant women (age 30+/-5 years). Platelet adhesion was studied in vitro by binding to fibrinogen-coated microwells, platelet morphology [mass and volume by flow cytometry], whole-platelet P-selectin (pPsel) by ELISA of the lysate of 2 x 10(8) cells, and the plasma markers soluble P-selectin (sP-sel) and beta-TG, by ELISA. The women with PIH had significantly raised sPsel, pPsel and (as expected) beta-TG (all p<0.05), when compared to the normotensive pregnant women and controls. However, in PIH platelet adhesion was similar to that in the normotensive pregnancy, but still higher than the normal controls (p<0.001). There was no difference among the three groups with respect to platelet mass and volume. pPsel and platelet adhesion correlated with gestational age and with systolic and diastolic blood pressure (all p<0.05). Increased platelet activation and adhesion develop during normal pregnancy, with some indices being further altered in PIH.

Platelet activation during preparation of platelet concentrates: a comparison of the platelet-rich plasma and the buffy coat methods

NARCIS (Netherlands)

Fijnheer, R.; Pietersz, R. N.; de Korte, D.; Gouwerok, C. W.; Dekker, W. J.; Reesink, H. W.; Roos, D.

1990-01-01

The activation of platelets during the preparation of platelet concentrates (PCs) by two methods was compared. To eliminate interdonor differences, 2 units of whole blood were pooled and subsequently divided into two batches. From one batch, the platelets were harvested as pelleted platelets from

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

Science.gov (United States)

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.

Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia.

Science.gov (United States)

Żmigrodzka, M; Guzera, M; Winnicka, A

2016-01-01

Platelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/l and group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogs in vitro varied depending on the platelet count and platelet activator.

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.

Lexipafant (BB-882), a platelet activating factor receptor antagonist, ameliorates mucosal inflammation in an animal model of colitis

NARCIS (Netherlands)

Meenan, J.; Grool, T. A.; Hommes, D. W.; Dijkhuizen, S.; ten Kate, F. J.; Wood, M.; Whittaker, M.; Tytgat, G. N.; van Deventer, S. J.

1996-01-01

To assess the anti-inflammatory action of lexipafant (BB-882), a platelet activating factor antagonist, in an animal model of acute colitis. An animal intervention study. Following the rectal instillation of formalin 0.75% into male New Zealand White (NZW) rabbits, 0.85 ml of aggregated

Effect of twenty minutes of aerobic exercise on in vivo platelet release in moderately trained females: radioimmunoassay of platelet factor 4 and beta-thromboglobulin

International Nuclear Information System (INIS)

Rudmann, S.V.

1986-01-01

Circulating blood platelets serve an important role in the physiological process of hemostasis. Physical exercise has been documented to result in alterations in many hemostatic parameters including platelet size, number and function. Most published research data support the hypotheses that both hemostasis and fibrinolysis become activated as a consequence of various levels of physical exercise. The purpose of this study was to determine the effect of twenty minutes aerobic exercise on platelet activation in vivo. Platelet activation in vivo is associated with the release of platelet granular contents. Platelet alpha granules contain two platelet specific proteins: platelet factor 4 (PF4) and beta-thromboglobulin (BTG). Elevated plasma levels of these proteins are a specific marker of in vivo platelet activation. Subjects were moderately trained female volunteers between the ages of 22 and 40 years. Subjects were exercised or twenty minutes on a bicycle ergometer at workloads that represented 65 to 75% of their functional capacity. Blood specimens were drawn within five minutes of exercise. Plasma samples from exercise and control subjects were assayed for PF4 and BTG using a sensitive competitive-binding radioimmunoassay procedure. The mean plasma levels of both proteins were significantly greater in the exercising subjects when compared with the non-exercising controls. Data from this study support the following research hypotheses: BTG plasma levels will be significantly higher in exercising subjects than in non-exercising controls, and PF4 plasma levels will be significantly higher in exercising subjects than in non-exercising controls

Does bipolar pacemaker current activate blood platelets?

DEFF Research Database (Denmark)

Gjesdal, Grunde; Hansen, Annebirthe Bo; Brandes, Axel

2009-01-01

OBJECTIVE: The aim of this study was to investigate whether bipolar pacemaker current lead can activate blood platelets. The null hypothesis was that 1 minute of electrical stimulation of platelets would not influence their subsequent reactivity to adenosine diphosphate (ADP). BACKGROUND: Both...... platelets and muscle cells contain actin and myosin filaments, and both cells are activated following calcium influx. Muscle cells open their calcium channels and contract when exposed to an electric current. Current through a bipolar pacemaker lead will expose a small volume of blood, including platelets......, to the depolarizing current. Platelet activation may ensue, resulting in aggregation, release reaction, and contraction. In contrast, a unipolar pacemaker system will not depolarize blood, but transmit current directly into the myocardium, and the current afterward passes through other tissues before returning...

Semi-synthetic preparation of 1-O-[1'-14C]hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) using plant cell cultures

International Nuclear Information System (INIS)

Weber, N.; Mangold, H.K.

1985-01-01

Incubation of photomixotrophic cell suspension cultures of rape (Brassica napus) and heterotrophic cell suspension cultures of soya (Glycine max) with 1-O-[1'- 14 C]hexadecyl-sn-glycerol or rac-1-O-[1'- 14 C]hexadecylglycerol leads in high yield (up to 78%) to labeled 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines. Alkaline hydrolysis of the choline glycerophospholipids yields pure 1-O-[1'- 14 C]hexadecyl-sn-glycero-3-phosphocholine. 1-O-[1'-14C]Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) is obtained by acetylating the lyso compound. The semi-synthetic preparation described leads to labeled platelet activating factor in an overall yield of 50-60% without loss of specific activity

Activated platelet-derived growth factor β receptor and Ras-mitogen-activated protein kinase pathway in natural bovine urinary bladder carcinomas.

Science.gov (United States)

Corteggio, Annunziata; Di Geronimo, Ornella; Roperto, Sante; Roperto, Franco; Borzacchiello, Giuseppe

2012-03-01

Bovine papillomavirus types 1 or 2 (BPV-1/2) are involved in the aetiopathogenesis of bovine urinary bladder cancer. BPV-1/2 E5 activates the platelet-derived growth factor β receptor (PDGFβR). The aim of this study was to analyse the Ras/mitogen-activated protein kinase (MAPK) pathway in relation to activation of PDGFβR in natural bovine urinary bladder carcinomas. Co-immunoprecipitation and Western blot analysis demonstrated that recruitment of growth factor receptor bound protein 2 (GRB-2) and Sos-1 to the activated PDGFβR was increased in carcinomas compared to normal tissues. Higher grade bovine urinary bladder carcinomas were associated with activation of Ras, but not with activation of downstream mitogen-activated protein kinase/extracellular signal-regulated kinase (Mek 1/2) or extracellular signal-regulated kinase (Erk 1/2). Copyright © 2011 Elsevier Ltd. All rights reserved.

Evaluation of two platelet-rich plasma processing methods and two platelet-activation techniques for use in llamas and alpacas.

Science.gov (United States)

Semevolos, Stacy A; Youngblood, Cori D; Grissom, Stephanie K; Gorman, M Elena; Larson, Maureen K

2016-11-01

OBJECTIVE To evaluate 2 processing methods (commercial kit vs conical tube centrifugation) for preparing platelet rich plasma (PRP) for use in llamas and alpacas. SAMPLES Blood samples (30 mL each) aseptically collected from 6 healthy llamas and 6 healthy alpacas. PROCEDURES PRP was prepared from blood samples by use of a commercial kit and by double-step conical tube centrifugation. A CBC was performed for blood and PRP samples. Platelets in PRP samples were activated by means of a freeze-thaw method with or without 23mM CaCl 2 , and concentrations of platelet-derived growth factor-BB and transforming growth factor-β 1 were measured. Values were compared between processing methods and camelid species. RESULTS Blood CBC values for llamas and alpacas were similar. The commercial kit yielded a significantly greater degree of platelet enrichment (mean increase, 8.5 fold vs 2.8 fold) and WBC enrichment (mean increase, 3.7 fold vs 1.9 fold) than did conical tube centrifugation. Llamas had a significantly greater degree of platelet enrichment than alpacas by either processing method. No difference in WBC enrichment was identified between species. Concentrations of both growth factors were significantly greater in PRP samples obtained by use of the commercial kit versus those obtained by conical tube centrifugation. CONCLUSIONS AND CLINICAL RELEVANCE For blood samples from camelids, the commercial kit yielded a PRP product with a higher platelet and WBC concentration than achieved by conical tube centrifugation. Optimal PRP platelet and WBC concentrations for various applications need to be determined for llamas and alpacas.

Echicetin coated polystyrene beads: a novel tool to investigate GPIb-specific platelet activation and aggregation.

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Alexey Navdaev

Full Text Available von Willebrand factor/ristocetin (vWF/R induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways.

Echicetin Coated Polystyrene Beads: A Novel Tool to Investigate GPIb-Specific Platelet Activation and Aggregation

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Petunin, Alexey; Clemetson, Kenneth J.; Gambaryan, Stepan; Walter, Ulrich

2014-01-01

von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways. PMID:24705415

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.

Nephropathy in type 1 diabetes is associated with increased circulating activated platelets and platelet hyperreactivity

DEFF Research Database (Denmark)

Tarnow, Inge; Michelson, Alan D.; Barnard, Marc R.

2009-01-01

Patients with diabetes mellitus (DM) have increased platelet activation compared to non-diabetic controls. Platelet hyperreactivity has been associated with adverse cardiovascular outcomes in Type 2 DM, and with diabetic nephropathy. We investigated the relationship between platelet activation...... and nephropathy in Type 1 DM. Patients with Type 1 DM and diabetic nephropathy (n = 35), age- and sex-matched Type 1 DM patients with persistent normoalbuminuria (n = 51), and healthy age- and sex-matched controls (n = 30) were studied. Platelet surface P-selectin, platelet surface activated GPIIb/IIIa, monocyte...... controls (P = 0.0075). There were no differences between groups in activated GPIIb/IIIa or in response to TRAP at any end-point. More patients with nephropathy received aspirin (71.4%) compared to normoalbuminuric patients (27.4%) (P Type 1 diabetic nephropathy, as compared with normoalbuminuria...

Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

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Hou Ssu-Yu

2010-06-01

Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP

Platelet activation and platelet-leukocyte interaction in β-thalassemia/hemoglobin E patients with marked nucleated erythrocytosis.

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Keawvichit, Rassamon; Khowawisetsut, Ladawan; Chaichompoo, Porntip; Polsrila, Korakot; Sukklad, Suchana; Sukapirom, Kasama; Khuhapinant, Archrob; Fucharoen, Suthat; Pattanapanyasat, Kovit

2012-11-01

Patients with thalassemia, an inherited hemolytic anemia, have increased risk of hypercoagulable complications. A whole blood flow cytometric (FCM) method has been used for studies of platelet activation and platelet-leukocyte aggregation in these patients. However, this FCM method presents technical difficulties because of the high proportion of immature red blood cells (RBCs) in these patients. A protocol for the simultaneous measurement of platelet activation and their aggregation with leukocyte populations in whole blood using four-color FCM which excluded immature RBC was devised, and evaluated for the evaluation of platelet function in patients with β-thalassemia/hemoglobin E (HbE). Whole blood from these patients and from healthy volunteers was stained for platelet activation and platelet-leukocyte aggregates using anti-CD42a, anti-CD62P, anti-CD45 and glycophorin A (GPA) conjugated with different fluorochromes. Our FCM method is simple, effective and based on the assumption that GPA is present on all immature RBCs, but is not expressed on CD45⁺ leukocytes. Results from the studies showed that blood samples from these patients contained a high frequency of circulating activated platelets (CD42a⁺/CD62P⁺) when compared to samples from healthy individuals. The percentage of platelet-neutrophil, platelet-monocyte-but not platelet-lymphocyte-aggregates were also elevated in both thalassemia genotypes with marked increase in patients who had undergone splenectomy. These findings suggest that platelets adhere to neutrophils and monocytes are activated which support the clinical observation that splenectomized thalassemia patients have an increased risk of arterial or venous thrombotic manifestations.

Clinical-grade quality platelet-rich plasma releasate (PRP-R/SRGF) from CaCl2 -activated platelet concentrates promoted expansion of mesenchymal stromal cells.

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Borghese, C; Agostini, F; Durante, C; Colombatti, A; Mazzucato, M; Aldinucci, D

2016-08-01

The aim of our study was to test a platelet-rich plasma releasate (PRP-R/SRGF) from CaCl2 -activated platelets as a source of growth factors for the expansion of mesenchymal stromal cells (MSCs). PRP-R/SRGF, obtained with a low-cost procedure, is characterized by a reduced variability of growth factor release. PRP-R/SRGF is a clinical-grade quality solution obtained from CaCl2 -activated platelets. Its activity was evaluated by measuring the proliferation, the phenotype, the differentiation potential and the immunosuppressive properties of MSCs derived from bone marrow (BM) and adipose tissue (AT). PRP-R/SRGF was more active than FBS to expand BM- and AT-derived MSCs. PRP-R/SRGF treatment did not affect the expression of typical MSCs surface markers, neither MSCs differentiation potential nor their capability to inhibit activated T-cell proliferation. The clinical-grade PRP-R/SRGF may be used in the clinical setting for the expansion of MSCs. © 2016 International Society of Blood Transfusion.

Effect of platelet activating factor on endothelial permeability to plasma macromolecules

International Nuclear Information System (INIS)

Handley, D.A.; Arbeeny, C.M.; Lee, M.L.; Van Valen, R.G.; Saunders, R.N.

1984-01-01

The effect of intrajugular administration of platelet activating factor (PAF-C16) on vascular permeability was examined in the guinea pig. To examine the loss of selective endothelial permeability, the extravasative effect of PAF was assessed by monitoring hemoconcentration and the plasma loss of 125 I-albumin (6.7 nm), 125 I-low density lipoproteins (22.0 nm) or 125 I-very low density lipoproteins (62.1 nm). Extravasation was dose-dependent and began 1 min after PAF administration, continuing for 5-7 min. During extravasation, there was no evidence for selective plasma retention of any of the labeled plasma tracers, as measured by plasma radioactivity. These results suggest that PAF-induced extravasation is dose-dependent, with increases in vascular permeability sufficient to permit similar plasma efflux rates of albumin, low density lipoproteins and very low density lipoproteins

The Effects of Smoking on Platelet Count, Mean Platelet Volume and Cardiovascular Risk Factors: A Case-control Study

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Ruhuşen Kutlu

2017-12-01

Full Text Available Aim: Smoking and cholesterol levels are two important components of atherosclerosis. Mean platelet volume (MPV is an indicator of platelet function and activation and a potential marker of cardiovascular disease. In this study, we aimed to investigate the effects of cigarette-smoking on platelet count, MPV and cardiovascular risk factors. Methods: This research was planned as a case-control study. Patients who attended our family medicine outpatient clinic were included in the study. Sociodemographic characteristics, smoking status, hematological and biochemical parameters of the patients were recorded. Results: The mean age of 880 patients who participated in the study was 35.85±11.6 years (17-77. 54.5% (n=480 of participants were smokers and 45.5% (n=400 were non-smokers. The number of smokers among working individuals was higher than in non-workers. The white blood cell, hemoglobin, hematocrit, red blood cell, mean corpuscular volume and MPV values in the smokers were higher than in the non-smokers, while platelet count was higher in non-smokers (p<0.001. There was a statistically significant relationship between MPV levels and the number of daily cigarette smoking among smokers (p=0.014. Conclusion: MPV levels in smokers were significantly higher than in non-smokers. Platelet count and MPV levels should be investigated in larger patient groups in terms of atherosclerosis and other defined cardiovascular risk factors. It is therefore should take its rightful place in clinical practice.

Platelets retain high levels of active plasminogen activator inhibitor 1.

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Helén Brogren

Full Text Available The vascular fibrinolytic system is crucial for spontaneous lysis of blood clots. Plasminogen activator inhibitor 1 (PAI-1, the principal inhibitor of the key fibrinolytic enzyme tissue-type plasminogen activator (tPA, is present in platelets at high concentrations. However, the majority of PAI-1 stored in platelets has been considered to be inactive. Our recent finding (Brogren H, et al. Blood 2004 that PAI-1 de novo synthesized in platelets remained active for over 24 h, suggested that PAI-1 stored in the α-granules might be active to a larger extent than previously reported. To re-evaluate this issue, we performed experiments where the fraction of active PAI-1 was estimated by analyzing the tPA-PAI-1 complex formation. In these experiments platelets were lysed with Triton X-100 in the presence of serial dilutions of tPA and subsequently the tPA-PAI-1 complex was evaluated by Western blot. Also, using a non-immunologic assay, tPA was labeled with (125I, and (125I-tPA and (125I-tPA-PAI-1 was quantified by scintigraphy. Interestingly, both methods demonstrated that the majority (>50% of platelet PAI-1 is active. Further analyses suggested that pre-analytical procedures used in previous studies (sonication or freezing/thawing may have substantially reduced the activity of platelet PAI-1, which has lead to an underestimation of the proportion of active PAI-1. Our in vitro results are more compatible with the role of PAI-1 in clot stabilization as demonstrated in physiological and pathophysiological studies.

Low platelet monoamine oxidase activity in pathological gambling

International Nuclear Information System (INIS)

Carrasco, J.L.; Saiz-Ruiz, J.; Hollander, E.; Cesar, J.; Lopez-Ibor, J.J. Jr.

1994-01-01

Decreased platelet monoamine oxidase (MAO) activity has been reported in association with sensation-seeking personality type and in some mental disorders associated with a lack of impulse control. Pathological gambling itself has been related with both sensation-seeking and reduced impulse control. Platelet MAO activity was investigated in 15 DSM-III-R pathological gamblers from our outpatient clinic. Gamblers had a significantly lower platelet MAO activity than a group of 25 healthy controls. The range of MAO levels in gamblers was also significantly shorter than in controls. In controls, platelet MAO levels showed the previously described negative correlations with sensation-seeking scores but not in gamblers. The findings are consistent with previous studies showing an association of low platelet MAO activity with impulse control disorders and raise some interesting therapeutic alternatives for pathological gambling. (au) (40 refs.)

Low platelet monoamine oxidase activity in pathological gambling

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Carrasco, J.L. [Department of Psychiatry, Centro de Salud Mental, Parla Madrid (Spain); Saiz-Ruiz, J. [Department of Psychiatry and Haematology, Hospital Ramon y Cajal, Madrid (Spain); Hollander, E. [Department of Psychiatry, Mount Sinai School of Medicine, Queens Hospital Center, New York (United States); Cesar, J. [Department of Haematology, Hospital Ramon y Cajal, Madrid (Spain); Lopez-Ibor, J.J. Jr. [Department of Psychiatry, Hospital San Carlos, Complutense University, Madrid (Spain)

1994-12-01

Decreased platelet monoamine oxidase (MAO) activity has been reported in association with sensation-seeking personality type and in some mental disorders associated with a lack of impulse control. Pathological gambling itself has been related with both sensation-seeking and reduced impulse control. Platelet MAO activity was investigated in 15 DSM-III-R pathological gamblers from our outpatient clinic. Gamblers had a significantly lower platelet MAO activity than a group of 25 healthy controls. The range of MAO levels in gamblers was also significantly shorter than in controls. In controls, platelet MAO levels showed the previously described negative correlations with sensation-seeking scores but not in gamblers. The findings are consistent with previous studies showing an association of low platelet MAO activity with impulse control disorders and raise some interesting therapeutic alternatives for pathological gambling. (au) (40 refs.).

Elevated thrombopoietin in plasma of burned patients without and with sepsis enhances platelet activation.

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Lupia, E; Bosco, O; Mariano, F; Dondi, A E; Goffi, A; Spatola, T; Cuccurullo, A; Tizzani, P; Brondino, G; Stella, M; Montrucchio, G

2009-06-01

Thrombopoietin (TPO) is a humoral growth factor that does not induce platelet aggregation per se, but enhances platelet activation in response to several agonists. Circulating levels of TPO are increased in patients with sepsis and are mainly related to sepsis severity. To investigate the potential contribution of elevated TPO levels in platelet activation during burn injury complicated or not by sepsis. We studied 22 burned patients, 10 without and 12 with sepsis, and 10 healthy subjects. We measured plasma levels of TPO, as well as leukocyte-platelet binding and P-selectin expression. The priming activity of plasma from burned patients or healthy subjects on platelet aggregation and leukocyte-platelet binding, and the role of TPO in these effects were also studied in vitro. Burned patients without and with sepsis showed higher circulating TPO levels and increased monocyte-platelet binding compared with healthy subjects. Moreover, TPO levels, monocyte-platelet binding and P-selectin expression were significantly higher in burned patients with sepsis than in burned patients without sepsis. In vitro, plasma from burned patients without and with sepsis, but not from healthy subjects, primed platelet aggregation, monocyte-platelet binding and platelet P-selectin expression. The effect of plasma from burned patients with sepsis was significantly higher than that of plasma from burned patients without sepsis. An inhibitor of TPO prevented the priming effect of plasma from burned patients. Increased TPO levels may enhance platelet activation during burn injury and sepsis, potentially participating in the pathogenesis of multi-organ failure in these diseases.

Alternatives to allogeneic platelet transfusion.

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Desborough, Michael J R; Smethurst, Peter A; Estcourt, Lise J; Stanworth, Simon J

2016-11-01

Allogeneic platelet transfusions are widely used for the prevention and treatment of bleeding in thrombocytopenia. Recent evidence suggests platelet transfusions have limited efficacy and are associated with uncertain immunomodulatory risks and concerns about viral or bacterial transmission. Alternatives to transfusion are a well-recognised tenet of Patient Blood Management, but there has been less focus on different strategies to reduce bleeding risk by comparison to platelet transfusion. Direct alternatives to platelet transfusion include agents to stimulate endogenous platelet production (thrombopoietin mimetics), optimising platelet adhesion to endothelium by treating anaemia or increasing von Willebrand factor levels (desmopressin), increasing formation of cross-linked fibrinogen (activated recombinant factor VII, fibrinogen concentrate or recombinant factor XIII), decreasing fibrinolysis (tranexamic acid or epsilon aminocaproic acid) or using artificial or modified platelets (cryopreserved platelets, lyophilised platelets, haemostatic particles, liposomes, engineered nanoparticles or infusible platelet membranes). The evidence base to support the use of these alternatives is variable, but an area of active research. Much of the current randomised controlled trial focus is on evaluation of the use of thrombopoietin mimetics and anti-fibrinolytics. It is also recognised that one alternative strategy to platelet transfusion is choosing not to transfuse at all. © 2016 John Wiley & Sons Ltd.

Identification of functional VEGF receptors on human platelets.

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Selheim, Frode; Holmsen, Holm; Vassbotn, Flemming S

2002-02-13

Platelets secrete platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) upon stimulation. We have demonstrated that platelets have functionally active PDGF alpha-receptors, a transmembrane tyrosine kinase involved in negative feedback regulation. Here we demonstrate the presence of the related VEGF receptors fms-like tyrosine kinase-1 and kinase-insert domain region on human platelets. VEGF itself did not cause platelet aggregation. However, addition of exogenous VEGF to SFRLLN or thrombin-stimulated platelets potentiated platelet aggregation. Moreover, thrombin-induced phosphoinositide 3-kinase and mitogen-activated protein kinase activity were enhanced in the presence of VEGF.

The Dynamics of Platelet Activation during the Progression of Streptococcal Sepsis.

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Sinead M Hurley

Full Text Available Platelets contribute to inflammation however, the role of platelet activation during the pathophysiological response to invasive bacterial infection and sepsis is not clear. Herein, we have investigated platelet activation in a mouse model of invasive Streptococcus pyogenes infection at 5, 12, and 18 hours post infection and correlated this to parameters of infection. The platelet population in ex-vivo blood samples showed no increased integrin activation or surface presentation of CD62P, however platelet-neutrophil complex formation and plasma levels of CD62P were increased during bacterial dissemination and the progression of sepsis, indicating that platelet activation had occurred in vivo. Platelet-neutrophil complex formation was the most discriminatory marker of platelet activation. Platelet-neutrophil complexes were increased above baseline levels during early sepsis but decreased to significantly lower levels than baseline during late sepsis. The removal of these complexes from the circulation coincided with a significant increase in organ damage and the accumulation of platelets in the liver sinusoids, suggesting that platelet activation in the circulation precedes accumulation of platelets in damaged organs. The results demonstrate that monitoring platelet activation using complementary methods may provide prognostic information during the pathogenesis of invasive S. pyogenes infection.

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

Platelets Express Activated P2Y12 Receptor in Patients With Diabetes Mellitus.

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Hu, Liang; Chang, Lin; Zhang, Yan; Zhai, Lili; Zhang, Shenghui; Qi, Zhiyong; Yan, Hongmei; Yan, Yan; Luo, Xinping; Zhang, Si; Wang, Yiping; Kunapuli, Satya P; Ye, Hongying; Ding, Zhongren

2017-08-29

Platelets from patients with diabetes mellitus are hyperactive. Hyperactivated platelets may contribute to cardiovascular complications and inadequate responses to antiplatelet agents in the setting of diabetes mellitus. However, the underlying mechanism of hyperactivated platelets is not completely understood. We measured P2Y 12 expression on platelets from patients with type 2 diabetes mellitus and on platelets from rats with diabetes mellitus. We also assayed platelet P2Y 12 activation by measuring cAMP and VASP phosphorylation. The antiplatelet and antithrombotic effects of AR-C78511 and cangrelor were compared in rats. Finally, we explored the role of the nuclear factor-κB pathway in regulating P2Y 12 receptor expression in megakaryocytes. Platelet P2Y 12 levels are 4-fold higher in patients with type 2 diabetes mellitus compared with healthy subjects. P2Y 12 expression correlates with ADP-induced platelet aggregation (r=0.89, P diabetes mellitus is constitutively activated. Although both AR-C78511, a potent P2Y 12 inverse agonist, and cangrelor have similar antiplatelet efficacy on platelets from healthy subjects, AR-C78511 exhibits more powerful antiplatelet effects on diabetic platelets than cangrelor (aggregation ratio 36±3% versus 49±5%, respectively, P diabetes mellitus than cangrelor (thrombus weight 4.9±0.3 mg versus 8.3±0.4 mg, respectively, P diabetes mellitus. Platelet P2Y 12 receptor expression is significantly increased and the receptor is constitutively activated in patients with type 2 diabetes mellitus, which contributes to platelet hyperactivity and limits antiplatelet drug efficacy in type 2 diabetes mellitus. © 2017 American Heart Association, Inc.

Platelet factor 4 activity against P. falciparum and its translation to nonpeptidic mimics as antimalarials.

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Love, Melissa S; Millholland, Melanie G; Mishra, Satish; Kulkarni, Swapnil; Freeman, Katie B; Pan, Wenxi; Kavash, Robert W; Costanzo, Michael J; Jo, Hyunil; Daly, Thomas M; Williams, Dewight R; Kowalska, M Anna; Bergman, Lawrence W; Poncz, Mortimer; DeGrado, William F; Sinnis, Photini; Scott, Richard W; Greenbaum, Doron C

2012-12-13

Plasmodium falciparum pathogenesis is affected by various cell types in the blood, including platelets, which can kill intraerythrocytic malaria parasites. Platelets could mediate these antimalarial effects through human defense peptides (HDPs), which exert antimicrobial effects by permeabilizing membranes. Therefore, we screened a panel of HDPs and determined that human platelet factor 4 (hPF4) kills malaria parasites inside erythrocytes by selectively lysing the parasite digestive vacuole (DV). PF4 rapidly accumulates only within infected erythrocytes and is required for parasite killing in infected erythrocyte-platelet cocultures. To exploit this antimalarial mechanism, we tested a library of small, nonpeptidic mimics of HDPs (smHDPs) and identified compounds that kill P. falciparum by rapidly lysing the parasite DV while sparing the erythrocyte plasma membrane. Lead smHDPs also reduced parasitemia in a murine malaria model. Thus, identifying host molecules that control parasite growth can further the development of related molecules with therapeutic potential. Copyright © 2012 Elsevier Inc. All rights reserved.

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

Platelet growth factors from allogeneic platelet-rich plasma for clinical improvement in split-thickness skin graft.

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Sonker, Atul; Dubey, Anju; Bhatnagar, Ankur; Chaudhary, Rajendra

2015-01-01

Platelets are a source of numerous growth factors which facilitate repair and healing. Thus platelet rich plasma has been increasingly used as a treatment modality in the field of reconstructive surgeries for wound healing. This preliminary study was carried out to explore whether platelet growth factors from platelet rich plasma could be used for enhancement of split thickness skin graft survival. Twenty patients (13 males and 7 females) requiring split thickness skin graft for various clinical reasons were enrolled in the study. Platelet rich plasma was collected by apheresis and frozen at -80° C. It was thawed at room temperature immediately before its intended application. PRP was applied only on one half of the wound, while another half served as control. Patient was followed for 6 weeks. The effect was assessed at first dressing in terms of graft uptake and subsequently as time taken for complete healing. There was 100% uptake of the graft in the area where platelet rich plasma was applied. In the control area, there was complete graft loss in 4 cases, partial loss in 7 cases and complete uptake in 9 cases. This study demonstrated promising results on application of PRP to split thickness skin grafts. Further randomized studies with greater sample size may be undertaken to establish platelet rich plasma as a validated treatment modality.

21 CFR 864.7695 - Platelet factor 4 radioimmunoassay.

Science.gov (United States)

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...

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.

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

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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.

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...

PPARγ ligands decrease hydrostatic pressure-induced platelet aggregation and proinflammatory activity.

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Fang Rao

Full Text Available Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ. We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg or increased (120, 180, 240 mmHg hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg. The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs. These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure.

PPARγ ligands decrease hydrostatic pressure-induced platelet aggregation and proinflammatory activity.

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Rao, Fang; Yang, Ren-Qiang; Chen, Xiao-Shu; Xu, Jin-Song; Fu, Hui-Min; Su, Hai; Wang, Ling

2014-01-01

Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ). We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg) or increased (120, 180, 240 mmHg) hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa) binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L) was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg). The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs). These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure.

Nitric oxide mediates angiogenesis induced in vivo by platelet-activating factor and tumor necrosis factor-alpha.

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Montrucchio, G.; Lupia, E.; de Martino, A.; Battaglia, E.; Arese, M.; Tizzani, A.; Bussolino, F.; Camussi, G.

1997-01-01

We evaluated the role of an endogenous production of nitric oxide (NO) in the in vitro migration of endothelial cells and in the in vivo angiogenic response elicited by platelet-activating factor (PAF), tumor necrosis factor-alpha (TNF), and basic fibroblast growth factor (bFGF). The NO synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME), but not its enantiomer D-NAME, prevented chemotaxis of endothelial cells induced in vitro by PAF and by TNF. The motogenic activity of TNF was also inhibited by WEB 2170, a specific PAF-receptor antagonist. In contrast, chemotaxis induced by bFGF was not prevented by L-NAME or by WEB 2170. Angiogenesis was studied in vivo in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model, the angiogenesis induced by PAF and TNF was inhibited by WEB 2170 and L-NAME but not by D-NAME. In contrast, angiogenesis induced by bFGF was not affected by L-NAME or by WEB 2170. TNF, but not bFGF, induced PAF synthesis within Matrigel. These results suggest that NO mediates the angiogenesis induced by PAF as well as that induced by TNF, which is dependent on the production of PAF. In contrast, the angiogenic effect of bFGF appears to be both PAF and NO independent. Images Figure 3 Figure 4 PMID:9250168

Antimicrobial effect of platelet-rich plasma and platelet-rich fibrin.

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Badade, Pallavi S; Mahale, Swapna A; Panjwani, Alisha A; Vaidya, Prutha D; Warang, Ayushya D

2016-01-01

Platelet concentrates have been extensively used in a variety of medical fields to promote soft- and hard-tissue regeneration. The significance behind their use lies in the abundance of growth factors (GFs) in platelets α-granules that promote wound healing. Other than releasing a pool of GFs upon activation, platelets also have many features that indicate their role in the anti-infective host defense. The aim of this study is to evaluate the antimicrobial activities of platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) against periodontal disease-associated bacteria. Blood samples were obtained from ten adult male patients. PRP and PRF were procured using centrifugation. The antimicrobial activity of PRP and PRF was evaluated by microbial culturing using bacterial strains of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. P. gingivalis and A. actinomycetemcomitans were inhibited by PRP but not by PRF. PRP is a potentially useful substance in the fight against periodontal pathogens. This might represent a valuable property in adjunct to the enhancement of tissue regeneration.

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).

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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.

Can mean platelet component be used as an index of platelet activity in stable coronary artery disease?

LENUS (Irish Health Repository)

Cooke, John

2012-01-31

Acute coronary syndrome is associated with intracoronary thrombosis secondary to platelet activation. Previous groups have investigated platelet activation in both stable and unstable vascular disease. Most measures of platelet activation are not routinely available or easily adaptable to large scale clinical use. Recently, measurement of the mean platelet component (MPC) has become part of the routine data provided by an automated full blood count analyser, the Advia 120. MPC measures platelet density which changes on platelet activation. Our objectives were to determine if platelet activation, as measured by MPC, is increased in patients with stable coronary artery disease (CAD) and to determine if MPC could be useful in differentiating people with stable CAD from controls on an everyday clinical basis. Three hundred and forty-five consecutive patients attending for elective coronary angiography had full blood count analysis and MPC measurement performed using an ADVIA-120 analyser. Three hundred and twenty-four were analysed in our final dataset. Two hundred and fifty-three (78%) had CAD. Patients with CAD were significantly (p<0.001) older than those without (63.8 versus 56.0 years). Results failed to demonstrate a difference (p=0.467) in MPC between patients with CAD and those with normal coronary arteries (25.8 versus 26.0). Likewise, there was no correlation between MPC and the severity of CAD (Kendall\\'s tau b=-0.086, p=0.04). MPC is not a useful index of platelet activity in stable CAD when used in everyday clinical practice.

Can mean platelet component be used as an index of platelet activity in stable coronary artery disease?

LENUS (Irish Health Repository)

Cooke, John

2009-04-01

Acute coronary syndrome is associated with intracoronary thrombosis secondary to platelet activation. Previous groups have investigated platelet activation in both stable and unstable vascular disease. Most measures of platelet activation are not routinely available or easily adaptable to large scale clinical use. Recently, measurement of the mean platelet component (MPC) has become part of the routine data provided by an automated full blood count analyser, the Advia 120. MPC measures platelet density which changes on platelet activation. Our objectives were to determine if platelet activation, as measured by MPC, is increased in patients with stable coronary artery disease (CAD) and to determine if MPC could be useful in differentiating people with stable CAD from controls on an everyday clinical basis. Three hundred and forty-five consecutive patients attending for elective coronary angiography had full blood count analysis and MPC measurement performed using an ADVIA-120 analyser. Three hundred and twenty-four were analysed in our final dataset. Two hundred and fifty-three (78%) had CAD. Patients with CAD were significantly (p<0.001) older than those without (63.8 versus 56.0 years). Results failed to demonstrate a difference (p=0.467) in MPC between patients with CAD and those with normal coronary arteries (25.8 versus 26.0). Likewise, there was no correlation between MPC and the severity of CAD (Kendall\\'s tau b=-0.086, p=0.04). MPC is not a useful index of platelet activity in stable CAD when used in everyday clinical practice.

Role of platelet-released growth factors in detoxification of reactive oxygen species in osteoblasts.

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Tohidnezhad, Mersedeh; Wruck, Christoph-Jan; Slowik, Alexander; Kweider, Nisreen; Beckmann, Rainer; Bayer, Andreas; Houben, Astrid; Brandenburg, Lars-Ove; Varoga, Deike; Sönmez, Tolga-Taha; Stoffel, Marcus; Jahr, Holger; Lippross, Sebastian; Pufe, Thomas

2014-08-01

Oxidative stress can impair fracture healing. To protect against oxidative damage, a system of detoxifying and antioxidative enzymes works to reduce the cellular stress. The transcription of these enzymes is regulated by antioxidant response element (ARE). The nuclear factor (erythroid-derived 2)-like2 (Nrf2) plays a major role in transcriptional activation of ARE-driven genes. Recently it has been shown that vascular endothelial growth factor (VEGF) prevents oxidative damage via activation of the Nrf2 pathway in vitro. Platelet-released growth factor (PRGF) is a mixture of autologous proteins and growth factors, prepared from a determined volume of platelet-rich plasma (PRP). It has already used to enhance fracture healing in vitro. The aim of the present study was to elucidate if platelets can lead to upregulation of VEGF and if platelets can regulate the activity of Nrf2-ARE system in primary human osteoblast (hOB) and in osteoblast-like cell line (SAOS-2). Platelets and PRGF were obtained from healthy human donors. HOB and SAOS-2 osteosarcoma cell line were used. The ARE activity was analysed using a dual luciferase reporter assay system. We used Western blot to detect the nuclear accumulation of Nrf2 and the amount of cytosolic antioxidant Thioredoxin Reductase-1 (TXNRD-1), Heme Oxygenase-1 (HO-1) and NAD(P)H quinine oxidoreductase-1 (NQO1). Gene expression analysis was performed by real-time RT PCR. ELISA was used for the quantification of growth factors. The activity of ARE was increased in the presence of PRGF up to 50%. Western blotting demonstrated enhanced nuclear accumulation of Nrf2. This was followed by an increase in the protein expression of the aforementioned downstream targets of Nrf2. Real-time RT PCR data showed an upregulation in the gene expression of the VEGF after PRGF treatment. This was confirmed by ELISA, where the treatment with PRGF induced the protein level of VEGF in both cells. These results provide a new insight into PRGF's mode of

[Platelet function in acute myeloid leukemia. II. Aggregation of isolated platelets].

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Zawilska, K; Komarnicki, M; Mańka, B

1978-01-01

In 22 patients with acute myeloid leukaemia (17 cases of myeloblastic leukaemia, 4 cases of myelomonocytic leukaemia and 1 case of undifferentiated-cell leukaemia) platelets were isolated from the plasma by the method of Nicholls and Hampton as modified by Levy-Toledano by centrifugation in albumin gradient. The aim of platelet isolation was their "concentration" in cases of thrombocytopenia to values making possible aggregation tests, and platelet separation from the influence of plasma factors. Then aggregation of isolated platelets caused by ADP was studied. In 16 out of 22 patients a fall of aggregation was observed, with the mean values of aggregation rate and intensity were significantly lower. Parallelly done determinations of aggregating activity released from the platelets by thrombin showed lower values as compared with platelets from healthy subjects. In might be thought, in this connection, that the demonstrated reduction of isolated platelets is associated with a diminution of the nucleotide pool or disturbances of the platelet release reaction. The disturbances of the platelet release reaction. The disturbances of aggregation of isolated platelets and reduction of the aggregating activity were most pronounced in acute myelomonocytic leukaemia.

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

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Söderström, Anna C; Nybo, Mads; Nielsen, Christian; Vinholt, Pernille J

2016-12-01

The results of laboratory analyses are affected by pre-analytical variables, and in particular can platelets be activated by shear handling stress and secrete granular substances. We therefore evaluated the effect of centrifugation speed and time on pre-analytical platelet activation. Citrate- and EDTA-anticoagulated blood from healthy volunteers were centrifuged at 80-10,000 g for 5-15 min to prepare plasma and platelet-rich plasma. Pre-analytical platelet activation was assessed by flow cytometric measurement of platelet P-selectin (CD62p) expression. Blood cell counts, mean platelet volume (MPV), immature platelet fraction (IPF), and platelet distribution width (PDW) were measured. Platelet aggregation in platelet-rich plasma induced by arachidonic acid (AA), ADP or thrombin receptor activator peptide-6 (TRAP) was tested by 96-well aggregometry. The median percentage of platelets expressing P-selectin in citrate- and EDTA-plasma centrifuged at 2000 g for 10 min were 43% [interquartile range (IQR), 38%-53%] and 56% (IQR, 31%-78%), respectively (p=0.82). Platelet-rich plasma prepared at 100-250 g for 10 min had significantly lower platelet P-selectin expression (11%-15%), pcentrifuged. In platelet-rich plasma, increasing centrifugation speed significantly increased platelet yield but decreased contamination from other blood cells, platelet composition was altered as platelet parameters (MPV, IPF, and PDW) was lowered. Platelet aggregation was not affected by the centrifugation speed platelet-rich plasma was prepared. Proportional to centrifugation speed, platelets in plasma and platelet-rich plasma were activated with centrifugation speed, cell content and composition changed while platelet aggregation was unaltered.

A novel role of sesamol in inhibiting NF-κB-mediated signaling in platelet activation

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Chang Chao-Chien

2011-12-01

Full Text Available Abstract Background Platelet activation is relevant to a variety of coronary heart diseases. Our previous studies revealed that sesamol possesses potent antiplatelet activity through increasing cyclic AMP formation. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of sesamol in NF-κB-mediated platelet function. Methods Platelet aggregation, Fura 2-AM fluorescence, and immunoblotting analysis were used in this study. Results NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were markedly activated by collagen (1 μg/ml in washed human platelets, and these signaling events were attenuated by sesamol (2.5~25 μM. Furthermore, SQ22536 and ODQ, inhibitors of adenylate cyclase and guanylate cyclase, respectively, strongly reversed the sesamol (25 μM-mediated inhibitory effects of IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation stimulated by collagen. The protein kinase A (PKA inhibitor, H89, also reversed sesamol-mediated inhibition of IκBα degradation. Moreover, BAY11-7082, an NF-κB inhibitor, abolished IκBα degradation, phospholipase C (PLCγ2 phosphorylation, protein kinase C (PKC activation, [Ca2+]i mobilization, and platelet aggregation stimulated by collagen. Preincubation of platelets with the inhibitors, SQ22536 and H89, both strongly reversed sesamol-mediated inhibition of platelet aggregation and [Ca2+]i mobilization. Conclusions Sesamol activates cAMP-PKA signaling, followed by inhibition of the NF-κB-PLC-PKC cascade, thereby leading to inhibition of [Ca2+]i mobilization and platelet aggregation. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our data demonstrating that inhibition of NF-κB interferes with platelet function may

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

Influence of platelet-activating factor receptor (PAFR) on Brucella abortus infection: implications for manipulating the phagocytic strategy of B. abortus.

Science.gov (United States)

Lee, Jin Ju; Simborio, Hannah Leah; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Arayan, Lauren Togonon; Lee, Hu Jang; Min, Wongi; Her, Moon; Rhee, Man Hee; Watarai, Masahisa; Chang, Hong Hee; Kim, Suk

2016-04-21

Brucella abortus is an intracellular pathogen which can infect and persist in host cells through multiple interactions. Above all, its interaction to host cell receptor is important to understand the pathogenic mechanisms of B. abortus. Accordingly, we demonstrated that platelet-activating factor receptor (PAFR) affects host cell response against B. abortus infection. First of all, B. abortus infection to macrophage induces secretion of platelet-activating factor (PAF), which is a PAFR agonist. The stimulation of PAFR by PAF remarkably increases B. abortus uptake into macrophages. It induces Janus kinase 2 (JAK2) and p38α phosphorylation, indicating that PAFR-mediated activation of JAK2 signaling leads to enhanced uptake of B. abortus. Moreover, the dynamics of F-actin polymerization revealed that PAFR-mediated B. abortus uptake is related with the reorganization of F-actin and JAK2. Upon B. abortus phagocytosis, reduced PAFR in the membrane and subsequently increased levels of PAFR colocalization with endosomes were observed which indicate that B. abortus uptake into macrophages allowed PAFR trafficking to endosomes. This study demonstrated that PAFR has a compelling involvement in B. abortus uptake as a promoter of phagocytosis, which is associated with JAK2 activation. Thus, our findings establish a novel insight into a receptor-related phagocytic mechanism of B. abortus.

Platelet activation by extracellular matrix proteins in haemostasis and thrombosis.

Science.gov (United States)

Watson, Steve P

2009-01-01

The prevention of excessive blood loss to avoid fatal haemorrhage is a pivotal process for all organisms possessing a circulatory system. Increased circulating blood volume and pressure, as required in larger animals, make this process all the more important and challenging. It is essential to have a powerful and rapid system to detect damage and generate an effective seal, and which is also exquisitely regulated to prevent unwanted, excessive or systemic activation so as to avoid blockage of vessels. Thus, a highly specialised and efficient haemostatic system has evolved that consists of cellular (platelets) and protein (coagulation factors) components. Importantly, this is able to support haemostasis in both the low shear environment of the venous system and the high shear environment of the arterial system. Endothelial cells, lining the entire circulation system, play a crucial role in the delicate balance between activation and inhibition of the haemostatic system. An intact and healthy endothelium supports blood flow by preventing attachment of cells and proteins which is required for initiation of coagulation and platelet activation. Endothelial cells produce and release the two powerful soluble inhibitors of platelet activation, nitric oxide and prostacyclin, and express high levels of CD39 which rapidly metabolises the major platelet feedback agonist, ADP. This antithrombotic environment however can rapidly change following activation or removal of endothelial cells through injury or rupture of atherosclerotic plaques. Loss of endothelial cells exposes the subendothelial extracellular matrix which creates strong signals for activation of the haemostatic system including powerful platelet adhesion and activation. Quantitative and qualitative changes in the composition of the subendothelial extracellular matrix influence these prothrombotic characteristics with life threatening thrombotic and bleeding complications, as illustrated by formation of

Discovery and evaluation of a series of 3-acylindole imidazopyridine platelet-activating factor antagonists.

Science.gov (United States)

Curtin, M L; Davidsen, S K; Heyman, H R; Garland, R B; Sheppard, G S; Florjancic, A S; Xu, L; Carrera, G M; Steinman, D H; Trautmann, J A; Albert, D H; Magoc, T J; Tapang, P; Rhein, D A; Conway, R G; Luo, G; Denissen, J F; Marsh, K C; Morgan, D W; Summers, J B

1998-01-01

Studies conducted with the goal of discovering a second-generation platelet-activating factor (PAF) antagonist have identified a novel class of potent and orally active antagonists which have high aqueous solubility and long duration of action in animal models. The compounds arose from the combination of the lipophilic indole portion of Abbott's first-generation PAF antagonist ABT-299 (2) with the methylimidazopyridine heterocycle moiety of British Biotechnology's BB-882 (1) and possess the positive attributes of both of these clinical candidates. Structure-activity relationship (SAR) studies indicated that modification of the indole and benzoyl spacer of lead compound 7b gave analogues that were more potent, longer-lived, and bioavailable and resulted in the identification of 1-(N, N-dimethylcarbamoyl)-4-ethynyl-3-[3-fluoro-4-[(1H-2-methylimidazo[4,5-c] pyrid-1-yl)methyl]benzoyl]indole hydrochloride (ABT-491, 22 m.HCl) which has been evaluated extensively and is currently in clinical development.

Bioactivity and stability of endogenous fibrogenic factors in platelet-rich fibrin

DEFF Research Database (Denmark)

Lundquist, R.; Dziegiel, M.H.; Agren, M.S.

2008-01-01

Platelet-rich fibrin (PRF) is an autologous fibrin sealant (FS) enriched with a platelet concentrate (> 1,000,000 platelets/microL) produced by the automated Vivostat system and used to enhance wound healing. The effects of PRF were compared with supernatant from thrombin-activated platelet conce...

Gallic Acid Attenuates Platelet Activation and Platelet-Leukocyte Aggregation: Involving Pathways of Akt and GSK3β

Science.gov (United States)

Chang, Shih-Sheng; Lee, Viola S. Y.; Tseng, Yu-Lun; Chang, Kuan-Cheng; Chen, Kuen-Bao; Chen, Yuh-Lien; Li, Chi-Yuan

2012-01-01

Platelet activation and its interaction with leukocytes play an important role in atherothrombosis. Cardiovascular diseases resulted from atherothrombosis remain the major causes of death worldwide. Gallic acid, a major constituent of red wine and tea, has been believed to have properties of cardiovascular protection, which is likely to be related to its antioxidant effects. Nonetheless, there were few and inconsistent data regarding the effects of gallic acid on platelet function. Therefore, we designed this in vitro study to determine whether gallic acid could inhibit platelet activation and the possible mechanisms. From our results, gallic acid could concentration-dependently inhibit platelet aggregation, P-selectin expression, and platelet-leukocyte aggregation. Gallic acid prevented the elevation of intracellular calcium and attenuated phosphorylation of PKCα/p38 MAPK and Akt/GSK3β on platelets stimulated by the stimulants ADP or U46619. This is the first mechanistic explanation for the inhibitory effects on platelets from gallic acid. PMID:22811749

Gallic Acid Attenuates Platelet Activation and Platelet-Leukocyte Aggregation: Involving Pathways of Akt and GSK3β

Directory of Open Access Journals (Sweden)

Shih-Sheng Chang

2012-01-01

Full Text Available Platelet activation and its interaction with leukocytes play an important role in atherothrombosis. Cardiovascular diseases resulted from atherothrombosis remain the major causes of death worldwide. Gallic acid, a major constituent of red wine and tea, has been believed to have properties of cardiovascular protection, which is likely to be related to its antioxidant effects. Nonetheless, there were few and inconsistent data regarding the effects of gallic acid on platelet function. Therefore, we designed this in vitro study to determine whether gallic acid could inhibit platelet activation and the possible mechanisms. From our results, gallic acid could concentration-dependently inhibit platelet aggregation, P-selectin expression, and platelet-leukocyte aggregation. Gallic acid prevented the elevation of intracellular calcium and attenuated phosphorylation of PKCα/p38 MAPK and Akt/GSK3β on platelets stimulated by the stimulants ADP or U46619. This is the first mechanistic explanation for the inhibitory effects on platelets from gallic acid.

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

Targeting factor VIII expression to platelets for hemophilia A gene therapy does not induce an apparent thrombotic risk in mice.

Science.gov (United States)

Baumgartner, C K; Mattson, J G; Weiler, H; Shi, Q; Montgomery, R R

2017-01-01

Essentials Platelet-Factor (F) VIII gene therapy is a promising treatment in hemophilia A. This study aims to evaluate if platelet-FVIII expression would increase the risk for thrombosis. Targeting FVIII expression to platelets does not induce or elevate thrombosis risk. Platelets expressing FVIII are neither hyper-activated nor hyper-responsive. Background Targeting factor (F) VIII expression to platelets is a promising gene therapy approach for hemophilia A, and is successful even in the presence of inhibitors. It is well known that platelets play important roles not only in hemostasis, but also in thrombosis and inflammation. Objective To evaluate whether platelet-FVIII expression might increase thrombotic risk and thereby compromise the safety of this approach. Methods In this study, platelet-FVIII-expressing transgenic mice were examined either in steady-state conditions or under prothrombotic conditions induced by inflammation or the FV Leiden mutation. Native whole blood thrombin generation assay, rotational thromboelastometry analysis and ferric chloride-induced vessel injury were used to evaluate the hemostatic properties. Various parameters associated with thrombosis risk, including D-dimer, thrombin-antithrombin complexes, fibrinogen, tissue fibrin deposition, platelet activation status and activatability, and platelet-leukocyte aggregates, were assessed. Results We generated a new line of transgenic mice that expressed 30-fold higher levels of platelet-expressed FVIII than are therapeutically required to restore hemostasis in hemophilic mice. Under both steady-state conditions and prothrombotic conditions induced by lipopolysaccharide-mediated inflammation or the FV Leiden mutation, supratherapeutic levels of platelet-expressed FVIII did not appear to be thrombogenic. Furthermore, FVIII-expressing platelets were neither hyperactivated nor hyperactivatable upon agonist activation. Conclusion We conclude that, in mice, more than 30-fold higher levels of

[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

On the Eulerian formulation of a stress induced platelet activation function

Czech Academy of Sciences Publication Activity Database

Bodnár, Tomáš

2014-01-01

Roč. 257, November (2014), s. 91-95 ISSN 0025-5564 Institutional support: RVO:61388998 Keywords : blood flow * platelet * stress * activation Subject RIV: BK - Fluid Dynamics Impact factor: 1.303, year: 2014 http://www.sciencedirect.com/science/article/pii/S0025556414001163

Combined aspirin and cilostazol treatment is associated with reduced platelet aggregation and prevention of exercise-induced platelet activation.

Science.gov (United States)

Cleanthis, M; Bhattacharya, V; Smout, J; Ashour, H; Stansby, G

2009-05-01

Cilostazol has proven efficacy in increasing walking distance in claudicants, but it has not been demonstrated to be more effective than placebo in secondary cardiovascular prevention. The direct effect of exercise on platelet function remains less well defined. We have investigated the effect of combination treatment with aspirin and cilostazol on platelet activity in claudicants subjected to repeated treadmill exercise. Nineteen claudicants completed a double-blind, randomised, controlled, cross-over trial. Each subject received a 2-week course of aspirin (75mg) and placebo and aspirin and cilostazol (100mg twice daily). Following each 2-week treatment period, patients participated in a standardised treadmill test (3.2kmh(-1), 10 degrees incline) walking to maximal claudication distance. The exercise was repeated thrice in total, and blood was sampled before and after exercise. Platelet activation was measured using free platelet counting aggregation, flow cytometry for surface markers of platelet activation and soluble P-selectin assay. Compared to aspirin and placebo, combination treatment with aspirin and cilostazol was associated with reduced arachidonic-acid-induced platelet aggregation (pWilcoxon signed-rank test). Aspirin and placebo treatment were associated with elevated P-selectin expression, platelet-monocyte aggregation and reduced CD42b expression (pWilcoxon signed-rank test) post-exercise. No difference was seen in spontaneous platelet aggregation whilst soluble P-selectin was reduced post-exercise with combination treatment with aspirin and cilostazol (pWilcoxon signed-rank test). Combination treatment with aspirin and cilostazol results in suppression of platelet activation and reduces the effect of exercise on platelets. The benefit seen may be a result of cilostazol enhancing the inhibitory effect of aspirin on the cyclo-oxygenase pathway.

Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts.

Science.gov (United States)

Kasahara, Kohji; Kaneda, Mizuho; Miki, Toshiaki; Iida, Kazuko; Sekino-Suzuki, Naoko; Kawashima, Ikuo; Suzuki, Hidenori; Shimonaka, Motoyuki; Arai, Morio; Ohno-Iwashita, Yoshiko; Kojima, Soichi; Abe, Mitsuhiro; Kobayashi, Toshihide; Okazaki, Toshiro; Souri, Masayoshi; Ichinose, Akitada; Yamamoto, Naomasa

2013-11-07

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-β-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbβ3-myosin complex is formed as a primary axis to promote platelet contraction.

Inhibition of platelet activation by lachrymatory factor synthase (LFS)-silenced (tearless) onion juice.

Science.gov (United States)

Thomson, Susan J; Rippon, Paula; Butts, Chrissie; Olsen, Sarah; Shaw, Martin; Joyce, Nigel I; Eady, Colin C

2013-11-06

Onion and garlic are renowned for their roles as functional foods. The health benefits of garlic are attributed to di-2-propenyl thiosulfinate (allicin), a sulfur compound found in disrupted garlic but not found in disrupted onion. Recently, onions have been grown with repressed lachrymatory factor synthase (LFS) activity, which causes these onions to produce increased amounts of di-1-propenyl thiosulfinate, an isomer of allicin. This investigation into the key health attributes of LFS-silenced (tearless) onions demonstrates that they have some attributes more similar to garlic and that this is likely due to the production of novel thiosulfinate or metabolites. The key finding was that collagen-induced in vitro platelet aggregation was significantly reduced by tearless onion extract over normal onion extract. Thiosulfinate or derived compounds were shown not to be responsible for the observed changes in the inflammatory response of AGS (stomach adenocarcinoma) cells to tumor necrosis factor alpha (TNFα) when pretreated with model onion juices. A preliminary rat feeding trial indicated that the tearless onions may also play a key role in reducing weight gain.

Autologous blood preparations rich in platelets, fibrin and growth factors.

Science.gov (United States)

Fioravanti, C; Frustaci, I; Armellin, E; Condò, R; Arcuri, C; Cerroni, L

2015-01-01

Bone regeneration is often needed prior to dental implant treatment due to the lack of adequate quantity and quality after infectious diseases. The greatest regenerative power was obtained with autologous tissue, primarily the bone alive, taken from the same site or adjacent sites, up to the use centrifugation of blood with the selection of the parts with the greatest potential regenerative. In fact, various techniques and technologies were chronologically successive to cope with an ever better preparation of these concentrates of blood. Our aim is to review these advances and discuss the ways in which platelet concentrates may provide such unexpected beneficial therapeutic effects. The research has been carried out in the MEDLINE and Cochrane Central Register of Controlled Trials database by choosing keywords as "platelet rich plasma", "platelet rich fibrin", "platelet growth factors", and "bone regeneration" and "dentistry". Autologous platelet rich plasma is a safe and low cost procedure to deliver growth factors for bone and soft tissue healing. The great heterogeneity of clinical outcomes can be explained by the different PRP products with qualitative and quantitative difference among substance.

Detection of activated platelets using activation-specific monoclonal antibody (SZ-51) in clinical disorders

International Nuclear Information System (INIS)

Wu Guoxin; Li Fugang; Li Jianyong; Ruan Changgeng

1991-10-01

A direct test for activated platelets in whole blood was developed by radioimmunoassay with 125 I labeled SZ-51, an antibody specific for an α-granule membrane protein (GMP-140) that associates with the platelet surface during secretion. The assay had sufficient sensitivity to detect as few as 2% activated platelets. In 50 normal subjects, minimal GMP-140 molecules per platelet were expressed on the surface of circulating platelets. Ten patients undergoing cardiopulmonary bypass had transiently increased expression of GMP-140 molecules during the bypass procedure, especially at the end of bypass. Evaluation of 18 patients with epidemic hemorrhagic fever (EHF) has shown that the number of GMP-140 molecules on the platelet surface was closely related to the four different phases of EHF. In six patients suffered from acute myocardial infarction (AMI), the number of GMP-140 molecules changed with the procession of AMI and the highest occurred 48 h after AMI. The GMP-140 molecules were also increased in patients with asthma attack (n = 14), but not in patients with idiopathic thrombocytopenic purpura (n = 11) and diabetic mellitus (n = 48). Taken together, these studies suggest that activated platelet can be reliably measured in whole blood using radiolabeled SZ-51 antibody and the detection of activated platelets is potentially useful in identifying patients with certain thrombotic disorders and others

Antagonism of Sorafenib and Regorafenib actions by platelet factors in hepatocellular carcinoma cell lines

International Nuclear Information System (INIS)

D’Alessandro, Rosalba; Refolo, Maria G; Lippolis, Catia; Giannuzzi, Grazia; Carella, Nicola; Messa, Caterina; Cavallini, Aldo; Carr, Brian I

2014-01-01

Platelets are frequently altered in hepatocellular carcinoma (HCC) patients. Platelet lysates (hPL) can enhance HCC cell growth and decrease apoptosis. The aims were to evaluate whether hPL can modulate the actions of Sorafenib or Regorafenib, two clinical HCC multikinase antagonists. Several human HCC cell lines were grown in the presence and absence of Sorafenib or Regorafenib, with or without hPL. Growth was measured by MTT assay, apoptosis was assessed by Annexin V and by western blot, and autophagy and MAPK growth signaling were also measured by western blot, and migration and invasion were measured by standard in vitro assays. Both Sorafenib and Regorafenib-mediated inhibition of cell growth, migration and invasion were all antagonized by hPL. Drug-mediated apoptosis and decrease in phospho-ERK levels were both blocked by hPL, which also increased anti-apoptotic phospho-STAT, Bax and Bcl-xL levels. Preliminary data, obtained with epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), included in hPL, revealed that these factors were able to antagonized Sorafenib in a proliferation assay, in particular when used in combination. Platelet factors can antagonize Sorafenib or Regorafenib-mediated growth inhibition and apoptosis in HCC cells. The modulation of platelet activity or numbers has the potential to enhance multikinase drug actions

Oxidative response of neutrophils to platelet-activating factor is altered during acute ruminal acidosis induced by oligofructose in heifers

OpenAIRE

Concha, Claudia; Carretta, María Daniella; Alarcón, Pablo; Conejeros, Ivan; Gallardo, Diego; Hidalgo, Alejandra Isabel; Tadich, Nestor; Cáceres, Dante Daniel; Hidalgo, María Angélica; Burgos, Rafael Agustín

2014-01-01

Reactive oxygen species (ROS) production is one of the main mechanisms used to kill microbes during innate immune response. D-lactic acid, which is augmented during acute ruminal acidosis, reduces platelet activating factor (PAF)-induced ROS production and L-selectin shedding in bovine neutrophils in vitro. This study was conducted to investigate whether acute ruminal acidosis induced by acute oligofructose overload in heifers interferes with ROS production and L-selectin shedding in blood ne...

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.

Redox Proteomics and Platelet Activation: Understanding the Redox Proteome to Improve Platelet Quality for Transfusion

Science.gov (United States)

Sonego, Giona; Abonnenc, Mélanie; Tissot, Jean-Daniel; Prudent, Michel; Lion, Niels

2017-01-01

Blood banks use pathogen inactivation (PI) technologies to increase the safety of platelet concentrates (PCs). The characteristics of PI-treated PCs slightly differ from those of untreated PCs, but the underlying reasons are not well understood. One possible cause is the generation of oxidative stress during the PI process. This is of great interest since reactive oxygen species (ROS) act as second messengers in platelet functions. Furthermore, there are links between protein oxidation and phosphorylation, another mechanism that is critical for cell regulation. Current research efforts focus on understanding the underlying mechanisms and identifying new target proteins. Proteomics technologies represent powerful tools for investigating signaling pathways involving ROS and post-translational modifications such as phosphorylation, while quantitative techniques enable the comparison of the platelet resting state versus the stimulated state. In particular, redox cysteine is a key player in platelet activation upon stimulation by different agonists. This review highlights the experiments that have provided insights into the roles of ROS in platelet function and the implications for platelet transfusion, and potentially in diseases such as inflammation and platelet hyperactivity. The review also describes the implication of redox mechanism in platelet storage considerations. PMID:28208668

The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays.

Directory of Open Access Journals (Sweden)

Marlien Pieters

Full Text Available Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g, platelet-containing (352 g and platelet-rich plasma (200 g were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation. Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly

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...

DREAM plays an important role in platelet activation and thrombogenesis

Science.gov (United States)

Kim, Kyungho; Tseng, Alan; Barazia, Andrew; Italiano, Joseph E.

2017-01-01

Downstream regulatory element antagonist modulator (DREAM), a transcriptional repressor, is known to modulate pain responses. However, it is unknown whether DREAM is expressed in anucleate platelets and plays a role in thrombogenesis. By using intravital microscopy with DREAM-null mice and their bone marrow chimeras, we demonstrated that both hematopoietic and nonhematopoietic cell DREAMs are required for platelet thrombus formation following laser-induced arteriolar injury. In a FeCl3-induced thrombosis model, we found that compared with wild-type (WT) control and nonhematopoietic DREAM knockout (KO) mice, DREAM KO control and hematopoietic DREAM KO mice showed a significant delay in time to occlusion. Tail bleeding time was prolonged in DREAM KO control mice, but not in WT or DREAM bone marrow chimeric mice. In vivo adoptive transfer experiments further indicated the importance of platelet DREAM in thrombogenesis. We found that DREAM deletion does not alter the ultrastructural features of platelets but significantly impairs platelet aggregation and adenosine triphosphate secretion induced by numerous agonists (collagen-related peptide, adenosine 5′-diphosphate, A23187, thrombin, or U46619). Biochemical studies revealed that platelet DREAM positively regulates phosphoinositide 3-kinase (PI3K) activity during platelet activation. Using DREAM-null platelets and PI3K isoform-specific inhibitors, we observed that platelet DREAM is important for α-granule secretion, Ca2+ mobilization, and aggregation through PI3K class Iβ (PI3K-Iβ). Genetic and pharmacological studies in human megakaryoblastic MEG-01 cells showed that DREAM is important for A23187-induced Ca2+ mobilization and its regulatory function requires Ca2+ binding and PI3K-Iβ activation. These results suggest that platelet DREAM regulates PI3K-Iβ activity and plays an important role during thrombus formation. PMID:27903531

A New Platelet-Aggregation-Inhibiting Factor Isolated from Bothrops moojeni Snake Venom

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Bruna Barbosa de Sousa

2017-01-01

Full Text Available This work reports the purification and functional characterization of BmooPAi, a platelet-aggregation-inhibiting factor from Bothrops moojeni snake venom. The toxin was purified by a combination of three chromatographic steps (ion-exchange on DEAE-Sephacel, molecular exclusion on Sephadex G-75, and affinity chromatography on HiTrap™ Heparin HP. BmooPAi was found to be a single-chain protein with an apparent molecular mass of 32 kDa on 14% SDS-PAGE, under reducing conditions. Sequencing of BmooPAi by Edman degradation revealed the amino acid sequence LGPDIVPPNELLEVM. The toxin was devoid of proteolytic, haemorrhagic, defibrinating, or coagulant activities and induced no significant oedema or hyperalgesia. BmooPAi showed a rather specific inhibitory effect on ristocetin-induced platelet aggregation in human platelet-rich plasma, whereas it had little or no effect on platelet aggregation induced by collagen and adenosine diphosphate. The results presented in this work suggest that BmooPAi is a toxin comprised of disintegrin-like and cysteine-rich domains, originating from autolysis/proteolysis of PIII SVMPs from B. moojeni snake venom. This toxin may be of medical interest because it is a platelet aggregation inhibitor, which could potentially be developed as a novel therapeutic agent to prevent and/or treat patients with thrombotic disorders.

Platelet lysate activates quiescent cell proliferation and reprogramming in human articular cartilage: Involvement of hypoxia inducible factor 1.

Science.gov (United States)

Nguyen, Van Thi; Cancedda, Ranieri; Descalzi, Fiorella

2018-03-01

The idea of rescuing the body self-repair capability lost during evolution is progressively gaining ground in regenerative medicine. In particular, growth factors and bioactive molecules derived from activated platelets emerged as promising therapeutic agents acting as trigger for repair of tissue lesions and restoration of tissue functions. Aim of this study was to assess the potential of a platelet lysate (PL) for human articular cartilage repair considering its activity on progenitor cells and differentiated chondrocytes. PL induced the re-entry in the cell cycle of confluent, growth-arrested dedifferentiated/progenitor cartilage cells. In a cartilage permissive culture environment, differentiated cells also resumed proliferation after exposure to PL. These findings correlated with an up-regulation of the proliferation/survival pathways ERKs and Akt and with an induction of cyclin D1. In short- and long-term cultures of articular cartilage explants, we observed a release of proliferating chondroprogenitors able to differentiate and form an "in vitro" tissue with properties of healthy articular cartilage. Moreover, in cultured cartilage cells, PL induced a hypoxia-inducible factor (HIF-1) alpha increase, its nuclear relocation and the binding to HIF-1 responsive elements. These events were possibly related to the cell proliferation because the HIF-1 inhibitor acriflavine inhibited HIF-1 binding to HIF-1 responsive elements and cell proliferation. Our study demonstrates that PL induces quiescent cartilage cell activation and proliferation leading to new cartilage formation, identifies PL activated pathways playing a role in these processes, and provides a rationale to the application of PL for therapeutic treatment of damaged articular cartilage. Copyright © 2017 John Wiley & Sons, Ltd.

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

DEFF Research Database (Denmark)

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

2016-01-01

BACKGROUND: The results of laboratory analyses are affected by pre-analytical variables, and in particular can platelets be activated by shear handling stress and secrete granular substances. We therefore evaluated the effect of centrifugation speed and time on pre-analytical platelet activation....... METHODS: Citrate- and EDTA-anticoagulated blood from healthy volunteers were centrifuged at 80-10,000 g for 5-15 min to prepare plasma and platelet-rich plasma. Pre-analytical platelet activation was assessed by flow cytometric measurement of platelet P-selectin (CD62p) expression. Blood cell counts, mean...... of platelets expressing P-selectin in citrate- and EDTA-plasma centrifuged at 2000 g for 10 min were 43% [interquartile range (IQR), 38%-53%] and 56% (IQR, 31%-78%), respectively (p=0.82). Platelet-rich plasma prepared at 100-250 g for 10 min had significantly lower platelet P-selectin expression (11%-15%), p...

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

Increased platelet expression of FcGammaRIIa and its potential impact on platelet reactivity in patients with end stage renal disease

Directory of Open Access Journals (Sweden)

Sobel Burton E

2007-06-01

Full Text Available Abstract Background Increased platelet reactivity has been implicated in cardiovascular disease – the major cause of death in patients with end stage renal disease (ESRD. FcGammaRIIA is a component of glycoprotein VI and Ib-IX-V that mediate activation of platelets by collagen and von Willebrand factor. To determine whether expression of FcGammaRIIA impacts platelet reactivity we quantified its expression and platelet reactivity in 33 patients with ESRD who were undergoing hemodialysis. Methods Blood samples were obtained from patients immediately before hemodialysis and before administration of heparin. Platelet expression of FcGammaRIIA and the activation of platelets in response to low concentrations of convulxin (1 ng/ml, selected to mimic effects of collagen, thrombin (1 nM, adenosine diphosphate (ADP, 0.2 uM, or platelet activating factor (PAF, 1 nM were determined with the use of flow cytometry in samples of whole blood anticoagulated with corn trypsin inhibitor (a specific inhibitor of Factor XIIa. Results Patients were stratified with respect to the median expression of FcGammaRIIA. Patients with high platelet expression of FcGammaRIIA exhibited 3-fold greater platelet reactivity compared with that in those with low expression in response to convulxin (p Conclusion Increased platelet reactivity in response to low concentrations of diverse agonists is associated with high expression of FcGammaRIIA and may contribute to an increased risk of thrombosis in patients with ESRD.

Relationship between ADAMTS13 activity, von Willebrand factor antigen levels and platelet function in the early and late phases after TIA or ischaemic stroke.

Science.gov (United States)

McCabe, Dominick J H; Murphy, Stephen J X; Starke, Richard; Harrison, Paul; Brown, Martin M; Sidhu, Paul S; Mackie, Ian J; Scully, Marie; Machin, Samuel J

2015-01-15

Reduced ADAMTS13 activity is seen in thrombotic thrombocytopenic purpura (TTP), and may lead to accumulation of prothrombotic ultra-large von Willebrand factor (ULVWF) multimers in vivo. ADAMTS13 activity and its relationship with VWF antigen (VWF:Ag) levels and platelet function in 'non-TTP related' TIA or ischaemic stroke has not been comprehensively studied. In this prospective pilot observational analytical case-control study, ADAMTS13 activity and VWF:Ag levels were quantified in platelet poor plasma in 53 patients in the early phase (≤ 4 weeks) and 34 of these patients in the late phase (≥ 3 months) after TIA or ischaemic stroke on aspirin. Data were compared with those from 22 controls not on aspirin. The impact of ADAMTS13 on platelet function in whole blood was quantified by measuring Collagen-ADP (C-ADP) and Collagen-Epinephrine closure times on a platelet function analyser (PFA-100(®)). Median ADAMTS13 activity was significantly reduced in the early phase (71.96% vs. 95.5%, P TIA or stroke compared with controls (86.3% vs. 95.5%, P=0.19). There was a significant inverse relationship between ADAMTS13 activity and VWF:Ag levels in the early phase (r=-0.31; P=0.024), but not in the late phase after TIA or stroke (P=0.74). There was a positive correlation between ADAMTS13 activity and C-ADP closure times in early phase patients only, likely mediated via VWF:Ag levels. ADAMTS13 activity is reduced and VWF:Ag expression is increased within 4 weeks of TIA or ischaemic stroke onset, and can promote enhanced platelet adhesion and aggregation in response to stimulation with collagen and ADP via VWF-mediated pathways. These data improve our understanding of the dynamic haemostatic and thrombotic profiles of ischaemic cerebrovascular disease (CVD) patients, and are important in view of the potential future role that ADAMTS13 may have to play as an anti-thrombotic agent in CVD. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression of Angiogenesis Regulatory Proteins and Epithelial-Mesenchymal Transition Factors in Platelets of the Breast Cancer Patients

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Hui Han

2014-01-01

Full Text Available Platelets play a role in tumor angiogenesis and growth and are the main transporters of several angiogenesis regulators. Here, we aimed to determine the levels of angiogenesis regulators and epithelial-mesenchymal transition factors sequestered by circulating platelets in breast cancer patients and age-matched healthy controls. Platelet pellets (PP and platelet-poor plasma (PPP were collected by routine protocols. Vascular endothelial growth factor (VEGF, platelet-derived growth factor BB (PDGF-BB, thrombospondin-1 (TSP-1, platelet factor 4 (PF4, and transforming growth factor-β1 (TGF-β1 were measured by enzyme-linked immunosorbent assay. Angiogenesis-associated expression of VEGF (2.1 pg/106 platelets versus 0.9 pg/106 platelets, P < 0.001, PF4 (21.2 ng/106 platelets versus 10.2 ng/106 platelets, P < 0.001, PDGF-BB (42.9 pg/106 platelets versus 19.1 pg/106 platelets, P < 0.001, and TGF-β1 (15.3 ng/106 platelets versus 4.3 ng/106 platelets, P < 0.001 differed in the PP samples of cancer and control subjects. In addition, protein concentrations were associated with clinical characteristics (P<0.05. Circulating platelets in breast cancer sequester higher levels of PF4, VEGF, PDGF-BB, and TGF-β1, suggesting a possible target for early diagnosis. VEGF, PDGF, and TGF-β1 concentrations in platelets may be associated with prognosis.

Sulfatides partition disabled-2 in response to platelet activation.

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Karen E Drahos

Full Text Available BACKGROUND: Platelets contact each other at the site of vascular injury to stop bleeding. One negative regulator of platelet aggregation is Disabled-2 (Dab2, which is released to the extracellular surface upon platelet activation. Dab2 inhibits platelet aggregation through its phosphotyrosine-binding (PTB domain by competing with fibrinogen for alphaIIbbeta3 integrin receptor binding by an unknown mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Using protein-lipid overlay and liposome-binding assays, we identified that the N-terminal region of Dab2, including its PTB domain (N-PTB, specifically interacts with sulfatides. Moreover, we determined that such interaction is mediated by two conserved basic motifs with a dissociation constant (K(d of 0.6 microM as estimated by surface plasmon resonance (SPR analysis. In addition, liposome-binding assays combined with mass spectroscopy studies revealed that thrombin, a strong platelet agonist, cleaved N-PTB at a site located between the basic motifs, a region that becomes protected from thrombin cleavage when bound to sulfatides. Sulfatides on the platelet surface interact with coagulation proteins, playing a major role in haemostasis. Our results show that sulfatides recruit N-PTB to the platelet surface, sequestering it from integrin receptor binding during platelet activation. This is a transient recruitment that follows N-PTB internalization by an actin-dependent process. CONCLUSIONS/SIGNIFICANCE: Our experimental data support a model where two pools of Dab2 co-exist at the platelet surface, in both sulfatide- and integrin receptor-bound states, and their balance controls the extent of the clotting response.

Effects of sodium citrate and acid citrate dextrose solutions on cell counts and growth factor release from equine pure-platelet rich plasma and pure-platelet rich gel.

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Giraldo, Carlos E; Álvarez, María E; Carmona, Jorge U

2015-03-14

There is a lack information on the effects of the most commonly used anticoagulants for equine platelet rich plasmas (PRPs) elaboration on cell counts and growth factor release from platelet rich gels (PRGs). The aims of this study were 1) to compare the effects of the anticoagulants sodium citrate (SC), acid citrate dextrose solution A (ACD-A) and ACD-B on platelet (PLT), leukocyte (WBC) and on some parameters associated to platelet activation including mean platelet volume (MPV) and platelet distribution width (PDW) between whole blood, pure PRP (P-PRP) and platelet-poor plasma (PPP); 2) to compare transforming growth factor beta 1 (TGF-β(1)) and platelet-derived growth factor isoform BB (PDGF-BB) concentrations in supernatants from pure PRG (P-PRG), platelet-poor gel (PPG), P-PRP lysate (positive control) and plasma (negative control); 3) to establish the possible correlations between all the studied cellular and molecular parameters. In all cases the three anticoagulants produced P-PRPs with significantly higher PLT counts compared with whole blood and PPP. The concentrations of WBCs were similar between P-PRP and whole blood, but significantly lower in PPP. The type of anticoagulant did not significantly affect the cell counts for each blood component. The anticoagulants also did not affect the MPV and PDW parameters. Independently of the anticoagulant used, all blood components presented significantly different concentrations of PDGF-BB and TGF-β(1). The highest growth factor (GF) concentrations were observed from P-PRP lysates, followed by PRG supernatants, PPP lysates, PPG supernatants and plasma. Significant correlations were observed between PLT and WBC counts (ρ = 0.80), PLT count and TGF-β(1) concentration (ρ = 0.85), PLT count and PDGF-BB concentration (ρ = 0.80) and PDGF-BB and TGF-β(1) concentrations (ρ = 0.75). The type of anticoagulant was not correlated with any of the variables evaluated. The anticoagulants did not

Kaempferol inhibits thrombosis and platelet activation.

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Choi, Jun-Hui; Park, Se-Eun; Kim, Sung-Jun; Kim, Seung

2015-08-01

The objectives of the present study were to investigate whether kaempferol affects pro-coagulant proteinase activity, fibrin clot formation, blood clot and thrombin (or collagen/epinephrine)-stimulated platelet activation, thrombosis, and coagulation in ICR (Imprinting Control Region) mice and SD (Sprague-Dawley) rats. Kaempferol significantly inhibited the enzymatic activities of thrombin and FXa by 68 ± 1.6% and 52 ± 2.4%, respectively. Kaempferol also inhibited fibrin polymer formation in turbidity. Microscopic analysis was performed using a fluorescent conjugate. Kaempferol completely attenuated phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38, c-Jun N-terminal kinase (JNK) 1/2, and phosphoinositide 3-kinase (PI3K)/PKB (AKT) in thrombin-stimulated platelets and delayed aggregation time (clotting) by 34.6% in an assay of collagen/epinephrine-stimulated platelet activation. Moreover, kaempferol protected against thrombosis development in 3 animal models, including collagen/epinephrine- and thrombin-induced acute thromboembolism models and an FeCl3-induced carotid arterial thrombus model. The ex vivo anticoagulant effect of kaempferol was further confirmed in ICR mice. This study demonstrated that kaempferol may be clinically useful due to its ability to reduce or prevent thrombotic challenge. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Platelet activation and platelet-leukocyte interaction in dogs naturally infected with Babesia rossi

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Goddard, Amelia; Leisewitz, Andrew L; Kristensen, Annemarie Thuri

2015-01-01

EDTA as anticoagulant. Activated platelets and PLA formation were detected by measuring surface expression of P-selectin (CD62P) on platelets, monocytes and neutrophils. Of the Babesia-infected dogs, 29 survived and seven died. The percentage of CD62P-positive monocytes was significantly higher (P = 0.......036) in the Babesia-infected dogs (54%) than in healthy control dogs (35.3%). However, there were no significant differences between the Babesia-infected and control groups for CD62P-positive platelets (4.9% and 1.2%, respectively) and CD62P-positive neutrophils (28.3% and 17.9%, respectively). The percentage of CD62...... groups for the percentage of CD62P-positive platelets (survivors 4.8%; non-survivors 5.3%; controls 1.2%) or CD62P-positive neutrophils (survivors 31.6%; non-survivors 5.6%; controls 17.9%). In conclusion, Babesia-infected dogs, specifically dogs that survived, had a significantly increased percentage...

Growth factor and proteinase profile of Vivostat® platelet-rich fibrin linked to tissue repair.

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Agren, M S; Rasmussen, K; Pakkenberg, B; Jørgensen, B

2014-07-01

Autologous platelet-rich fibrin (PRF(®)) is prepared by the automatic Vivostat(®) system. Conflicting results with Vivostat PRF in acute wound healing prompted us to examine its cellular and biomolecular composition. Specifically, platelets, selected growth factors and matrix metalloproteinase (MMP)-9 were quantified using novel analytical methods. Ten healthy non-thrombocytopenic volunteers donated blood for generation of intermediate fibrin-I and final PRF. Anticoagulated whole blood and serum procured in parallel served as baseline controls. Leucocyte, erythrocyte and platelet counts in whole blood and fibrin-I were determined by automated haematology analyser. Platelet concentration in PRF was quantified manually by stereologic analysis of Giemsa-stained tissue sections, and the total content of five growth factors and MMP-9 by enzyme-linked immunosorbent assays. The number of leucocytes and erythrocytes was reduced (P platelets increased (P fibrin-I versus whole blood. PRF contained 982 ± 206 × 10(9) platelets/l representing 3·9-fold (P platelet-derived growth factor (PDGF)-AB [2·5-fold, P PDGF-BB [1·6-fold, P vascular endothelial growth factor > basic fibroblast growth factor [75-fold, P platelet enrichment and biomolecular constituents may guide clinicians in their optimal use of Vivostat PRF for tissue regenerative applications. © 2013 International Society of Blood Transfusion.

Platelet activating factor induces transient blood-brain barrier opening to facilitate edaravone penetration into the brain.

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Fang, Weirong; Zhang, Rui; Sha, Lan; Lv, Peng; Shang, Erxin; Han, Dan; Wei, Jie; Geng, Xiaohan; Yang, Qichuan; Li, Yunman

2014-03-01

The blood-brain barrier (BBB) greatly limits the efficacy of many neuroprotective drugs' delivery to the brain, so improving drug penetration through the BBB has been an important focus of research. Here we report that platelet activating factor (PAF) transiently opened BBB and facilitated neuroprotectant edaravone penetration into the brain. Intravenous infusion with PAF induced a transient BBB opening in rats, reflected by increased Evans blue leakage and mild edema formation, which ceased within 6 h. Furthermore, rat regional cerebral blood flow (rCBF) declined acutely during PAF infusion, but recovered slowly. More importantly, this transient BBB opening significantly increased the penetration of edaravone into the brain, evidenced by increased edaravone concentrations in tissue interstitial fluid collected by microdialysis and analyzed by Ultra-performance liquid chromatograph combined with a hybrid quadrupole time-of-flight mass spectrometer (UPLC-MS/MS). Similarly, incubation of rat brain microvessel endothelial cells monolayer with 1 μM PAF for 1 h significantly increased monolayer permeability to (125)I-albumin, which recovered 1 h after PAF elimination. However, PAF incubation with rat brain microvessel endothelial cells for 1 h did not cause detectable cytotoxicity, and did not regulate intercellular adhesion molecule-1, matrix-metalloproteinase-9 and P-glycoprotein expression. In conclusion, PAF could induce transient and reversible BBB opening through abrupt rCBF decline, which significantly improved edaravone penetration into the brain. Platelet activating factor (PAF) transiently induces BBB dysfunction and increases BBB permeability, which may be due to vessel contraction and a temporary decline of regional cerebral blood flow (rCBF) triggered by PAF. More importantly, the PAF induced transient BBB opening facilitates neuroprotectant edaravone penetration into brain. The results of this study may provide a new approach to improve drug delivery into

Overview of platelet physiology and laboratory evaluation of platelet function.

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Rodgers, G M

1999-06-01

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

Platelet activation, adhesion, inflammation, and aggregation potential are altered in the presence of electronic cigarette extracts of variable nicotine concentrations.

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Hom, Sarah; Chen, Li; Wang, Tony; Ghebrehiwet, Berhane; Yin, Wei; Rubenstein, David A

2016-11-01

Tobacco smoke extracts prepared from both mainstream and sidestream smoking have been associated with heightened platelet activation, aggregation, adhesion, and inflammation. Conversely, it has been shown that pure nicotine inhibits similar platelet functions. In this work, we 1) evaluated the effects of e-cigarette extracts on platelet activities and 2) elucidated the differences between the nicotine-dependent and non-nicotine dependent (e.g. fine particulate matter or toxic compounds) effects of tobacco and e-cigarette products on platelet activities. To accomplish these goals, platelets from healthy volunteers (n = 50) were exposed to tobacco smoke extracts, e-cigarette vapor extracts, and pure nicotine and changes in platelet activation, adhesion, aggregation, and inflammation were evaluated, using optical aggregation, flow cytometry, and ELISA methods. Interestingly, the exposure of platelets to e-vapor extracts induced a significant up-regulation in the expression of the pro-inflammatory gC1qR and cC1qR and induced a marked increase in the deposition of C3b as compared with traditional tobacco smoke extracts. Similarly, platelet activation, as measured by a prothrombinase based assay, and platelet aggregation were also significantly enhanced after exposure to e-vapor extracts. Finally, platelet adhesion potential toward fibrinogen, von Willebrand factor, and other platelets was also enhanced after exposure to e-cigarette vapor extracts. In the presence of pure nicotine, platelet functions were observed to be inhibited, which further suggests that other constituents of tobacco smoke and electronic vapor can antagonize platelet functions, however, the presence of nicotine in extracts somewhat perpetuated the platelet functional changes in a dose-dependent manner.

Vasoactive side effects of intravenous immunoglobulin preparations in a rat model and their treatment with recombinant platelet-activating factor acetylhydrolase

NARCIS (Netherlands)

Bleeker, W. K.; Teeling, J. L.; Verhoeven, A. J.; Rigter, G. M.; Agterberg, J.; Tool, A. T.; Koenderman, A. H.; Kuijpers, T. W.; Hack, C. E.

2000-01-01

Previously, we observed in a rat model that intravenous administration of intramuscular immunoglobulin preparations induced a long-lasting hypotension, which appeared to be associated with the presence of IgG polymers and dimers in the preparations, but unrelated to complement activation. We found

Platelet-vessel wall interaction in health and disease

NARCIS (Netherlands)

Löwenberg, E. C.; Meijers, J. C. M.; Levi, M. [=Marcel M.

2010-01-01

Upon vessel wall injury platelets rapidly adhere to the exposed subendothelial matrix which is mediated by several cellular receptors present on platelets or endothelial cells and various adhesive proteins such as von Willebrand factor, collagen and fibrinogen. Subsequent platelet activation results

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

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Véronique Ollivier

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

The novel role of platelet-activating factor in protecting mice against lipopolysaccharide-induced endotoxic shock.

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Young-Il Jeong

Full Text Available BACKGROUND: Platelet-activating factor (PAF has been long believed to be associated with many pathophysiological processes during septic shock. Here we present novel activities for PAF in protecting mice against LPS-mediated endotoxic shock. PRINCIPAL FINDINGS: In vivo PAF treatment immediately after LPS challenge markedly improved the survival rate against mortality from endotoxic shock. Administration of PAF prominently attenuated LPS-induced organ injury, including profound hypotension, excessive polymorphonuclear neutrophil infiltration, and severe multiple organ failure. In addition, PAF treatment protects against LPS-induced lymphocytes apoptosis. These protective effects of PAF was correlated with significantly decreases in the production of the inflammatory mediators such as TNF-alpha, IL-1beta, IL-12, and IFN-gamma, while increasing production of the anti-inflammatory cytokine IL-10 in vivo and in vitro. CONCLUSIONS: Taken together, these results suggest that PAF may protect mice against endotoxic shock via a complex mechanism involving modulation of inflammatory and anti-inflammatory mediators.

Salivary Platelet Activating Factor Levels in Periodontal Disease

Science.gov (United States)

1991-05-01

toxins may be present (Madri, 1990). In addition, endogenous mediators, such as C5a, may attract polymorphonuclear leukocytes (PMN) to the site of...relatively harmless to many cells in vitro and in vivo with most pathology resulting from release of endogenous mediators from inflammatory cells (Beutler and...and lipids dissolved in pyrogen -free saline containing 0.25% bovine serum albumin (BSA; Miles Laboratories, Elkhart, IN). Rabbit platelets were

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

Science.gov (United States)

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.

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.

Effect of exercise on platelet activation during aspirin or clopidogrel intake in healthy men

DEFF Research Database (Denmark)

Hjorth Madsen, Esben; Christiansen, Morten Krogh; Schmidt, Erik Berg

2009-01-01

aggregometry. A significant increase in plasma von Willebrand Factor was also found in response to exercise. In conclusion, platelet activation occurs during exercise in healthy individuals. This activation is not prevented by use of aspirin or clopidogrel, and may partly be explained by an increase in plasma...

Metabolomics analyses identify platelet activating factors and heme breakdown products as Lassa fever biomarkers.

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Trevor V Gale

2017-09-01

Full Text Available Lassa fever afflicts tens of thousands of people in West Africa annually. The rapid progression of patients from febrile illness to fulminant syndrome and death provides incentive for development of clinical prognostic markers that can guide case management. The small molecule profile of serum from febrile patients triaged to the Viral Hemorrhagic Fever Ward at Kenema Government Hospital in Sierra Leone was assessed using untargeted Ultra High Performance Liquid Chromatography Mass Spectrometry. Physiological dysregulation resulting from Lassa virus (LASV infection occurs at the small molecule level. Effects of LASV infection on pathways mediating blood coagulation, and lipid, amino acid, nucleic acid metabolism are manifest in changes in the levels of numerous metabolites in the circulation. Several compounds, including platelet activating factor (PAF, PAF-like molecules and products of heme breakdown emerged as candidates that may prove useful in diagnostic assays to inform better care of Lassa fever patients.

Nanodiamonds activate blood platelets and induce thromboembolism.

Science.gov (United States)

Kumari, Sharda; Singh, Manoj K; Singh, Sunil K; Grácio, José J A; Dash, Debabrata

2014-03-01

Nanodiamonds (NDs) have been evaluated for a wide range of biomedical applications. Thus, thorough investigation of the biocompatibility of NDs has become a research priority. Platelets are highly sensitive and are one of the most abundant cell types found in blood. They have a central role in hemostasis and arterial thrombosis. In this study, we aim to investigate the direct and acute effects of carboxylated NDs on platelet function. In this study, pro-coagulant parameters such as platelet aggregability, intracellular Ca(2+) flux, mitochondrial transmembrane potential (ΔΨm), generation of reactive oxygen species, surface exposure of phosphatidylserine, electron microscopy, cell viability assay and in vivo thromboembolism were analyzed in great detail. Carboxylated NDs evoked significant activation of human platelets. When administered intravenously in mice, NDs were found to induce widespread pulmonary thromboembolism, indicating the remarkable thrombogenic potential of this nanomaterial. Our findings raise concerns regarding the putative biomedical applications of NDs pertaining to diagnostics and therapeutics, and their toxicity and prothrombotic properties should be critically evaluated.

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

Science.gov (United States)

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

Factor H C-Terminal Domains Are Critical for Regulation of Platelet/Granulocyte Aggregate Formation

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Adam Z. Blatt

2017-11-01

Full Text Available Platelet/granulocyte aggregates (PGAs increase thromboinflammation in the vasculature, and PGA formation is tightly controlled by the complement alternative pathway (AP negative regulator, Factor H (FH. Mutations in FH are associated with the prothrombotic disease atypical hemolytic uremic syndrome (aHUS, yet it is unknown whether increased PGA formation contributes to the thrombosis seen in patients with aHUS. Here, flow cytometry assays were used to evaluate the effects of aHUS-related mutations on FH regulation of PGA formation and characterize the mechanism. Utilizing recombinant fragments of FH spanning the entire length of the protein, we mapped the regions of FH most critical for limiting AP activity on the surface of isolated human platelets and neutrophils, as well as the regions most critical for regulating PGA formation in human whole blood stimulated with thrombin receptor-activating peptide (TRAP. FH domains 19–20 were the most critical for limiting AP activity on platelets, neutrophils, and at the platelet/granulocyte interface. The role of FH in PGA formation was attributed to its ability to regulate AP-mediated C5a generation. AHUS-related mutations in domains 19–20 caused differential effects on control of PGA formation and AP activity on platelets and neutrophils. Our data indicate FH C-terminal domains are key for regulating PGA formation, thus increased FH protection may have a beneficial impact on diseases characterized by increased PGA formation, such as cardiovascular disease. Additionally, aHUS-related mutations in domains 19–20 have varying effects on control of TRAP-mediated PGA formation, suggesting that some, but not all, aHUS-related mutations may cause increased PGA formation that contributes to excessive thrombosis in patients with aHUS.

Rac1 is essential for phospholipase C-gamma2 activation in platelets

DEFF Research Database (Denmark)

Pleines, Irina; Elvers, Margitta; Strehl, Amrei

2008-01-01

isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCgamma2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC......Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cbeta or PLCgamma2. Active PLCs trigger Ca(2+) mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCbeta...... regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation...

Mechanism study of endothelial protection and inhibits platelet activation of low molecular weight fucoidan from Laminaria japonica

Science.gov (United States)

Chen, Anjin; Zhang, Fang; Shi, Jie; Zhao, Xue; Yan, Meixing

2016-10-01

Several studies have indicated that fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica could inhibit the activation of platelets directly by reducing the platelet aggregation. To explore the direct effect of LMW fucoidan on the platelet system furthermore and examine the possible mechanism, the endothelial protection and inhibits platelet activation effects of two LMW fucoidan were investigated. In the present study, Endothelial injury model of rats was made by injection of adrenaline (0.4 mg kg-1) and human umbilical vein endothelial cells were cultured. vWF level was be investigated in vivo and in vitro as an important index of endothelial injury. LMW fucoidan could significantly reduce vWF level in vascular endothelial injury rats and also significantly reduce vWF level in vitro. The number of EMPs was be detected as another important index of endothelial injury. The results showed that LMW fucoidan reduced EMPs stimulated by tumor necrosis factor. In this study, it was found that by inhibiting platelet adhesion, LMW fucoidan played a role in anti-thrombosis and the specific mechanism of action is to inhibit the flow of extracellular Ca2+. All in a word, LMW fucoidan could inhibit the activation of platelets indirectly by reducing the concentration of EMPs and vWF, at the same time; LMW fucoidan inhibited the activation of platelets directly by inhibiting the flow of extracellular Ca2+.

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.

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

International Nuclear Information System (INIS)

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

Safety of recombinant human platelet-derived growth factor-BB in Augment® Bone Graft

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Luis A Solchaga

2012-12-01

Full Text Available This article discusses nonclinical and clinical data regarding the safety of recombinant human platelet-derived growth factor-BB as a component of the Augment® Bone Graft (Augment. Augment is a bone graft substitute intended to be used as an alternative to autologous bone graft in the fusion of hindfoot and ankle joints. Nonclinical studies included assessment of the pharmacokinetic profile of intravenously administered recombinant human platelet-derived growth factor-BB in rat and dog, effects of intravenous administration of recombinant human platelet-derived growth factor-BB in a reproductive and development toxicity study in rats, and chronic toxicity and carcinogenicity of Augment in a 12-month implantation model. These studies showed that systemic exposure was brief and clearance was rapid. No signs of toxicity, carcinogenicity, or tumor promotion were observed even with doses far exceeding the maximum clinical dose. Results of clinical trials (605 participants and commercial use of recombinant human platelet-derived growth factor-BB containing products indicate that these products are not associated with increased incidence of adverse events or cancer. The safety data presented provide evidence that recombinant human platelet-derived growth factor-BB is a safe therapeutic when used in combination products as a single administration during surgical procedures for bone repair and fusion. There is no evidence associating use of recombinant human platelet-derived growth factor-BB in Augment with chronic toxicity, carcinogenicity, or tumor promotion.

[3H]52770 RP, a platelet-activating factor receptor antagonist, and tritiated platelet-activating factor label a common specific binding site in human polymorphonuclear leukocytes

International Nuclear Information System (INIS)

Marquis, O.; Robaut, C.; Cavero, I.

1988-01-01

In human polymorphonuclear leukocytes (PMNs), the tritiated platelet activating factor ([ 3 H]PAF) labels in a saturable manner a single class of binding sites with a Kd of 3.5 +/- 0.5 nM (n = 7) and a maximum binding capacity (Bmax) of 206 +/- 13 fmol/2.5 X 10(6) PMNs (n = 7). 52770 RP, a nonphospholipid antagonist of PAF receptors, fully and competitively displaced the [ 3 H]PAF from its binding sites with a Ki of 7.0 +/- 0.7 nM (n = 4). The high potency and the low solubility in cellular membranes of this compound led us to prepare [ 3 H]52770 RP. This ligand was characterized by a binding which was rapid, reversible, confined to a single site, saturable, specific and stereoselective. Its Kd and Bmax were 4.2 +/- 0.3 nM and 181 +/- 11 fmol/2.5 X 10(6) PMNs, respectively. The stereoselectivity of the binding was suggested by the 600- and 1050-fold higher potency of the d-enantiomer with respect to l-52770 RP in displacing [ 3 H]52770 RP or [ 3 H]PAF, respectively. Several PAF analogs (e.g., lyso-PAF, 2-O-methyl-lyso-PAF), which are poorly active as PAF receptor agonists in functional tests, were weak displacers of [ 3 H]PAF and [ 3 H]52770 RP. Furthermore, for a series of 14 known PAF receptor agonists or antagonists belonging to different chemical families, there was an excellent correlation (r = 0.98) between their ability to displace [ 3 H]PAF and [ 3 H]52770 RP. Thus, [ 3 H]52770 RP and [ 3 H]PAF appear to interact with the same binding site on human PMNs which is proposed to be the PAF receptor mediating functional responses

Activated platelets enhance IL-10 secretion and reduce TNF-α secretion by monocytes

DEFF Research Database (Denmark)

Gudbrandsdottir, Sif; Hasselbalch, Hans C; Nielsen, Claus H

2013-01-01

), Escherichia coli LPS, or intact Porphyromonas gingivalis. Addition of platelets activated by thrombin-receptor-activating peptide enhanced IL-10 production induced by LPS (p gingivalis (p ....05), and P. gingivalis (p gingivalis-stimulated cultures (p ... of activated platelets. Adherence of platelets increased TG- and TT-induced IL-10 secretion by monocytes (p gingivalis (p

Effects of pathogen reduction systems on platelet microRNAs, mRNAs, activation, and function.

Science.gov (United States)

Osman, Abdimajid; Hitzler, Walter E; Meyer, Claudius U; Landry, Patricia; Corduan, Aurélie; Laffont, Benoit; Boilard, Eric; Hellstern, Peter; Vamvakas, Eleftherios C; Provost, Patrick

2015-01-01

Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen+ ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin+ UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p < 0.05) and an impaired platelet aggregation response to ADP (p < 0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established.

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

Science.gov (United States)

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.

Comparison of platelet activation through hinge vs bulk flow in mechanical heart valves

Science.gov (United States)

Hedayat, Mohammadali; Borazjani, Iman

2017-11-01

Bileaflet mechanical heart valves increase the risk of thrombus formation in patients which is believed to be initiated by platelet activation. Platelets can be activated by the elevated shear stresses in the bulk flow during the systole phase or the flow through the hinge during the diastole. However, the importance of platelet activation by the bulk flow vs the hinge in MHVs has yet to be studied. Here, we investigate the contribution of each of the above mechanisms to the activation of platelets in MHs by performing simulation of the flow through a 25mm St. Jude Medical valve placed in a straight aorta. Two different gap sizes (250 and 150 micrometer) are used in this study. The simulations are done using a sharp interface curvilinear immersed boundary method along with a strong-coupling algorithm for FSI solver on overset grids. The platelet activation through the hinge for different gap sizes is compared to the activation in the bulk flow using two platelet activation models to ensure the consistency of the results. Our results for all gap sizes using different activation models show that the integration of platelet activation caused by the bulk flow is several times higher in comparison to the activation through the hinge. This work is supported by the American Heart Association Grant 13SDG17220022, and the computational resources were partly provided by Center for Computational Research (CCR) at University at Buffalo.

The Platelet Aggregation-Inducing Factor Aggrus/Podoplanin Promotes Pulmonary Metastasis

Science.gov (United States)

Kunita, Akiko; Kashima, Takeshi G.; Morishita, Yasuyuki; Fukayama, Masashi; Kato, Yukinari; Tsuruo, Takashi; Fujita, Naoya

2007-01-01

Tumor cell-induced platelet aggregation has been reported to facilitate hematogenous metastasis. Aggrus/podoplanin is a platelet aggregation-inducing factor that is up-regulated in a number of human cancers and has been implicated in tumor progression. We studied herein the role of Aggrus in tumor growth, metastasis, and survival in vivo. Aggrus expression in Chinese hamster ovary cells promoted pulmonary metastasis in both an experimental and a spontaneous mouse model. No differences in the size of metastatic foci or in primary tumor growth were found in either set of mice. Aggrus-expressing cells, which were covered with platelets, arrested in the lung microvasculature 30 minutes after injection. In addition, lung metastasis resulting from Aggrus expression decreased the survival of the mice. By generating several Aggrus point mutants, we revealed that point mutation at the platelet aggregation-stimulating domain of Aggrus (Thr34 and Thr52) obliterated both platelet aggregation and metastasis. Furthermore, administration of aspirin to mice reduced the number of metastatic foci. These results indicate that Aggrus contributes to the establishment of metastasis by promoting platelet aggregation without affecting subsequent growth. Thus, Aggrus could serve as an ideal therapeutic target for drug development to block metastasis. PMID:17392172

Platelet-activating factor dilates efferent arterioles through glomerulus-derived nitric oxide.

Science.gov (United States)

Arima, S; Ren, Y; Juncos, L A; Ito, S

1996-01-01

Despite evidence that platelet-activating factor (PAF) is produced by the glomerulus, its direct action on the glomerular microcirculation is poorly understood. It was recently reported that at picomolar concentrations, PAF dilates isolated microperfused afferent arterioles (Af-Art) via nitric oxide (NO). The present study tested the hypothesis that PAF acts on the glomerulus to release NO, which in turn controls the resistance of the efferent arteriole (Ef-Art). Rabbit Ef-Art were perfused from the distal end (retrograde perfusion [RP]) to eliminate the influence of the glomerulus, or through the glomerulus from the end of the Af-Art (orthograde perfusion [OP]) to maintain the influence of the glomerulus. Ef-Art were preconstricted by approximately 40% with norepinephrine and increasing doses of PAF were added to both the arteriolar perfusate and bath. Only with OP did PAF at picomolar concentrations cause significant dilation: at 400 pmol, the diameter increased by 64 +/- 11% from the preconstricted level (N = 6, P Art. At nanomolar concentrations, PAF constricted Ef-Art similarly in both RP and OP: at 40 nM, the diameter decreased by 24 +/- 4% (N = 6, P OP (10 +/- 2%, N = 6; P Art; and (2) at nanomolar concentrations, PAF constricts the Ef-Art partly through release of cyclooxygenase metabolites. Thus, PAF may play a role in glomerular hemodynamics under various physiological and pathological conditions.

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

Calidad del plasma rico en plaquetas: estudio de la activación plaquetaria Platelet-rich plasma quality: a study on platelet activation

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C. Sáez-Torres Barroso

2007-08-01

Full Text Available Objetivo. El plasma rico en plaquetas (PRP es utilizado de forma cada vez más frecuente en técnicas quirúrgicas de regeneración tisular. No obstante, el procesamiento de la sangre hasta obtener PRP puede desencadenar la activación prematura de las plaquetas y la pérdida de los factores bioactivos. En este trabajo estudiamos la calidad de los concentrados de plaquetas obtenidos siguiendo la técnica de doble centrifugación en tubo. Método. Se someten 50 ml de sangre a una primera centrifugación a 200g 10 minutos, se recoge el sobrenadante y se centrifuga a 700g 15 minutos. Posteriormente, tras eliminar las 2/3 partes del plasma, se resuspenden las plaquetas y se analiza el grado de enriquecimiento, el estado de activación y la reserva funcional de las plaquetas. Resultados. El enriquecimiento en plaquetas del PRP fue de 364±177% (n=45 respecto de los niveles presentes en sangre total. Mediante el estudio de la expresión de CD62 por citometría de flujo se determinó el porcentaje de plaquetas activadas en las muestras de 8 donantes. Mientras que en la sangre no procesada se detectó un 2,7% de plaquetas activadas, tras la preparación del PRP éste era sólo de 3,6%, aumentando hasta el 16% en el concentrado almacenado toda la noche a 22º C. Tras la estimulación con trombina el porcentaje de plaquetas activadas fue de 96,2%. Conclusión. Este protocolo de preparación de PRP no produce una activación significativa de las plaquetas. La respuesta a la estimulación con trombina de los concentrados indica un buen estado de reserva plaquetaria.Objective. Platelet Rich Plasma (PRP is an autologous preparation currently used in oral and maxillofacial reconstructive surgery. Blood collection and preparation of platelet concentrates may lead to platelet activation and the premature loss of their granular load. In this study, we have analyzed the quality of the PRP obtained from a small volume of whole blood through a double centrifugation

Increased platelet activation in early symptomatic versus asymptomatic carotid stenosis and relationship with microembolic status: Results from the Platelets And Carotid Stenosis (PACS) Study.

LENUS (Irish Health Repository)

Kinsella, Ja

2013-04-26

BACKGROUND: Cerebral microembolic signals (MES) may predict increased stroke risk in carotid stenosis. However, the relationship between platelet counts or platelet activation status and MES in symptomatic versus asymptomatic carotid stenosis has not been comprehensively assessed. SETTING: University teaching hospitals. METHODS: This prospective, pilot observational study assessed platelet counts and platelet activation status, and the relationship between platelet activation and MES in asymptomatic versus early (≤4 weeks after TIA\\/stroke) and late phase (≥3 months) symptomatic moderate or severe (≥50%) carotid stenosis patients. Full blood count measurements were performed, and whole blood flow cytometry was used to quantify platelet surface activation marker expression (CD62P and CD63) and circulating leucocyte-platelet complexes. Bilateral simultaneous transcranial Doppler ultrasound monitoring of the middle cerebral arteries was performed for 1 hour to classify patients as MES-positive or MES-negative. RESULTS: Data from 31 asymptomatic patients were compared with 46 symptomatic patients in the early phase, and 35 of these patients followed up to the late phase after symptom onset. The median platelet count (211 vs. 200 x 10(9) \\/L; p=0.03) and the median% lymphocyte-platelet complexes were higher in early symptomatic than asymptomatic patients (2.8 vs. 2.4%, p=0.001). The% lymphocyte-platelet complexes was higher in early symptomatic than asymptomatic patients with ≥70% carotid stenosis (p=0.0005), and in symptomatic patients recruited within 7 days of symptom onset (p=0.028). Complete TCD data were available in 25 asymptomatic and 31 early phase symptomatic, and 27 late phase symptomatic patients. 12% of asymptomatic versus 32% of early phase symptomatic (p=0.02) and 19% of late phase symptomatic patients (p=0.2) were MES-positive. Early symptomatic MES-negative patients had a higher% lymphocyte-platelet complexes than asymptomatic MES

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.

Clot lysis time in platelet-rich plasma: method assessment, comparison with assays in platelet-free and platelet-poor plasmas, and response to tranexamic acid.

Science.gov (United States)

Panes, Olga; Padilla, Oslando; Matus, Valeria; Sáez, Claudia G; Berkovits, Alejandro; Pereira, Jaime; Mezzano, Diego

2012-01-01

Fibrinolysis dysfunctions cause bleeding or predisposition to thrombosis. Platelets contain several factors of the fibrinolytic system, which could up or down regulate this process. However, the temporal relationship and relative contributions of plasma and platelet components in clot lysis are mostly unknown. We developed a clot lysis time (CLT) assay in platelet-rich plasma (PRP-CLT, with and without stimulation) and compared it to a similar one in platelet-free plasma (PFP) and to another previously reported test in platelet-poor plasma (PPP). We also studied the differential effects of a single dose of tranexamic acid (TXA) on these tests in healthy subjects. PFP- and PPP-CLT were significantly shorter than PRP-CLT, and the three assays were highly correlated (p plasma PAI-1, von Willebrand factor, fibrinogen, LDL-cholesterol, and triglycerides (p platelet aggregation/secretion, platelet counts, and pro-coagulant tests to explore factor X activation by platelets, PRP clotting time, and thrombin generation in PRP. Among all the studied variables, PFP-CLT was independently associated with plasma PAI-1, LDL-cholesterol, and triglycerides and, additionally, stimulated PRP-CLT was also independently associated with plasma fibrinogen. A single 1 g dose of TXA strikingly prolonged all three CLTs, but in contrast to the results without the drug, the lysis times were substantially shorter in non-stimulated or stimulated PRP than in PFP and PPP. This standardized PRP-CLT may become a useful tool to study the role of platelets in clot resistance and lysis. Our results suggest that initially, the platelets enmeshed in the clot slow down the fibrinolysis process. However, the increased clot resistance to lysis induced by TXA is overcome earlier in platelet-rich clots than in PFP or PPP clots. This is likely explained by the display of platelet pro-fibrinolytic effects. Focused research is needed to disclose the mechanisms for the relationship between CLT and plasma

Platelet activation and aggregation

DEFF Research Database (Denmark)

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

2013-01-01

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

Dabigatran reduces thrombin-induced platelet aggregation and activation in a dose-dependent manner

DEFF Research Database (Denmark)

Vinholt, Pernille Just; Nielsen, Christian; Söderström, Anna Cecilia

2017-01-01

Dabigatran is an oral anticoagulant and a reversible inhibitor of thrombin. Further, dabigatran might affect platelet function through a direct effect on platelet thrombin receptors. The aim was to investigate the effect of dabigatran on platelet activation and platelet aggregation. Healthy donor...

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

International Nuclear Information System (INIS)

Torr, S.R.; Winters, K.J.; Santoro, S.A.; Sobel, B.E.

1990-01-01

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

Effect of steroids on the activation status of platelets in patients with Immune thrombocytopenia (ITP).

Science.gov (United States)

Bhoria, Preeti; Sharma, Saniya; Varma, Neelam; Malhotra, Pankaj; Varma, Subhash; Luthra-Guptasarma, Manni

2015-01-01

The activation status of platelets in Immune Thrombocytopenia (ITP) patients--which is still somewhat controversial--is of potential interest, because activated platelets tend to aggregate (leading to excessive clotting or thromboembolic events) but cannot do so when platelet numbers are low, as in ITP. Although corticosteroids are the first line of therapy in ITP, the effect of steroids on activation of platelets has not been evaluated so far. We examined the status of platelet activation (with and without stimulation with ADP) in ITP patients, at the start of therapy (pre-steroid treatment, naive) and post-steroid treatment (classified on the basis of steroid responsiveness). We used flow cytometry to evaluate the levels of expression of P-selectin, and PAC-1 binding to platelets of 55 ITP patients and a similar number of healthy controls, treated with and without ADP. We found that platelets in ITP patients exist in an activated state. In patients who are responsive to steroids, the treatment reverses this situation. Also, the fold activation of platelets upon treatment with ADP is more in healthy controls than in ITP patients; treatment with steroids causes platelets in steroid-responsive patients to become more responsive to ADP-activation, similar to healthy controls. Thus steroids may cause changes in the ability of platelets to get activated with an agonist like ADP. Our results provide new insights into how, and why, steroid therapy helps in the treatment of ITP.

Exogenous modification of platelet membranes with the omega-3 fatty acids EPA and DHA reduces platelet procoagulant activity and thrombus formation.

Science.gov (United States)

Larson, Mark K; Tormoen, Garth W; Weaver, Lucinda J; Luepke, Kristen J; Patel, Ishan A; Hjelmen, Carl E; Ensz, Nicole M; McComas, Leah S; McCarty, Owen J T

2013-02-01

Several studies have implicated the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in inhibition of normal platelet function, suggesting a role for platelets in EPA- and DHA-mediated cardioprotection. However, it is unclear whether the cardioprotective mechanisms arise from alterations to platelet-platelet, platelet-matrix, or platelet-coagulation factor interactions. Our previous results led us to hypothesize that EPA and DHA alter the ability of platelets to catalyze the generation of thrombin. We tested this hypothesis by exogenously modifying platelet membranes with EPA and DHA, which resulted in compositional changes analogous to increased dietary EPA and DHA intake. Platelets treated with EPA and DHA showed reductions in the rate of thrombin generation and exposure of platelet phosphatidylserine. In addition, treatment of platelets with EPA and DHA decreased thrombus formation and altered the processing of thrombin precursor proteins. Furthermore, treatment of whole blood with EPA and DHA resulted in increased occlusion time and a sharply reduced accumulation of fibrin under flow conditions. These results demonstrate that EPA and DHA inhibit, but do not eliminate, the ability of platelets to catalyze thrombin generation in vitro. The ability of EPA and DHA to reduce the procoagulant function of platelets provides a possible mechanism behind the cardioprotective phenotype in individuals consuming high levels of EPA and DHA.

Glaucocalyxin A inhibits platelet activation and thrombus formation preferentially via GPVI signaling pathway.

Directory of Open Access Journals (Sweden)

Wei Li

Full Text Available Platelets play a pivotal role in atherothrombosis and the antiplatelet agents have been proved to be useful in preventing onset of acute clinical events including myocardial infarction and stroke. Increasing number of natural compounds has been identified to be potential antiplatelet agents. Here we report the antiplatelet effect of glaucocalyxin A (GLA, an ent-diterpenoid that we isolated and purified from the aerial parts of Rabdosia japonica (Burm. f. var. glaucocalyx (Maxim. Hara, and investigate the molecular mechanisms by which GLA inhibits platelet activation and thrombus formation. The effect of GLA on platelet activation was measured using platelets freshly isolated from peripheral blood of healthy donors. Results showed that pretreatment of human platelets with lower concentrations of GLA (0.01 μg/ml, 0.1 μg/ml significantly inhibited platelet aggregation induced by collagen (P<0.001 and CRP (P<0.01, a synthetic GPVI ligand, but not by ADP and U46619. Accordingly, GLA inhibited collagen-stimulated tyrosine phosphorylation of Syk, LAT, and phospholipase Cγ2, the signaling events in collagen receptor GPⅥ pathway. GLA also inhibited platelet p-selectin secretion and integrin activation by convulxin, a GPVI selective ligand. Additionally, GLA was found to inhibit low-dose thrombin-induced platelet activation. Using a flow chamber device, GLA was found to attenuate platelet adhesion on collagen surfaces in high shear condition. In vivo studies showed that GLA administration increased the time for complete occlusion upon vascular injury in mice, but did not extend tail-bleeding time when mice were administered with relatively lower doses of GLA. Therefore, the present results provide the molecular basis for the inhibition effect of GLA on platelet activation and its in vivo effect on thrombus formation, suggesting that GLA could potentially be developed as an antiplatelet and antithrombotic agent.

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.

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

The Phosphatase Inhibitor Calyculin-A Impairs Clot Retraction, Platelet Activation, and Thrombin Generation

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Renáta Hudák

2017-01-01

Full Text Available The aim of this study was to investigate the effect of the serine/threonine protein phosphatase inhibitor, calyculin-A (CLA, on clot formation and on the procoagulant activity of human platelets. Platelet-rich plasma (PRP samples were preincubated with buffer or CLA and subsequently platelets were activated by the protease-activated receptor 1 (PAR-1 activator, thrombin receptor activating peptide (TRAP. Clot retraction was detected by observing clot morphology up to 1 hour, phosphatidylserine- (PS- expression was studied by flow cytometry, and thrombin generation was measured by a fluorimetric assay. For the intracellular Ca2+ assay, platelets were loaded with calcium-indicator dyes and the measurements were carried out using a ratiometric method with real-time confocal microscopy. CLA preincubation inhibited clot retraction, PS-expression, and thrombin formation. TRAP activation elicited Ca2+ response and PS-expression in a subset of platelets. The activated PRP displayed significantly faster and enhanced thrombin generation compared to nonactivated samples. CLA pretreatment abrogated PS-exposure and clot retraction also in TRAP-activated samples. As a consequence of the inhibitory effect on calcium elevation and PS-expression, CLA significantly downregulated thrombin generation in PRP. Our results show that CLA pretreatment may be a useful tool to investigate platelet activation mechanisms that contribute to clot formation and thrombin generation.

Correlation between Platelet Gelsolin and Platelet Activation Level in Acute Myocardial Infarction Rats and Intervention Effect of Effective Components of Chuanxiong Rhizome and Red Peony Root

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Yue Liu

2013-01-01

Full Text Available The biological role of platelet gelsolin in platelet activation of acute myocardial infarction is not defined. In order to provide a potential new antiplatelet target for Chinese medicine and to elucidate the contribution of Xiongshao capsule, the effective components of Chuanxiong rhizome and red peony root, in this study, we randomly allocated Sprague Dawley rats to left anterior descending coronary artery ligation or sham surgery and different drug prophylaxis as control. We found that gelsolin is highly expressed in platelet rich plasma and lowly expressed in platelet poor plasma, accompanied by the high platelet activation level in model rats; plasma actin filaments and mean fluorescence intensity (MFI of platelet calcium ion increased and plasma vitamin D binding protein decreased in model rats. Xiongshao capsule could inhibit the gelsolin expression in platelet rich plasma and ischemic heart tissue simultaneously and reduce the level of plasma F-actin and MFI of platelet calcium ion. Our study concludes that platelet gelsolin is an important contributor to platelet activation, and platelet gelsolin inhibition may form a novel target for antiplatelet therapy. Xiongshao capsule may be a promising Chinese medicine drug for antiplatelet and aspirin-like cardioprotection effect.

Treatment with a histone deacetylase inhibitor, valproic acid, is associated with increased platelet activation in a large animal model of traumatic brain injury and hemorrhagic shock

DEFF Research Database (Denmark)

Dekker, Simone E; Sillesen, Martin; Bambakidis, Ted

2014-01-01

synergistic benefits. In this study, we hypothesized that VPA administration would be associated with a conservation of platelet function as measured by increased platelet activation after resuscitation. MATERIALS AND METHODS: Ten swine (42-50 kg) were subjected to TBI and HS (40% blood loss). Animals were...... neuroprotective effects of VPA may be due to a conservation of platelet function as measured by a higher platelet activation response after resuscitation....... left in shock for 2 h before resuscitation with either FFP or FFP + VPA (300 mg/kg). Serum levels of platelet activation markers transforming growth factor beta, CD40 L, P-selectin, and platelet endothelial cell adhesion molecule (PECAM) 1 were measured at baseline, postresuscitation, and after a 6-h...

Lysophosphatidylcholine acyltransferase1 overexpression promotes oral squamous cell carcinoma progression via enhanced biosynthesis of platelet-activating factor.

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Shida-Sakazume, Tomomi; Endo-Sakamoto, Yosuke; Unozawa, Motoharu; Fukumoto, Chonji; Shimada, Ken; Kasamatsu, Atsushi; Ogawara, Katsunori; Yokoe, Hidetaka; Shiiba, Masashi; Tanzawa, Hideki; Uzawa, Katsuhiro

2015-01-01

The relevance of lysophosphatidylcholine acyltransferase1 (LPCAT1), a cytosolic enzyme in the remodeling pathway of phosphatidylcholine metabolism, in oral squamous cell carcinoma (OSCC) is unknown. We investigated LPCAT1 expression and its functional mechanism in OSCCs. We analyzed LPCAT1 mRNA and protein expression levels in OSCC-derived cell lines. Immunohistochemistry was performed to identify correlations between LPCAT1 expression levels and primary OSCCs clinicopathological status. We established LPCAT1 knockdown models of the OSCC-derived cell lines (SAS, Ca9-22) for functional analysis and examined the association between LPCAT1 expression and the platelet-activating factor (PAF) concentration and PAF-receptor (PAFR) expression. LPCAT1 mRNA and protein were up-regulated significantly (poral keratinocytes. Immunohistochemistry showed significantly (poral cancer.

Considerations on the Use of Platelet-Rich Plasma, Specifically for Burn Treatment

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Marck, R.E.; Middelkoop, E.; Breederveld, R.S.

2014-01-01

Platelet-rich plasma (PRP) is a fraction of blood plasma with a platelet concentration above baseline. After activation of the platelets, growth factors are released, which are involved in wound-healing processes. Application of a multitude of growth factors seems to boost the healing process. In

Release kinetics of platelet-derived and plasma-derived growth factors from autologous plasma rich in growth factors.

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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.

Intra-plaque production of platelet-activating factor correlates with neoangiogenesis in human carotid atherosclerotic lesions.

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Lupia, Enrico; Pucci, Angela; Peasso, Paolo; Merlo, Maurizio; Baron, Paolo; Zanini, Cristina; Del Sorbo, Lorenzo; Rizea-Savu, Simona; Silvestro, Luigi; Forni, Marco; Emanuelli, Giorgio; Camussi, Giovanni; Montrucchio, Giuseppe

2003-09-01

Platelet-activating factor (PAF) is a phospholipid mediator synthesized by activated inflammatory and endothelial cells. Recently PAF has been shown to contribute to neoangiogenesis in several experimental models. Here we evaluated the presence of PAF and its potential role in neovascularization within human atherosclerotic plaques. The amount of PAF extracted from 18 carotid plaques (266.65+/-40.07 pg/100 mg dry tissue; mean +/- SE) was significantly higher than that extracted from 18 normal arterial specimens (6 from carotid artery and 12 from aorta) (4.72+/-2.31 pg/100 mg dry tissue; mean +/- SE). The levels of PAF significantly correlated with the infiltration of CD68-positive monocytes and the extent of neovascularization, detected as von Willebrand Factor-positive cells. The amount of PAF also correlated with the area occupied by TNF-alpha-expressing cells. The absence of enhanced level of PAF in the circulation of atherosclerotic patients suggests a local production of this mediator within the plaque. The lipid extracts of atherosclerotic plaques containing high levels of PAF-bioactivity, but not those of control arteries, were angiogenic in a murine Matrigel model. WEB 2170, a specific PAF receptor antagonist, significantly prevented angiogenesis induced by the lipid extracts of atherosclerotic plaques. Our results indicate a local production of PAF within the atherosclerotic plaques and suggest that it may contribute to intra-plaque neoangiogenesis.

Comparison of the effect of calcium gluconate and batroxobin on the release of transforming growth factor beta 1 in canine platelet concentrates

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Silva Raul F

2012-07-01

Full Text Available Abstract Background The clinical use of autologous platelet concentrates (also known as platelet-rich plasma on the field of regenerative therapy, in the last decade has been the subject of several studies especially in equine medicine and surgery. The objectives of this study was: 1 to describe and compare the cellular population in whole blood, lower fraction (A and upper fraction (B of platelet concentrates, 2 to measure and compare the transforming growth factor beta 1 (TGF-β1 concentration in plasma and both platelet concentrates after be activated with calcium gluconate or batroxobin plus calcium gluconate and, 3 to determine correlations between cell counts in platelet concentrates and concentrations of TGF-β1. Blood samples were taken from 16 dogs for complete blood count, plasma collection and platelet concentrates preparation. The platelet concentrates (PC were arbitrarily divided into two fractions, specifically, PC-A (lower fraction and PC-B (upper fraction. The Platelet concentrates were analyzed by hemogram. After activated with calcium gluconate or batroxobin plus calcium gluconate, TGF-β1 concentration was determined in supernatants of platelet concentrates and plasma. Results There were differences statistically significant (P 1 concentration between whole blood, plasma and both platelet concentrates. A significant correlation was found between the number of platelets in both platelet concentrates and TGF-β1 concentration. Platelet collection efficiency was 46.34% and 28.16% for PC-A and PC-B, respectively. TGF-β1 concentration efficiency for PC activated with calcium gluconate was 47.75% and 31.77%, for PC-A and PC-B, respectively. PC activated with batroxobin plus CG showed 46.87% and 32.24% for PC-A and PC-B, respectively. Conclusions The methodology used in this study allows the concentration of a number of platelets and TGF-β1 that might be acceptable for a biological effect for clinical or experimental use as a

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

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Daniela eWeth

2015-04-01

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

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 ...

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

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Taisuke Watanabe

2017-01-01

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

Enhancement by platelets of oxygen radical responses of human neutrophils

International Nuclear Information System (INIS)

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

1986-01-01

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- 2 and H 2 O 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- 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- 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

Granulocyte-platelet interactions and platelet fibrinogen receptor exposure

International Nuclear Information System (INIS)

Kornecki, E.; Ehrlich, Y.H.; Egbring, R.; Gramse, M.; Seitz, R.; Eckardt, A.; Lukasiewicz, H.; Niewiarowski, S.

1988-01-01

The authors have examined the interaction of human granulocyte elastase with human platelets. Incubation of human platelets with human granulocyte elastase exposed active fibrinogen-binding sites as evidenced by 125 I-labeled fibrinogen binding and spontaneous fibrinogen-induced platelet aggregation. The aggregation of platelets by fibrinogen occurred at low concentrations of human granulocyte elastase. Platelets pretreated with human granulocyte elastase exposed an average of 10,500 fibrinogen-binding sites per platelet, i.e., about one-third the number of binding sites exposed by optimal concentrations of ADP. With the use of a polyclonal antiplatelet membrane antibody, the glycoproteins IIb (GPIIb), IIIa (GPIIIa), and a 60,000-Da (60 kDa) protein (66 kDa in a reduced system) derived from GPIIIa were immunoprecipitated from the surface of detergent extracts of human 125 I-radiolabeled platelets pretreated with increasing concentrations of human granulocyte elastase. They conclude that (1) the proteolytic action of human granulocyte elastase on platelet GPIIIa results in the formation of two major hydrolytic products, and (2) human granulocyte elastase exposes active fibrongen-binding sites associated with the GPIIb/GPIIIa complex, resulting in direct platelet aggregation by fibrinogen

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.

The feed gas composition determines the degree of physical plasma-induced platelet activation for blood coagulation

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Bekeschus, Sander; Brüggemeier, Janik; Hackbarth, Christine; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Partecke, Lars-Ivo; van der Linde, Julia

2018-03-01

Cold atmospheric (physical) plasma has long been suggested to be a useful tool for blood coagulation. However, the clinical applicability of this approach has not been addressed sufficiently. We have previously demonstrated the ability of a clinically accepted atmospheric pressure argon plasma jet (kINPen® MED) to coagulate liver incisions in mice with similar performance compared to the gold standard electrocauterization. We could show that plasma-mediated blood coagulation was dependent on platelet activation. In the present work, we extended on this by investigating kINPen®-mediated platelet activation in anticoagulated human donor blood ex vivo. With focus on establishing high-throughput, multi-parametric platelet activation assays and performing argon feed gas parameter studies we achieved the following results: (i) plasma activated platelets in heparinized but not in EDTA-anticoagulated blood; (ii) plasma decreased total platelet counts but increased numbers of microparticles; (iii) plasma elevated the expression of several surface activation markers on platelets (CD62P, CD63, CD69, and CD41/61); (iv) in platelet activation, wet and dry argon plasma outperformed feed gas admixtures with oxygen and/or nitrogen; (v) plasma-mediated platelet activation was accompanied by platelet aggregation. Platelet aggregation is a necessary requirement for blood clot formation. These findings are important to further elucidate molecular details and clinical feasibility of cold physical plasma-mediated blood coagulation.

Potential Angiogenic Role of Platelet-Activating Factor in Human Breast Cancer

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Montrucchio, Giuseppe; Sapino, Anna; Bussolati, Benedetta; Ghisolfi, Gianpiero; Rizea-Savu, Simona; Silvestro, Luigi; Lupia, Enrico; Camussi, Giovanni

1998-01-01

This study investigated the presence of platelet-activating factor (PAF) in the lipid extracts of 18 primary breast carcinomas and 20 control breast tissues. The amount of PAF detected in breast carcinomas was significantly higher than in controls. The mass spectrometric analysis of PAF-bioactive lipid extract from breast carcinomas showed the presence of several molecular species of PAF, including C16-alkylPAF, C18-lysophosphatidylcholine (LPC), C16-LPC, lyso-PAF, and C16-acylPAF. The amount of bioactive PAF extracted from breast specimens significantly correlated with tumor vascularization revealed by the number of CD34- and CD31-positive cells. As C16-alkylPAF was previously shown to induce angiogenesis in vivo, we evaluated whether the thin layer chromatography-purified lipid extracts of breast specimens elicited neoangiogenesis in a murine model of subcutaneous Matrigel injection. The lipid extracts from specimens of breast carcinoma containing high levels of PAF bioactivity, but not from breast carcinomas containing low levels of PAF bioactivity or from normal breast tissue, induced a significant angiogenic response. This angiogenic response was significantly inhibited by the PAF receptor antagonist WEB 2170. T47D and MCF7 breast cancer cell lines, but not an immortalized nontumor breast cell line (MCF10), released PAF in the culture medium. A significant in vivo neoangiogenic response, inhibited by WEB 2170, was elicited by T47D and MCF7 but not by MCF10 culture medium. These results indicate that an increased concentration of PAF is present in tumors with high microvessel density and that PAF may account for the neoangiogenic activity induced in mice by the lipid extracts obtained from breast cancer. A contribution of PAF in the neovascularization of human breast cancer is suggested. PMID:9811351

Insomnia, platelet serotonin and platelet monoamine oxidase in chronic alcoholism.

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Nenadic Sviglin, Korona; Nedic, Gordana; Nikolac, Matea; Mustapic, Maja; Muck-Seler, Dorotea; Borovecki, Fran; Pivac, Nela

2011-08-18

Insomnia is a common sleep disorder frequently occurring in chronic alcoholic patients. Neurobiological basis of insomnia, as well as of alcoholism, is associated with disrupted functions of the main neurotransmitter systems, including the serotonin (5-hydroxytryptamine, 5-HT) system. Blood platelets are considered a limited peripheral model for the central 5-HT neurons, since both platelets and central 5-HT synaptosomes have similar dynamics of 5-HT. Platelet 5-HT concentration and platelet monoamine oxidase type B (MAO-B) are assumed to represent biomarkers for particular symptoms and behaviors in psychiatric disorders. The hypothesis of this study was that platelet 5-HT concentration and platelet MAO-B activity will be altered in chronic alcoholic patients with insomnia compared to comparable values in patients without insomnia. The study included 498 subjects: 395 male and 103 female medication-free patients with alcohol dependence and 502 healthy control subjects: 325 men and 177 women. The effects of early, middle and late insomnia (evaluated using the Hamilton Depression Rating Scale), as well as sex, age and smoking on platelet 5-HT concentration and platelet MAO-B activity were evaluated using one-way ANOVA and multiple regression analysis by the stepwise method. Platelet 5-HT concentration, but not platelet MAO-B activity, was significantly reduced in alcoholic patients with insomnia compared to patients without insomnia. Multiple regression analysis revealed that platelet 5-HT concentration was affected by middle insomnia, smoking and sex, while platelet MAO activity was affected only by sex and age. The present and previous data suggest that platelet 5-HT concentration might be used, after controlling for sex and smoking, as a biomarker for insomnia in alcoholism, PTSD and in rotating shift workers. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Effect of red blood cells on platelet activation and thrombus formation in tortuous arterioles

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Jennifer K. W. Chesnutt

2013-12-01

Full Text Available Thrombosis is a major contributor to cardiovascular disease, which can lead to myocardial infarction and stroke. Thrombosis may form in tortuous microvessels, which are often seen throughout the human body, but the microscale mechanisms and processes are not well understood. In straight vessels, the presence of red blood cells (RBCs is known to push platelets toward walls, which may affect platelet aggregation and thrombus formation. However in tortuous vessels, the effects of RBC interactions with platelets in thrombosis are largely unknown. Accordingly, the objective of this work was to determine the physical effects of RBCs, platelet size, and vessel tortuosity on platelet activation and thrombus formation in tortuous arterioles. A discrete element computational model was used to simulate the transport, collision, adhesion, aggregation, and shear-induced platelet activation of hundreds of individual platelets and RBCs in thrombus formation in tortuous arterioles. Results showed that high shear stress near the inner sides of curved arteriole walls activated platelets to initiate thrombosis. RBCs initially promoted platelet activation, but then collisions of RBCs with mural thrombi reduced the amount of mural thrombus and the size of emboli. In the absence of RBCs, mural thrombus mass was smaller in a highly tortuous arteriole compared to a less tortuous arteriole. In the presence of RBCs however, mural thrombus mass was larger in the highly tortuous arteriole compared to the less tortuous arteriole. As well, smaller platelet size yielded less mural thrombus mass and smaller emboli, either with or without RBCs. This study shed light on microscopic interactions of RBCs and platelets in tortuous microvessels, which have implications in various pathologies associated with thrombosis and bleeding.

An inhibition of p38 mitogen activated protein kinase delays the platelet storage lesion.

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Andrey Skripchenko

Full Text Available BACKGROUND AND OBJECTIVES: Platelets during storage undergo diverse alterations collectively known as the platelet storage lesion, including metabolic, morphological, functional and structural changes. Some changes correlate with activation of p38 mitogen activated protein kinase (p38 MAPK. Another MAPK, extracellular signal-related kinase (ERK, is involved in PLT activation. The aim of this study was to compare the properties of platelets stored in plasma in the presence or absence of p38 and ERK MAPK inhibitors. MATERIALS AND METHODS: A single Trima apheresis platelet unit (n = 12 was aliquoted into five CLX storage bags. Two aliquots were continuously agitated with or without MAPK inhibitors. Two aliquots were subjected to 48 hours of interruption of agitation with or without MAPK inhibitors. One aliquot contained the same amount of solvent vehicle used to deliver the inhibitor. Platelets were stored at 20-24°C for 7 days and sampled on Days 1, 4, and 7 for 18 in vitro parameters. RESULTS: Inhibition of p38 MAPK by VX-702 leads to better maintenance of all platelet in vitro storage parameters including platelet mitochondrial function. Accelerated by interruption of agitation, the platelet storage lesion of units stored with VX-702 was diminished to that of platelets stored with continuous agitation. Inhibition of ERK MAPK did not ameliorate decrements in any in vitro platelet properties. CONCLUSION: Signaling through p38 MAPK, but not ERK, is associated with platelet deterioration during storage.

Platelet activating factor-induced ceramide micro-domains drive endothelial NOS activation and contribute to barrier dysfunction.

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Sanda Predescu

Full Text Available The spatial and functional relationship between platelet activating factor-receptor (PAF-R and nitric oxide synthase (eNOS in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles.

Structural and functional analysis of a platelet-activating lysophosphatidylcholine of Trypanosoma cruzi.

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Felipe Gazos-Lopes

2014-08-01

Full Text Available Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR. Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive.Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS. Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC, namely sn-1 C18:1(delta 9-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF.Taken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the progression of Chagas disease and its biosynthesis could

Markers for vulnerability to psychopathology: Temperament traits associated with platelet MAO activity

International Nuclear Information System (INIS)

Schalling, D.; Aasberg, M.; Edman, G.; Oreland, L.

1987-01-01

The functional linkage between platelet MAO activity and psychopathology was explored by analyzing temperamental correlates in 40 male subjects by means of scales from the Eysenck Personality Questionnaire (EPQ), the Zuckerman Sensation Seeking Inventory, and the Karolinska Scales of Personality (KSP). Linear correlation were found with two sensation seeking scales, replicating earlier findings. However, nonlinear correlations predominated. Subjects with intemediate platelet MAO activity had higher scores in conformity scales and lower scores in anxiety and hostility scales than low and high MAO subgroups. Low MAO subjects showed a pattern of higher scores in KSP Impulsiveness, EPQ Neuroticism, and KSP Somatic Anxiety and Irritability and lower scores in KSP Socialization, in line with personality profiles found in alcoholics, phychopaths, and suicide attempters who also tend to have low platelet MAO activity. High MAO subject scored lower in sensation seeking and conformity scales and higher in KSP Phychasthenia, Muscular Tension and Suspicion scales, consistent with clinical links between high platelet MAO activity and anxiety and paranoia. (author)

Platelets and infections—complex interactions with bacteria

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Hind eHAMZEH-COGNASSE

2015-02-01

Full Text Available Platelets can be considered sentinels of vascular system due to their high number in the circulation and to the range of functional immunoreceptors they express. Platelets express a wide range of potential bacterial receptors, including complement receptors, FcγRII, Toll-Like Receptors but also integrins conventionally described in the hemostatic response, such as GPIIb-IIIa or GPIb. Bacteria bind these receptors either directly, or indirectly via fibrinogen, fibronectin, the first complement C1q, the von Willebrand Factor, etc. The fate of platelet bound bacteria is questioned. Several studies reported the ability of activated platelets to internalize bacteria such as Staphylococcus aureus or Porphyromonas gingivalis, though there is no clue on what happens thereafter. Are they sheltered from the immune system in the cytoplasm of platelets or are they lysed? Indeed, while the presence of phagolysosome has not been demonstrated in platelets, they contain antimicrobial peptides that were shown to be efficient on S. aureus. Besides, the fact that bacteria can bind to platelets via receptors involved in hemostasis suggests that they may induce aggregation; this has indeed been described for Streptococcus sanguinis, S. epidermidis or C. pneumoniae. On the other hand, platelets are able to display an inflammatory response to an infectious triggering. We, and others, have shown that platelet release soluble immunomodulatory factors upon stimulation by bacterial components. Moreover, interactions between bacteria and platelets are not limited to only these two partners. Indeed, platelets are also essential for the formation of Neutrophil Extracellular Traps by neutrophils, resulting in bacterial clearance by trapping bacteria and concentrating antibacterial factors but in enhancing thrombosis. In conclusion, the platelet-bacteria interplay is a complex game; its fine analysis is complicated by the fact that the inflammatory component adds to the

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbβ3 and activated platelets.

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Marcinkiewicz, Cezary; Gerstenhaber, Jonathan A; Sternberg, Mark; Lelkes, Peter I; Feuerstein, Giora

2017-01-01

Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the α IIb β 3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP-Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, α IIb β 3 receptor antagonist, specifically blocked F-NDP-Bit-PFR complex formation. Moreover, F-NDP-Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP-Bit particles could serve as noninvasive, "real-time" optical diagnostics for clots present in blood vessels.

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

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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

Synthesis of Analogues of Gingerol and Shogaol, the Active Pungent Principles from the Rhizomes of Zingiber officinale and Evaluation of Their Anti-Platelet Aggregation Effects

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Shih, Hung-Cheng; Chern, Ching-Yuh; Kuo, Ping-Chung; Wu, You-Cheng; Chan, Yu-Yi; Liao, Yu-Ren; Teng, Che-Ming; Wu, Tian-Shung

2014-01-01

The present study was aimed at discovering novel biologically active compounds based on the skeletons of gingerol and shogaol, the pungent principles from the rhizomes of Zingiber officinale. Therefore, eight groups of analogues were synthesized and examined for their inhibitory activities of platelet aggregation induced by arachidonic acid, collagen, platelet activating factor, and thrombin. Among the tested compounds, [6]-paradol (5b) exhibited the most significant anti-platelet aggregation activity. It was the most potent candidate, which could be used in further investigation to explore new drug leads. PMID:24599082

Synthesis of Analogues of Gingerol and Shogaol, the Active Pungent Principles from the Rhizomes of Zingiber officinale and Evaluation of Their Anti-Platelet Aggregation Effects

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Hung-Cheng Shih

2014-03-01

Full Text Available The present study was aimed at discovering novel biologically active compounds based on the skeletons of gingerol and shogaol, the pungent principles from the rhizomes of Zingiber officinale. Therefore, eight groups of analogues were synthesized and examined for their inhibitory activities of platelet aggregation induced by arachidonic acid, collagen, platelet activating factor, and thrombin. Among the tested compounds, [6]-paradol (5b exhibited the most significant anti-platelet aggregation activity. It was the most potent candidate, which could be used in further investigation to explore new drug leads.

Platelet RNA as a circulating biomarker trove for cancer diagnostics.

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Best, M G; Vancura, A; Wurdinger, T

2017-07-01

Platelets are multifunctional cell fragments, circulating in blood in high abundance. Platelets assist in thrombus formation, sensing of pathogens entering the blood stream, signaling to immune cells, releasing vascular remodeling factors, and, negatively, enhancing cancer metastasis. Platelets are 'educated' by their environment, including in patients with cancer. Cancer cells appear to initiate intraplatelet signaling, resulting in splicing of platelet pre-mRNAs, and enhance secretion of cytokines. Platelets can induce leukocyte and endothelial cell modeling factors, for example, through adenine nucleotides (ATP), thereby facilitating extravasation of cancer cells. Besides releasing factors, platelets can also sequester RNAs and proteins released by cancer cells. Thus, platelets actively respond to queues from local and systemic conditions, thereby altering their transcriptome and molecular content. Platelets contain a rich repertoire of RNA species, including mRNAs, small non-coding RNAs and circular RNAs; although studies regarding the functionality of the various platelet RNA species require more attention. Recent advances in high-throughput characterization of platelet mRNAs revealed 10 to > 1000 altered mRNAs in platelets in the presence of disease. Hence, platelet RNA appears to be dynamically affected by pathological conditions, thus possibly providing opportunities to use platelet RNA as diagnostic, prognostic, predictive, or monitoring biomarkers. In this review, we cover the literature regarding the platelet RNA families, processing of platelet RNAs, and the potential application of platelet RNA as disease biomarkers. © 2017 International Society on Thrombosis and Haemostasis.

Platelets and the innate immune system: Mechanisms of bacterial-induced platelet activation.

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Cox, Dermot; Kerrigan, Steven W; Watson, Steve

2011-01-01

It has become clear that platelets are not simply cell fragments that can plug the leak in a damaged blood vessel, they are in fact key components in the innate immune system which is supported by the presence of Toll-like receptors (TLRs) on platelets. As the first responding cell to a site of injury they are well placed to direct the immune response to deal with any resulting exposure to pathogens. The response is triggered by bacteria binding to platelets which usually triggers platelet ac...

Amarogentin, a Secoiridoid Glycoside, Abrogates Platelet Activation through PLCγ2-PKC and MAPK Pathways

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Ting-Lin Yen

2014-01-01

Full Text Available Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 μM inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (MAPKs. It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLCγ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts.

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Kakudo, Natsuko; Minakata, Tatsuya; Mitsui, Toshihito; Kushida, Satoshi; Notodihardjo, Frederik Zefanya; Kusumoto, Kenji

2008-11-01

This study evaluated changes in platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets by platelet-rich plasma activation, and the proliferation potential of activated platelet-rich plasma and platelet-poor plasma on human adipose-derived stem cells and human dermal fibroblasts. Platelet-rich plasma was prepared using a double-spin method, with the number of platelets counted in each preparation stage. Platelet-rich and platelet-poor plasma were activated with autologous thrombin and calcium chloride, and levels of platelet-released PDGF-AB and TGF-beta1 were determined by enzyme-linked immunosorbent assay. Cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 5% whole blood plasma, nonactivated platelet-rich plasma, nonactivated platelet-poor plasma, activated platelet-rich plasma, or activated platelet-poor plasma. In parallel, these cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 1%, 5%, 10%, or 20% activated platelet-rich plasma. The cultured human adipose-derived stem cells and human dermal fibroblasts were assayed for proliferation. Platelet-rich plasma contained approximately 7.9 times as many platelets as whole blood, and its activation was associated with the release of large amounts of PDGF-AB and TGF-beta1. Adding activated platelet-rich or platelet-poor plasma significantly promoted the proliferation of human adipose-derived stem cells and human dermal fibroblasts. Adding 5% activated platelet-rich plasma to the medium maximally promoted cell proliferation, but activated platelet-rich plasma at 20% did not promote it. Platelet-rich plasma can enhance the proliferation of human adipose-derived stem cells and human dermal fibroblasts. These results support clinical platelet-rich plasma application for cell-based, soft-tissue engineering and wound healing.

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.

Enhancement by platelets of oxygen radical responses of human neutrophils

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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.

The use of autologous 111In-labelled platelets and scintigraphy to illustrate enhanced platelet activity during erection in the chacma baboon

International Nuclear Information System (INIS)

Dormehl, I.C.; Du Plessis, M.; Maree, M.; Bornman, M.S.; Du Plessis, D.J.

1984-01-01

The demonstration of thrombelastographic hypercoagulability in the penile blood during erection, and the accompanying deposition of fibrin onto the endothelial layer of the deep penile artery and trabecular surface inspired this investigation of the possible role that platelets might play in the process. The bloodpooling pattern in the penis during and after erection from electro-stimulation was studied in 9 male adult baboons (Papio ursinus) using in vivo sup(99m)Tc-labelled red blood cells and scintigraphy. Platelet activity was similarly investigated after administering autologous 111 In-labelled platelets to the baboons. The results indicate an enhanced platelet concentration with respect to blood-pooling during erection, and an entrapment of platelets after erection. (orig.) [de

Effects of a single bout of strenuous exercise on platelet activation in female ApoE/LDLR-/- mice.

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Przyborowski, K; Kassassir, H; Wojewoda, M; Kmiecik, K; Sitek, B; Siewiera, K; Zakrzewska, A; Rudolf, A M; Kostogrys, R; Watala, C; Zoladz, J A; Chlopicki, S

2017-11-01

Strenuous physical exercise leads to platelet activation that is normally counterbalanced by the production of endothelium-derived anti-platelet mediators, including prostacyclin (PGI 2 ) and nitric oxide (NO). However, in the case of endothelial dysfunction, e.g. in atherosclerosis, there exists an increased risk for intravascular thrombosis during exercise that might be due to an impairment in endothelial anti-platelet mechanisms. In the present work, we evaluated platelet activation at rest and following a single bout of strenuous treadmill exercise in female ApoE/LDLR - /- mice with early (3-month-old) and advanced (7-month-old) atherosclerosis compared to female age-matched WT mice. In sedentary and post-exercise groups of animals, we analyzed TXB 2 generation and the expression of platelet activation markers in the whole blood ex vivo assay. We also measured pre- and post-exercise plasma concentration of 6-keto-PGF 1α , nitrite/nitrate, lipid profile, and blood cell count. Sedentary 3- and 7-month-old ApoE/LDLR - /- mice displayed significantly higher activation of platelets compared to age-matched wild-type (WT) mice, as evidenced by increased TXB 2 production, expression of P-selectin, and activation of GPIIb/IIIa receptors, as well as increased fibrinogen and von Willebrand factor (vWf) binding. Interestingly, in ApoE/LDLR - /- but not in WT mice, strenuous exercise partially inhibited TXB 2 production, the expression of activated GPIIb/IIIa receptors, and fibrinogen binding, with no effect on the P-selectin expression and vWf binding. Post-exercise down-regulation of the activated GPIIb/IIIa receptor expression and fibrinogen binding was not significantly different between 3- and 7-month-old ApoE/LDLR - /- mice; however, only 7-month-old ApoE/LDLR - /- mice showed lower TXB 2 production after exercise. In female 4-6-month-old ApoE/LDLR - /- but not in WT mice, an elevated pre- and post-exercise plasma concentration of 6-keto-PGF 1α was observed. In turn

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

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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.

Responsiveness of platelets during storage studied with flow cytometry--formation of platelet subpopulations and LAMP-1 as new markers for the platelet storage lesion.

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Södergren, A L; Tynngård, N; Berlin, G; Ramström, S

2016-02-01

Storage lesions may prevent transfused platelets to respond to agonists and arrest bleeding. The aim of this study was to evaluate and quantify the capacity of platelet activation during storage using flow cytometry and new markers of platelet activation. Activation responses of platelets prepared by apheresis were measured on days 1, 5, 7 and 12. In addition, comparisons were made for platelet concentrates stored until swirling was affected. Lysosome-associated membrane protein-1 (LAMP-1), P-selectin and phosphatidylserine (PS) exposure were assessed by flow cytometry on platelets in different subpopulations in resting state or following stimulation with platelet agonists (cross-linked collagen-related peptide (CRP-XL), PAR1- and PAR4-activating peptides). The ability to form subpopulations upon activation was significantly decreased already at day 5 for some agonist combinations. The agonist-induced exposure of PS and LAMP-1 also gradually decreased with time. Spontaneous exposure of P-selectin and PS increased with time, while spontaneous LAMP-1 exposure was unchanged. In addition, agonist-induced LAMP-1 expression clearly discriminated platelet concentrates with reduced swirling from those with retained swirling. This suggests that LAMP-1 could be a good marker to capture changes in activation capacity in stored platelets. The platelet activation potential seen as LAMP-1 exposure and fragmentation into platelet subpopulations is potential sensitive markers for the platelet storage lesion. © 2015 International Society of Blood Transfusion.

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

Effects of hormones on platelet aggregation.

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

2014-04-01

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

Application of platelet-rich plasma and platelet-rich fibrin in fat grafting: basic science and literature review.

Science.gov (United States)

Liao, Han-Tsung; Marra, Kacey G; Rubin, J Peter

2014-08-01

Due to the natural properties of fat, fat grafting remains a popular procedure for soft tissue volume augmentation and reconstruction. However, clinical outcome varies and is technique dependent. Platelet-rich plasma (PRP) contains α-granules, from which multiple growth factors such as platelet-derived growth factor, transforming growth factor-β, vascular endothelial growth factor, and epidermal growth factor can be released after activation. In recent years, the scope of PRP therapies has extended from bone regeneration, wound healing, and healing of musculoskeletal injuries, to enhancement of fat graft survival. In this review, we focus on the definition of PRP, the different PRP preparation and activation methods, and growth factor concentrations. In addition, we discuss possible mechanisms for the role of PRP in fat grafting by reviewing in vitro studies with adipose-derived stem cells, preadipocytes, and adipocytes, and preclinical and clinical research. We also review platelet-rich fibrin, a so-called second generation PRP, and its slow-releasing biology and effects on fat grafts compared to PRP in both animal and clinical research. Finally, we provide a general foundation on which to critically evaluate earlier studies, discuss the limitations of previous research, and direct plans for future experiments to improve the optimal effects of PRP in fat grafting.

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.

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

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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.

Quality of harvested autologous platelets compared with stored donor platelets for use after cardiopulmonary bypass procedures.

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Crowther, M; Ford, I; Jeffrey, R R; Urbaniak, S J; Greaves, M

2000-10-01

Platelet dysfunction has a major contribution in bleeding after cardiopulmonary bypass (CPB) and transfusion of platelets is frequently used to secure haemostasis. Allogeneic platelets prepared for transfusion are functionally impaired. Autologous platelets harvested preoperatively require a shorter storage time before transfusion and their use also avoids the risks associated with transfusion of allogeneic blood products. For the first time, we have compared the functional quality of autologous platelets with allogeneic platelets prepared by two methods, immediately before infusion. Platelet activation was assessed by P-selectin expression and fibrinogen binding using flow cytometry. We also monitored the effects of CPB surgery and re-infusion of autologous platelets on platelet function. Autologous platelet-rich plasma (PRP) contained a significantly lower (P platelets compared with allogeneic platelet preparations, and also contained a significantly higher (P platelets. Allogeneic platelets prepared by donor apheresis were more activated and less responsive than those produced by centrifugation of whole blood. In patients' blood, the percentage of platelets expressing P-selectin or binding fibrinogen increased significantly after CPB (P platelets responsive to in vitro agonists was decreased (P platelet activation during the procedure. The percentage of activated platelets decreased (statistically not significant) after re-infusion of autologous PRP. P-selectin expression had returned to pre-CPB levels 24 h post-operatively. Autologous platelet preparations display minimal activation, but remain responsive. Conservation of platelet function may contribute to the potential clinical benefits of autologous transfusion in cardiopulmonary bypass.

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

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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.

Renal cells activate the platelet receptor CLEC-2 through podoplanin

Science.gov (United States)

Christou, Charita M.; Pearce, Andrew C.; Watson, Aleksandra A.; Mistry, Anita R.; Pollitt, Alice Y.; Fenton-May, Angharad E.; Johnson, Louise A.; Jackson, David G.; Watson, Steve P.; O'Callaghan, Chris A.

2009-01-01

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin, rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing human immunodeficiency virus type 1 (HIV-1). This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of this study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to 293T cells in which the HIV can be grown. Further, 293T cells activate both platelets and CLEC-2-transfected DT-40 B cells. The transmembrane protein podoplanin was identified on 293T cells and demonstrated to mediate both binding of 293T cells to CLEC-2 and 293T cell activation of CLEC-2-transfected DT-40 B cells. Podoplanin is expressed on renal cells (podocytes). Further, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5 ± 3.7μM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells. PMID:18215137

Three-dimentional simulation of flow-induced platelet activation in artificial heart valves

Science.gov (United States)

Hedayat, Mohammadali; Asgharzadeh, Hafez; Borazjani, Iman

2015-11-01

Since the advent of heart valve, several valve types such as mechanical and bio-prosthetic valves have been designed. Mechanical Heart Valves (MHV) are durable but suffer from thromboembolic complications that caused by shear-induced platelet activation near the valve region. Bio-prosthetic Heart Valves (BHV) are known for better hemodynamics. However, they usually have a short average life time. Realistic simulations of heart valves in combination with platelet activation models can lead to a better understanding of the potential risk of thrombus formation in such devices. In this study, an Eulerian approach is developed to calculate the platelet activation in three-dimensional simulations of flow through MHV and BHV using a parallel overset-curvilinear immersed boundary technique. A curvilinear body-fitted grid is used for the flow simulation through the anatomic aorta, while the sharp-interface immersed boundary method is used for simulation of the Left Ventricle (LV) with prescribed motion. In addition, dynamics of valves were calculated numerically using under-relaxed strong-coupling algorithm. Finally, the platelet activation results for BMV and MHV are compared with each other.

Platelet activation, function, and reactivity in atherosclerotic carotid artery stenosis: a systematic review of the literature.

LENUS (Irish Health Repository)

Kinsella, J A

2012-09-27

An important proportion of transient ischemic attack or ischemic stroke is attributable to moderate or severe (50-99%) atherosclerotic carotid stenosis or occlusion. Platelet biomarkers have the potential to improve our understanding of the pathogenesis of vascular events in this patient population. A detailed systematic review was performed to collate all available data on ex vivo platelet activation and platelet function\\/reactivity in patients with carotid stenosis. Two hundred thirteen potentially relevant articles were initially identified; 26 manuscripts met criteria for inclusion in this systematic review. There was no consistent evidence of clinically informative data from urinary or soluble blood markers of platelet activation in patients with symptomatic moderate or severe carotid stenosis who might be considered suitable for carotid intervention. Data from flow cytometry studies revealed evidence of excessive platelet activation in patients in the early, sub-acute, or late phases after transient ischemic attack or stroke in association with moderate or severe carotid stenosis and in asymptomatic moderate or severe carotid stenosis compared with controls. Furthermore, pilot data suggest that platelet activation may be increased in recently symptomatic than in asymptomatic severe carotid stenosis. Excessive platelet activation and platelet hyperreactivity may play a role in the pathogenesis of first or subsequent transient ischemic attack or stroke in patients with moderate or severe carotid stenosis. Larger longitudinal studies assessing platelet activation status with flow cytometry and platelet function\\/reactivity in symptomatic vs. asymptomatic carotid stenosis are warranted to improve our understanding of the mechanisms responsible for transient ischemic attack or stroke.

The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays

NARCIS (Netherlands)

M. Pieters (Marlien); S.A. Barnard (Sunelle A.); D.T. Loots (Du Toit); D.C. Rijken (Dingeman)

2017-01-01

textabstractDue to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen

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

OpenAIRE

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...

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbβ3 and activated platelets

Directory of Open Access Journals (Sweden)

Marcinkiewicz C

2017-05-01

Full Text Available Cezary Marcinkiewicz,1,2 Jonathan A Gerstenhaber,1 Mark Sternberg,2 Peter I Lelkes,1 Giora Feuerstein1,2 1Department of Bioengineering, College of Engineering, Temple University, Philadelphia, 2Debina Diagnostic, Inc., Newton Square, PA, USA Abstract: Thromboembolic events (TEE underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs functionalized with bitistatin (Bit, a disintegrin that specifically binds to the αIIbβ3 integrin, platelet fibrinogen receptor (PFR on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP–Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, αIIbβ3 receptor antagonist, specifically blocked F-NDP–Bit–PFR complex formation. Moreover, F-NDP–Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP. Our results suggest that engineered F-NDP–Bit particles could serve as noninvasive, “real-time” optical diagnostics for clots present in blood vessels. Keywords: carbon nanoparticles, blood clots, imaging, platelet fibrinogen receptor, fluorescence, disintegrin, thromboembolic complications, thrombosis

Reduction of relative centrifugation force within injectable platelet-rich-fibrin (PRF) concentrates advances patients' own inflammatory cells, platelets and growth factors: the first introduction to the low speed centrifugation concept.

Science.gov (United States)

Choukroun, J; Ghanaati, S

2018-02-01

The aim of this study was to analyze systematically the influence of the relative centrifugation force (RCF) on leukocytes, platelets and growth factor release within fluid platelet-rich fibrin matrices (PRF). Systematically using peripheral blood from six healthy volunteers, the RCF was reduced four times for each of the three experimental protocols (I-III) within the spectrum (710-44 g), while maintaining a constant centrifugation time. Flow cytometry was applied to determine the platelets and leukocyte number. The growth factor concentration was quantified 1 and 24 h after clotting using ELISA. Reducing RCF in accordance with protocol-II (177 g) led to a significantly higher platelets and leukocytes numbers compared to protocol-I (710 g). Protocol-III (44 g) showed a highly significant increase of leukocytes and platelets number in comparison to -I and -II. The growth factors' concentration of VEGF and TGF-β1 was significantly higher in protocol-II compared to -I, whereas protocol-III exhibited significantly higher growth factor concentration compared to protocols-I and -II. These findings were observed among 1 and 24 h after clotting, as well as the accumulated growth factor concentration over 24 h. Based on the results, it has been demonstrated that it is possible to enrich PRF-based fluid matrices with leukocytes, platelets and growth factors by means of a single alteration of the centrifugation settings within the clinical routine. We postulate that the so-called low speed centrifugation concept (LSCC) selectively enriches leukocytes, platelets and growth factors within fluid PRF-based matrices. Further studies are needed to evaluate the effect of cell and growth factor enrichment on wound healing and tissue regeneration while comparing blood concentrates gained by high and low RCF.

Antimicrobial activity of pure platelet-rich plasma against microorganisms isolated from oral cavity.

Science.gov (United States)

Drago, Lorenzo; Bortolin, Monica; Vassena, Christian; Taschieri, Silvio; Del Fabbro, Massimo

2013-02-25

Autologous platelet concentrates (PCs) have been extensively used in a variety of medical fields to promote soft and hard tissue regeneration. The significance behind their use lies in the abundance of growth factors in platelets α-granules that promotes wound healing. In addition, antibacterial properties of PCs against various bacteria have been recently pointed out. In this study, the antimicrobial effect of pure platelet-rich plasma (P-PRP) was evaluated against oral cavity microorganisms such as Enterococcus faecalis, Candida albicans, Streptococcus agalactiae, Streptococcus oralis and Pseudomonas aeruginosa. Blood samples were obtained from 17 patients who underwent oral surgery procedures involving the use of P-PRP. The antibacterial activity of P-PRP, evaluated as the minimum inhibitory concentration (MIC), was determined through the microdilution twofold serial method. P-PRP inhibited the growth of Enterococcus faecalis, Candida albicans, Streptococcus agalactiae and Streptococcus oralis, but not of Pseudomonas aeruginosa strains. P-PRP is a potentially useful substance in the fight against postoperative infections. This might represent a valuable property in adjunct to the enhancement of tissue regeneration.

Platelet degranulation and monocyte-platelet complex formation are increased in the acute and convalescent phases after ischaemic stroke or transient ischaemic attack.

LENUS (Irish Health Repository)

McCabe, Dominick J H

2004-06-01

Flow cytometric studies suggest that platelets are activated in ischaemic stroke or transient ischaemic attack (TIA). However, few studies have measured circulating leucocyte-platelet complexes in this patient population. Whole blood flow cytometry was used to quantify the expression of CD62P-, CD63-, and PAC1-binding, and the percentages of leucocyte-platelet complexes in acute (1-27 d, n = 79) and convalescent (79-725 d, n = 70) ischaemic cerebrovascular disease (CVD) patients compared with controls without CVD (n = 27). We performed a full blood count, and measured plasma levels of soluble P-selectin, soluble E-selectin, and von Willebrand factor antigen (VWF:Ag) as additional markers of platelet and\\/or endothelial cell activation. The median percentage CD62P expression and the median percentage monocyte-platelet complexes were higher in both acute and convalescent CVD patients than controls (P <\\/= 0.02). The mean white cell count and mean VWF:Ag levels were significantly elevated in the acute and convalescent phases after ischaemic stroke or TIA (P <\\/= 0.02). Otherwise, there was no significant increase in any other marker of platelet or endothelial activation in CVD patients. There was a positive correlation between the percentage expression of CD62P and the percentages of both neutrophil-platelet and monocyte-platelet complexes in the acute phase, and the percentages of all leucocyte-platelet complexes in the convalescent phase after ischaemic CVD. This study provides evidence for ongoing excessive platelet and\\/or endothelial activation in ischaemic CVD patients despite treatment with antithrombotic therapy.

Accurate measurement of volume and shape of resting and activated blood platelets from light scattering.

Science.gov (United States)

Moskalensky, Alexander E; Yurkin, Maxim A; Konokhova, Anastasiya I; Strokotov, Dmitry I; Nekrasov, Vyacheslav M; Chernyshev, Andrei V; Tsvetovskaya, Galina A; Chikova, Elena D; Maltsev, Valeri P

2013-01-01

We introduce a novel approach for determination of volume and shape of individual blood platelets modeled as an oblate spheroid from angle-resolved light scattering with flow-cytometric technique. The light-scattering profiles (LSPs) of individual platelets were measured with the scanning flow cytometer and the platelet characteristics were determined from the solution of the inverse light-scattering problem using the precomputed database of theoretical LSPs. We revealed a phenomenon of parameter compensation, which is partly explained in the framework of anomalous diffraction approximation. To overcome this problem, additional a priori information on the platelet refractive index was used. It allowed us to determine the size of each platelet with subdiffraction precision and independent of the particular value of the platelet aspect ratio. The shape (spheroidal aspect ratio) distributions of platelets showed substantial differences between native and activated by 10 μM adenosine diphosphate samples. We expect that the new approach may find use in hematological analyzers for accurate measurement of platelet volume distribution and for determination of the platelet activation efficiency.

Responsiveness of platelets during storage studied with flow cytometry - formation of platelet subpopulations and LAMP-1 as new markers for the platelet storage lesion

OpenAIRE

Södergren, Anna; Tynngård, Nahreen; Berlin, Gösta; Ramström, Sofia

2016-01-01

Background and ObjectivesStorage lesions may prevent transfused platelets to respond to agonists and arrest bleeding. The aim of this study was to evaluate and quantify the capacity of platelet activation during storage using flow cytometry and new markers of platelet activation. Materials and MethodsActivation responses of platelets prepared by apheresis were measured on days 1, 5, 7 and 12. In addition, comparisons were made for platelet concentrates stored until swirling was affected. Lyso...

Amarogentin, a secoiridoid glycoside, abrogates platelet activation through PLC γ 2-PKC and MAPK pathways.

Science.gov (United States)

Yen, Ting-Lin; Lu, Wan-Jung; Lien, Li-Ming; Thomas, Philip Aloysius; Lee, Tzu-Yin; Chiu, Hou-Chang; Sheu, Joen-Rong; Lin, Kuan-Hung

2014-01-01

Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60  μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC) γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLC γ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

Combination of recombinant factor VIIa and fibrinogen corrects clot formation in primary immune thrombocytopenia at very low platelet counts

DEFF Research Database (Denmark)

Larsen, Ole H; Stentoft, Jesper; Radia, Deepti

2013-01-01

Haemostatic treatment modalities alternative to platelet transfusion are desirable to control serious acute bleeds in primary immune thrombocytopenia (ITP). This study challenged the hypothesis that recombinant activated factor VII (rFVIIa) combined with fibrinogen concentrate may correct whole b...

Development of pro-inflammatory phenotype in monocytes after engulfing Hb-activated platelets in hemolytic disorders.

Science.gov (United States)

Singhal, Rashi; Chawla, Sheetal; Rathore, Deepak K; Bhasym, Angika; Annarapu, Gowtham K; Sharma, Vandana; Seth, Tulika; Guchhait, Prasenjit

2017-02-01

Monocytes and macrophage combat infections and maintain homeostatic balance by engulfing microbes and apoptotic cells, and releasing inflammatory cytokines. Studies have described that these cells develop anti-inflammatory properties upon recycling the free-hemoglobin (Hb) in hemolytic conditions. While investigating the phenotype of monocytes in two hemolytic disorders-paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease (SCD), we observed a high number of pro-inflammatory (CD14 + CD16 hi ) monocytes in these patients. We further investigated in vitro the phenotype of these monocytes and found an estimated 55% of CD14 + cells were transformed into the CD14 + CD16 hi subset after engulfing Hb-activated platelets. The CD14 + CD16 hi monocytes, which were positive for both intracellular Hb and CD42b (platelet marker), secreted significant amounts of TNF-α and IL-1β, unlike monocytes treated with only free Hb, which secreted more IL-10. We have shown recently the presence of a high number of Hb-bound hyperactive platelets in patients with both diseases, and further investigated if the monocytes engulfed these activated platelets in vivo. As expected, we found 95% of CD14 + CD16 hi monocytes with both intracellular Hb and CD42b in both diseases, and they expressed high TNF-α. Furthermore our data showed that these monocytes whether from patients or developed in vitro after treatment with Hb-activated platelets, secreted significant amounts of tissue factor. Besides, these CD14 + CD16 hi monocytes displayed significantly decreased phagocytosis of E. coli. Our study therefore suggests that this alteration of monocyte phenotype may play a role in the increased propensity to pro-inflammatory/coagulant complications observed in these hemolytic disorders-PNH and SCD. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Antimicrobial activity of pure platelet-rich plasma against microorganisms isolated from oral cavity

OpenAIRE

Drago, Lorenzo; Bortolin, Monica; Vassena, Christian; Taschieri, Silvio; Del Fabbro, Massimo

2013-01-01

Background Autologous platelet concentrates (PCs) have been extensively used in a variety of medical fields to promote soft and hard tissue regeneration. The significance behind their use lies in the abundance of growth factors in platelets ?-granules that promotes wound healing. In addition, antibacterial properties of PCs against various bacteria have been recently pointed out. In this study, the antimicrobial effect of pure platelet-rich plasma (P-PRP) was evaluated against oral cavity mic...

Reciprocal regulation of platelet responses to P2Y and thromboxane receptor activation.

Science.gov (United States)

Barton, J F; Hardy, A R; Poole, A W; Mundell, S J

2008-03-01

Thromboxane A(2) and ADP are two major platelet agonists that stimulate two sets of G protein-coupled receptors to activate platelets. Although aggregation responses to ADP and thromboxane desensitize, there are no reports currently addressing whether activation by one agonist may heterologously desensitize responses to the other. To demonstrate whether responses to ADP or U46619 may be modulated by prior treatment of platelets with the alternate agonist, revealing a level of cross-desensitization between receptor systems. Here we show that pretreatment of platelets with either agonist substantially desensitizes aggregation responses to the other agonist. Calcium responses to thromboxane receptor activation are desensitized by preactivation of P2Y(1) but not P2Y(12) receptors. This heterologous desensitization is mediated by a protein kinase C (PKC)-independent mechanism. Reciprocally, calcium responses to ADP are desensitized by pretreatment of platelets with the thromboxane analogue, U46619, and P2Y(12)-mediated inhibition of adenylate cyclase is also desensitized by pretreatment with U46619. In this direction, desensitization is comprised of two components, a true heterologous component that is PKC-independent, and a homologous component that is mediated through stimulated release of dense granule ADP. This study reveals cross-desensitization between ADP and thromboxane receptor signaling in human platelets. Cross-desensitization is mediated by protein kinases, involving PKC-dependent and independent pathways, and indicates that alterations in the activation state of one receptor may have effects upon the sensitivity of the other receptor system.

Thrombus imaging in a primate model with antibodies specific for an external membrane protein of activated platelets

International Nuclear Information System (INIS)

Palabrica, T.M.; Furie, B.C.; Konstam, M.A.; Aronovitz, M.J.; Connolly, R.; Brockway, B.A.; Ramberg, K.L.; Furie, B.

1989-01-01

The activated platelet is a potential target for the localization of thrombi in vivo since, after stimulation and secretion of granule contents, activated platelets are concentrated at sites of blood clot formation. In this study, we used antibodies specific for a membrane protein of activated platelets to detect experimental thrombi in an animal model. PADGEM (platelet activation-dependent granule-external membrane protein), a platelet alpha-granule membrane protein, is translocated to the plasma membrane during platelet activation and granule secretion. Since PADGEM is internal in unstimulated platelets, polyclonal anti-PADGEM and monoclonal KC4 antibodies do not bind to circulating resting platelets but do interact with activated platelets. Dacron graft material incubated with radiolabeled KC4 or anti-PADGEM antibodies in the presence of thrombin-activated platelet-rich plasma bound most of the antibody. Imaging experiments with 123I-labeled anti-PADGEM in baboons with an external arterial-venous Dacron shunt revealed rapid uptake in the thrombus induced by the Dacron graft; control experiments with 123I-labeled nonimmune IgG exhibited minimal uptake. Deep venous thrombi, formed by using percutaneous balloon catheters to stop blood flow in the femoral vein of baboons, were visualized with 123I-labeled anti-PADGEM. Thrombi were discernible against blood pool background activity without subtraction techniques within 1 hr. No target enhancement was seen with 123I-labeled nonimmune IgG. 123I-labeled anti-PADGEM cleared the blood pool with an initial half-disappearance time of 6 min and did not interfere with hemostasis. These results indicate that radioimmunoscintigraphy with anti-PADGEM antibodies can visualize thrombi in baboon models and is a promising technique for clinical thrombus detection in humans

Cytokine, chemokine, and growth factor profile of platelet-rich plasma.

Science.gov (United States)

Mussano, F; Genova, T; Munaron, L; Petrillo, S; Erovigni, F; Carossa, S

2016-07-01

During wound healing, biologically active molecules are released from platelets. The rationale of using platelet-rich plasma (PRP) relies on the concentration of bioactive molecules and subsequent delivery to healing sites. These bioactive molecules have been seldom simultaneously quantified within the same PRP preparation. In the present study, the flexible Bio-Plex system was employed to assess the concentration of a large range of cytokines, chemokines, and growth factors in 16 healthy volunteers so as to determine whether significant baseline differences may be found. Besides IL-1b, IL-1ra, IL-4, IL-6, IL-8, IL-12, IL-13, IL-17, INF-γ, TNF-α, MCP-1, MIP-1a, RANTES, bFGF, PDGF, and VEGF that were already quantified elsewhere, the authors reported also on the presence of IL-2, IL-5, IL-7, IL-9, IL-10, IL-15 G-CSF, GM-CSF, Eotaxin, CXCL10 chemokine (IP-10), and MIP 1b. Among the most interesting results, it is convenient to mention the high concentrations of the HIV-suppressive and inflammatory cytokine RANTES and a statistically significant difference between males and females in the content of PDGF-BB. These data are consistent with previous reports pointing out that gender, diet, and test system affect the results of platelet function in healthy subjects, but seem contradictory when compared to other quantification assays in serum and plasma. The inconsistencies affecting the experimental results found in literature, along with the variability found in the content of bioactive molecules, urge further research, hopefully in form of randomized controlled clinical trials, in order to find definitive evidence of the efficacy of PRP treatment in various pathologic and regenerative conditions.

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

Dansyl (5-dimethylaminonaphthalene-1-sulphonyl)-heparin binds antithrombin III and platelet factor 4 at separate sites

Science.gov (United States)

Piepkorn, Michael W.

1981-01-01

Antithrombin III binds to, and thereby augments the fluorescence of, dansyl-(5-dimethylaminonaphthalene-1-sulphonyl)-heparin; platelet factor 4 binding to the fluorescent heparin has little of this effect. Competition studies in which antithrombin III competes with platelet factor 4 for heparin binding demonstrate that heparin can simultaneously bind both proteins. PMID:7317004

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.

Piperine Inhibits the Activities of Platelet Cytosolic Phospholipase A2 and Thromboxane A2 Synthase without Affecting Cyclooxygenase-1 Activity: Different Mechanisms of Action Are Involved in the Inhibition of Platelet Aggregation and Macrophage Inflammatory Response

Directory of Open Access Journals (Sweden)

Dong Ju Son

2014-08-01

Full Text Available PURPOSE: Piperine, a major alkaloid of black pepper (Piper nigrum and long pepper (Piper longum, was shown to have anti-inflammatory activity through the suppression of cyclooxygenase (COX-2 gene expression and enzyme activity. It is also reported to exhibit anti-platelet activity, but the mechanism underlying this action remains unknown. In this study, we investigated a putative anti-platelet aggregation mechanism involving arachidonic acid (AA metabolism and how this compares with the mechanism by which it inhibits macrophage inflammatory responses; METHODS: Rabbit platelets and murine macrophage RAW264.7 cells were treated with piperine, and the effect of piperine on the activity of AA-metabolizing enzymes, including cytosolic phospholipase A2 (cPLA2, COX-1, COX-2, and thromboxane A2 (TXA2 synthase, as well as its effect on AA liberation from the plasma membrane components, were assessed using isotopic labeling methods and enzyme immunoassay kit; RESULTS: Piperine significantly suppressed AA liberation by attenuating cPLA2 activity in collagen-stimulated platelets. It also significantly inhibited the activity of TXA2 synthase, but not of COX-1, in platelets. These results suggest that piperine inhibits platelet aggregation by attenuating cPLA2 and TXA2 synthase activities, rather than through the inhibition of COX-1 activity. On the other hand, piperine significantly inhibited lipopolysaccharide-induced generation of prostaglandin (PGE2 and PGD2 in RAW264.7 cells by suppressing the activity of COX-2, without effect on cPLA2; CONCLUSION: Our findings indicate that piperine inhibits platelet aggregation and macrophage inflammatory response by different mechanisms.

Antibody production of wild-type and enzyme V279F variants of PAF-AH as a risk factor for Cardiovascular disease

Science.gov (United States)

Ramadhani, Anggia N.; Puspitarini, Sapti; Sari, Anissa N.; Widodo

2017-11-01

Coronary artery disease (CAD) has emerged as a leading cause of death in Indonesia nowadays. WHO data in 2012 revealed that 37% of the Indonesian population died from this disease. CAD occurs because of endothelial dysfunction in the arteries. Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH), is a phospholipase A2 enzyme, encoded by the PLA2G7 gene. This protein is predicted to be involved in inflammatory phospholipid metabolism so it can be used as a biomarker of CAD in the early phase. Thus, the purpose of this research is to discover the difference in antibody production between wild-type and mutant V279F. The PAF-AH enzyme was isolated from mice lymphocyte cells in order to develop this enzyme as a biomarker of cardiovascular disease. PAF-AH migrates at 55kDa according to SDS-PAGE analysis. Flow cytometry analysis showed that mutant PAF-AH (V279F) is more antigenic than wild-type PAF-AH. The missense mutation of V279F PAF-AH means this enzyme cannot catabolize the acetyl group at the sn-2 position of PAF.

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

OpenAIRE

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

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 us...

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....

Effect of supine exercise on platelet aggregation and fibrinolytic activity

DEFF Research Database (Denmark)

Dag, B; Fornitz, Gitte Gleerup; Bak, A M

1994-01-01

In 12 healthy young men, strenuous cycling exercise in the supine position, caused platelet aggregability to decrease and the ADP threshold to rise from 7.0 microM resting, to 9.5 exercising (P ... from 178 to 68 min, PAI-1 fell from 8.91 to 5.16 IU ml-1, and t-PA rose from 0.56 to 3.95 IU ml-1, all three values were significant to P exercise, it did not increase platelet activity as expected, but caused a modest increase...... of fibrinolytic activity. These results suggest that supine exercise will not affect the haemostatic system adversely....

Association of Factor V Secretion with Protein Kinase B Signaling in Platelets from Horses with Atypical Equine Thrombasthenia.

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Norris, J W; Pombo, M; Shirley, E; Blevins, G; Tablin, F

2015-01-01

Two congenital bleeding diatheses have been identified in Thoroughbred horses: Glanzmann thrombasthenia (GT) and a second, novel diathesis associated with abnormal platelet function in response to collagen and thrombin stimulation. Platelet dysfunction in horses with this second thrombasthenia results from a secretory defect. Two affected and 6 clinically normal horses. Ex vivo study. Washed platelets were examined for (1) expression of the αIIb-β3 integrin; (2) fibrinogen binding capacity in response to ADP and thrombin; (3) secretion of dense and α-granules; (4) activation of the mammalian target of rapamycin (mTOR)-protein kinase B (AKT) signaling pathway; and (5) cellular distribution of phosphatidylinositol-4-phosphate-3-kinase, class 2B (PIK3C2B) and SH2 containing inositol-5'-phosphatase 1 (SHIP1). Platelets from affected horses expressed normal amounts of αIIb-β3 integrin and bound fibrinogen normally in response to ADP, but bound 80% less fibrinogen in response to thrombin. α-granules only released 50% as much Factor V as control platelets, but dense granules released their contents normally. Protein kinase B (AKT) phosphorylation was reduced after thrombin activation, but mTOR Complex 2 (mTORC2) and phosphoinositide-dependent kinase 1 (PDK1) signaling were normal. SH2-containing inositol-5'-phosphatase 1 (SHIP1) did not localize to the cytoskeleton of affected platelets and was decreased overall consistent with reduced AKT phosphorylation. Defects in fibrinogen binding, granule secretion, and signal transduction are unique to this thrombasthenia, which we designate as atypical equine thrombasthenia. Copyright © The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of American College of Veterinary Internal Medicine.

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.

Optimisation of a double-centrifugation method for preparation of canine platelet-rich plasma.

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Shin, Hyeok-Soo; Woo, Heung-Myong; Kang, Byung-Jae

2017-06-26

Platelet-rich plasma (PRP) has been expected for regenerative medicine because of its growth factors. However, there is considerable variability in the recovery and yield of platelets and the concentration of growth factors in PRP preparations. The aim of this study was to identify optimal relative centrifugal force and spin time for the preparation of PRP from canine blood using a double-centrifugation tube method. Whole blood samples were collected in citrate blood collection tubes from 12 healthy beagles. For the first centrifugation step, 10 different run conditions were compared to determine which condition produced optimal recovery of platelets. Once the optimal condition was identified, platelet-containing plasma prepared using that condition was subjected to a second centrifugation to pellet platelets. For the second centrifugation, 12 different run conditions were compared to identify the centrifugal force and spin time to produce maximal pellet recovery and concentration increase. Growth factor levels were estimated by using ELISA to measure platelet-derived growth factor-BB (PDGF-BB) concentrations in optimised CaCl 2 -activated platelet fractions. The highest platelet recovery rate and yield were obtained by first centrifuging whole blood at 1000 g for 5 min and then centrifuging the recovered platelet-enriched plasma at 1500 g for 15 min. This protocol recovered 80% of platelets from whole blood and increased platelet concentration six-fold and produced the highest concentration of PDGF-BB in activated fractions. We have described an optimised double-centrifugation tube method for the preparation of PRP from canine blood. This optimised method does not require particularly expensive equipment or high technical ability and can readily be carried out in a veterinary clinical setting.

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

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

2011-02-01

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). MP were double stained with annexin V and CD61 (platelet-derived MP; PMP), P-selectin or CD63 (MP from activated platelets) and CD144 plus E-selectin (endothelial cell-derived MP; EMP) and detected by flow cytometry in platelet donors (n=36) and apheresis PC (n=11; Trima™). PC contained MP, mainly from resting platelets [93% (90-95)], and minor fractions of PMP from activated platelets [P-selectin(+) or CD63(+); 4·8% (3·2-7·7) and 2·6% (2·0-4·0)]. Compared to donors, levels of annexin V+ MP, PMP, P-selectin(+) and CD63(+) MP were 1·7-, 2·3-, 8·6- and 3·1-fold higher in PC (all P<0·05). During storage (1-5 days), levels of annexin V+ MP and PMP did not increase, although small increases in the fraction of P-selectin(+) or CD63(+) MP occurred (both P<0·05). PC also contained EMP, which were 2·6- to 3·7-fold enriched in PC compared to donors (P<0·05). Transfusion of apheresis PC also results in transfusion of HLA-carrying PMP and EMP. This might counteract the aim of reducing transfused HLA load by leucodepletion. The increases in PMP exposing P-selectin or CD63 reflect mild platelet activation during storage. We conclude that in leucodepleted platelet apheresis using fluidized particle bed technology, MP are harvested mainly from the donor by apheresis. Improvement in apheresis technology might reduce MP load. © 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.

Flow cytometric analysis reveals the high levels of platelet activation parameters in circulation of multiple sclerosis patients.

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Morel, Agnieszka; Rywaniak, Joanna; Bijak, Michał; Miller, Elżbieta; Niwald, Marta; Saluk, Joanna

2017-06-01

The epidemiological studies confirm an increased risk of cardiovascular disease in multiple sclerosis, especially prothrombotic events directly associated with abnormal platelet activity. The aim of our study was to investigate the level of blood platelet activation in the circulation of patients with chronic phase of multiple sclerosis (SP MS) and their reactivity in response to typical platelets' physiological agonists. We examined 85 SP MS patients diagnosed according to the revised McDonald's criteria and 50 healthy volunteers as a control group. The platelet activation and reactivity were assessed using flow cytometry analysis of the following: P-selectin expression (CD62P), activation of GP IIb/IIIa complex (PAC-1 binding), and formation of platelet microparticles (PMPs) and platelet aggregates (PA) in agonist-stimulated (ADP, collagen) and unstimulated whole blood samples. Furthermore, we measured the level of soluble P-selectin (sP-selectin) in plasma using ELISA method, to evaluate the in vivo level of platelet activation, both in healthy and SP MS subjects. We found a statistically significant increase in P-selectin expression, GP IIb/IIIa activation, and formation of PMPs and PA, as well as in unstimulated and agonist-stimulated (ADP, collagen) platelets in whole blood samples from patients with SP MS in comparison to the control group. We also determined the higher sP-selectin level in plasma of SP MS subjects than in the control group. Based on the obtained results, we might conclude that during the course of SP MS platelets are chronically activated and display hyperreactivity to physiological agonists, such as ADP or collagen.

Platelet activating factor enhances synaptic vesicle exocytosis via PKC, elevated intracellular calcium, and modulation of synapsin 1 dynamics and phosphorylation

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Jennetta W Hammond

2016-01-01

Full Text Available Platelet activating factor (PAF is an inflammatory phospholipid signaling molecule implicated in synaptic plasticity, learning and memory and neurotoxicity during neuroinflammation. However, little is known about the intracellular mechanisms mediating PAF’s physiological or pathological effects on synaptic facilitation. We show here that PAF receptors are localized at the synapse. Using fluorescent reporters of presynaptic activity we show that a non-hydrolysable analogue of PAF (cPAF enhances synaptic vesicle release from individual presynaptic boutons by increasing the size or release of the readily releasable pool and the exocytosis rate of the total recycling pool. cPAF also activates previously silent boutons resulting in vesicle release from a larger number of terminals. The underlying mechanism involves elevated calcium within presynaptic boutons and protein kinase C (PKC activation. Furthermore, cPAF increases synapsin I phosphorylation at sites 1 and 3, and increases dispersion of synapsin I from the presynaptic compartment during stimulation, freeing synaptic vesicles for subsequent release. These findings provide a conceptual framework for how PAF, regardless of its cellular origin, can modulate synapses during normal and pathologic synaptic activity.

Subcutaneous Administration of Low-Molecular-Weight Heparin to Horses Inhibits Ex Vivo Equine Herpesvirus Type 1-Induced Platelet Activation

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Tracy Stokol

2018-05-01

Full Text Available Equine herpesvirus type 1 (EHV-1 is a major cause of infectious respiratory disease, abortion and neurologic disease. Thrombosis in placental and spinal vessels and subsequent ischemic injury in EHV-1-infected horses manifests clinically as abortion and myeloencephalopathy. We have previously shown that addition of heparin anticoagulants to equine platelet-rich plasma (PRP can abolish ex vivo EHV-1-induced platelet activation. The goal of this study was to test whether platelets isolated from horses treated with unfractionated heparin (UFH or low-molecular-weight heparin (LMWH were resistant to ex vivo EHV-1-induced activation. In a masked, block-randomized placebo-controlled cross-over trial, 9 healthy adult horses received 4 subcutaneous injections at q. 12 h intervals of one of the following treatments: UFH (100 U/kg loading dose, 3 maintenance doses of 80 U/kg, 2 doses of LMWH (enoxaparin 80 U/kg 24 h apart with saline at the intervening 12 h intervals, or 4 doses of saline. Blood samples were collected before treatment and after 36 h, 40 h (4 h after the last injection and 60 h (24 h after the last injection. Two strains of EHV-1, Ab4 and RacL11, were added to PRP ex vivo and platelet membrane expression of P selectin was measured as a marker of platelet activation. Drug concentrations were monitored in a Factor Xa inhibition (anti-Xa bioassay. We found that LMWH, but not UFH, inhibited platelet activation induced by low concentrations (1 × 106 plaque forming units/mL of both EHV-1 strains at 40 h. At this time point, all horses had anti-Xa activities above 0.1 U/ml (range 0.15–0.48 U/ml with LMWH, but not UFH. By 60 h, a platelet inhibitory effect was no longer detected and anti-Xa activity had decreased (range 0.03 to 0.07 U/ml in LMWH-treated horses. Neither heparin inhibited platelet activation induced by high concentrations (5 × 106 plaque forming units/mL of the RacL11 strain. We found substantial between horse

Platelet receptor polymorphisms do not influence Staphylococcus aureus–platelet interactions or infective endocarditis

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Daga, Shruti; Shepherd, James G.; Callaghan, J. Garreth S.; Hung, Rachel K.Y.; Dawson, Dana K.; Padfield, Gareth J.; Hey, Shi Y.; Cartwright, Robyn A.; Newby, David E.; Fitzgerald, J. Ross

2011-01-01

Cardiac vegetations result from bacterium–platelet adherence, activation and aggregation, and are associated with increased morbidity and mortality in infective endocarditis. The GPIIb/IIIa and FcγRIIa platelet receptors play a central role in platelet adhesion, activation and aggregation induced by endocarditis pathogens such as Staphylococcus aureus, but the influence of known polymorphisms of these receptors on the pathogenesis of infective endocarditis is unknown. We determined the GPIIIa platelet antigen PlA1/A2 and FcγRIIa H131R genotype of healthy volunteers (n = 160) and patients with infective endocarditis (n = 40), and investigated the influence of these polymorphisms on clinical outcome in infective endocarditis and S. aureus–platelet interactions in vitro. Platelet receptor genotype did not correlate with development of infective endocarditis, vegetation characteristics on echocardiogram or the composite clinical end-point of embolism, heart failure, need for surgery or mortality (P > 0.05 for all), even though patients with the GPIIIa PlA1/A1 genotype had increased in vivo platelet activation (P = 0.001). Furthermore, neither GPIIIa PlA1/A2 nor FcγRIIa H131R genotype influenced S. aureus-induced platelet adhesion, activation or aggregation in vitro (P > 0.05). Taken together, our data suggest that the GPIIIa and FcγRIIa platelet receptor polymorphisms do not influence S. aureus–platelet interactions in vitro or the clinical course of infective endocarditis. PMID:21044892

Platelet biomechanics, platelet bioenergetics, and applications to clinical practice and translational research.

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George, Mitchell J; Bynum, James; Nair, Prajeeda; Cap, Andrew P; Wade, Charles E; Cox, Charles S; Gill, Brijesh S

2018-07-01

The purpose of this review is to explore the relationship between platelet bioenergetics and biomechanics and how this relationship affects the clinical interpretation of platelet function devices. Recent experimental and technological advances highlight platelet bioenergetics and biomechanics as alternative avenues for collecting clinically relevant data. Platelet bioenergetics drive energy production for key biomechanical processes like adhesion, spreading, aggregation, and contraction. Platelet function devices like thromboelastography, thromboelastometry, and aggregometry measure these biomechanical processes. Platelet storage, stroke, sepsis, trauma, or the activity of antiplatelet drugs alters measures of platelet function. However, the specific mechanisms governing these alterations in platelet function and how they relate to platelet bioenergetics are still under investigation.

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

Subpopulations in purified platelets adhering on glass.

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Donati, Alessia; Gupta, Swati; Reviakine, Ilya

2016-06-22

Understanding how platelet activation is regulated is important in the context of cardiovascular disorders and their management with antiplatelet therapy. Recent evidence points to different platelet subpopulations performing different functions. In particular, procoagulant and aggregating subpopulations have been reported in the literature in platelets treated with the GPVI agonists. How the formation of platelet subpopulations upon activation is regulated remains unclear. Here, it is shown that procoagulant and aggregating platelet subpopulations arise spontaneously upon adhesion of purified platelets on clean glass surfaces. Calcium ionophore treatment of the adhering platelets resulted in one platelet population expressing both the procoagulant and the adherent population markers phosphatidylserine and the activated form of GPIIb/IIIa, while all of the platelets expressed CD62P independently of the ionophore treatment. Therefore, all platelets have the capacity to express all three activation markers. It is concluded that platelet subpopulations observed in various studies reflect the dynamics of the platelet activation process.

Dual-specificity phosphatase 3 deficiency or inhibition limits platelet activation and arterial thrombosis.

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Musumeci, Lucia; Kuijpers, Marijke J; Gilio, Karen; Hego, Alexandre; Théâtre, Emilie; Maurissen, Lisbeth; Vandereyken, Maud; Diogo, Catia V; Lecut, Christelle; Guilmain, William; Bobkova, Ekaterina V; Eble, Johannes A; Dahl, Russell; Drion, Pierre; Rascon, Justin; Mostofi, Yalda; Yuan, Hongbin; Sergienko, Eduard; Chung, Thomas D Y; Thiry, Marc; Senis, Yotis; Moutschen, Michel; Mustelin, Tomas; Lancellotti, Patrizio; Heemskerk, Johan W M; Tautz, Lutz; Oury, Cécile; Rahmouni, Souad

2015-02-17

A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase Cγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug. © 2014 American Heart Association, Inc.

Dependence of plasmin-mediated degradation of platelet adhesive receptors on temperature and Ca2+

International Nuclear Information System (INIS)

Winters, K.J.; Eisenberg, P.R.; Jaffe, A.S.; Santoro, S.A.

1990-01-01

The effects of activation of plasminogen by streptokinase and tissue-type-plasminogen activator on platelet activation and the membrane glycoproteins (GPs) that mediate platelet adhesion and aggregation are not yet fully defined. To clarify effects on platelets during activation of plasminogen in vitro, we used monoclonal antibodies (MoAbs), flow cytometry, and platelets surface-labeled with 125 I to characterize changes in receptors for fibrinogen (GPIIb-IIIa), von Willebrand factor (GPIb), and collagen (GPIa-IIa). Activation of plasminogen in plasma with pharmacologic concentrations of plasminogen activators did not degrade GPIIb-IIIa or GPIb, and caused only a modest decrease in GPIa. In washed platelets GPIIb-IIIa was extensively degraded by plasmin at 37 degrees C in the absence of exogenous Ca 2+ , conditions that destabilize the IIb-IIIa complex. Degradation of GPIb in washed platelets displayed a similar although less-marked dependence on temperature and the absence of Ca 2+ . The binding of activation-specific MoAbs did not increase during activation of plasminogen in plasma. We conclude that during pharmacologic fibrinolysis, reported inhibition of platelet function in plasma is not due to degradation of platelet-adhesive receptors. In addition, platelet activation observed during thrombolytic therapy does not appear to be a direct consequence of plasminogen activation

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

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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

Procoagulant expression in platelets and defects leading to clinical disorders.

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Solum, N O

1999-12-01

Hemostasis is a result of interactions between fibrillar structures in the damaged vessel wall, soluble components in plasma, and cellular elements in blood represented mainly by platelets and platelet-derived material. During formation of a platelet plug at the damaged vessel wall, factors IXa and VIIIa form the "tenase" complex, leading to activation of factor X on the surface of activated platelets. Subsequently, factors Xa and Va form the "prothrombinase" complex, which catalyzes the formation of thrombin from prothrombin, leading to fibrin formation. An enhanced expression of negatively charged phosphatidylserine in the outer membrane leaflet resulting from a breakdown of the phospholipid asymmetry is essential for the formation of the procoagulant surface. An ATP-driven and inward-acting aminophospholipid "translocase" and a "floppase" counterbalancing this have been postulated to maintain the dynamic state of phospholipid asymmetry. A phospholipid-nonspecific "scramblase," believed to be responsible for the fast breakdown of the asymmetry during cell activation, has recently been isolated from erythrocytes, cloned, and characterized. An intracellular calcium-binding segment and one or more thioesterified fatty acids are probably of importance for calcium-induced activation of this transporter protein. Cytosolic calcium ions also activate the calcium-dependent protease calpain associated with shedding of microvesicles from the transformed platelet membrane. These are shed with a procoagulant surface and with surface-exposed P-selectin from the alpha-granules. Theoretically, therefore, microvesicles can be involved in both coagulation and inflammation. Scott syndrome is probably caused by a defect in the activation of an otherwise normal scramblase, resulting in a relatively severe bleeding tendency. In Stormorken syndrome, the patients demonstrate a spontaneous surface expression of aminophospholipids. Activated platelets and the presence of procoagulant

Flavanols and Platelet Reactivity

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Debra A. Pearson

2005-01-01

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

UV-C irradiation disrupts platelet surface disulfide bonds and activates the platelet integrin alphaIIbbeta3

NARCIS (Netherlands)

Verhaar, Robin; Dekkers, David W. C.; de Cuyper, Iris M.; Ginsberg, Mark H.; de Korte, Dirk; Verhoeven, Arthur J.

2008-01-01

UV-C irradiation has been shown to be effective for pathogen reduction in platelet concentrates, but preliminary work indicated that UV-C irradiation of platelets can induce platelet aggregation. In this study, the mechanism underlying this phenomenon was investigated. Irradiation of platelets with

Role of tumor necrosis factor-alpha and platelet-activating factor in neoangiogenesis induced by synovial fluids of patients with rheumatoid arthritis.

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Lupia, E; Montrucchio, G; Battaglia, E; Modena, V; Camussi, G

1996-08-01

The aim of the present study was to investigate in vivo in a mouse model the stimulation of neoangiogenesis by synovial fluids of patients with rheumatoid arthritis (RA) and to determine the role of tumor necrosis factor (TNF)-alpha and platelet-activating factor (PAF) in the formation of new vessels. Angiogenesis was studied in a mouse model in which Matrigel, injected subcutaneously, was used as a vehicle for the delivery of potential angiogenic stimuli. Synovial fluids of patients with RA but not with osteoarthritis (OA) were shown to induce neoangiogenesis. Since synovial fluid of patients with RA contained significantly higher levels of TNF-alpha-like bioactivity and of PAF than that of patients with OA, the role of these mediators was evaluated by using an anti-TNF-alpha neutralizing monoclonal antibody (mAb) and a PAF receptor antagonist, WEB 2170. When added to Matrigel, anti-TNF-alpha mAb and particularly WEB 2170 significantly reduced neoangiogenesis induced by synovial fluids of RA patients. Moreover, PAF extracted and purified from synovial fluid induced angiogenesis. These results suggest that the neoangiogenesis observed in rheumatoid synovitis may be due, at least in part, to the angiogenic effect of locally produced TNF-alpha and PAF.

Parameters of platelet activity in patients with thyrotoxicosis in the presence of chronic hyperglycemia and atrial fibrillation

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G G Petrik

2011-06-01

Full Text Available Object. The study of platelet hemostasis in patients with thyrotoxicosis in the presence of chronic hyperglycemia and atrial fibrillation to prevent coagulation disorders. Material and methods. A comprehensive analysis of the parameters of platelet and coagulation hemostasis in 91 patients (67 women and 24 men with uncomplicated thyrotoxicosis, as well as with the atrial fibrillation or a combination of hyperthyroidism with diabetes has been completed. Results. The aggregation activity of platelets in uncomplicated thyrotoxicosis comparable with that of healthy people. atrial fibrillation, as well as chronic hyperglycemia, modify platelet activity by increasing the average volume of platelets, their ability to aggregate intensification of the release reaction and along with the decrease of disaggregation properties. Regardless of the presence of diabetes or atrial fibrillation, hyperthyroidism is accompanied by the activation of coagulation. Conclusion. The presence of transforming effects of chronic hyperglycemia and atrial fibrillation on the platelet activity determines the feasibility of differentiated approach to the prevent of coagulation disorders in patients with thyrotoxicosis.

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.

Prevotella intermedia induces severe bacteremic pneumococcal pneumonia in mice with upregulated platelet-activating factor receptor expression.

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Nagaoka, Kentaro; Yanagihara, Katsunori; Morinaga, Yoshitomo; Nakamura, Shigeki; Harada, Tatsuhiko; Hasegawa, Hiroo; Izumikawa, Koichi; Ishimatsu, Yuji; Kakeya, Hiroshi; Nishimura, Masaharu; Kohno, Shigeru

2014-02-01

Streptococcus pneumoniae is the leading cause of respiratory infection worldwide. Although oral hygiene has been considered a risk factor for developing pneumonia, the relationship between oral bacteria and pneumococcal infection is unknown. In this study, we examined the synergic effects of Prevotella intermedia, a major periodontopathic bacterium, on pneumococcal pneumonia. The synergic effects of the supernatant of P. intermedia (PiSup) on pneumococcal pneumonia were investigated in mice, and the stimulation of pneumococcal adhesion to human alveolar (A549) cells by PiSup was assessed. The effects of PiSup on platelet-activating factor receptor (PAFR) transcript levels in vitro and in vivo were analyzed by quantitative real-time PCR, and the differences between the effects of pneumococcal infection induced by various periodontopathic bacterial species were verified in mice. Mice inoculated with S. pneumoniae plus PiSup exhibited a significantly lower survival rate, higher bacterial loads in the lungs, spleen, and blood, and higher inflammatory cytokine levels in the bronchoalveolar lavage fluid (macrophage inflammatory protein 2 and tumor necrosis factor alpha) than those infected without PiSup. In A549 cells, PiSup increased pneumococcal adhesion and PAFR transcript levels. PiSup also increased lung PAFR transcript levels in mice. Similar effects were not observed in the supernatants of Porphyromonas gingivalis or Fusobacterium nucleatum. Thus, P. intermedia has the potential to induce severe bacteremic pneumococcal pneumonia with enhanced pneumococcal adhesion to lower airway cells.

Dissecting Bacterial Cell Wall Entry and Signaling in Eukaryotic Cells: an Actin-Dependent Pathway Parallels Platelet-Activating Factor Receptor-Mediated Endocytosis.

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Loh, Lip Nam; Gao, Geli; Tuomanen, Elaine I

2017-01-03

The Gram-positive bacterial cell wall (CW) peptidoglycan-teichoic acid complex is released into the host environment during bacterial metabolism or death. It is a highly inflammatory Toll-like receptor 2 (TLR2) ligand, and previous in vivo studies have demonstrated its ability to recapitulate pathological features of pneumonia and meningitis. We report that an actin-dependent pathway is involved in the internalization of the CW by epithelial and endothelial cells, in addition to the previously described platelet-activating factor receptor (PAFr)-dependent uptake pathway. Unlike the PAFr-dependent pathway, which is mediated by clathrin and dynamin and does not lead to signaling, the alternative pathway is sensitive to 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and engenders Rac1, Cdc42, and phosphatidylinositol 3-kinase (PI3K) signaling. Upon internalization by this macropinocytosis-like pathway, CW is trafficked to lysosomes. Intracellular CW trafficking is more complex than previously recognized and suggests multiple points of interaction with and without innate immune signaling. Streptococcus pneumoniae is a major human pathogen infecting the respiratory tract and brain. It is an established model organism for understanding how infection injures the host. During infection or bacterial growth, bacteria shed their cell wall (CW) into the host environment and trigger inflammation. A previous study has shown that CW enters and crosses cell barriers by interacting with a receptor on the surfaces of host cells, termed platelet-activating factor receptor (PAFr). In the present study, by using cells that are depleted of PAFr, we identified a second pathway with features of macropinocytosis, which is a receptor-independent fluid uptake mechanism by cells. Each pathway contributes approximately the same amount of cell wall trafficking, but the PAFr pathway is silent, while the new pathway appears to contribute to the host inflammatory response to CW insult. Copyright © 2017

Abnormal megakaryocyte development and platelet function in Nbeal2(-/-) mice.

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Kahr, Walter H A; Lo, Richard W; Li, Ling; Pluthero, Fred G; Christensen, Hilary; Ni, Ran; Vaezzadeh, Nima; Hawkins, Cynthia E; Weyrich, Andrew S; Di Paola, Jorge; Landolt-Marticorena, Carolina; Gross, Peter L

2013-11-07

Gray platelet syndrome (GPS) is an inherited bleeding disorder associated with macrothrombocytopenia and α-granule-deficient platelets. GPS has been linked to loss of function mutations in NEABL2 (neurobeachin-like 2), and we describe here a murine GPS model, the Nbeal2(-/-) mouse. As in GPS, Nbeal2(-/-) mice exhibit splenomegaly, macrothrombocytopenia, and a deficiency of platelet α-granules and their cargo, including von Willebrand factor (VWF), thrombospondin-1, and platelet factor 4. The platelet α-granule membrane protein P-selectin is expressed at 48% of wild-type levels and externalized upon platelet activation. The presence of P-selectin and normal levels of VPS33B and VPS16B in Nbeal2(-/-) platelets suggests that NBEAL2 acts independently of VPS33B/VPS16B at a later stage of α-granule biogenesis. Impaired Nbeal2(-/-) platelet function was shown by flow cytometry, platelet aggregometry, bleeding assays, and intravital imaging of laser-induced arterial thrombus formation. Microscopic analysis detected marked abnormalities in Nbeal2(-/-) bone marrow megakaryocytes, which when cultured showed delayed maturation, decreased survival, decreased ploidy, and developmental abnormalities, including abnormal extracellular distribution of VWF. Our results confirm that α-granule secretion plays a significant role in platelet function, and they also indicate that abnormal α-granule formation in Nbeal2(-/-) mice has deleterious effects on megakaryocyte survival, development, and platelet production.

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.

Impact of local anaesthetics and needle calibres used for painless PRP injections on platelet functionality.

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Bausset, Olivier; Magalon, Jeremy; Giraudo, Laurent; Louis, Marie-Laure; Serratrice, Nicolas; Frere, Corrine; Magalon, Guy; Dignat-George, Françoise; Sabatier, Florence

2014-01-01

The platelet-rich plasma (PRP) is an autologous biotherapy commonly used for its healing properties. Once activated, platelets released a real "cocktail" of growth factor and cytokines implied in numerous regenerative processes. However the impact of medical practices associated to PRP therapeutic use on platelets functionality remains poorly known. we evaluated the in vitro effects of two commonly used local anesthetics (Xylocaine(*) and Naropin(*)) on PRP functionality. We also investigated the quantity and quality of PRP that passed through the smallest gauge needle commercialized. PRP from 9 healthy volunteers were prepared using our previously described home made purification protocol. Platelet aggregation capacity was evaluated by aggregometry assays and the growth factor release was determined by ELISA after platelet activation. We also evaluated the platelet activation status, reactivity and stability of platelets by flow cytometry using the P-selectin expression marker. the association of local anaesthetics with PRP injections resulted in a significant decrease of platelets functionality, assessed by their capacity of aggregating. Local anaesthetics did not interfere with the growth factor release. The different needle sizes and calibres tested for PRP injections did not influence the platelet functionality. the use of local anaesthetics to prevent pain during PRP injections could compromise the therapeutic potential of PRP. These results suggest using carefully local anaesthetics or limiting their use as often is possible. To minimize injection pain, we recommend using 30 G needles. These data will lead to clinical recommendations for painless and controlled PRP injections.

Platelet transfusions reduce fibrinolysis but do not restore platelet function during trauma hemorrhage.

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Vulliamy, Paul; Gillespie, Scarlett; Gall, Lewis S; Green, Laura; Brohi, Karim; Davenport, Ross A

2017-09-01

Platelets play a critical role in hemostasis with aberrant function implicated in trauma-induced coagulopathy. However, the impact of massive transfusion protocols on platelet function during trauma hemorrhage is unknown. The aim of this study was to characterize the effects of platelet transfusion on platelet aggregation and fibrinolytic markers during hemostatic resuscitation. Trauma patients enrolled into the prospective Activation of Coagulation and Inflammation in Trauma study between January 2008 and November 2015 who received at least four units of packed red blood cells (PRBCs) were included. Blood was drawn in the emergency department within 2 hours of injury and at intervals after every four units of PRBCs transfused. Platelet aggregation was assessed in whole blood with multiple electrode aggregometry. Plasma proteins were quantified by enzyme-linked immunosorbent assay. Of 161 patients who received four or more PRBCs as part of their initial resuscitation, 44 received 8 to 11 units and 28 received 12 units or more. At each timepoint during bleeding, platelet aggregation was similar in patients who had received a platelet transfusion compared with those who had only received other blood products (p > 0.05 for all timepoints). Platelet transfusion during the four PRBC intervals was associated with a decrease in maximum lysis on rotational thromboelastometry (start of interval, 6% [2-12] vs. end of interval, 2% [0-5]; p = 0.001), an increase in plasminogen activator inhibitor-1 (start of interval, 35.9 ± 14.9 vs. end of interval, 66.7 ± 22.0; p = 0.007) and a decrease in tissue plasminogen activator (start of interval, 26.2 ± 10.5 vs. end of interval, 19.0 +/- 5.1; p = 0.04). No statistically significant changes in these parameters occurred in intervals which did not contain platelets. Current hemostatic resuscitation strategies do not appear to restore platelet aggregation during active hemorrhage. However, stored platelets may attenuate fibrinolysis

Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation.

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Metcalfe, Clive; Ramasubramoni, Anjana; Pula, Giordano; Harper, Matthew T; Mundell, Stuart J; Coxon, Carmen H

2016-01-01

Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.

Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation.

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Clive Metcalfe

Full Text Available Thioredoxin (Trx is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12 to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase. In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb. This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.