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

Platelets Drive Thrombus Propagation in a Hematocrit and Glycoprotein VI-Dependent Manner in an In Vitro Venous Thrombosis Model.

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Lehmann, Marcus; Schoeman, Rogier M; Krohl, Patrick J; Wallbank, Alison M; Samaniuk, Joseph R; Jandrot-Perrus, Martine; Neeves, Keith B

2018-05-01

The objective of this study was to measure the role of platelets and red blood cells on thrombus propagation in an in vitro model of venous valvular stasis. A microfluidic model with dimensional similarity to human venous valves consists of a sinus distal to a sudden expansion, where for sufficiently high Reynolds numbers, 2 countercurrent vortices arise because of flow separation. The primary vortex is defined by the points of flow separation and reattachment. A secondary vortex forms in the deepest recess of the valve pocket characterized by low shear rates. An initial fibrin gel formed within the secondary vortex of a tissue factor-coated valve sinus. Platelets accumulated at the interface of the fibrin gel and the primary vortex. Red blood cells at physiological hematocrits were necessary to provide an adequate flux of platelets to support thrombus growth out of the valve sinus. A subpopulation of platelets that adhered to fibrin expose phosphatidylserine. Platelet-dependent thrombus growth was attenuated by inhibition of glycoprotein VI with a blocking Fab fragment or D-dimer. A 3-step process regulated by hemodynamics was necessary for robust thrombus propagation: First, immobilized tissue factor initiates coagulation and fibrin deposition within a low flow niche defined by a secondary vortex in the pocket of a model venous valve. Second, a primary vortex delivers platelets to the fibrin interface in a red blood cell-dependent manner. Third, platelets adhere to fibrin, activate through glycoprotein VI, express phosphatidylserine, and subsequently promote thrombus growth beyond the valve sinus and into the bulk flow. © 2018 American Heart Association, Inc.

Increased platelet expression of glycoprotein IIIa following aspirin treatment in aspirin-resistant but not aspirin-sensitive subjects

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Floyd, Christopher N; Goodman, Timothy; Becker, Silke; Chen, Nan; Mustafa, Agnesa; Schofield, Emma; Campbell, James; Ward, Malcolm; Sharma, Pankaj; Ferro, Albert

2014-01-01

Aims Aspirin is widely used as an anti-platelet agent for cardiovascular prophylaxis. Despite aspirin treatment, many patients experience recurrent thrombotic events, and aspirin resistance may contribute to this. We examined the prevalence of aspirin resistance in a healthy population, and investigated whether the platelet proteome differed in aspirin-resistant subjects. Methods Ninety-three healthy subjects received aspirin 300 mg daily for 28 days. Before and at the end of treatment, urine was taken to determine 11-dehydrothromboxane B2, and blood was taken to measure arachidonic acid (AA)-induced aggregation of platelet-rich plasma and to interrogate the platelet proteome by mass spectrometric analysis with further confirmation of findings using Western blotting. Results In two of the 93 subjects, neither AA-induced aggregation nor urinary 11-dehydrothromboxane B2 was effectively suppressed by aspirin, despite measurable plasma salicylate concentrations, suggesting the presence of true aspirin resistance. Despite no detectable differences in the platelet proteome at baseline, following aspirin a marked increase was seen in platelet glycoprotein IIIa expression in the aspirin-resistant but not aspirin-sensitive subjects. An increase in platelet glycoprotein IIIa expression with aspirin resistance was confirmed in a separate cohort of 17 patients with stable coronary artery disease on long term aspirin treatment, four of whom exhibited aspirin resistance. Conclusions In a healthy population, true aspirin resistance is uncommon but exists. Resistance is associated with an increase in platelet glycoprotein IIIa expression in response to aspirin. These data shed new light on the mechanism of aspirin resistance, and provide the potential to identify aspirin-resistant subjects using a novel biomarker. PMID:25099258

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

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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

Genomic clone encoding the α chain of the OKM1, LFA-1, and platelet glycoprotein IIb-IIIa molecules

International Nuclear Information System (INIS)

Cosgrove, L.J.; Sandrin, M.S.; Rajasekariah, P.; McKenzie, I.F.C.

1986-01-01

LFA-1, an antigen involved in cytolytic T lymphocyte-mediated killing, and Mac-1, the receptor for complement component C3bi, constitute a family of structurally and functionally related cell surface glycoproteins involved in cellular interactions. In both mouse and man, Mac-1 (OKM1) and LFA-1 share a common 95-kDa β subunit but are distinguished by their α chains, which have different cellular distributions, apparent molecular masses (165 and 177 kDa, respectively), and peptide maps. The authors report the isolation of a genomic clone from a human genomic library that on transfection into mouse fibroblasts produced a molecule(s) reactive with monoclonal antibodies to OKM1, to LFA-1, and to platelet glycoprotein IIb-IIIa. This gene was cloned by several cycles of transfection of L cells with a human genomic library cloned in λ phase Charon 4A and subsequent rescue of the λ phage. Transfection with the purified recombinant λ DNA yielded a transfectant that expressed the three human α chains of OKM1, LFA-1, and glycoprotein IIb-IIIa, presumably in association with the murine β chain

Pregnancy-specific glycoproteins bind integrin αIIbβ3 and inhibit the platelet-fibrinogen interaction.

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Shanley, Daniel K; Kiely, Patrick A; Golla, Kalyan; Allen, Seamus; Martin, Kenneth; O'Riordan, Ronan T; Ball, Melanie; Aplin, John D; Singer, Bernhard B; Caplice, Noel; Moran, Niamh; Moore, Tom

2013-01-01

Pregnancy-specific glycoproteins (PSGs) are immunoglobulin superfamily members encoded by multigene families in rodents and primates. In human pregnancy, PSGs are secreted by the syncytiotrophoblast, a fetal tissue, and reach a concentration of up to 400 ug/ml in the maternal bloodstream at term. Human and mouse PSGs induce release of anti-inflammatory cytokines such as IL-10 and TGFβ1 from monocytes, macrophages, and other cell types, suggesting an immunoregulatory function. RGD tri-peptide motifs in the majority of human PSGs suggest that they may function like snake venom disintegrins, which bind integrins and inhibit interactions with ligands. We noted that human PSG1 has a KGD, rather than an RGD motif. The presence of a KGD in barbourin, a platelet integrin αIIbβ3 antagonist found in snake venom, suggested that PSG1 may be a selective αIIbβ3 ligand. Here we show that human PSG1 binds αIIbβ3 and inhibits the platelet - fibrinogen interaction. Unexpectedly, however, the KGD is not critical as multiple PSG1 domains independently bind and inhibit αIIbβ3 function. Human PSG9 and mouse Psg23 are also inhibitory suggesting conservation of this function across primate and rodent PSG families. Our results suggest that in species with haemochorial placentation, in which maternal blood is in direct contact with fetal trophoblast, the high expression level of PSGs reflects a requirement to antagonise abundant (3 mg/ml) fibrinogen in the maternal circulation, which may be necessary to prevent platelet aggregation and thrombosis in the prothrombotic maternal environment of pregnancy.

Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation.

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Mammadova-Bach, Elmina; Ollivier, Véronique; Loyau, Stéphane; Schaff, Mathieu; Dumont, Bénédicte; Favier, Rémi; Freyburger, Geneviève; Latger-Cannard, Véronique; Nieswandt, Bernhard; Gachet, Christian; Mangin, Pierre H; Jandrot-Perrus, Martine

2015-07-30

Fibrin, the coagulation end product, consolidates the platelet plug at sites of vascular injury and supports the recruitment of circulating platelets. In addition to integrin αIIbβ3, another as-yet-unidentified receptor is thought to mediate platelet interaction with fibrin. Platelet glycoprotein VI (GPVI) interacts with collagen and several other adhesive macromolecules. We evaluated the hypothesis that GPVI could be a functional platelet receptor for fibrin. Calibrated thrombin assays using platelet-rich plasma (PRP) showed that tissue factor-triggered thrombin generation was impaired in GPVI-deficient patients and reduced by the anti-GPVI Fab 9O12. Assays on reconstituted PRP and PRP from fibrinogen-deficient patients revealed a fibrinogen-dependent enhancement of thrombin generation, which relied on functional GPVI. The effect of GPVI was found to depend on fibrin polymerization. A binding assay showed a specific interaction between GPVI-Fc and fibrin, inhibited by the Fab 9O12. This Fab also reduced platelet adhesion to fibrin at low (300 s(-1)) and high (1500 s(-1)) wall shear rates. Platelets adherent to fibrin displayed shape change, exposure of procoagulant phospholipids, and the formation of small clots. When hirudinated blood was perfused at 1500 s(-1) over preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 85%. This study identifies GPVI as a platelet receptor for polymerized fibrin with 2 major functions: (1) amplification of thrombin generation and (2) recruitment of circulating platelets to clots. These so-far-unrecognized properties of GPVI confer on it a key role in thrombus growth and stabilization. © 2015 by The American Society of Hematology.

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

OpenAIRE

Landau, Meytal; Rosenberg, Nurit

2011-01-01

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

Effect of 60Co γ-ray irradiation on human platelets

International Nuclear Information System (INIS)

Wu Guoxin; Zhao Yiming; Ruan Changgeng

1992-01-01

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

Effects of 60Co γ-ray irradiation on human platelets

International Nuclear Information System (INIS)

Wu Guoxin; Zhao Yiming; Ruan Changgeng

1991-02-01

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

Inhibition of platelet [3H]- imipramine binding by human plasma protein fractions

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Strijewski, A.; Chudzik, J.; Tang, S.W.

1988-01-01

Inhibition of high-affinity [ 3 H]-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha 1 acid glycoprotein, high density and low density lipoprotein, IgG and α 1 -antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of [ 3 H]-imipramine binding site

Proteolysis of platelet receptors in humans and other species.

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

2010-08-01

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

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

International Nuclear Information System (INIS)

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

2007-01-01

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

The prevalence of the platelet glycoprotein VI polymorphisms in patients with sticky platelet syndrome and ischemic stroke.

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Kubisz, Peter; Ivanková, Jela; Škereňová, Mária; Staško, Ján; Hollý, Pavol

2012-11-01

The aim of the study was to evaluate the genetic variability of the GP6 gene in patients with sticky platelet syndrome (SPS), a disorder characterized by platelet hyperaggregability, and thus to identify the genetic changes of the glycoprotein VI with possible relation to the platelet hyperaggregability. Seventy-one patients with SPS, clinically manifested as ischemic stroke, and 77 controls without SPS and with negative personal history of thromboembolic events were involved. SPS was diagnosed by platelet aggregometry (PACKS-4 aggregometer, Helena Laboratories) according to the method and criteria described by Mammen and Bick. Seven single-nucleotide polymorphisms (SNPs) of the GP6 gene (rs1654410, rs1671153, rs1654419, rs11669150, rs1613662, rs12610286, and rs1654431) were evaluated with the use of restriction-fragment-length polymorphism analysis. All allele and genotype frequencies were comparable between both SPS patients and the control group with no statistically significant differences. The haplotype analysis showed a higher occurrence of the one major haplotype (TTGTGA, 0.228 vs. 0.174; odds ratio (OR) 1.421; confidence interval (CI) 0.799-2.526) and two minor haplotypes (CGATAA, 0,026 vs. 0,006; OR 4.117; CI 0.443-38.25; TTGTGG, 0.018 vs. 0.009; OR 2.107; CI 0.259-17.12) in patients with SPS. None of haplotype differences was statistically significant. However, both the allele G of SNP rs12610286 (P = 0.029; OR 2.411; CI 1.134-5.123) and one major haplotype (TTGTGA; P = 0.012; OR 2.749; CI 1.223-6.174) were found significantly more frequent in patients with SPS type I in comparison with controls. Our results, especially higher occurrence of four haplotypes in SPS patients, can support an idea that variability of the GP6 gene may be associated with the platelet hyperaggregability in SPS.

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

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

2016-02-11

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

Comparative Studies of Vertebrate Platelet Glycoprotein 4 (CD36

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Roger S. Holmes

2012-09-01

Full Text Available Platelet glycoprotein 4 (CD36 (or fatty acyl translocase [FAT], or scavenger receptor class B, member 3 [SCARB3] is an essential cell surface and skeletal muscle outer mitochondrial membrane glycoprotein involved in multiple functions in the body. CD36 serves as a ligand receptor of thrombospondin, long chain fatty acids, oxidized low density lipoproteins (LDLs and malaria-infected erythrocytes. CD36 also influences various diseases, including angiogenesis, thrombosis, atherosclerosis, malaria, diabetes, steatosis, dementia and obesity. Genetic deficiency of this protein results in significant changes in fatty acid and oxidized lipid uptake. Comparative CD36 amino acid sequences and structures and CD36 gene locations were examined using data from several vertebrate genome projects. Vertebrate CD36 sequences shared 53–100% identity as compared with 29–32% sequence identities with other CD36-like superfamily members, SCARB1 and SCARB2. At least eight vertebrate CD36 N-glycosylation sites were conserved which are required for membrane integration. Sequence alignments, key amino acid residues and predicted secondary structures were also studied. Three CD36 domains were identified including cytoplasmic, transmembrane and exoplasmic sequences. Conserved sequences included N- and C-terminal transmembrane glycines; and exoplasmic cysteine disulphide residues; TSP-1 and PE binding sites, Thr92 and His242, respectively; 17 conserved proline and 14 glycine residues, which may participate in forming CD36 ‘short loops’; and basic amino acid residues, and may contribute to fatty acid and thrombospondin binding. Vertebrate CD36 genes usually contained 12 coding exons. The human CD36 gene contained transcription factor binding sites (including PPARG and PPARA contributing to a high gene expression level (6.6 times average. Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate CD36 gene with vertebrate

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

Energy Technology Data Exchange (ETDEWEB)

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

2007-10-15

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

Non-redundant roles of phosphoinositide 3-kinase isoforms alpha and beta in glycoprotein VI-induced platelet signaling and thrombus formation.

Science.gov (United States)

Gilio, Karen; Munnix, Imke C A; Mangin, Pierre; Cosemans, Judith M E M; Feijge, Marion A H; van der Meijden, Paola E J; Olieslagers, Servé; Chrzanowska-Wodnicka, Magdalena B; Lillian, Rivka; Schoenwaelder, Simone; Koyasu, Shigeo; Sage, Stewart O; Jackson, Shaun P; Heemskerk, Johan W M

2009-12-04

Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cgamma2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3Kalpha or -beta strongly and selectively suppressed GPVI-induced Ca(2+) mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cgamma2 by PI3Kalpha/beta. That PI3Kalpha and -beta have a non-redundant function in GPVI-induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca(2+) increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3Kalpha/beta was blocked or p85alpha was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kdelta or -gamma. Furthermore, PI3Kalpha/beta, but not PI3Kgamma, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kalpha and -beta isoforms are required for full GPVI-dependent platelet Ca(2+) signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kalpha an interesting new target for anti-platelet therapy.

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

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Robert, Amélie; Boyer, Lucie; Pineault, Nicolas

2011-03-01

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

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.

Radioimmune assay of human platelet prostaglandin synthetase

International Nuclear Information System (INIS)

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

1982-01-01

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

Frequency of anti-glycoprotein Ia/IIa (anti-HPA-5b,-5a and anti-glycoprotein IIb/IIIa (anti-HPA-1a,-3a,-4a alloantibodies in multiparous women of African descent

Directory of Open Access Journals (Sweden)

Zaccheaus A Jeremiah

2010-05-01

Full Text Available Zaccheaus A Jeremiah1, Justina E Oburu2, Osaro Erhabor1, Fiekumo I Buseri1, Teddy C Adias31Haematology and Blood Transfusion Science Unit, Department of Medical Laboratory Sciences, College of Health Sciences, Niger Delta University, Wilberforce Island, Nigeria; 2Department of Haematology and Blood Transfusion, University of Port Harcourt Teaching Hospital, Port Harcourt, Nigeria; 3Rivers State University of Science and Technology, Port Harcourt, NigeriaBackground: Human platelet antibodies are often implicated in some disease conditions, such as neonatal alloimmune thrombocytopenia (NAIT, idiopathic thrombocytopenic purpura (ITP and platelet refractoriness. The frequencies of these alloantibodies have not been reported in Nigeria and West Africa.Methods: Screening for allontibodies to human platelet antigens (HPA was undertaken using the GTI PakPlus® qualitative solid phase ELISA reagent. Platelet count was done using the ICSH approved procedure using 1% ammonium oxalate reagent.Study design: A cross-section of apparently healthy adult Nigerian multiparous non-pregnant women, who were staff of a tertiary health facility in the Niger Delta, Nigeria, were screened for alloantibodies to human platelet antigens.Results: Of the one hundred (100 women screened, the prevalence of anti-glycoprotein IIb/IIIa (anti-HPA-Ia,-3a,-4a was zero percent (0%, anti-glycoprotein Ia/IIa (anti-HPA-5b accounted for 30% of results, while anti-glycoprotein Ia/IIa (anti-HPA-5a was 18%. Parity was found to exert significant influence on the development to HPA antibodies (Fisher’s Exact Test = 11.683, P < 0.05; 13.577, P < 0.01. The platelet count of the women did not appear to exert any influence on the development of the antibodies (P > 0.05.Conclusion: This study has observed a high prevalence of anti-HPA-5b in our sample population. The prevalence of alloantibodies to HPA antigens was found to associate strongly with parity. These results indicate that there is a

Glycoprotein VI/Fc receptor γ chain-independent tyrosine phosphorylation and activation of murine platelets by collagen

OpenAIRE

Jarvis, Gavin E.; Best, Denise; Watson, Steve P.

2004-01-01

We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRγ (Fc receptor γ) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRγ chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induc...

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

International Nuclear Information System (INIS)

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

1990-01-01

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

Humanizing recombinant glycoproteins from Chinese hamster ovary cells

DEFF Research Database (Denmark)

Hansen, Anders Holmgaard; Amann, Thomas; Kol, Stefan

With new tools for gene-editing like zinc-fingers, TALENS and CRISPR, it is now feasible totailor-make the N-Glycoforms for therapeutic glycoproteins that have previously been almost impossible. We here demonstrate a case of humanizing a recombinant human glycoprotein that in Wild type (WT) Chinese...

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

Roles of platelet STIM1 and Orai1 in glycoprotein VI- and thrombin-dependent procoagulant activity and thrombus formation.

Science.gov (United States)

Gilio, Karen; van Kruchten, Roger; Braun, Attila; Berna-Erro, Alejandro; Feijge, Marion A H; Stegner, David; van der Meijden, Paola E J; Kuijpers, Marijke J E; Varga-Szabo, David; Heemskerk, Johan W M; Nieswandt, Bernhard

2010-07-30

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca(2+) entry (SOCE) with Orai1 as principal Ca(2+) entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca(2+) entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1(-/-) and Orai1(-/-) platelets had greatly impaired glycoprotein (GP) VI-dependent Ca(2+) signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2(-/-) platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca(2+) signals of Stim1(-/-) and Orai1(-/-) platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1(-/-) and Orai1(-/-) platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca(2+) entry, inhibited Ca(2+) and procoagulant responses even in Stim1(-/-) and Orai1(-/-) platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca(2+) entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca(2+) entry and PS exposure, only one relying on STIM1-Orai1 interaction.

The role of platelet and endothelial GARP in thrombosis and hemostasis.

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Vermeersch, Elien; Denorme, Frederik; Maes, Wim; De Meyer, Simon F; Vanhoorelbeke, Karen; Edwards, Justin; Shevach, Ethan M; Unutmaz, Derya; Fujii, Hodaka; Deckmyn, Hans; Tersteeg, Claudia

2017-01-01

Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.

The role of platelet and endothelial GARP in thrombosis and hemostasis.

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Elien Vermeersch

Full Text Available Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32 is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish.To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice.Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice.Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected.Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.

Superoxide dismutase of human platelets

International Nuclear Information System (INIS)

Kimura, Akiro; Fujimura, Kingo; Kuramoto, Atsushi

1979-01-01

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

Soluble glycoprotein VI, a specific marker of platelet activation is increased in the plasma of subjects with seropositive rheumatoid arthritis.

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John R Stack

Full Text Available Anti-citrullinated protein antibodies (ACPA have been shown to cause platelet activation in vitro, through the low-affinity immunoglobulin G (IgG receptor (FcγRIIa on platelets. Platelet activation via engagement of FcγRIIa results in proteolytic cleavage and shedding of platelet specific glycoprotein VI (GPVI which can be detected in the plasma as soluble GPVI (sGPVI. We hypothesized that plasma levels of sGPVI would be increased among patients with seropositive RA as a consequence of antibody-induced platelet activation and GPVI shedding.Samples from 84 patients with RA (65 seropositive and 19 seronegative and 67 healthy controls were collected prospectively and analysed for sGPVI using a standardised ELISA.Patients with seropositive RA had significantly higher levels of sGPVI compared to seronegative RA and controls. Median (IQR sGPVI levels were 4.2 ng/ml (3.2, 8.0 in seropositve RA, 2.2 ng/ml (1.5, 3.5 in seronegative RA and 2.2 ng/ml (1.6, 3.4 in controls (p<0.0001. sGPVI levels correlated with ACPA titres (r = 0.32, p = 0.0026 and with RF titres (r = 0.48, p<0.0001.Plasma sGPVI, a specific marker of platelet activation is increased among patients with seropositive RA.

Platelet Glycoprotein lb-1X and Malignancy

Science.gov (United States)

2011-09-01

initiate coagulation, resulting in the formation of a fibrin - rich tumor cell- platelet emboli (Figure 1). Many of these coagulation factor-tumor cell...the tumor cell in a fibrin - rich web. (13;23;24) During this process, the platelet integrin receptor, aIIb 3, serves as a receptor linking fibrin ... platelets , and tumor cells into a fibrin rich clot normally associated with a thrombus. (25;25) Indeed, it can be speculated the fibrin - rich clot

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation*

Science.gov (United States)

Gilio, Karen; van Kruchten, Roger; Braun, Attila; Berna-Erro, Alejandro; Feijge, Marion A. H.; Stegner, David; van der Meijden, Paola E. J.; Kuijpers, Marijke J. E.; Varga-Szabo, David; Heemskerk, Johan W. M.; Nieswandt, Bernhard

2010-01-01

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca2+ entry (SOCE) with Orai1 as principal Ca2+ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca2+ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1−/− and Orai1−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca2+ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2−/− platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca2+ signals of Stim1−/− and Orai1−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1−/− and Orai1−/− platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca2+ entry, inhibited Ca2+ and procoagulant responses even in Stim1−/− and Orai1−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca2+ entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca2+ entry and PS exposure, only one relying on STIM1-Orai1 interaction. PMID:20519511

Platelet glycoprotein IaC807T polymorphisms and ischemic stroke in young Chinese Han population.

Science.gov (United States)

Zhang, J; Huang, D; Yang, J; An, H; Ojha, R; DU, C; Liu, R

2012-11-01

The objective of this study was to investigate the association between platelet glycoprotein (GP) Ia C807T polymorphisms and ischemic stroke in young Chinese Han Population. We conducted a case-control study in 92 consecutive young (ischemic stroke inpatients and outpatients, 86 elder ischemic stroke control (> 50 years), and 160 age- and sex-matched healthy control. Genotyping of platelet GP Ia C807Tpolymorphisms was performed by polymerase chain reaction followed by sequencing nucleic acid with dideoxy chain-termination method and an ABI PRISM3100 (Perkin-Elmer Co) genetic analyzer. Student's t-test, chi-square test, and logistic regression modeling were used for data significance analyses. Hypertension and smoking were found to be the independent risk factors for ischemic stroke patients (aged ischemic stroke patients (aged > 50 years). There was no significant difference observed in the T allele frequency of GPIa C807T polymorphisms between young stroke patients and corresponding controls. These findings suggest that there is no role of GPIa C807T polymorphisms in the development of young first-ever ischemic stroke in Chinese Han Population.

Lea blood group antigen on human platelets

International Nuclear Information System (INIS)

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

1985-01-01

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

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.

Platelet fibrinogen binding in Basset Hound Hereditary Thrombopathy

International Nuclear Information System (INIS)

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

1986-01-01

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

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

Science.gov (United States)

Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

2000-09-01

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets.

Novel agents for anti-platelet therapy

Directory of Open Access Journals (Sweden)

Ji Xuebin

2011-11-01

Full Text Available Abstract Anti-platelet therapy plays an important role in the treatment of patients with thrombotic diseases. The most commonly used anti-platelet drugs, namely, aspirin, ticlopidine, and clopidogrel, are effective in the prevention and treatment of cardio-cerebrovascular diseases. Glycoprotein IIb/IIIa antagonists (e.g., abciximab, eptifibatide and tirofiban have demonstrated good clinical benefits and safety profiles in decreasing ischemic events in acute coronary syndrome. However, adverse events related to thrombosis or bleeding have been reported in cases of therapy with glycoprotein IIb/IIIa antagonists. Cilostazol is an anti-platelet agent used in the treatment of patients with peripheral ischemia, such as intermittent claudication. Presently, platelet adenosine diphosphate P2Y(12 receptor antagonists (e.g., clopidogrel, prasugrel, cangrelor, and ticagrelor are being used in clinical settings for their pronounced protective effects. The new protease-activated receptor antagonists, vorapaxar and atopaxar, potentially decrease the risk of ischemic events without significantly increasing the rate of bleeding. Some other new anti-platelet drugs undergoing clinical trials have also been introduced. Indeed, the number of new anti-platelet drugs is increasing. Consequently, the efficacy of these anti-platelet agents in actual patients warrants scrutiny, especially in terms of the hemorrhagic risks. Hopefully, new selective platelet inhibitors with high anti-thrombotic efficiencies and low hemorrhagic side effects can be developed.

Stent parameters predict major adverse clinical events and the response to platelet glycoprotein IIb/IIIa blockade: findings of the ESPRIT trial.

Science.gov (United States)

Tcheng, James E; Lim, Ing Haan; Srinivasan, Shankar; Jozic, Joseph; Gibson, C Michael; O'Shea, J Conor; Puma, Joseph A; Simon, Daniel I

2009-02-01

Only limited data describe relationships between stent parameters (length and diameter), adverse events after percutaneous coronary intervention, and effects of platelet glycoprotein IIb/IIIa blockade by stent parameters. In this post hoc analysis of the 1983 patients receiving a stent in the Enhanced Suppression of the Platelet Glycoprotein IIb/IIIa Receptor with Integrilin Therapy randomized percutaneous coronary intervention trial of eptifibatide versus placebo, rates of the major adverse cardiac event (MACE) end point (death, myocardial infarction, urgent target-vessel revascularization, or thrombotic bailout) at 48 hours and 1 year were correlated with stent parameters and then analyzed by randomization to eptifibatide versus placebo. In the placebo group, MACE increased with number of stents implanted, total stent length (by quartiles of or=30 mm), and total stented vessel area (by quartiles of area or=292 mm(2)). By stent parameters, MACE at 48 hours was reduced in the eptifibatide group at stent lengths of 18 to or=30 mm (OR, 0.43; 95% CI, 0.25 to 0.75; P=0.003), stent diameters of >2.5 to <3.5 mm (OR, 0.56; 95% CI, 0.39 to 0.82; P=0.002), and with 2 stents implanted (OR, 0.39; 95% CI, 0.22 to 0.69; P=0.001). In the placebo group, near-linear relationships were observed between both increasing stent length and increasing stented vessel area and MACE at 48 hours and 1 year (all, P<0.001); these gradients were flattened in the eptifibatide group (P=0.005 for stent length). Stent parameters predict MACE after percutaneous coronary intervention. Glycoprotein IIb/IIIa blockade mitigates much of the hazard of increasing procedural complexity.

Rapid resensitization of purinergic receptor function in human platelets.

Science.gov (United States)

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

2008-08-01

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

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

Science.gov (United States)

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.

Convergent evolution of pregnancy-specific glycoproteins in human and horse.

Science.gov (United States)

Aleksic, Denis; Blaschke, Lisa; Mißbach, Sophie; Hänske, Jana; Weiß, Wiebke; Handler, Johannes; Zimmermann, Wolfgang; Cabrera-Sharp, Victoria; Read, Jordan E; de Mestre, Amanda M; O'Riordan, Ronan; Moore, Tom; Kammerer, Robert

2016-09-01

Pregnancy-specific glycoproteins (PSGs) are members of the carcinoembryonic antigen cell adhesion molecule (CEACAM) family that are secreted by trophoblast cells. PSGs may modulate immune, angiogenic and platelet responses during pregnancy. Until now, PSGs are only found in species that have a highly invasive (hemochorial) placentation including humans, mice and rats. Surprisingly, analyzing the CEACAM gene family of the horse, which has a non-invasive epitheliochorial placenta, with the exception of the transient endometrial cups, we identified equine CEACAM family members that seem to be related to PSGs of rodents and primates. We identified seven genes that encode secreted PSG-like CEACAMs Phylogenetic analyses indicate that they evolved independently from an equine CEACAM1-like ancestor rather than from a common PSG-like ancestor with rodents and primates. Significantly, expression of PSG-like genes (CEACAM44, CEACAM48, CEACAM49 and CEACAM55) was found in non-invasive as well as invasive trophoblast cells such as purified chorionic girdle cells and endometrial cup cells. Chorionic girdle cells are highly invasive trophoblast cells that invade the endometrium of the mare where they form endometrial cups and are in close contact with maternal immune cells. Therefore, the microenvironment of invasive equine trophoblast cells has striking similarities to the microenvironment of trophoblast cells in hemochorial placentas, suggesting that equine PSG-like CEACAMs and rodent and primate PSGs have undergone convergent evolution. This is supported by our finding that equine PSG-like CEACAM49 exhibits similar activity to certain rodent and human PSGs in a functional assay of platelet-fibrinogen binding. Our results have implications for understanding the evolution of PSGs and their functions in maternal-fetal interactions. © 2016 Society for Reproduction and Fertility.

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

Science.gov (United States)

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.

Selective elution of HLA antigens and beta 2-microglobulin from human platelets by chloroquine diphosphate

International Nuclear Information System (INIS)

Kao, K.J.

1988-01-01

To determine whether chloroquine can specifically elute HLA antigens and beta 2-microglobulin (beta 2-M) from the platelet surface, quantitative immunofluorescence flow cytometry and monoclonal antibodies were used to show that HLA antigens and beta 2-M were proportionally eluted from the platelet surface without affecting the membrane glycoproteins IIb and IIIa. Second, an autoradiogram of electrophoresed I-125-labeled platelets showed that only beta 2-M but not other I-125-labeled membrane proteins could be eluted. Although HLA antigens were poorly labeled by I-125 and could not be detected on the autoradiogram, the eluted HLA antigens could be detected by anti-HLA monoclonal antibody and immunoblotting techniques. No loss of plasma membrane integrity was observed by transmission electron microscopy after chloroquine treatment of platelets. The results indicate that chloroquine selectively elutes HLA antigens and their noncovalently associated beta 2-M without affecting other integral platelet membrane proteins

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

Identification of functional VEGF receptors on human platelets.

Science.gov (United States)

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.

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

Relationship between the ability of oral streptococci to interact with platelet glycoprotein Ibalpha and with the salivary low-molecular-weight mucin, MG2.

Science.gov (United States)

Plummer, Christopher; Douglas, Charles William Ian

2006-12-01

The oral streptococci Streptococcus sanguinis, Streptococcus gordonii and Streptococcus oralis are common aetiological agents of infective endocarditis, and their ability to adhere to and induce the aggregation of platelets is thought to be a virulence trait. The platelet glycoprotein GPIbalpha has been implicated as the adhesion receptor for S. sanguinis and S. gordonii, but it is not known if this is the case for S. oralis and other species. The aim of this study was to determine the GPIbalpha-interactive capability of a range of oral streptococci and to determine the relationship between this capability and their ability to interact with the salivary constituents that they would encounter in their normal habitat. All platelet-adhesive S. sanguinis strains and most S. gordonii strains adhered in a GPIbalpha-dependent manner, but strains of S. oralis, Streptococcus cristatus, Streptococcus parasanguinis and Streptococcus mitis had no direct affinity for platelets. Those strains that were able to bind GPIbalpha also bound to the low-molecular-weight submandibular salivary mucin, MG2, and this interaction was sialic acid-dependent. The data suggest that S. sanguinis and S. gordonii may be efficient colonizers of platelet vegetations because of their adaptation to recognize sialylated salivary mucins. In contrast, S. oralis does not interact with platelets and so is likely to colonize vegetations through an as yet unidentified mechanism.

Scalable Generation of Universal Platelets from Human Induced Pluripotent Stem Cells

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Qiang Feng

2014-11-01

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

Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation?

Science.gov (United States)

Burnouf, Thierry; Strunk, Dirk; Koh, Mickey B C; Schallmoser, Katharina

2016-01-01

The essential physiological role of platelets in wound healing and tissue repair builds the rationale for the use of human platelet derivatives in regenerative medicine. Abundant growth factors and cytokines stored in platelet granules can be naturally released by thrombin activation and clotting or artificially by freeze/thaw-mediated platelet lysis, sonication or chemical treatment. Human platelet lysate prepared by the various release strategies has been established as a suitable alternative to fetal bovine serum as culture medium supplement, enabling efficient propagation of human cells under animal serum-free conditions for a multiplicity of applications in advanced somatic cell therapy and tissue engineering. The rapidly increasing number of studies using platelet derived products for inducing human cell proliferation and differentiation has also uncovered a considerable variability of human platelet lysate preparations which limits comparability of results. The main variations discussed herein encompass aspects of donor selection, preparation of the starting material, the possibility for pooling in plasma or additive solution, the implementation of pathogen inactivation and consideration of ABO blood groups, all of which can influence applicability. This review outlines the current knowledge about human platelet lysate as a powerful additive for human cell propagation and highlights its role as a prevailing supplement for human cell culture capable to replace animal serum in a growing spectrum of applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Isoforms of purified methyltransferase from human blood platelets ...

African Journals Online (AJOL)

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

Oral streptococci utilize a Siglec-like domain of serine-rich repeat adhesins to preferentially target platelet sialoglycans in human blood.

Directory of Open Access Journals (Sweden)

Lingquan Deng

2014-12-01

Full Text Available Damaged cardiac valves attract blood-borne bacteria, and infective endocarditis is often caused by viridans group streptococci. While such bacteria use multiple adhesins to maintain their normal oral commensal state, recognition of platelet sialoglycans provides an intermediary for binding to damaged valvular endocardium. We use a customized sialoglycan microarray to explore the varied binding properties of phylogenetically related serine-rich repeat adhesins, the GspB, Hsa, and SrpA homologs from Streptococcus gordonii and Streptococcus sanguinis species, which belong to a highly conserved family of glycoproteins that contribute to virulence for a broad range of Gram-positive pathogens. Binding profiles of recombinant soluble homologs containing novel sialic acid-recognizing Siglec-like domains correlate well with binding of corresponding whole bacteria to arrays. These bacteria show multiple modes of glycan, protein, or divalent cation-dependent binding to synthetic glycoconjugates and isolated glycoproteins in vitro. However, endogenous asialoglycan-recognizing clearance receptors are known to ensure that only fully sialylated glycans dominate in the endovascular system, wherein we find these particular streptococci become primarily dependent on their Siglec-like adhesins for glycan-mediated recognition events. Remarkably, despite an excess of alternate sialoglycan ligands in cellular and soluble blood components, these adhesins selectively target intact bacteria to sialylated ligands on platelets, within human whole blood. These preferred interactions are inhibited by corresponding recombinant soluble adhesins, which also preferentially recognize platelets. Our data indicate that circulating platelets may act as inadvertent Trojan horse carriers of oral streptococci to the site of damaged endocardium, and provide an explanation why it is that among innumerable microbes that gain occasional access to the bloodstream, certain viridans group

Increase in density and accumulation of serotonin by human aging platelets

International Nuclear Information System (INIS)

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

1984-01-01

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

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

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Bert Rutten

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

Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production

International Nuclear Information System (INIS)

Jarvis, Donald L.

2003-01-01

The baculovirus-insect cell expression system is widely used to produce recombinant glycoproteins for many different biomedical applications. However, due to the fundamental nature of insect glycoprotein processing pathways, this system is typically unable to produce recombinant mammalian glycoproteins with authentic oligosaccharide side chains. This minireview summarizes our current understanding of insect protein glycosylation pathways and our recent efforts to address this problem. These efforts have yielded new insect cell lines and baculoviral vectors that can produce recombinant glycoproteins with humanized oligosaccharide side chains

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

Science.gov (United States)

Landau, Meytal; Rosenberg, Nurit

2011-03-01

Human platelet antigens (HPAs) are polymorphisms in platelet membrane glycoproteins (GPs) that can stimulate production of alloantibodies once exposed to foreign platelets (PLTs) with different HPAs. These antibodies can cause neonatal alloimmune thrombocytopenia, posttransfusion purpura, and PLT transfusion refractoriness. Most HPAs are localized on the main PLT receptors: 1) integrin αIIbβ3, known as the fibrinogen receptor; 2) the GPIb-IX-V complex that functions as the receptor for von Willebrand factor; and 3) integrin α2β1, which functions as the collagen receptor. We analyzed the structural location and the evolutionary conservation of the residues associated with the HPAs to characterize the features that induce immunologic responses but do not cause inherited diseases. We found that all HPAs reside in positions located on the protein surface, apart from the ligand-binding site, and are evolutionary variable. Disease-causing mutations often reside in highly conserved and buried positions. In contrast, the HPAs affect residues on the protein surface that were not conserved throughout evolution; this explains their naive effect on the protein function. Nonetheless, the HPAs involve substitutions of solvent-exposed positions that lead to altered interfaces on the surface of the protein and might present epitopes foreign to the immune system. © 2010 American Association of Blood Banks.

Storage of human platelets by freezing

Energy Technology Data Exchange (ETDEWEB)

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

1976-01-01

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

Annotating Human P-Glycoprotein Bioassay Data.

Science.gov (United States)

Zdrazil, Barbara; Pinto, Marta; Vasanthanathan, Poongavanam; Williams, Antony J; Balderud, Linda Zander; Engkvist, Ola; Chichester, Christine; Hersey, Anne; Overington, John P; Ecker, Gerhard F

2012-08-01

Huge amounts of small compound bioactivity data have been entering the public domain as a consequence of open innovation initiatives. It is now the time to carefully analyse existing bioassay data and give it a systematic structure. Our study aims to annotate prominent in vitro assays used for the determination of bioactivities of human P-glycoprotein inhibitors and substrates as they are represented in the ChEMBL and TP-search open source databases. Furthermore, the ability of data, determined in different assays, to be combined with each other is explored. As a result of this study, it is suggested that for inhibitors of human P-glycoprotein it is possible to combine data coming from the same assay type, if the cell lines used are also identical and the fluorescent or radiolabeled substrate have overlapping binding sites. In addition, it demonstrates that there is a need for larger chemical diverse datasets that have been measured in a panel of different assays. This would certainly alleviate the search for other inter-correlations between bioactivity data yielded by different assay setups.

Generation of Megakaryocytes and Platelets from Human Pluripotent Stem Cells.

Science.gov (United States)

Pick, Marjorie

2016-01-01

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

EMMPRIN (CD147) is a novel receptor for platelet GPVI and mediates platelet rolling via GPVI-EMMPRIN interaction.

Science.gov (United States)

Seizer, Peter; Borst, Oliver; Langer, Harald F; Bültmann, Andreas; Münch, Götz; Herouy, Yared; Stellos, Konstantinos; Krämer, Björn; Bigalke, Boris; Büchele, Berthold; Bachem, Max G; Vestweber, Dietmar; Simmet, Thomas; Gawaz, Meinrad; May, Andreas E

2009-04-01

The Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147, basigin) is an immunoglobulin-like receptor expressed in various cell types. During cellular interactions homotypic EMMPRIN-EMMPRIN interactions are known to induce the synthesis of matrix metalloproteinases. Recently, we have identified EMMPRIN as a novel receptor on platelets. To our knowledge EMMPRIN has not been shown to serve as adhesion receptor, yet. Here we characterise platelet glycoprotein VI (GPVI) as a novel adhesion receptor for EMMPRIN. Human platelets were prestimulated with ADP and perfused over immobilised recombinant EMMPRIN-Fc or Fc-fragments under arterial shear conditions. ADP-stimulated platelets showed significantly enhanced rolling (but not enhanced firm adhesion) on immobilised EMMPRIN-Fc compared to Fc. Pretreatment of platelets with blocking mAbs anti-EMMPRIN or anti-GPVI leads to a significant reduction of rolling platelets on immobilised EMMPRIN-Fc, whereas pretreatment with blocking mAbs anti-p-selectin, anti-alpha4-integrin or anti-GPIIb/IIIa complex (20 microg/ml each) had no effect. Consistently, chinese hamster ovary (CHO) cells stably transfected with GPVI showed enhanced rolling (but not adhesion) on immobilised EMMPRIN-Fc in comparison to non-transfected CHO cells. Similarly, CHO cells stably transfected with EMMPRIN showed enhanced rolling on immobilised GPVI-Fc (or EMMPRIN-Fc) compared to non transfected CHO-cells. Finally, specific binding of EMMPRIN to GPVI was demonstrated by a modified ELISA and surface plasmon resonance technology with a dissociation constant of 88 nM. Platelet GPVI is a novel receptor for EMMPRIN and can mediate platelet rolling via GPVI-EMMPRIN interaction.

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

Science.gov (United States)

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.

Monoclonal antibody to an external epitope of the human mdr1 P-glycoprotein

NARCIS (Netherlands)

Arceci, R. J.; Stieglitz, K.; Bras, J.; Schinkel, A.; Baas, F.; Croop, J.

1993-01-01

A membrane glycoprotein, termed P-glycoprotein, has been shown to be responsible for cross-resistance to a broad range of structurally and functionally distinct cytotoxic agents. P-glycoprotein, encoded in humans by the mdr1 gene, functions as an energy-dependent efflux pump to exclude these

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.

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

Human Milk Glycoproteins Protect Infants Against Human Pathogens

Science.gov (United States)

Liu, Bo

2013-01-01

Abstract Breastfeeding protects the neonate against pathogen infection. Major mechanisms of protection include human milk glycoconjugates functioning as soluble receptor mimetics that inhibit pathogen binding to the mucosal cell surface, prebiotic stimulation of gut colonization by favorable microbiota, immunomodulation, and as a substrate for bacterial fermentation products in the gut. Human milk proteins are predominantly glycosylated, and some biological functions of these human milk glycoproteins (HMGPs) have been reported. HMGPs range in size from 14 kDa to 2,000 kDa and include mucins, secretory immunoglobulin A, bile salt-stimulated lipase, lactoferrin, butyrophilin, lactadherin, leptin, and adiponectin. This review summarizes known biological roles of HMGPs that may contribute to the ability of human milk to protect neonates from disease. PMID:23697737

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

International Nuclear Information System (INIS)

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

1987-01-01

Human platelet concentrate from the American Red Cross Blood Center was stored at 24 degree C in a shaker and aliquots were taken out at time intervals aseptically. Platelet activating factor (PAF) stimulated turnover of phosphoinositide (PPI) was monitored by assaying 32 P incorporation into phosphoinositides using platelet rich plasma (PRP). Platelets in PRP were incubated with 1 x 10 -7 M PAF at 37 degree C with gentle shaking and after 5 min their lipids were extracted and analysed by TLC for 32 P-phosphoinositides. The percent stimulation of 32 P incorporation by PAF (over control) into PPI was approximately 250, 100, 60, 25 and 20 on days 1, 2, 3, 5 and 6, respectively. This indicated a dramatic decrease in PAF responsive turnover of platelet PPI during storage. These findings have important implications in relation to PAF receptor activity and viability of platelets at different periods of storage

Enhancement by platelets of oxygen radical responses of human neutrophils

Energy Technology Data Exchange (ETDEWEB)

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

1986-03-01

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

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.

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

DEFF Research Database (Denmark)

Song, Wen-Liang; Stubbe, Jane; Ricciotti, Emanuela

2012-01-01

during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis....... Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1-derived PGD₂ biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased...... thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD₂ was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD₂, like PGI₂, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular...

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

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

2015-10-01

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

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

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

2017-01-01

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

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

International Nuclear Information System (INIS)

Hwang, S.B.

1987-01-01

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

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

Science.gov (United States)

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

2000-04-01

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

Improved Human Erythropoiesis and Platelet Formation in Humanized NSGW41 Mice

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Susann Rahmig

2016-10-01

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

Mechanobiology of Platelets: Techniques to Study the Role of Fluid Flow and Platelet Retraction Forces at the Micro- and Nano-Scale

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Nathan J. Sniadecki

2011-12-01

Full Text Available Coagulation involves a complex set of events that are important in maintaining hemostasis. Biochemical interactions are classically known to regulate the hemostatic process, but recent evidence has revealed that mechanical interactions between platelets and their surroundings can also play a substantial role. Investigations into platelet mechanobiology have been challenging however, due to the small dimensions of platelets and their glycoprotein receptors. Platelet researchers have recently turned to microfabricated devices to control these physical, nanometer-scale interactions with a higher degree of precision. These approaches have enabled exciting, new insights into the molecular and biomechanical factors that affect platelets in clot formation. In this review, we highlight the new tools used to understand platelet mechanobiology and the roles of adhesion, shear flow, and retraction forces in clot formation.

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

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Ye-Ming Lee

2012-01-01

Full Text Available Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulus L.. Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation stimulated by collagen. Xanthohumol inhibited platelet activation accompanied by relative [Ca2+]i mobilization, thromboxane A2 formation, hydroxyl radical (OH● formation, and phospholipase C (PLCγ2, protein kinase C (PKC, mitogen-activated protein kinase (MAPK, and Akt phosphorylation. Neither SQ22536, an inhibitor of adenylate cyclase, nor ODQ, an inhibitor of guanylate cyclase, reversed the xanthohumol-mediated inhibitory effect on platelet aggregation. Furthermore, xanthohumol did not significantly increase nitrate formation in platelets. This study demonstrates for the first time that xanthohumol possesses potent antiplatelet activity which may initially inhibit the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, followed by inhibition of the thromboxane A2 formation, thereby leading to inhibition of [Ca2+]i and finally inhibition of platelet aggregation. Therefore, this novel role of xanthohumol may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.

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

Science.gov (United States)

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

2017-08-01

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

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

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

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Irina V. Gorudko

2013-07-01

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

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

International Nuclear Information System (INIS)

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

1980-01-01

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

Electrophoretic demonstration of glycoproteins, lipoproteins, and phosphoproteins in human and bovine enamel

DEFF Research Database (Denmark)

Kirkeby, S; Moe, D; Bøg-Hansen, T C

1990-01-01

Enamel proteins from fully mineralized human molars and from bovine tooth germs were separated by electrophoresis. The gels were stained for detection of glycoproteins, lipoproteins, and phosphoproteins. Glycoproteins were shown by periodic acid-Schiff staining and lectin blotting. In mature human...... enamel a number of high molecular weight proteins could be demonstrated after ethylenediaminetetra-acetic acid demineralization and subsequent Triton X-100 extraction. These proteins are suggested to be lipoproteins. Phosphoproteins could only be visualized in enamel matrix from the tooth germs....

Role of the serine-rich surface glycoprotein Srr1 of Streptococcus agalactiae in the pathogenesis of infective endocarditis.

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Ho Seong Seo

Full Text Available The binding of bacteria to fibrinogen and platelets are important events in the pathogenesis of infective endocarditis. Srr1 is a serine-rich repeat glycoprotein of Streptococcus agalactiae that binds directly to the Aα chain of human fibrinogen. To assess the impact of Srr1 on the pathogenesis of endocarditis due to S. agalactiae, we first examined the binding of this organism to immobilized human platelets. Strains expressing Srr1 had significantly higher levels of binding to human platelets in vitro, as compared with isogenic Δsrr1 mutants. In addition, platelet binding was inhibited by pretreatment with anti-fibrinogen IgG or purified Srr1 binding region. To assess the contribution of Srr1 to pathogenicity, we compared the relative virulence of S. agalactiae NCTC 10/84 strain and its Δsrr1 mutant in a rat model of endocarditis, where animals were co-infected with the WT and the mutant strains at a 1:1 ratio. At 72 h post-infection, bacterial densities (CFU/g of the WT strain within vegetations, kidneys, and spleens were significantly higher, as compared with the Δsrr1 mutant. These results indicate that Srr1 contributes to the pathogenesis of endocarditis due to S. agalactiae, at least in part through its role in fibrinogen-mediated platelet binding.

Evaluation of amotosalem treated platelets over 7 days of storage with an automated cytometry assay panel.

Science.gov (United States)

Diquattro, M; De Francisci, G; Bonaccorso, R; Tagliavia, A M; Marcatti, M; Palma, B; Agliastro, R

2013-12-01

Pathogen Inactivation allows to overcome microbial contamination and growth related to storage of platelets concentrates (PC) at room temperature. The aim of our study was to evaluate the platelet storage lesion extending the storage period of pathogen inactivated platelet concentrates over 7 days using an automated cytometry assay panel. We analyzed 43 concentrates subjected to pathogen inactivation (CPPI) at 3, 5 and 7 days evaluating: platelet count, mean platelet volume, platelets at low optical density, platelets at high density, GPIIb-IIIa glycoprotein, platelet microparticles, lactate dehydrogenase. The collection bags (Fenwal) and the IBS kit made in PL2410/PL2411 are approved for the conservation of PC up to 7 days. Data analysis was performed with anova test. All the parameters except small platelets and PMP were statistically different among day 7 vs. 3 and day 7 vs. 5. Our study showed a progressive modification of pathogen inactivated platelet concentrates observed up to 7 days. The persistence of the secretory pool and the presence of the platelet membrane fibrinogen receptor suggest the persistence of a potential hemostatic efficacy. Clinical studies are necessary to directly correlate this type of analysis to 24 h recovery or survival of transfused platelets in humans. © 2013 John Wiley & Sons Ltd.

Global Proteome Analysis Identifies Active Immunoproteasome Subunits in Human Platelets*

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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

Of von Willebrand factor and platelets.

Science.gov (United States)

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.

Aluminum induces lipid peroxidation and aggregation of human blood platelets

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T.J.C. Neiva

1997-05-01

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

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

International Nuclear Information System (INIS)

Walker, G.; Bourguignon, L.Y.

1990-01-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

Energy Technology Data Exchange (ETDEWEB)

Walker, G.; Bourguignon, L.Y. (Univ. of Miami Medical School, FL (USA))

1990-08-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

Role of the membrane skeleton in preventing the shedding of procoagulant-rich microvesicles from the platelet plasma membrane

OpenAIRE

1990-01-01

The platelet plasma membrane is lined by a membrane skeleton that appears to contain short actin filaments cross-linked by actin-binding protein. Actin-binding protein is in turn associated with specific plasma membrane glycoproteins. The aim of this study was to determine whether the membrane skeleton regulates properties of the plasma membrane. Platelets were incubated with agents that disrupted the association of the membrane skeleton with membrane glycoproteins. The consequences of this c...

Cross-Linking GPVI-Fc by Anti-Fc Antibodies Potentiates Its Inhibition of Atherosclerotic Plaque- and Collagen-Induced Platelet Activation

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Janina Jamasbi, RPh

2016-04-01

Full Text Available To enhance the antithrombotic properties of recombinant glycoprotein VI fragment crystallizable (GPVI-Fc, the authors incubated GPVI-Fc with anti-human Fc antibodies to cross-link the Fc tails of GPVI-Fc. Cross-linking potentiated the inhibition of human plaque- and collagen-induced platelet aggregation by GPVI-Fc under static and flow conditions without increasing bleeding time in vitro. Cross-linking with anti-human-Fc Fab2 was even superior to anti-human-Fc immunoglobulin G (IgG. Advanced optical imaging revealed a continuous sheath-like coverage of collagen fibers by cross-linked GPVI-Fc complexes. Cross-linking of GPVI into oligomeric complexes provides a new, highly effective, and probably safe antithrombotic treatment as it suppresses platelet GPVI-plaque interaction selectively at the site of acute atherothrombosis.

Global proteome analysis identifies active immunoproteasome subunits in human platelets.

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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.

Niacin and biosynthesis of PGD₂by platelet COX-1 in mice and humans.

Science.gov (United States)

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

2012-04-01

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

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

Allele frequencies of human platelet antigens in Banjar, Bugis, Champa, Jawa and Kelantan Malays in Peninsular Malaysia.

Science.gov (United States)

Wan Syafawati, W U; Norhalifah, H K; Zefarina, Z; Zafarina, Z; Panneerchelvam, S; Norazmi, M N; Chambers, G K; Edinur, H A

2015-10-01

The major aims of this study are to characterise and compile allelic data of human platelet antigen (HPA)-1 to -6 and -15 systems in five Malay sub-ethnic groups in Peninsular Malaysia. HPAs are polymorphic glycoproteins expressed on the surface of platelet membranes and are genetically differentiated across ethnogeographically unrelated populations. Blood samples were obtained with informed consent from 192 volunteers: Banjar (n = 30), Bugis (n = 37), Champa (n = 51), Jawa (n = 39) and Kelantan (n = 35). Genotyping was done using polymerase chain reaction-sequence specific primer method. In general, frequencies of HPAs in the Malay sub-ethnic groups are more similar to those in Asian populations compared with other more distinct populations such as Indians, Australian Aborigines and Europeans. This study provides the first HPA datasets for the selected Malay sub-ethnic groups. Subsequent analyses including previously reported HPA data of Malays, Chinese and Indians revealed details of the genetic relationships and ancestry of various sub-populations in Peninsular Malaysia. Furthermore, the comprehensive HPA allele frequency information from Peninsular Malaysia provided in this report has potential applications for future study of diseases, estimating risks associated with HPA alloimmunization and for developing an efficient HPA-typed donor recruitment strategy. © 2015 British Blood Transfusion Society.

The peanut lectin-binding glycoproteins of human epidermal keratinocytes

International Nuclear Information System (INIS)

Morrison, A.I.; Keeble, S.; Watt, F.M.

1988-01-01

The peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes. In order to investigate the significance of this change in cell-surface carbohydrate authors have identified the PNA-binding glycoproteins of cultured human keratinocytes and antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of [ 14 C]galactose- or [ 14 C]mannose- and [ 14 C]glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification

Imaging thrombus with radiolabelled monoclonal antibody to platelets

Energy Technology Data Exchange (ETDEWEB)

Peters, A.M.; Lavender, J.P.; Needham, S.G.; Loutfi, I.; Snook, D.; Epenetos, A.A.; Lumley, P.; Keery, R.J.; Hogg, N.

1986-12-13

A study was conducted evaluating a method of imaging thrombus with platelets radiolabelled with a /sup 111/In labelled monoclonal antibody, P256, directed to the platelet surface glycoprotein complex IIb/IIIa. when the number of receptors occupied by P256 was less than 3% of the total available on the platelet surface, platelet function was undisturbed. P256 was radiolabelled with /sup 111/In using diethylenetriaminepenta-acetic acid, which achieved a specific activity of 185 MBq (5 mCi)/mg. No impairment of immunoreactivity was detected at this specific activity. Platelets were labelled with radiolabelled monoclonal antibody in vitro in two patients at a receptor occupancy of 6% and in vivo in six patients at a receptor occupancy of 1%. In vivo recovery and biodistribution kinetics suggested that after in vitro labelling platelets were minimally activated. The /sup 111/In kinetics recorded after intravenous P256 suggested rapid and efficient radiolabelling of platelets and gave no indication of platelet activation. Of the six patients who received intravenous P256, three had documented thrombus, two of whom gave positive results on P256 platelet scintigraphy. The third had chronic deep venous thrombosis and was scintigraphically negative.

Release of a human platelet specific protein measured by a radioimmunoassay

Energy Technology Data Exchange (ETDEWEB)

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

1975-06-01

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

The hemostatic agent ethamsylate promotes platelet/leukocyte aggregate formation in a model of vascular injury.

Science.gov (United States)

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

2004-08-01

The hemostatic agent ethamsylate enhances membrane expression of P-selectin in human platelets, but whether this promotes platelet-leukocyte aggregate formation is unknown. Here we investigated this point by flow cytometry determination of human platelet-leukocyte aggregates under basal conditions and after whole-blood perfusion through a damaged rabbit aorta segment. Actions of ethamsylate on adhesive molecules of platelets and leukocytes were investigated in parallel. Under basal conditions, ethamsylate was unable to modify whole-blood platelet-leukocyte aggregation, but following whole-blood perfusion through a damaged vessel, ethamsylate produced a modest, but significant increase in platelet-leukocyte aggregates (48+/-21 and 45+/-26% above control levels at ethamsylate 20 and 40 microm respectively). In isolated leukocyte plasma membranes, 14C-ethamsylate specifically bound up to an amount of 660 pmol/mg protein. Moreover, at concentrations > or =1 microm, ethamsylate induced an important (100-200%) and significant increase in the P-selectin glycoprotein ligand 1 (PSGL-1) fluorescence signal in isolated leukocytes and was unable to significantly modify the percentage of CD11b-positive cells. However, no significant changes in aggregate formation were found when ethamsylate was incubated with isolated leukocytes and blood was reconstituted and perfused. In isolated platelet cell membranes, anti-P-selectin antibody and the anti-integrin RGD-containing pentapeptide (GRDGS) were unable to displace 14C-ethamsylate binding. In conclusion, ethamsylate specifically binds to plasma membranes of leukocytes, enhances membrane PSGL-1 expression and promotes leukocyte-platelet aggregation in whole-blood perfused through a damaged vascular segment. These results together with the previously observed enhancement of platelet P-selectin membrane expression [Thromb. Res. (2002)107:329-335] confirms and extends the view that ethamsylate acts on the first step of hemostasis, by

Antiaggregant effects of Arbutus unedo extracts in human platelets.

Science.gov (United States)

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

2007-09-05

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

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

Science.gov (United States)

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

2013-11-01

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

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

International Nuclear Information System (INIS)

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

1989-01-01

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

Exosomes: novel effectors of human platelet lysate activity

Directory of Open Access Journals (Sweden)

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.

Comparative Analysis of Whey N-Glycoproteins in Human Colostrum and Mature Milk Using Quantitative Glycoproteomics.

Science.gov (United States)

Cao, Xueyan; Song, Dahe; Yang, Mei; Yang, Ning; Ye, Qing; Tao, Dongbing; Liu, Biao; Wu, Rina; Yue, Xiqing

2017-11-29

Glycosylation is a ubiquitous post-translational protein modification that plays a substantial role in various processes. However, whey glycoproteins in human milk have not been completely profiled. Herein, we used quantitative glycoproteomics to quantify whey N-glycosylation sites and their alteration in human milk during lactation; 110 N-glycosylation sites on 63 proteins and 91 N-glycosylation sites on 53 proteins were quantified in colostrum and mature milk whey, respectively. Among these, 68 glycosylation sites on 38 proteins were differentially expressed in human colostrum and mature milk whey. These differentially expressed N-glycoproteins were highly enriched in "localization", "extracellular region part", and "modified amino acid binding" according to gene ontology annotation and mainly involved in complement and coagulation cascades pathway. These results shed light on the glycosylation sites, composition and biological functions of whey N-glycoproteins in human colostrum and mature milk, and provide substantial insight into the role of protein glycosylation during infant development.

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.

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

Science.gov (United States)

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

2012-01-01

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

Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

International Nuclear Information System (INIS)

Peerschke, E.I.

1991-01-01

Previous studies indicated a correlation between the formation of EDTA-resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA-resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA-resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding

Pharmacological modulation of human platelet leukotriene C4-synthase.

Science.gov (United States)

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

1997-03-21

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

Design and synthesis of novel platelet fibrinogen receptor antagonists with 2H-1,4-benzoxazine-3(4H)-one scaffold. A systematic study.

Science.gov (United States)

Anderluh, Marko; Cesar, Jozko; Stefanic, Petra; Kikelj, Danijel; Janes, Damjan; Murn, Jernej; Nadrah, Kristina; Tominc, Mojca; Addicks, Elisabeth; Giannis, Athanassios; Stegnar, Mojca; Dolenc, Marija Sollner

2005-01-01

New platelet glycoprotein IIb/IIIa (GP IIb/IIIa, integrin alpha(IIb)beta3) antagonists were prepared on a 2H-1,4-benzoxazine-3(4H)-one scaffold. Their anti-aggregatory activities in human platelet rich plasma and their affinity towards alpha(IIb)beta3 and alpha(V)beta3 integrins were assessed. Various substitution positions and side chain variations were studied. In contrast to the generally accepted model, compounds containing ethyl esters as aspartate mimetics were in general more active than the corresponding free acids. We suggest an explanation for the observed behaviour of these new compounds.

Platelet apoptosis by cld-induced glycoportein Ibα clustering

DEFF Research Database (Denmark)

van der Wal, Dianne E; Du, V X; Lo, KS

2010-01-01

Summary Background:  Cold-storage of platelets followed by rewarming induces changes in Glycoprotein (GP) Ibα-distribution indicative of receptor clustering and initiates thromboxane A2-formation. GPIbα is associated with 14-3-3 proteins, which contribute to GPIbα-signaling and in nucleated cells...

Podoplanin expression in primary brain tumors induces platelet aggregation and increases risk of venous thromboembolism.

Science.gov (United States)

Riedl, Julia; Preusser, Matthias; Nazari, Pegah Mir Seyed; Posch, Florian; Panzer, Simon; Marosi, Christine; Birner, Peter; Thaler, Johannes; Brostjan, Christine; Lötsch, Daniela; Berger, Walter; Hainfellner, Johannes A; Pabinger, Ingrid; Ay, Cihan

2017-03-30

Venous thromboembolism (VTE) is common in patients with brain tumors, and underlying mechanisms are unclear. We hypothesized that podoplanin, a sialomucin-like glycoprotein, increases the risk of VTE in primary brain tumors via its ability to induce platelet aggregation. Immunohistochemical staining against podoplanin and intratumoral platelet aggregates was performed in brain tumor specimens of 213 patients (mostly high-grade gliomas [89%]) included in the Vienna Cancer and Thrombosis Study, a prospective observational cohort study of patients with newly diagnosed cancer or progressive disease aimed at identifying patients at risk of VTE. Platelet aggregation in response to primary human glioblastoma cells was investigated in vitro. During 2-year follow-up, 29 (13.6%) patients developed VTE. One-hundred fifty-one tumor specimens stained positive for podoplanin (33 high expression, 47 medium expression, 71 low expression). Patients with podoplanin-positive tumors had lower peripheral blood platelet counts ( P < .001) and higher D-dimer levels ( P < .001). Podoplanin staining intensity was associated with increasing levels of intravascular platelet aggregates in tumor specimens ( P < .001). High podoplanin expression was associated with an increased risk of VTE (hazard ratio for high vs no podoplanin expression: 5.71; 95% confidence interval, 1.52-21.26; P = 010), independent of age, sex, and tumor type. Podoplanin-positive primary glioblastoma cells induced aggregation of human platelets in vitro, which could be abrogated by an antipodoplanin antibody. In conclusion, high podoplanin expression in primary brain tumors induces platelet aggregation, correlates with hypercoagulability, and is associated with increased risk of VTE. Our data indicate novel insights into the pathogenesis of VTE in primary brain tumors. © 2017 by The American Society of Hematology.

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

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Anahita Jenab

2015-11-01

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

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

Science.gov (United States)

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

2002-09-15

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

Defining glycoprotein cancer biomarkers by MS in conjunction with glycoprotein enrichment.

Science.gov (United States)

Song, Ehwang; Mechref, Yehia

2015-01-01

Protein glycosylation is an important and common post-translational modification. More than 50% of human proteins are believed to be glycosylated to modulate the functionality of proteins. Aberrant glycosylation has been correlated to several diseases, such as inflammatory skin diseases, diabetes mellitus, cardiovascular disorders, rheumatoid arthritis, Alzheimer's and prion diseases, and cancer. Many approved cancer biomarkers are glycoproteins which are not highly abundant proteins. Therefore, effective qualitative and quantitative assessment of glycoproteins entails enrichment methods. This chapter summarizes glycoprotein enrichment methods, including lectin affinity, immunoaffinity, hydrazide chemistry, hydrophilic interaction liquid chromatography, and click chemistry. The use of these enrichment approaches in assessing the qualitative and quantitative changes of glycoproteins in different types of cancers are presented and discussed. This chapter highlights the importance of glycoprotein enrichment techniques for the identification and characterization of new reliable cancer biomarkers.

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...

Platelet HPA-1 a/HPA-1 b polymorphism and the risk of periprocedural myocardial infarction in patients undergoing elective PCI

NARCIS (Netherlands)

Verdoia, M.; Secco, G.G.; Barbieri, L.; Cassetti, E.; Schaffer, A.; Sinigaglia, F.; Marino, P.; Suryapranata, H.; Luca, G. De

2014-01-01

Periprocedural myocardial infarction (PMI) represents a relatively common complication of percutaneous coronary intervention (PCI) and large interests have been focused on platelets in order to prevent such a complication. The single nucleotide polymorphism Leu33Pro of platelet glycoprotein IIIa has

Development of glycoprotein capture-based label-free method for the high-throughput screening of differential glycoproteins in hepatocellular carcinoma.

Science.gov (United States)

Chen, Rui; Tan, Yexiong; Wang, Min; Wang, Fangjun; Yao, Zhenzhen; Dong, Liwei; Ye, Mingliang; Wang, Hongyang; Zou, Hanfa

2011-07-01

A robust, reproducible, and high throughput method was developed for the relative quantitative analysis of glycoprotein abundances in human serum. Instead of quantifying glycoproteins by glycopeptides in conventional quantitative glycoproteomics, glycoproteins were quantified by nonglycosylated peptides derived from the glycoprotein digest, which consists of the capture of glycoproteins in serum samples and the release of nonglycopeptides by trypsin digestion of captured glycoproteins followed by two-dimensional liquid chromatography-tandem MS analysis of released peptides. Protein quantification was achieved by comparing the spectrum counts of identified nonglycosylated peptides of glycoproteins between different samples. This method was demonstrated to have almost the same specificity and sensitivity in glycoproteins quantification as capture at glycopeptides level. The differential abundance of proteins present at as low as nanogram per milliliter levels was quantified with high confidence. The established method was applied to the analysis of human serum samples from healthy people and patients with hepatocellular carcinoma (HCC) to screen differential glycoproteins in HCC. Thirty eight glycoproteins were found with substantial concentration changes between normal and HCC serum samples, including α-fetoprotein, the only clinically used marker for HCC diagnosis. The abundance changes of three glycoproteins, i.e. galectin-3 binding protein, insulin-like growth factor binding protein 3, and thrombospondin 1, which were associated with the development of HCC, were further confirmed by enzyme-linked immunosorbent assay. In conclusion, the developed method was an effective approach to quantitatively analyze glycoproteins in human serum and could be further applied in the biomarker discovery for HCC and other cancers.

The interaction of thrombin with platelet protease nexin

International Nuclear Information System (INIS)

Knupp, C.L.

1989-01-01

Thrombin interacts with a platelet protein which is immunologically related to fibroblast protease nexin and has been termed platelet protease nexin I (PNI). Conflicting hypotheses about the relationship of the thrombin-PNI complex formation to platelet activation have been proposed. The studies presented here demonstrate that the platelet-associated and supernatant complexes with added 125I-thrombin are formed only under conditions which produce platelet activation in normal and chymotrypsin-modified platelets. The platelet-associated complex is formed prior to the appearance of complexes in supernatants. Appearance of the supernatant complex coincides with the appearance of thrombospondin in the reaction supernatants. Excess native thrombin, dansylarginine N-(3-ethyl-1,5-pentanediyl) amide or hirudin can prevent radiolabeled platelet-associated complex formation if added before 125I-thrombin. DAPA or hirudin can prevent or dissociate complex formation if added up to one minute after thrombin but not at later time points. The surface associated complex is accessible to trypsin although a portion remains with the cytoskeletal proteins when thrombin-activated platelets are solubilized with Triton X 100. The surface-associated complex formation parallels many aspects of the specific measurable thrombin binding, yet it does not appear to involve other identified surface glycoprotein thrombin receptors or substrates. Although the time course of appearance of the complexes in supernatants is consistent with other data which suggest that PNI may be released from platelet granules during platelet activation, other explanations for the appearance of PNI on the platelet surface and in supernatants during platelet activation are possible

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

Science.gov (United States)

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.

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

Science.gov (United States)

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

1975-02-27

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

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

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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.

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

NARCIS (Netherlands)

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

2013-01-01

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

High-performance liquid chromatography of human glycoprotein hormones.

Science.gov (United States)

Chlenov, M A; Kandyba, E I; Nagornaya, L V; Orlova, I L; Volgin, Y V

1993-02-12

The chromatographic behavior of the glycoprotein hormones from human pituitary glands and of placental origin [thyroid-stimulating hormone, luteinizing hormone and chorionic gonadotropin (CG)] was studied. It was shown that hydrophobic interaction chromatography on a microparticulate packing and anion-exchange HPLC can be applied for the purification of these hormones. Reversed-phase HPLC on wide-pore C4-bonded silica at neutral pH can be applied for the determination of the above hormones and for the isolation of pure CG and its subunits.

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

Directory of Open Access Journals (Sweden)

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.

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

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Sandra Mjoll Jonsdottir-Buch

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

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

Science.gov (United States)

Peluso, Ilaria; Palmery, Maura; Serafini, Mauro

2015-07-01

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

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

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Carolina Bonet Bub

2016-02-01

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

EMMPRIN (CD147/basigin) mediates platelet-monocyte interactions in vivo and augments monocyte recruitment to the vascular wall.

Science.gov (United States)

Schulz, C; von Brühl, M-L; Barocke, V; Cullen, P; Mayer, K; Okrojek, R; Steinhart, A; Ahmad, Z; Kremmer, E; Nieswandt, B; Frampton, J; Massberg, S; Schmidt, R

2011-05-01

Platelets play a central role in hemostasis, in inflammatory diseases such as atherosclerosis, and during thrombus formation following vascular injury. Thereby, platelets interact intensively with monocytes and enhance their recruitment to the vascular wall. To investigate the role of the extracellular matrix metalloproteinase inducer (EMMPRIN) in platelet-monocyte interactions. Isolated human monocytes were perfused in vitro over firmly adherent platelets to allow investigation of the role of EMMPRIN in platelet-monocyte interactions under flow conditions. Monocytes readily bound to surface-adherent platelets. Both antibody blockade and gene silencing of monocyte EMMPRIN substantially attenuated firm adhesion of monocytes to platelets at arterial and venous shear rates. In vivo, platelet interactions with the murine monocyte cell line ANA-1 were significantly decreased when ANA-1 cells were pretreated with EMMPRIN-silencing small interfering RNA prior to injection into wild-type mice. Using intravital microscopy, we showed that recruitment of EMMPRIN-silenced ANA-1 to the injured carotid artery was significantly reduced as compared with control cells. Further silencing of EMMPRIN resulted in significantly fewer ANA-1-platelet aggregates in the mouse circulation as determined by flow cytometry. Finally, we identified glycoprotein (GP)VI as a critical corresponding receptor on platelets that mediates interaction with monocyte EMMPRIN. Thus, blocking of GPVI inhibited the effect of EMMPRIN on firm monocyte adhesion to platelets under arterial flow conditions in vitro, and abrogated EMMPRIN-mediated platelet-monocyte aggregate formation in vivo. EMMPRIN supports platelet-monocyte interactions and promotes monocyte recruitment to the arterial wall. Therefore, EMMPRIN might represent a novel target to reduce vascular inflammation and atherosclerotic lesion development. © 2011 International Society on Thrombosis and Haemostasis.

Imaging thrombus with radiolabelled monoclonal antibody to platelets

International Nuclear Information System (INIS)

Loutfi, I.; Peters, A.M.; Lavender, J.P.; Epenetos, A.A.

1988-01-01

Indium-111-hydroxyquinoline labelled platelets, though useful in the detection of thrombus, have not gained widespread use owing to the time and technical skill required for their preparation. A study was therefore conducted evaluating a new method of imaging thrombus with platelets radiolabelled with a 111 In labelled monoclonal antibody, P 256 , directed to the platelet surface glycoprotein complex IIb/IIIa. When the number of receptors occupied by P 256 was less than 3% of the total available on the platelet surface platelet function, as assessed by platelet aggregometry, was undisturbed. P 256 was radiolabelled with 111 In using diethylenetriaminepenta-acetic acid, which achieved a specific activity of 185 MBq (5 mCi)/mg. No impairment of immunoreactivity was detected at this specific activity. Platelets were labelled with radiolabelled monoclonal antibody in vitro in two patients at a receptor occupancy of 6% and in vivo - that is, by direct intravenous injection of P 256 - in six patients at a receptor occupancy of 1%. In vivo recovery and biodistribution kinetics suggested that after in vitro labelling platelets were minimally activated. The 111 In kinetics recorded after intravenous P 256 suggested rapid and efficient radiolabelling of platelets and gave no indication of platelet activation. Of the six patients who received intravenous P 256 , three had documented thrombus, tow of whom gave positive results on P 256 platelet scintigraphy. The third subject had chromic deep venous thrombosis and was scintigraphically negative. Imaging thrombus using a radiolabelled monoclonal antibody directed to platelets appears to offer great potential as a simple, non-invasive approach to the diagnosis of thrombosis. 3 refs. (Author)

Human CRISP-3 binds serum alpha(1)B-glycoprotein across species

DEFF Research Database (Denmark)

Udby, Lene; Johnsen, Anders H; Borregaard, Niels

2010-01-01

CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular...

3,3′,4,4′,5-Pentachlorobiphenyl Inhibits Drug Efflux Through P-Glycoprotein in KB-3 Cells Expressing Mutant Human P-Glycoprotein

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Hiroshi Fujise

2004-01-01

Full Text Available The effects on the drug efflux of 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126, the most toxic of all coplanar polychlorinated biphenyls (Co-PCBs, were examined in KB-3 cells expressing human wild-type and mutant P-glycoprotein in which the 61st amino acid was substituted for serine or phenylalanine (KB3-Phe61. In the cells expressing P-glycoproteins, accumulations of vinblastine and colchicine decreased form 85% to 92% and from 62% to 91%, respectively, and the drug tolerances for these chemicals were increased. In KB3-Phe61, the decreases in drug accumulation were inhibited by adding PCB-126 in a way similar to that with cyclosporine A: by adding 1 μM PCB-126, the accumulations of vinblastine and colchicine increased up to 3.3- and 2.3-fold, respectively. It is suggested that PCB-126 decreased the drug efflux by inhibiting the P-glycoprotein in KB3-Phe61. Since there were various P-glycoproteins and many congeners of Co-PCBs, this inhibition has to be considered a new cause of the toxic effects of Co-PCBs.

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

Science.gov (United States)

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

2018-05-28

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

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

International Nuclear Information System (INIS)

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

1991-01-01

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

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...

Covalent modification of platelet proteins by palmitate

International Nuclear Information System (INIS)

Muszbek, L.; Laposata, M.

1989-01-01

Covalent attachment of fatty acid to proteins plays an important role in association of certain proteins with hydrophobic membrane structures. In platelets, the structure of many membrane glycoproteins (GPs) has been examined in detail, but the question of fatty acid acylation of platelet proteins has not been addressed. In this study, we wished to determine (a) whether platelet proteins could be fatty acid acylated; and, if so, (b) whether these modified proteins were present in isolated platelet membranes and cytoskeletal fractions; and (c) if the pattern of fatty acid acylated proteins changed on stimulation of the platelets with the agonist thrombin. We observed that in platelets allowed to incorporate 3H-palmitate, a small percentage (1.37%) of radioactivity incorporated into the cells became covalently bound to protein. Selective cleavage of thioester, thioester plus O-ester, and amide-linked 3H-fatty acids from proteins, and their subsequent analysis by high-performance liquid chromatography (HPLC) indicated that the greatest part of 3H-fatty acid covalently bound to protein was thioester-linked 3H-palmitate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography, at least ten major radiolabeled proteins were detected. Activation of platelets by thrombin greatly increased the quantity of 3H-palmitoylated proteins associated with the cytoskeleton. Nearly all radiolabeled proteins were recovered in the membrane fraction, indicating that these proteins are either integral or peripheral membrane proteins or proteins tightly associated to membrane constituents. Components of the GPIIb-IIIa complex were not palmitoylated. Thus, platelet proteins are significantly modified posttranslationally by 3H-palmitate, and incorporation of palmitoylated proteins into the cytoskeleton is a prominent component of the platelet response to thrombin stimulation

Improved platelet survival after cold storage by prevention of glycoprotein Ibα clustering in lipid rafts

NARCIS (Netherlands)

Gitz, E.; Koekman, C.A.; van den Heuvel, D.J.|info:eu-repo/dai/nl/355331861; Deckmyn, H.; Akkerman, J.W.N.; Gerritsen, H.C.|info:eu-repo/dai/nl/071548777; Urbanus, R.T

2012-01-01

ABSTRACT Background Room temperature storage of platelets for transfusion increases the risk of microbial infection and decreases platelet functionality, leading to out-date discard rates of up to 20%. Cold storage may be a better alternative, but this treatment leads to rapid platelet clearance

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

Science.gov (United States)

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

2018-04-01

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

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

Platelet-derived growth factor inhibits platelet activation in heparinized whole blood.

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Selheim, F; Holmsen, H; Vassbotn, F S

1999-08-15

We previously have demonstrated that human platelets have functionally active platelet-derived growth factor alpha-receptors. Studies with gel-filtered platelets showed that an autocrine inhibition pathway is transduced through this tyrosine kinase receptor during platelet activation. The physiological significance of this inhibitory effect of platelet-derived growth factor on gel-filtered platelets activation is, however, not known. In the present study, we investigated whether platelet-derived growth factor inhibits platelet activation under more physiological conditions in heparinized whole blood, which represents a more physiological condition than gel-filtered platelets. Using flow cytometric assays, we demonstrate here that platelet-derived growth factor inhibits thrombin-, thrombin receptor agonist peptide SFLLRN-, and collagen-induced platelet aggregation and shedding of platelet-derived microparticles from the platelet plasma membrane during platelet aggregation in stirred heparinized whole blood. The inhibitory effect of platelet-derived growth factor was dose dependent. However, under nonaggregating conditions (no stirring), we could not demonstrate any significant effect of platelet-derived growth factor on thrombin- and thrombin receptor agonist peptide-induced platelet surface expression of P-selectin. Our results demonstrate that platelet-derived growth factor appears to be a true antithrombotic agent only under aggregating conditions in heparinized whole blood.

MAC-1 Glycoprotein Family mediates adherence of neutrophils to endothelial cells stimulated by leukotriene B/sub 4/ and platelet activating factor

Energy Technology Data Exchange (ETDEWEB)

Tonnesen, M.G.; Anderson, D.C.; Springer, T.A.; Knedler, A.; Avdi, N.; Henson, P.M.

1986-03-01

The process of neutrophil (N) adhesion to and migration through endothelium (EC), an early event in the induction of the acute inflammatory response, has been attributed to the generation of extravascular chemoattractant peptides and lipids. Although both leukotriene B/sub 4/ (LTB/sub 4/) and platelet activating factor (PAF) enhance N adherence to EC, the mechanisms involved in this interaction are still not completely understood. Since the MAC-1 Glycoprotein (GP) Family has recently been shown to be required for a variety of adherence-dependent functions of stimulated N, the authors questioned whether these adherence-associated GP might be involved in N adherence to EC stimulated by LTB/sub 4/ or PAF. Using a microtiter adherence assay with /sup 111/In labeled N, they assessed the ability of N from patients with MAC-1, LFA-1 Deficiency to adhere to monolayers of human omental microvascular or umbilical vein EC as well as to serum-coated plastic. Patient N exhibited markedly diminished adherence in response to LTB/sub 4/ or PAF compared to normal controls. LTB/sub 4/ and PAF enhanced expression of the MAC-1 GP Family on the surface of normal N as determined by flow cytofluorimetry using a monoclonal antibody (TS1/18) to the GP common beta subunit. In addition TS1/18 (20 ..mu..g/ml) completely inhibited N adherence stimulated by either LTB/sub 4/ (10/sup -8/M) or PAF(10/sup -11/M). Thus, the MAC-1 GP Family appears to be important in chemotactic factor regulation of N adherence to EC.

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

Science.gov (United States)

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

2016-08-01

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

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

International Nuclear Information System (INIS)

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

1982-01-01

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

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

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

2012-01-01

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

Quantitative Glycoproteomic Analysis Identifies Platelet-Induced Increase of Monocyte Adhesion via the Up-Regulation of Very Late Antigen 5.

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Huang, Jiqing; Kast, Juergen

2015-08-07

Physiological stimuli, such as thrombin, or pathological stimuli, such as lysophosphatidic acid (LPA), activate platelets circulating in blood. Once activated, platelets bind to monocytes via P-selectin-PSGL-1 interactions but also release the stored contents of their granules. These platelet releasates, in addition to direct platelet binding, activate monocytes and facilitate their recruitment to atherosclerotic sites. Consequently, understanding the changes platelet releasates induce in monocyte membrane proteins is critical. We studied the glyco-proteome changes of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasates. We employed lectin affinity chromatography combined with filter aided sample preparation to achieve high glyco- and membrane protein and protein sequence coverage. Using stable isotope labeling by amino acids in cell culture, we quantified 1715 proteins, including 852 membrane and 500 glycoproteins, identifying the up-regulation of multiple proteins involved in monocyte extracellular matrix binding and transendothelial migration. Flow cytometry indicated expression changes of integrin α5, integrin β1, PECAM-1, and PSGL-1. The observed increase in monocyte adhesion to fibronectin was determined to be mediated by the up-regulation of very late antigen 5 via a P-selectin-PSGL-1 independent mechanism. This novel aspect could be validated on CD14+ human primary monocytes, highlighting the benefits of the improved enrichment method regarding high membrane protein coverage and reliable quantification.

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

International Nuclear Information System (INIS)

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

1989-01-01

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

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

Directory of Open Access Journals (Sweden)

Lea M Beaulieu

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

Oxidative alterations during human platelet storage

OpenAIRE

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

2014-01-01

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

Study on platelet kinetics

International Nuclear Information System (INIS)

Yui, Tokuo

1981-01-01

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

Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance.

Science.gov (United States)

Muñoz-Martínez, Francisco; Lu, Peihua; Cortés-Selva, Fernando; Pérez-Victoria, José María; Jiménez, Ignacio A; Ravelo, Angel G; Sharom, Frances J; Gamarro, Francisco; Castanys, Santiago

2004-10-01

Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer. Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains. However, their molecular mechanism of reversion has not been characterized. In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action. Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine. Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters. Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of [(3)H]colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target. Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations. Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential

A modified assay method for determining serotonin uptake in human platelets

International Nuclear Information System (INIS)

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

1981-01-01

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

Role of sialic acid for platelet life span: exposure of beta-galactose results in the rapid clearance of platelets from the circulation by asialoglycoprotein receptor-expressing liver macrophages and hepatocytes

DEFF Research Database (Denmark)

Sørensen, Anne Louise; Rumjantseva, Viktoria; Nayeb-Hashemi, Sara

2009-01-01

Although surface sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet circulation lifetime is not fully clarified. We show that thrombocytopenia in mice deficient in the St3gal4 sialyltransferase gene (St3Gal-IV(-/-) mice...

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

International Nuclear Information System (INIS)

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

1991-01-01

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

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

Directory of Open Access Journals (Sweden)

Monique Ramos de Oliveira Trugilho

2017-05-01

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

Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway

Science.gov (United States)

Gardner, Thomas J.

2016-01-01

SUMMARY The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. PMID:27307580

Studies on the subunits of human glycoprotein hormones in relation to reproduction

International Nuclear Information System (INIS)

Hagen, C.

1977-01-01

In this review summarising present knowledge of the biological and immunological activity of the subunits of human glycoprotein hormones, the specificity of the α-subunit and β-subunit radioimmunoassays are discussed. The crossreaction studies performed with the α-subunit radioimmunoassays are aummarised in one table while those with the β-subunit radioimmunoassays are presented in a second table. (JIW)

Platelets subvert T cell immunity against cancer via GARP-TGFβ axis.

Science.gov (United States)

Rachidi, Saleh; Metelli, Alessandra; Riesenberg, Brian; Wu, Bill X; Nelson, Michelle H; Wallace, Caroline; Paulos, Chrystal M; Rubinstein, Mark P; Garrett-Mayer, Elizabeth; Hennig, Mirko; Bearden, Daniel W; Yang, Yi; Liu, Bei; Li, Zihai

2017-05-05

Cancer-associated thrombocytosis has long been linked to poor clinical outcome, but the underlying mechanism is enigmatic. We hypothesized that platelets promote malignancy and resistance to therapy by dampening host immunity. We show that genetic targeting of platelets enhances adoptive T cell therapy of cancer. An unbiased biochemical and structural biology approach established transforming growth factor β (TGFβ) and lactate as major platelet-derived soluble factors to obliterate CD4 + and CD8 + T cell functions. Moreover, we found that platelets are the dominant source of functional TGFβ systemically as well as in the tumor microenvironment through constitutive expression of the TGFβ-docking receptor glycoprotein A repetitions predominant (GARP) rather than secretion of TGFβ per se. Platelet-specific deletion of the GARP-encoding gene Lrrc32 blunted TGFβ activity at the tumor site and potentiated protective immunity against both melanoma and colon cancer. Last, this study shows that T cell therapy of cancer can be substantially improved by concurrent treatment with readily available antiplatelet agents. We conclude that platelets constrain T cell immunity through a GARP-TGFβ axis and suggest a combination of immunotherapy and platelet inhibitors as a therapeutic strategy against cancer. Copyright © 2017, American Association for the Advancement of Science.

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

Science.gov (United States)

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

2017-10-01

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

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

International Nuclear Information System (INIS)

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

1990-01-01

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

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

Science.gov (United States)

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

2018-06-01

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

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

Science.gov (United States)

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.

Platelets are required for enhanced activation of the endothelium and fibrinogen in a mouse thrombosis model of APS.

Science.gov (United States)

Proulle, Valerie; Furie, Richard A; Merrill-Skoloff, Glenn; Furie, Barbara C; Furie, Bruce

2014-07-24

Antiphospholipid syndrome (APS) is defined by thrombosis, fetal loss, and the presence of antiphospholipid antibodies, including anti-β2-glycoprotein-1 autoantibodies (anti-β2GP1) that have a direct role in the pathogenesis of thrombosis in vivo. The cellular targets of the anti-β2GP1 autoantibody/β2GP1 complex in vivo were studied using a laser-induced thrombosis model of APS in a live mouse and human anti-β2GP1 autoantibodies affinity-purified from APS patients. Cell binding of fluorescently labeled β2GP1 and anti-β2GP1 autoantibodies revealed their colocalization on the platelet thrombus but not the endothelium. Anti-β2GP1 autoantibodies enhanced platelet activation, monitored by calcium mobilization, and endothelial activation, monitored by intercellular adhesion molecule-1 expression. When eptifibatide was infused to block platelet thrombus formation, enhanced fibrin generation and endothelial cell activation were eliminated. Thus, the anti-β2GP1 autoantibody/β2GP1 complex binds to the thrombus, enhancing platelet activation, and platelet secretion leads to enhanced endothelium activation and fibrin generation. These results lead to a paradigm shift away from the concept that binding of the anti-β2GP1 autoantibody/β2GP1 complex activates both endothelial cells and platelets toward one in which activation of platelets in response to anti-β2GP1 autoantibody/β2GP1 complex binding leads to subsequent enhanced endothelium activation and fibrin generation. © 2014 by The American Society of Hematology.

Human platelet vasopressin receptor identification by direct ultraviolet photoaffinity labeling

International Nuclear Information System (INIS)

Thibonnier, M.

1987-01-01

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

Imipramine binding in subpopulations of normal human blood platelets

International Nuclear Information System (INIS)

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

1984-01-01

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

Red Wine Inhibits Aggregation and Increases ATP-diphosphohydrolase (CD39) Activity of Rat Platelets in Vitro.

Science.gov (United States)

Caiazzo, Elisabetta; Tedesco, Idolo; Spagnuolo, Carmela; Russo, Gian Luigi; Ialenti, Armando; Cicala, Carla

2016-06-01

Moderate consumption of red wine has been shown to exert a peculiar cardioprotective effect compared with other alcoholic beverages; inhibition of platelet aggregation seems to be one of the mechanisms underlying this beneficial effect. CD39/ATP-diphosphohydrolase is an integral membrane glycoprotein metabolizing ATP and ADP to AMP; in concert with CD73/ecto-5'-nucleotidase, it contributes to extracellular adenosine accumulation. CD39 is considered a key modulator of thrombus formation; it inhibits platelet aggregation by promoting ADP hydrolysis. There is evidence that red wine consumption increases CD39 activity in platelets from streptozotocin-induced diabetic rats. Here we show that two kinds of Aglianico red wines inhibit aggregation and increase ATP--and ADPase activity in rat platelets.

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

Science.gov (United States)

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

2017-10-01

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

Max(a), a new low-frequency platelet-specific antigen localized on glycoprotein IIb, is associated with neonatal alloimmune thrombocytopenia

NARCIS (Netherlands)

Noris, P.; Simsek, S.; de Bruijne-Admiraal, L. G.; Porcelijn, L.; Huiskes, E.; van der Vlist, G. J.; van Leeuwen, E. F.; van der Schoot, C. E.; von dem Borne, A. E.

1995-01-01

We have identified a new platelet-specific alloantigen, Max(a), responsible for a typical case of neonatal alloimmune thrombocytopenic purpura. The maternal serum reacted strongly with paternal platelets in the platelet immunofluorescence test, whereas platelet alloantigen typing showed that no

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.

Science.gov (United States)

O'Connor, Marie N; Salles, Isabelle I; Cvejic, Ana; Watkins, Nicholas A; Walker, Adam; Garner, Stephen F; Jones, Chris I; Macaulay, Iain C; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H; Deckmyn, Hans; Stemple, Derek L; Ouwehand, Willem H

2009-05-07

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Inhibition of the plasma SCUBE1, a novel platelet adhesive protein, protects mice against thrombosis.

Science.gov (United States)

Wu, Meng-Ying; Lin, Yuh-Charn; Liao, Wei-Ju; Tu, Cheng-Fen; Chen, Ming-Huei; Roffler, Steve R; Yang, Ruey-Bing

2014-07-01

Signal peptide-CUB-EGF domain-containing protein 1 (SCUBE1), a secreted and surface-exposed glycoprotein on activated platelets, promotes platelet-platelet interaction and supports platelet-matrix adhesion. Its plasma level is a biomarker of platelet activation in acute thrombotic diseases. However, the exact roles of plasma SCUBE1 in vivo remain undefined. We generated new mutant (Δ) mice lacking the soluble but retaining the membrane-bound form of SCUBE1. Plasma SCUBE1-depleted Δ/Δ mice showed normal hematologic and coagulant features and expression of major platelet receptors, but Δ/Δ platelet-rich plasma showed impaired platelet aggregation in response to ADP and collagen treatment. The addition of purified recombinant SCUBE1 protein restored the aggregation of platelets in Δ/Δ platelet-rich plasma and further enhanced platelet aggregation in +/+ platelet-rich plasma. Plasma deficiency of SCUBE1 diminished arterial thrombosis in mice and protected against lethal thromboembolism induced by collagen-epinephrine treatment. Last, antibodies directed against the epidermal growth factor-like repeats of SCUBE1, which are involved in trans-homophilic protein-protein interactions, protected mice against fatal thromboembolism without causing bleeding in vivo. We conclude that plasma SCUBE1 participates in platelet aggregation by bridging adjacent activated platelets in thrombosis. Blockade of soluble SCUBE1 might represent a novel antithrombotic strategy. © 2014 American Heart Association, Inc.

Taurine and platelet aggregation

International Nuclear Information System (INIS)

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

1986-01-01

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

Complementary effects of thienopyridine pretreatment and platelet glycoprotein IIb/IIIa integrin blockade with eptifibatide in coronary stent intervention; results from the ESPRIT trial.

Science.gov (United States)

Dery, Jean-Pierre; Campbell, Mark E; Mathias, Jasmine; Pieper, Karen S; Harrington, Robert A; Madan, Mina; Gibson, C Michael; Tolleson, Thaddeus R; O'Shea, J Conor; Tcheng, James E

2007-07-01

This analysis sought to investigate the complementary effect of thienopyridine pretreatment and platelet glycoprotein (GP) IIb/IIIa integrin blockade in coronary stent intervention. Definitive evidence supporting combined antiplatelet therapy consisting of thienopyridine pretreatment and GP IIb/IIIa receptor blockade in patients undergoing percutaneous coronary intervention (PCI) with stent implantation is limited. We retrospectively analyzed clinical outcomes by thienopyridine use in the 2,040 patients randomized to eptifibatide or placebo who underwent PCI in the ESPRIT trial. A total of 901 patients received a loading dose of thienopyridine before PCI (group 1), 123 received thienopyridine pretreatment without a loading dose (group 2), and 1,016 were not treated with thienopyridine before PCI (group 3). The composite incidence of death or myocardial infarction at 30 days was significantly lower in group 1 than in groups 2 and 3 combined (OR, 0.71 [95%CI, 0.52-0.99]; P = 0.0417). A similar trend was seen for the composite of death, myocardial infarction, or urgent target vessel revascularization (unadjusted OR, 0.77 [0.57-1.05]; P = 0.1025). After adjusting for baseline characteristics, these differences were no longer significant. No interactions were identified with eptifibatide assignment for any of the group comparisons. Pretreatment with a loading dose of thienopyridine lowers the rate of ischemic complications regardless of treatment with a GP IIb/IIIa inhibitor. Conversely, the efficacy of eptifibatide is maintained whether or not a loading dose of a thienopyridine is administered. Optimal outcomes are achieved in patients receiving thienopyridine pretreatment along with platelet GP IIb/IIIa inhibitor therapy. (c) 2007 Wiley-Liss, Inc.

Carcinoma-specific Ulex europaeus agglutinin-I binding glycoproteins of human colorectal carcinoma and its relation to carcinoembryonic antigen.

Science.gov (United States)

Matsushita, Y; Yonezawa, S; Nakamura, T; Shimizu, S; Ozawa, M; Muramatsu, T; Sato, E

1985-08-01

Glycoproteins binding to Ulex europaeus agglutinin-I (UEA-I) lectin, which recognizes the terminal alpha-L-fucose residue, were analyzed in 18 cases of human colorectal carcinoma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by the Western blotting method. In the distal large bowel (descending and sigmoid colon and rectum), high-molecular-weight glycoproteins binding to UEA-I existed in carcinoma tissue but not in normal mucosa. In the proximal large bowel (ascending and transverse colon), high-molecular-weight glycoproteins binding to UEA-I were found both in normal mucosa and in carcinoma tissue, whereas those from the carcinoma tissue had an apparently lower molecular weight as compared to the weight of those from the normal mucosa. Thus there is a biochemical difference in UEA-I binding glycoproteins between the normal mucosa and the carcinoma tissue, although in our previous histochemical study no difference was observed in UEA-I binding glycoproteins of the proximal large bowel between the carcinoma tissue and the normal mucosa. Furthermore, carcinoembryonic antigen from the carcinoma tissue was found to have the same electrophoretical mobility as the UEA-I binding glycoproteins.

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

Energy Technology Data Exchange (ETDEWEB)

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

1989-08-01

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

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

OpenAIRE

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

2013-01-01

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

An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

International Nuclear Information System (INIS)

Tiwari, Vaibhav; Darmani, Nissar A.; Thrush, Gerald R.; Shukla, Deepak

2009-01-01

Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

Energy Technology Data Exchange (ETDEWEB)

Tiwari, Vaibhav [Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766 (United States); Darmani, Nissar A.; Thrush, Gerald R. [Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766 (United States); Shukla, Deepak, E-mail: dshukla@uic.edu [Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States)

2009-12-18

Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

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

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

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

Science.gov (United States)

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

2012-01-01

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

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

Directory of Open Access Journals (Sweden)

Venkateswarlu Kanamarlapudi

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

Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

DEFF Research Database (Denmark)

Nieswandt, B; Brakebusch, C; Bergmeier, W

2001-01-01

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subs...

An unusual dependence of human herpesvirus-8 Glycoproteins-induced cell-to-cell fusion on heparan sulfate

Science.gov (United States)

Tiwari, Vaibhav; Darmani, Nissar A.; Thrush, Gerald R.; Shukla, Deepak

2009-01-01

Human herpes virus 8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: 1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; 2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, 3) coexpression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis. PMID:19747451

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

Directory of Open Access Journals (Sweden)

Mathieu Barbier

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

GMP-140 binds to a glycoprotein receptor on human neutrophils: Evidence for a lectin-like interaction

International Nuclear Information System (INIS)

Moore, K.L.; Varki, A.; McEver, R.P.

1991-01-01

GMP-140 is a rapidly inducible receptor for neutrophils and monocytes expressed on activated platelets and endothelial cells. It is a member of the selectin family of lectin-like cell surface molecules that mediate leukocyte adhesion. We used a radioligand binding assay to characterize the interaction of purified GMP-140 with human neutrophils. Unstimulated neutrophils rapidly bound [125I]GMP-140 at 4 degrees C, reaching equilibrium in 10-15 min. Binding was Ca2+ dependent, reversible, and saturable at 3-6 nM free GMP-140 with half-maximal binding at approximately 1.5 nM. Receptor density and apparent affinity were not altered when neutrophils were stimulated with 4 beta-phorbol 12-myristate 13-acetate. Treatment of neutrophils with proteases abolished specific binding of [125I]GMP-140. Binding was also diminished when neutrophils were treated with neuraminidase from Vibrio cholerae, which cleaves alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids, or from Newcastle disease virus, which cleaves only alpha 2-3- and alpha 2-8-linked sialic acids. Binding was not inhibited by an mAb to the abundant myeloid oligosaccharide, Lex (CD15), or by the neoglycoproteins Lex-BSA and sialyl-Lex-BSA. We conclude that neutrophils constitutively express a glycoprotein receptor for GMP-140, which contains sialic acid residues that are essential for function. These findings support the concept that GMP-140 interacts with leukocytes by a lectin-like mechanism

Fabricating bio-inspired micro/nano-particles by polydopamine coating and surface interactions with blood platelets

Energy Technology Data Exchange (ETDEWEB)

Ye, Wei [Jiangsu Provincial Key Lab for Interventional Medical Devices, Huaiyin Institute of Technology, Huaian 223003 (China); State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China); Shi, Qiang, E-mail: shiqiang@ciac.ac.cn [State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China); Hou, Jianwen; Gao, Jian; Li, Chunming; Jin, Jing; Shi, Hengchong [State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China); Yin, Jinghua, E-mail: yinjh@ciac.ac.cn [State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China)

2015-10-01

Graphical abstract: The particles or particle aggregations activate the blood platelets and provide the physical adhesive sites for platelets adhesion. - Highlights: • Particles with varied sizes and surface properties were fabricated by facile polydopamine (PDA) coating on polystyrene microsphere. • The direct interaction between PDA particles and blood platelets was qualitatively investigated. • The knowledge on platelet–particle interactions provided the basic principle to select biocompatible micro/nano-particles in biomedical field. - Abstract: Although bio-inspired polydopamine (PDA) micro/nano-particles show great promise for biomedical applications, the knowledge on the interactions between micro/nano-particles and platelets is still lacking. Here, we fabricate PDA-coated micro/nano-particles and investigate the platelet–particle surface interactions. Our strategy takes the advantage of facile PDA coating on polystyrene (PS) microsphere to fabricate particles with varied sizes and surface properties, and the chemical reactivity of PDA layers to immobilize fibrinogen and bovine serum albumin to manipulate platelet activation and adhesion. We demonstrate that PS particles activate the platelets in the size-dependent manner, but PDA nanoparticles have slight effect on platelet activation; PS particles promote platelet adhesion while PDA particles reduce platelet adhesion on the patterned surface; Particles interact with platelets through activating the glycoprotein integrin receptor of platelets and providing physical sites for initial platelet adhesion. Our work sheds new light on the interaction between platelets and particles, which provides the basic principle to select biocompatible micro/nano-particles in biomedical field.

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

Science.gov (United States)

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

2012-08-10

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

Efficacy and Safety of Platelet Glycoprotein Receptor Blockade in Aged and Comorbid Mice With Acute Experimental Stroke.

Science.gov (United States)

Kraft, Peter; Schuhmann, Michael K; Fluri, Felix; Lorenz, Kristina; Zernecke, Alma; Stoll, Guido; Nieswandt, Bernhard; Kleinschnitz, Christoph

2015-12-01

Despite the medical and socioeconomic effect of ischemic stroke and extensive preclinical research, treatment options for ischemic stroke are limited. We recently identified and characterized essential steps of thrombus formation in stroke and demonstrated that inhibition of the platelet glycoprotein (GP) receptors Ib and VI, but not IIb/IIIa, protects young and healthy mice from ischemic neurodegeneration. Whether these findings translate to the clinic remains unclear. Considering that the typical stroke patient is elderly with comorbidity, we aimed to analyze the efficacy and safety of novel preclinical antithrombotics in adult and comorbid mice with acute experimental stroke. We subjected adult, healthy, atherosclerotic (Ldlr(-/-)), diabetic (streptozotocin treated), and hypertensive (RenTgMK) mice to a 60-minute transient middle cerebral artery occlusion. Animals were pretreated with anti-GPVI antibodies or treated 1 hour after stroke induction with anti-GPIb or anti-GPIIb/IIIa antigen-binding fragments, respectively. Isotype treatment served as control. Twenty-four hours after transient middle cerebral artery occlusion, we visually assessed the intracerebral hemorrhage rate and measured infarct volumes (using 2,3,5-triphenyltetrazolium chloride-stained brain slices) and functional outcome (using Bederson and grip-test scores). GPIb and GPVI inhibition protected the mice from ischemic stroke without increasing bleeding complications. In contrast, GPIIb/IIIa inhibition was not protective but increased the intracerebral hemorrhage rate. Inhibition of early steps of thrombus formation protects adult and comorbid mice from ischemic stroke. The use of clinically meaningful mouse strains might improve the translation of preclinical stroke research to the clinic. © 2015 American Heart Association, Inc.

Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow-derived mesenchymal stromal cells.

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Bernardi, Martina; Albiero, Elena; Alghisi, Alberta; Chieregato, Katia; Lievore, Chiara; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

2013-08-01

A medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed. Platelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells. After 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations. The proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

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

International Nuclear Information System (INIS)

Morinelli, T.A.

1987-01-01

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

Peroxiredoxin II is an antioxidant enzyme that negatively regulates collagen-stimulated platelet function.

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Jang, Ji Yong; Wang, Su Bin; Min, Ji Hyun; Chae, Yun Hee; Baek, Jin Young; Yu, Dae-Yeul; Chang, Tong-Shin

2015-05-01

Collagen-induced platelet signaling is mediated by binding to the primary receptor glycoprotein VI (GPVI). Reactive oxygen species produced in response to collagen have been found to be responsible for the propagation of GPVI signaling pathways in platelets. Therefore, it has been suggested that antioxidant enzymes could down-regulate GPVI-stimulated platelet activation. Although the antioxidant enzyme peroxiredoxin II (PrxII) has emerged as having a role in negatively regulating signaling through various receptors by eliminating H2O2 generated upon receptor stimulation, the function of PrxII in collagen-stimulated platelets is not known. We tested the hypothesis that PrxII negatively regulates collagen-stimulated platelet activation. We analyzed PrxII-deficient murine platelets. PrxII deficiency enhanced GPVI-mediated platelet activation through the defective elimination of H2O2 and the impaired protection of SH2 domain-containing tyrosine phosphatase 2 (SHP-2) against oxidative inactivation, which resulted in increased tyrosine phosphorylation of key components for the GPVI signaling cascade, including Syk, Btk, and phospholipase Cγ2. Interestingly, PrxII-mediated antioxidative protection of SHP-2 appeared to occur in the lipid rafts. PrxII-deficient platelets exhibited increased adhesion and aggregation upon collagen stimulation. Furthermore, in vivo experiments demonstrated that PrxII deficiency facilitated platelet-dependent thrombus formation in injured carotid arteries. This study reveals that PrxII functions as a protective antioxidant enzyme against collagen-stimulated platelet activation and platelet-dependent thrombosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Mouse podoplanin supports adhesion and aggregation of platelets under arterial shear: A novel mechanism of haemostasis.

Science.gov (United States)

Lombard, Stephanie E; Pollitt, Alice Y; Hughes, Craig E; Di, Ying; Mckinnon, Tom; O'callaghan, Chris A; Watson, Steve P

2017-11-01

The podoplanin-CLEC-2 axis is critical in mice for prevention of hemorrhage in the cerebral vasculature during mid-gestation. This raises the question as to how platelets are captured by podoplanin on neuroepithelial cells in a high shear environment. In this study, we demonstrate that mouse platelets form stable aggregates on mouse podoplanin at arterial shear through a CLEC-2 and Src kinase-dependent pathway. Adhesion and aggregation are also dependent on the platelet glycoprotein (GP) receptors, integrin αIIbβ3 and GPIb, and the feedback agonists ADP and thromboxane A 2 (TxA 2 ). CLEC-2 does not bind to von Willebrand factor (VWF) suggesting that the interaction with podoplanin is sufficient to both tether and activate platelets. Consistent with this, the surface plasmon resonance measurements reveal that mouse CLEC-2 binds to mouse podoplanin with nanomolar affinity. The present findings demonstrate a novel pathway of hemostasis in which podoplanin supports platelet capture and activation at arteriolar rates of shear.

Human plasma fibrinogen adsorption and platelet adhesion to polystyrene.

Science.gov (United States)

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

1999-02-01

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

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

Science.gov (United States)

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

2015-10-01

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

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

International Nuclear Information System (INIS)

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

1986-01-01

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

Evaluation of electrical aggregometry: comparison with optical aggregometry, secretion of ATP, and accumulation of radiolabeled platelets

International Nuclear Information System (INIS)

Ingerman-Wojenski, C.; Smith, J.B.; Silver, M.J.

1983-01-01

Platelet aggregation has been most commonly studied in vitro by measuring increases in light transmission as platelets aggregate in PRP (platelet-rich plasma). Recently, an electrical impedance method for measuring platelet aggregation has been introduced. This method can be used with either PRP or whole blood and measures an increase in impedance across electrodes placed in the blood samples as platelets accumulate on them. Results obtained by the two methods were compared using ADP and collagen as aggregating agents, and also have measured the secretion of platelet ATP simultaneously. Although the aggregometry results were similar, recordings obtained by the electrical method did not distinguish two waves of platelet aggregation or correlate with secretion as well as recordings obtained by the optical method. When PGI 2 (prostacyclin) or PGE 1 (prostaglandin E 1 ) was added to the PRP, both the rate and extent of the increase in light transmittance were inhibited, but the main effect on the increase in impedance was a decrease in its rate and not in its extent. Increases in impedance and secretion of ATP were also measured in whole blood after the platelets had been labeled with a 125 I-containing antibody specific for platelet surface glycoproteins. It appeared that the increases in impedance lagged several minutes behind the formation of platelet aggregates and the secretion of platelet ATP

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...

Delayed-onset of procoagulant signalling revealed by kinetic analysis of COAT platelet formation.

Science.gov (United States)

Alberio, Lorenzo; Ravanat, Catherine; Hechler, Béatrice; Mangin, Pierre H; Lanza, François; Gachet, Christian

2017-06-02

The combined action of collagen and thrombin induces the formation of COAT platelets, which are characterised by a coat of procoagulant and adhesive molecules on their surface. Although recent work has started to highlight their clinical relevance, the exact mechanisms regulating the formation of procoagulant COAT platelets remain unclear. Therefore, we employed flow cytometry in order to visualise in real time surface and intracellular events following simultaneous platelet activation with convulxin and thrombin. After a rapid initial response pattern characterised by the homogenous activation of the fibrinogen receptor glycoprotein IIb/IIIa in all platelets, starting with a delay of about 2 minutes an increasing fraction transforms to procoagulant COAT platelets. Their surface is characterised by progressive loss of PAC-1 binding, expression of negative phospholipids and retention of α-granule von Willebrand factor. Intracellular events in procoagulant COAT platelets are a marked increase of free calcium into the low micromolar range, concomitantly with early depolarisation of the mitochondrial membrane and activation of caspase-3, while non-COAT platelets keep the intracellular free calcium in the nanomolar range and maintain an intact mitochondrial membrane. We show for the first time that the flow-cytometrically distinct fractions of COAT and non-COAT platelets differentially phosphorylate two signalling proteins, PKCα and p38MAPK, which may be involved in the regulation of the different calcium fluxes observed in COAT versus non-COAT platelets. This study demonstrates the utility of concomitant cellular and signalling evaluation using flow cytometry in order to further dissect the mechanisms underlying the dichotomous platelet response observed after collagen/thrombin stimulation.

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

DEFF Research Database (Denmark)

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

2013-01-01

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

Serotonin binding in vitro by releasable proteins from human blood platelets

International Nuclear Information System (INIS)

Heemstra, V.L.

1983-11-01

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

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

Science.gov (United States)

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

2014-06-01

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

Glycoproteins and sialyl transferase of human B lymphoblastoid cell lines

International Nuclear Information System (INIS)

Lui, S.W.L.; Ng, M.H.

1980-01-01

We used two radiolabeling methods to study glycoproteins on the surface of lymphoblastoid cells. One of the methods affects tritiation of residues which are oxidized with galactose oxidase and the other causes tritiation of neuraminic acid residues. This approach was shown to allow a better resolution of cell surface glycoproteins than if either method were used alone. Glycoproteins of B 1 - 19 cells which harbor the Epstein-Barr virus genomes were compared with those of its parental cell line, BJAB, which does not harbor the viral genomes. These studies did not reveal a unique viral protein. A 28,000 mol. wt. glycoprotein was found to be the most prominent neuraminic acidlabeled product of B 1 - 19 cells and also of the two other cell lines, Raji and Ly38, which harbor the EBV genomes. A similar molecular weight species from BJAB cells identified by galactose oxidase labeling might be deficient in neuraminic acid residues as it was poorly labeled by the periodate oxidation method. The neuraminic acid content and level of sialyl transferase of BJAB cells were found to be lower than those of the other cell lines studied. (auth.)

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

Science.gov (United States)

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

2017-10-01

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

Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus

DEFF Research Database (Denmark)

Giang, Erick; Dorner, Marcus; Prentoe, Jannick C

2012-01-01

, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human m......Abs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81......bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies...

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

Directory of Open Access Journals (Sweden)

Beata Olas

2016-01-01

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

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

International Nuclear Information System (INIS)

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

2010-01-01

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

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

International Nuclear Information System (INIS)

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

1984-01-01

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

Serological responses in chimpanzees inoculated with human immunodeficiency virus glycoprotein (gp120) subunit vaccine

International Nuclear Information System (INIS)

Arthur, L.O.; Pyle, S.W.; Nara, P.L.

1987-01-01

The major envelope glycoprotein of a human immunodeficiency virus (HIV) has been purified and was utilized as a prototype vaccine in chimpanzees. The 120,000-dalton glycoprotein (gp120) was purified from membranes of human T-lymphotropic virus (HTLV)-IIIB-infected cells and the final preparation contained low levels to no detectable HTLV-IIIB core antigen (p24) and low levels of endotoxin. Chimpanzees inoculated with gp120 responded by developing antibodies that precipitated radiolabeled gp120 and neutralized in vitro infection of HTLV-IIIB. Antibodies to HTLV-IIIB p24 were not detected in the gp120-immunized chimpanzees. Peripheral blood leukocytes from the vaccinated animals were examined for T4 + and T8 + cells, and no decrease in the T4/T8 ratio was found, indicating that immunization with a ligand (gp120) that binds to T4 has not detectable adverse effect on the population of T4 + cells. The only current animal model that can be reproducibly infected with HIV is the chimpanzee. Immunization of chimpanzees with HIV proteins will provide an experimental system for testing the effectiveness of prototype vaccines for preventing HIV infection in vivo

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

Science.gov (United States)

Takayama, Naoya; Eto, Koji

2012-10-01

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

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

International Nuclear Information System (INIS)

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

1983-01-01

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

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

Science.gov (United States)

Carnevale, Roberto; Bartimoccia, Simona; Nocella, Cristina; Di Santo, Serena; Loffredo, Lorenzo; Illuminati, Giulio; Lombardi, Elisabetta; Boz, Valentina; Del Ben, Maria; De Marco, Luigi; Pignatelli, Pasquale; Violi, Francesco

2014-11-01

Platelets generate oxidized LDL (ox-LDL) via NOX2-derived oxidative stress. We investigated if once generated by activated platelets ox-LDL can propagate platelet activation. Experiments were performed in platelets from healthy subjects (HS), hyper-cholesterolemic patients and patients with NOX2 hereditary deficiency. Agonist-stimulated platelets from HS added with LDL were associated with a dose-dependent increase of reactive oxidant species and ox-LDL. Agonist-stimulated platelets from HS added with a fixed dose of LDL (57.14 μmol/L) or added with homogenized human atherosclerotic plaque showed enhanced ox-LDL formation (approximately +50% and +30% respectively), which was lowered by a NOX2 inhibitor (approximately -35% and -25% respectively). Compared to HS, ox-LDL production was more pronounced in agonist-stimulated platelet rich plasma (PRP) from hyper-cholesterolemic patients but was almost absent in PRP from NOX2-deficient patients. Platelet aggregation and 8-iso-PGF2α-ΙΙΙ formation increased in LDL-treated washed platelets (+42% and +53% respectively) and PRP (+31% and +53% respectively). Also, LDL enhanced platelet-dependent thrombosis at arterial shear rate (+33%) but did not affect platelet activation in NOX2-deficient patients. Platelet activation by LDL was significantly inhibited by CD36 or LOX1 blocking peptides, two ox-LDL receptor antagonists, or by a NOX2 inhibitor. LDL-added platelets showed increased p38MAPK (+59%) and PKC (+51%) phosphorylation, p47(phox) translocation to platelet membrane (+34%) and NOX2 activation (+30%), which were inhibited by ox-LDL receptor antagonists. Platelets oxidize LDL, which in turn amplify platelet activation via specific ox-LDL receptors; both effects are mediated by NOX2 activation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

Science.gov (United States)

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

2013-12-01

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

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

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

Science.gov (United States)

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

2012-02-15

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

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

Science.gov (United States)

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

2017-02-01

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

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

International Nuclear Information System (INIS)

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

1981-01-01

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

Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus

DEFF Research Database (Denmark)

Meunier, Jean-Christophe; Russell, Rodney S.; Goossens, Vera

2008-01-01

The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human...... monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly...... neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a...

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

International Nuclear Information System (INIS)

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

1986-01-01

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

Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13.

Science.gov (United States)

Kraus, Emma; Kraus, Kristina; Obser, Tobias; Oyen, Florian; Klemm, Ulrike; Schneppenheim, Reinhard; Brehm, Maria A

2014-12-01

The multimeric form of von Willebrand factor (VWF), is the largest soluble protein in mammals and exhibits a multidomain structure resulting in multiple functions. Upon agonist stimulation endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream where VWF plays an essential role in platelet-dependent primary hemostasis. Elongation of VWF strings on the cells' surface leads to accessibility of VWF binding sites for proteins, such as platelet membrane glycoprotein Ib. The prothrombotic strings are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage. VWF string formation was induced by histamine stimulation of HUVEC cells under unidirectional shear flow and VWF strings were detected employing the VWF binding peptide of platelet glycoprotein Ib coupled to latex beads. VWF strings were then used as substrate for kinetic studies of recombinant and plasma ADAMTS13. To investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13, we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13 without the need for isolated platelets. Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semi-automated, quantitative assessment of recombinant and plasma ADAMTS13 activity. Our assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of von Willebrand Disease and Thrombotic Thrombocytopenic Purpura as it allows detection of shear flow-dependent dysfunction of VWD-associated VWF mutants as well as TTP-associated ADAMTS13 mutants. Copyright © 2014 Elsevier Ltd. All rights reserved.

Analysis of glycoprotein E-selectin ligANDs on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells

KAUST Repository

Merzaban, Jasmeen S.

2011-06-09

Although well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using E-selectin-Ig chimera (E-Ig) shows that human marrow cells enriched for HSPCs (CD34+ cells) display greater E-selectin binding than those obtained from mouse (lin-/Sca-1+/c-kit+ [LSK] cells). To define the relevant glycoprotein E-selectin ligands, lysates from human CD34+ and KG1a cells and from mouse LSK cells were immunoprecipitated using E-Ig and resolved byWestern blot using E-Ig. In both human and mouse cells, E-selectin ligand reactivity was observed at ∼ 120- to 130-kDa region, which contained two E-selectin ligands, the P-selectin glycoprotein ligand- 1 glycoform "CLA," and CD43. Human, but not mouse, cells displayed a prominent ∼ 100-kDa band, exclusively comprising the CD44 glycoform "HCELL."E-Ig reactivity was most prominent on CLA in mouse cells and on HCELL in human cells. To further assess HCELL\\'s contribution to E-selectin adherence, complementary studies were performed to silence (via CD44 siRNA) or enforce its expression (via exoglycosylation). Under physiologic shear conditions, CD44/HCELL-silenced human cells showed striking decreases (> 50%) in E-selectin binding. Conversely, enforced HCELL expression of LSK cells profoundly increased E-selectin adherence, yielding > 3-fold more marrow homing in vivo. These data define the key glycoprotein E-selectin ligands of human and mouse HSPCs, unveiling critical species-intrinsic differences in both the identity and activity of these structures. © 2011 by The American Society of Hematology.

Evaluation of electrical aggregometry: comparison with optical aggregometry, secretion of ATP, and accumulation of radiolabeled platelets

International Nuclear Information System (INIS)

Ingerman-Wojenski, C.; Smith, J.B.; Silver, M.J.

1983-01-01

Platelet aggregation has been most commonly studied in vitro by measuring increases in light transmission as platelets aggregate in PRP. Recently, an electrical impedance method for measuring platelet aggregation has been introduced. This method can be used with either PRP or whole blood and measures an increase in impedance across electrodes placed in the blood samples as platelets accumulate on them. We have compared the results obtained by the two methods, using ADP and collagen as aggregating agents, and also have measured the secretion of platelet ATP simultaneously. Although the aggregometry results were similar, recordings obtained by the electrical method did not distinguish two waves of platelet aggregation or correlate with secretion as well as recordings obtained by the optical method. When PGI2 or PGE1 was added to the PRP, both the rate and extent of the increase in light transmittance were inhibited, but the main effect on the increase in impedance was a decrease in its rate and not in its extent. Increases in impedance and secretion of ATP were also measured in whole blood after the platelets had been labeled with a 125 I-containing antibody specific for platelet surface glycoproteins. It appeared that the increases in impedance lagged several minutes behind the formation of platelet aggregates and the secretion of platelet ATP. It is suggested that there are advantages and disadvantages to both methods of measurement of platelet aggregation and that the parameters being measured must be clearly understood to properly interpret the results

Staphylococcal superantigen-like 5 activates platelets and supports platelet adhesion under flow conditions, which involves glycoprotein Ib alpha and alpha(IIb)beta(3)

NARCIS (Netherlands)

De Haas, C. J. C.; Weeterings, C.; Vughs, M. M.; De Groot, P. G.; Van Strijp, J. A.; Lisman, T.

2009-01-01

Objectives: Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus aureus that has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). Methods and Results: When purified

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

Science.gov (United States)

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

2013-03-01

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

The role of platelets during reproduction.

Science.gov (United States)

Isermann, Berend; Nawroth, Peter P

2006-01-01

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

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

Directory of Open Access Journals (Sweden)

Serena Valsami

2015-01-01

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

Human platelet as an independent unit for sulfate conjugation

International Nuclear Information System (INIS)

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

1988-01-01

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

Demonstration of a specific C3a receptor on guinea pig platelets

International Nuclear Information System (INIS)

Fukuoka, Y.; Hugli, T.E.

1988-01-01

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

Profiling of Concanavalin A-Binding Glycoproteins in Human Hepatic Stellate Cells Activated with Transforming Growth Factor-β1

Directory of Open Access Journals (Sweden)

Yannan Qin

2014-11-01

Full Text Available Glycoproteins play important roles in maintaining normal cell functions depending on their glycosylations. Our previous study indicated that the abundance of glycoproteins recognized by concanavalin A (ConA was increased in human hepatic stellate cells (HSCs following activation by transforming growth factor-β1 (TGF-β1; however, little is known about the ConA-binding glycoproteins (CBGs of HSCs. In this study, we employed a targeted glycoproteomics approach using lectin-magnetic particle conjugate-based liquid chromatography-tandem mass spectrometry to compare CBG profiles between LX-2 HSCs with and without activation by TGF-β1, with the aim of discovering novel CBGs and determining their possible roles in activated HSCs. A total of 54 and 77 proteins were identified in the quiescent and activated LX-2 cells, respectively. Of the proteins identified, 14.3% were glycoproteins and 73.3% were novel potential glycoproteins. Molecules involved in protein processing in the endoplasmic reticulum (e.g., calreticulin and calcium signaling (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase β-2 [PLCB2] were specifically identified in activated LX-2 cells. Additionally, PLCB2 expression was upregulated in the cytoplasm of the activated LX-2 cells, as well as in the hepatocytes and sinusoidal cells of liver cirrhosis tissues. In conclusion, the results of this study may aid future investigations to find new molecular mechanisms involved in HSC activation and antifibrotic therapeutic targets.

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

Science.gov (United States)

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

2016-01-01

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

Human Platelet Senescence Study.

Science.gov (United States)

1980-03-01

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

Use of an anti-platelet monoclonal antibody F (ab')2 fragment for imaging thrombus

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Loutfi, I.; Stuttle, A.W.J.; Peters, A.M.; George, P.; Lavender, J.P.; Lumley, P.

1990-01-01

Ten patients with suspected thrombus have been studied using 111 In-labelled F (ab')2 fragments of P256, a monoclonal antibody which recognizes an epitope on the platelet membrane glycoprotein IIb/IIIa complex. The F (ab')2 fragment was radiolabelled with 111 In via diethylenetri-aminepentamacetic acid to give a specific activity of up to 190 MBq (5mCi) mg - 1 without impairment of immunoreactivity. In vitro platelet aggregation studies showed that the F (ab')2 fragment caused less platelet aggregation than the whole antibody on a molar ratio and was without significant effect upon the sensitivity of platelets to a range of aggregating agents. Platalets were labelled in ten patients by intravenous injection of approximately 100 μg P256 F (ab')2. Of the ten patients studies, six showed localization of activity consistent with platelet accumulation. Localization was clearly seen when associated with thrombus of the lower limbs (three patients: deep vein thrombosis; one patient: aortofemoral graft), and was apparent although less marked in two other cases, one of aortic aneurysm and one of carotid stenosis. Use of radiolabelled P256 F (ab')2 offers a means of non-invasive detection of thrombus which, from in vitro studies, would appear to have less direct effect of platelet behaviour than the whole antibody. (author). 9 refs. 8 figs. 1 tab

Herpes simplex virus (HSV)-specific proliferative and cytotoxic T-cell responses in humans immunized with an HSF type 2 glycoprotein subunit vaccine

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Zarling, J.M.; Moran, P.A.; Brewer, L.; Ashley, R.; Corey, L.

1988-12-01

Studies were undertaken to determine whether immunization of humans with a herpes simplex virus type 2 (HSV-2) glycoprotein-subunit vaccine would result in the priming of both HSV-specific proliferating cells and cytotoxic T cells. Peripheral blood lymphocytes (PBL) from all eight vaccinees studied responded by proliferating after stimulation with HSV-2, HSV-1, and glycoprotein gB-1. The PBL of five of these eight vaccinees proliferated following stimulation with gD-2, whereas stimulation with Gd-1 resulted in relatively low or no proliferative responses. T-cell clones were generated from HSV-2-stimulated PBL of three vaccinees who demonstrated strong proliferative responses to HSV-1 and HSV-2. Of 12 clones studied in lymphoproliferative assays, 9 were found to be cross-reactive for HSV-1 and HSV-2. Of the approximately 90 T-cell clones isolated, 14 demonstrated HSV-specific cytotoxic activity. Radioimmunoprecipitation-polyacrylamide gel electrophoresis analyses confirmed that the vaccinees had antibodies only to HSV glycoproteins, not to proteins which are absent in the subunit vaccine, indicating that these vaccinees had not become infected with HSV. Immunization of humans with an HSV-2 glycoprotein-subunit vaccine thus results in the priming of T cells that proliferate in response to stimulation with HSV and its glycoproteins and T cells that have cytotoxic activity against HSV-infected cells. Such HSV-specific memory T cells were detected as late as 2 years following the last boost with the subunit vaccine.

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

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

1987-01-01

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

Effects of delayed laboratory processing on platelet serotonin levels.

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

2013-01-01

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

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

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Sandgren P

2014-12-01

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

Platelet function in brown bear (Ursus arctos compared to man

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Särndahl Eva

2010-06-01

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

Ca 125 and Ca 19-9: two cancer-associated sialylsaccharide antigens on a mucus glycoprotein from human milk.

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Hanisch, F G; Uhlenbruck, G; Dienst, C; Stottrop, M; Hippauf, E

1985-06-03

The cancer-associated antigens Ca 125 and Ca 19-9 were demonstrated by radioimmunoassay to form structural units of a mucus glycoprotein in human milk taken from healthy women four days after parturition. The glycoprotein precipitated with the casein fraction at pH 4.6 and was completely absent in the whey as judged from Ca 19-9 assay. It could be effectively enriched by phenol-saline extraction from soluble milk proteins and further purified by gel filtration on Sephacryl S300 and Sephacryl S400. The active component with a bouyant density of 1.41 g/ml in isopycnic density gradient centrifugation (CsCl) shared common physico-chemical and chemical characteristics of mucus glycoproteins. Carbohydrates representing about 68% by weight were conjugated to protein by alkali-labile linkages, exclusively and were essentially free of D-mannose. Activities of Ca 125 and Ca 19-9 were both destroyed by treatment with periodate, mild alkali or neuraminidase suggesting the antigens are sialylated saccharides bound to protein by alkali-labile linkages. The fraction of monosialylated saccharide alditols isolated after reductive beta-elimination from the mucus glycoprotein was shown to inhibit monoclonal antibodies anti-(Ca 125) and anti-(Ca 19-9) in radioimmunoassay.

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

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Yoshito, Daniele

2011-01-01

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

Platelet Immunology in China: Research and Clinical Applications.

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

2017-04-01

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

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

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

2013-01-01

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

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

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Antheia Kissopoulou

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

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

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

2014-01-01

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

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

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Mustafa Tunalı

2014-01-01

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

Molecular cloning of the α subunit of human and guinea pig leukocyte adhesion glycoprotein Mo1: Chromosomal localization and homology to the α subunits of integrins

International Nuclear Information System (INIS)

Arnaout, M.A.; Remold-O'Donnell, E.; Pierce, M.W.; Harris, P.; Tenen, D.G.

1988-01-01

The cell surface-glycoprotein Mo1 is a member of the family of leukocyte cell adhesion molecules (Leu-CAMs) that includes lymphocyte function-associated antigen 1 (LFA-1) and p150,95. Each Leu-CAM is a heterodimer with a distinct α subunit noncovalently associated with a common β subunit. The authors describe the isolation and analysis of two partial cDNA clones encoding the α subunit of the Leu-CAM Mo1 in humans and guinea pigs. A monoclonal antibody directed against an epitope in the carboxyl-terminal portion of the guinea pig α chain was used for immunoscreening a λgt11 expression library. The sequence of a 378-base-pair insert from one immunoreactive clone revealed a single continuous open reading frame encoding 126 amino acids including a 26-amino acid tryptic peptide isolated from the purified guinea pig α subunit. A cDNA clone of identical size was isolated from a human monocyte/lymphocyte cDNA library by using the guinea pig clone as a probe. The human clone also encoded a 126-amino acid peptide including the sequence of an additional tryptic peptide present in purified human Mo1α chain. Southern analysis of DNA from hamster-human hybrids localized the human Mo1α chain to chromosome 16, which has been shown to contain the gene for the α chain of lymphocyte function-associated antigen 1. These data suggest that the α subunits of Leu-CAMs evolved by gene duplication from a common ancestral gene and strengthen the hypothesis that the α subunits of these heterodimeric cell adhesion molecules on myeloid and lymphoid cells, platelets, and fibroblasts are evolutionary related

Important role of platelets in modulating endotoxin-induced lung inflammation in CFTR-deficient mice.

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Caiqi Zhao

Full Text Available Mutation of CFTR (cystic fibrosis transmembrane conductance regulator leads to cystic fibrosis (CF. Patients with CF develop abnormalities of blood platelets and recurrent lung inflammation. However, whether CFTR-mutated platelets play a role in the development of lung inflammation is elusive. Therefore, we intratracheally challenged wildtype and F508del (a common type of CFTR mutation mice with LPS to observe changes of F508del platelets in the peripheral blood and indexes of lung inflammation (BAL neutrophils and protein levels. Furthermore, we investigated whether or not and how F508del platelets modulate the LPS-induced acute lung inflammation by targeting anti-platelet aggregation, depletion of neutrophils, reconstitution of bone marrow or neutrophils, blockade of P-selectin glycoprotein ligand-1 (PSGL-1, platelet activating factor (PAF, and correction of mutated CFTR trafficking. We found that LPS-challenged F508del mice developed severe thrombocytopenia and had higher levels of plasma TXB2 coincided with neutrophilic lung inflammation relative to wildtype control. Inhibition of F508del platelet aggregation or depletion of F508del neutrophils diminished the LPS-induced lung inflammation in the F508del mice. Moreover, wildtype mice reconstituted with either F508del bone marrow or neutrophils developed worse thrombocytopenia. Blocking PSGL-1, platelet activating factor (PAF, or rectifying trafficking of mutated CFTR in F508del mice diminished and alveolar neutrophil transmigration in the LPS-challenged F508del mice. These findings suggest that F508del platelets and their interaction with neutrophils are requisite for the development of LPS-induced lung inflammation and injury. As such, targeting platelets might be an emerging strategy for dampening recurrent lung inflammation in cystic fibrosis patients.

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

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

2010-01-01

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

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

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Hashemi SS

2016-08-01

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

Detection of von Willebrand factor and tissue factor in platelets-fibrin rich coronary thrombi in acute myocardial infarction.

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Yamashita, Atsushi; Sumi, Takahiro; Goto, Shinya; Hoshiba, Yasunari; Nishihira, Kensaku; Kawamoto, Riichirou; Hatakeyama, Kinta; Date, Haruhiko; Imamura, Takuroh; Ogawa, Hisao; Asada, Yujiro

2006-01-01

The rapid closure of coronary arteries due to occlusive thrombi is the major cause of acute myocardial infarction. However, the mechanisms of coronary thrombus formation have not been elucidated. We immunohistochemically assessed the localizations and their changes over time of glycoprotein IIb/IIIa, fibrin, von Willebrand factor (vWF), and tissue factor (TF), after the onset of chest pain (platelets, fibrin, vWF, and TF from the early phase of onset, and glycoprotein IIb/IIIa and fibrin were closely associated with vWF and TF, respectively. vWF and/or TF may contribute to occlusive thrombus formation and be novel therapeutic candidates for treating patients with coronary thrombosis.

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

International Nuclear Information System (INIS)

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

1983-01-01

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

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

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Whiss, P A; Andersson, R G G

2002-07-01

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

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

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S Era Dutta

2017-01-01

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

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

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Mohammad Mizanur Rahman

2017-02-01

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

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

Science.gov (United States)

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

2017-07-01

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

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

International Nuclear Information System (INIS)

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

1985-01-01

The platelet activation during radiolabeling in vitro with Cr-51 and In-111 may affect the platelet life-span (PLS) in vivo. A new RIA method to measure PLS is being evaluated. Aspirin inhibits platelet thromboxane (TxA/sub 2/) by acetylating cyclooxygenase. The time required for the TxA/sub 2/ levels to return towards control values depends on the rate of new platelets entering circulation and is a measure of PLS. A single dose of aspirin (150mg) was given to 5 normal human subjects. Blood samples were collected for 2 days before aspirin and daily for 10 days. TxA/sub 2/ production in response to endogenous thrombin was studied by allowing 1 ml blood sample to clot at 37 0 C for 90 min. Serum TxB/sub 2/ (stable breakdown product of Tx-A/sub 2/) levels determined by RIA technique. The plot of TxB/sub 2/ levels (% control) against time showed a gradual increase. The PLS calculated by linear regression analysis assuming a 2-day lag period before cyclooxygenase recovery is 9.7 +- 2.37. In the same 5 subjects, platelets from a 50ml blood sample were labeled with /sup 111/In-tropolone in 2 ml autologous plasma. Starting at 1 hr after injection of labeled platelets, 10 blood samples were obtained over a 8 day period. The PLS calculated based on a linear regression analysis is 10.2 +. 1.4. The PLS measured from the rate of platelet disappearance from circulation and the rate of platelet regeneration into circulation are quite comparable in normal subjects. TxA/sub 2/ regeneration RIA may provide a method to measure PLS without administering radioactivity to patient

O-acetylation of the serine-rich repeat glycoprotein GspB is coordinated with accessory Sec transport.

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Ravin Seepersaud

2017-08-01

Full Text Available The serine-rich repeat (SRR glycoproteins are a family of adhesins found in many Gram-positive bacteria. Expression of the SRR adhesins has been linked to virulence for a variety of infections, including streptococcal endocarditis. The SRR preproteins undergo intracellular glycosylation, followed by export via the accessory Sec (aSec system. This specialized transporter is comprised of SecA2, SecY2 and three to five accessory Sec proteins (Asps that are required for export. Although the post-translational modification and transport of the SRR adhesins have been viewed as distinct processes, we found that Asp2 of Streptococcus gordonii also has an important role in modifying the SRR adhesin GspB. Biochemical analysis and mass spectrometry indicate that Asp2 is an acetyltransferase that modifies N-acetylglucosamine (GlcNAc moieties on the SRR domains of GspB. Targeted mutations of the predicted Asp2 catalytic domain had no effect on transport, but abolished acetylation. Acetylated forms of GspB were only detected when the protein was exported via the aSec system, but not when transport was abolished by secA2 deletion. In addition, GspB variants rerouted to export via the canonical Sec pathway also lacked O-acetylation, demonstrating that this modification is specific to export via the aSec system. Streptococci expressing GspB lacking O-acetylated GlcNAc were significantly reduced in their ability bind to human platelets in vitro, an interaction that has been strongly linked to virulence in the setting of endocarditis. These results demonstrate that Asp2 is a bifunctional protein involved in both the post-translational modification and transport of SRR glycoproteins. In addition, these findings indicate that these processes are coordinated during the biogenesis of SRR glycoproteins, such that the adhesin is optimally modified for binding. This requirement for the coupling of modification and export may explain the co-evolution of the SRR

An alternative conformation of the gp41 heptad repeat 1 region coiled coil exists in the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor

International Nuclear Information System (INIS)

Mische, Claudia C.; Yuan Wen; Strack, Bettina; Craig, Stewart; Farzan, Michael; Sodroski, Joseph

2005-01-01

The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate

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

International Nuclear Information System (INIS)

Baldini, M.G.

1975-01-01

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

Human Platelets Exhibit Chemotaxis using Functional N-Formyl Peptide Receptors

National Research Council Canada - National Science Library

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

2005-01-01

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

Stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1

NARCIS (Netherlands)

Sanders, Rogier W.; Vesanen, Mika; Schuelke, Norbert; Master, Aditi; Schiffner, Linnea; Kalyanaraman, Roopa; Paluch, Maciej; Berkhout, Ben; Maddon, Paul J.; Olson, William C.; Lu, Min; Moore, John P.

2002-01-01

The envelope glycoprotein (Env) complex of human immunodeficiency virus type I has evolved a structure that is minimally immunogenic while retaining its natural function of receptor-mediated virus-cell fusion. The Env complex is trimeric; its six individual subunits (three gp120 and three gp41

Association between platelet glycoprotein Ia C807T gene polymorphism and ischemic stroke: a meta-analysis in a separate ethnic group.

Science.gov (United States)

Huang, Xiao-Yun; Fu, Wen-Jin; Mei, Zhi-Zhong; Yu, Ying-Li; Huang, Yi-Hong; Lin, Han; Chen, Jian-Jun; Wang, Ming-Xia; Guan, Shao-Bing; Fang, Hao-Wei

2017-11-30

Many studies have been examined the association of platelet glycoprotein (GP) Ia C807T polymorphism with ischemic stroke (IS) susceptibility. However, the results of these studies are inconsistent. To further assess the effects of GP Ia C807T polymorphism on the risk of IS, a meta-analysis was performed in a separate ethnic group. Relevant studies were identified using PubMed and Chinese databases through January 2017. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the associations. Finally, 13 studies contained 2438 IS cases and 2308 controls included. In the total analyses, a significantly elevated risk of IS was associated with all variants of GP Ia C807T in the Chinese population (T vs C: OR = 1.24, 95% CI = 1.09-1.40; TT vs CC: OR = 1.59, 95% CI = 1.17-2.15; TT and CT combined vs CC: OR = 1.32, 95% CI = 1.09-1.59; TT vs CC and CT: OR = 1.35, 95% CI = 1.04-1.76). In the subgroup analyses stratified by ethnicity and geographic areas, it revealed the significant results in Chinese Han and in South China. This meta-analysis provides the evidence that GP Ia C807T polymorphism may contribute to the IS development in the Chinese population, especially in South China, and further studies in other ethic groups are required for definite conclusions.

Orthobunyavirus ultrastructure and the curious tripodal glycoprotein spike.

Directory of Open Access Journals (Sweden)

Thomas A Bowden

Full Text Available The genus Orthobunyavirus within the family Bunyaviridae constitutes an expanding group of emerging viruses, which threaten human and animal health. Despite the medical importance, little is known about orthobunyavirus structure, a prerequisite for understanding virus assembly and entry. Here, using electron cryo-tomography, we report the ultrastructure of Bunyamwera virus, the prototypic member of this genus. Whilst Bunyamwera virions are pleomorphic in shape, they display a locally ordered lattice of glycoprotein spikes. Each spike protrudes 18 nm from the viral membrane and becomes disordered upon introduction to an acidic environment. Using sub-tomogram averaging, we derived a three-dimensional model of the trimeric pre-fusion glycoprotein spike to 3-nm resolution. The glycoprotein spike consists mainly of the putative class-II fusion glycoprotein and exhibits a unique tripod-like arrangement. Protein-protein contacts between neighbouring spikes occur at membrane-proximal regions and intra-spike contacts at membrane-distal regions. This trimeric assembly deviates from previously observed fusion glycoprotein arrangements, suggesting a greater than anticipated repertoire of viral fusion glycoprotein oligomerization. Our study provides evidence of a pH-dependent conformational change that occurs during orthobunyaviral entry into host cells and a blueprint for the structure of this group of emerging pathogens.

21 CFR 864.6675 - Platelet aggregometer.

Science.gov (United States)

2010-04-01

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

Platelet adhesion studies on dipyridamole coated polyurethane surfaces

Directory of Open Access Journals (Sweden)

Aldenhoff Y. B.J.

2003-06-01

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

A Functional Henipavirus Envelope Glycoprotein Pseudotyped Lentivirus Assay System

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Broder Christopher C

2010-11-01

Full Text Available Abstract Background Hendra virus (HeV and Nipah virus (NiV are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4 containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP gene encoding human immunodeficiency virus type-1 (HIV-1 genome in conjunction with the HeV and NiV fusion (F and attachment (G glycoproteins. Results Functional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2 peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F

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

International Nuclear Information System (INIS)

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

2014-01-01

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

Blood platelet kinetics and platelet transfusion.

Science.gov (United States)

Aster, Richard H

2013-11-01

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

A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis

DEFF Research Database (Denmark)

Dütting, Sebastian; Gaits-Iacovoni, Frederique; Stegner, David

2017-01-01

Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown...

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

Science.gov (United States)

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

2017-08-01

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

Pseudo (Platelet-type von Willebrand disease in pregnancy: a case report

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Grover Neetu

2013-01-01

Full Text Available Abstract Background Pseudo (platelet-type-von Willebrand disease is a rare autosomal dominant bleeding disorder caused by an abnormal function of the glycoprotein lb protein; the receptor for von Willebrand factor. This leads to an increased removal of VWF multimers from the circulation as well as platelets and this results in a bleeding diathesis. Worldwide, less than 50 patients are reported with platelet type von Willebrand disease (PT-VWD. Case presentation We describe the management of platelet type von Willebrand disease in pregnancy of a 26 year old Caucasian primigravida. The initial diagnosis was made earlier following a significant haemorrhage post tonsillectomy several years prior to pregnancy. The patient was managed under a multidisciplinary team which included obstetricians, haematologists, anaesthetists and neonatologists. Care plans were made for the ante- natal, intra-partum and post-partum periods in partnership with the patient. The patient’s platelet count levels dropped significantly during the antenatal period. This necessitated the active exclusion of other causes of thrombocytopenia in pregnancy. A vaginal delivery was desired and plans were made for induction of labour at 38 weeks of gestation with platelet cover in view of the progressive fall of the platelet count. The patient however went into spontaneous labour on the day of induction. She was transfused two units of platelets before delivery. She had an unassisted vaginal delivery of a healthy baby. The successful antenatal counselling has encouraged the diagnosis of the same condition in her mother and sister. We found this to be a particularly interesting case as well as challenging to manage due to its rarity. Psuedo von Willebrand disease in pregnancy can be confused with a number of other differential diagnoses, such as gestational thrombocutopenia, idiopathatic thrombocytopenia, thrombotic thrombocytopenic purpura and pre-eclampsia; all need consideration

Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors.

Directory of Open Access Journals (Sweden)

Yumiko Urano-Tashiro

Full Text Available Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2 of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N or Arg365 to Asn (R365N substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins.

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

Science.gov (United States)

Balazy, M

1991-12-15

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

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

Adipokine zinc-α2-glycoprotein regulated by growth hormone and linked to insulin sensitivity.

Science.gov (United States)

Balaz, Miroslav; Ukropcova, Barbara; Kurdiova, Timea; Gajdosechova, Lucia; Vlcek, Miroslav; Janakova, Zuzana; Fedeles, Jozef; Pura, Mikulas; Gasperikova, Daniela; Smith, Steven R; Tkacova, Ruzena; Klimes, Iwar; Payer, Juraj; Wolfrum, Christian; Ukropec, Jozef

2015-02-01

Hypertrophic obesity is associated with impaired insulin sensitivity and lipid-mobilizing activity of zinc-α2-glycoprotein. Adipose tissue (AT) of growth hormone (GH) -deficient patients is characterized by extreme adipocyte hypertrophy due to defects in AT lipid metabolism. It was hypothesized that zinc-α2-glycoprotein is regulated by GH and mediates some of its beneficial effects in AT. AT from patients with GH deficiency and individuals with obesity-related GH deficit was obtained before and after 5-year and 24-month GH supplementation therapy. GH action was tested in primary human adipocytes. Relationships of GH and zinc-α2-glycoprotein with adipocyte size and insulin sensitivity were evaluated in nondiabetic patients with noncancerous cachexia and hypertrophic obesity. AT in GH-deficient adults displayed a substantial reduction of zinc-α2-glycoprotein. GH therapy normalized AT zinc-α2-glycoprotein. Obesity-related relative GH deficit was associated with almost 80% reduction of zinc-α2-glycoprotein mRNA in AT. GH increased zinc-α2-glycoprotein mRNA in both AT of obese men and primary human adipocytes. Interdependence of GH and zinc-α2-glycoprotein in regulating AT morphology and metabolic phenotype was evident from their relationship with adipocyte size and AT-specific and whole-body insulin sensitivity. The results demonstrate that GH is involved in regulation of AT zinc-α2-glycoprotein; however, the molecular mechanism linking GH and zinc-α2-glycoprotein in AT is yet unknown. © 2014 The Obesity Society.

PlA polymorphism and platelet reactivity following clopidogrel loading dose in patients undergoing coronary stent implantation.

Science.gov (United States)

Angiolillo, Dominick J; Fernandez-Ortiz, Antonio; Bernardo, Esther; Alfonso, Fernando; Sabaté, Manel; Fernández, Cristina; Stranieri, Chiara; Trabetti, Elisabetta; Pignatti, Pier Franco; Macaya, Carlos

2004-01-01

The PlA polymorphism (Leu33Pro) of the platelet glycoprotein (GP) IIIa gene has been suggested to play an important role in coronary thrombosis. In vitro studies have shown differences for this polymorphism in platelet sensitivity towards antiplatelet drugs (aspirin and abciximab), suggesting a pharmacogenetic modulation. The aim of the study was to assess the modulatory effect of the PlA polymorphism on clopidogrel-induced antiplatelet effects in 38 patients undergoing coronary stent implantation receiving a 300 mg clopidogrel loading-dose. Platelet reactivity was assessed as GPIIb/IIIa activation and P-selectin expression in platelets stimulated with 2 micromol/l adenosine diphosphate using whole blood flow cytometry. The distribution of the homozygous PlA1/A1 and heterozygous PlA1/A2 genotypes were 74 and 26%, respectively. PlA2 carriers had a higher degree of GPIIb/IIIa activation (P = 0.05) and P-selectin expression (P = 0.02) during the overall study time course and a lower antiplatelet effect to a 300 mg clopidogrel loading-dose up to 24 h following intervention (P < 0.05). In conclusion, the Pl polymorphism of the GPIIIa gene modulates platelet reactivity towards clopidogrel front loading in patients undergoing coronary stenting. This suggests the need for individualized antithrombotic regimens to optimally inhibit platelet reactivity. Copyright 2004 Lippincott Williams and Wilkins

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

Directory of Open Access Journals (Sweden)

Maceler Aldrovandi

2017-04-01

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

Ingestion of onion soup high in quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in man: a pilot study.

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Hubbard, Gary P; Wolffram, Siegfried; de Vos, Ric; Bovy, Arnaud; Gibbins, Jonathan M; Lovegrove, Julie A

2006-09-01

Epidemiological data suggest that those who consume a diet rich in quercetin-containing foods may have a reduced risk of CVD. Furthermore, in vitro and ex vivo studies have observed the inhibition of collagen-induced platelet activation by quercetin. The aim of the present study was to investigate the possible inhibitory effects of quercetin ingestion from a dietary source on collagen-stimulated platelet aggregation and signalling. A double-blind randomised cross-over pilot study was undertaken. Subjects ingested a soup containing either a high or a low amount of quercetin. Plasma quercetin concentrations and platelet aggregation and signalling were assessed after soup ingestion. The high-quercetin soup contained 69 mg total quercetin compared with the low-quercetin soup containing 5 mg total quercetin. Plasma quercetin concentrations were significantly higher after high-quercetin soup ingestion than after low-quercetin soup ingestion and peaked at 2.59 (sem 0.42) mumol/l. Collagen-stimulated (0.5 mug/ml) platelet aggregation was inhibited after ingestion of the high-quercetin soup in a time-dependent manner. Collagen-stimulated tyrosine phosphorylation of a key component of the collagen-signalling pathway via glycoprotein VI, Syk, was significantly inhibited by ingestion of the high-quercetin soup. The inhibition of Syk tyrosine phosphorylation was correlated with the area under the curve for the high-quercetin plasma profile. In conclusion, the ingestion of quercetin from a dietary source of onion soup could inhibit some aspects of collagen-stimulated platelet aggregation and signalling ex vivo. This further substantiates the epidemiological data suggesting that those who preferentially consume high amounts of quercetin-containing foods have a reduced risk of thrombosis and potential CVD risk.

USE OF VITAL STAINING IN STORED HUMAN PLATELETS MORPHOFUNCTIONAL ANALYSIS

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M. S. Makarov

2014-01-01

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

Platelet transfusion therapy: from 1973 to 2005.

NARCIS (Netherlands)

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

2006-01-01

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

Three-Dimensionally Functionalized Reverse Phase Glycoprotein Array for Cancer Biomarker Discovery and Validation.

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Pan, Li; Aguilar, Hillary Andaluz; Wang, Linna; Iliuk, Anton; Tao, W Andy

2016-11-30

Glycoproteins have vast structural diversity that plays an important role in many biological processes and have great potential as disease biomarkers. Here, we report a novel functionalized reverse phase protein array (RPPA), termed polymer-based reverse phase glycoprotein array (polyGPA), to capture and profile glycoproteomes specifically, and validate glycoproteins. Nitrocellulose membrane functionalized with globular hydroxyaminodendrimers was used to covalently capture preoxidized glycans on glycoproteins from complex protein samples such as biofluids. The captured glycoproteins were subsequently detected using the same validated antibodies as in RPPA. We demonstrated the outstanding specificity, sensitivity, and quantitative capabilities of polyGPA by capturing and detecting purified as well as endogenous α-1-acid glycoprotein (AGP) in human plasma. We further applied quantitative N-glycoproteomics and the strategy to validate a panel of glycoproteins identified as potential biomarkers for bladder cancer by analyzing urine glycoproteins from bladder cancer patients or matched healthy individuals.

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

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Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

2014-02-01

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

Platelets and Multi-Organ Failure in Sepsis

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Elisabetta Greco

2017-10-01

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

Platelets and Multi-Organ Failure in Sepsis.

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

2017-10-20

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

Platelet-Rich Plasma Increases Pigmentation.

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Uysal, Cagri A; Ertas, Nilgun Markal

2017-11-01

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

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.

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

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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

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

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

2017-09-09

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

Molecular insight into conformational transmission of human P-glycoprotein

International Nuclear Information System (INIS)

Chang, Shan-Yan; Liu, Fu-Feng; Dong, Xiao-Yan; Sun, Yan

2013-01-01

P-glycoprotein (P-gp), a kind of ATP-binding cassette transporter, can export candidates through a channel at the two transmembrane domains (TMDs) across the cell membranes using the energy released from ATP hydrolysis at the two nucleotide-binding domains (NBDs). Considerable evidence has indicated that human P-gp undergoes large-scale conformational changes to export a wide variety of anti-cancer drugs out of the cancer cells. However, molecular mechanism of the conformational transmission of human P-gp from the NBDs to the TMDs is still unclear. Herein, targeted molecular dynamics simulations were performed to explore the atomic detail of the conformational transmission of human P-gp. It is confirmed that the conformational transition from the inward- to outward-facing is initiated by the movement of the NBDs. It is found that the two NBDs move both on the two directions (x and y). The movement on the x direction leads to the closure of the NBDs, while the movement on the y direction adjusts the conformations of the NBDs to form the correct ATP binding pockets. Six key segments (KSs) protruding from the TMDs to interact with the NBDs are identified. The relative movement of the KSs along the y axis driven by the NBDs can be transmitted through α-helices to the rest of the TMDs, rendering the TMDs to open towards periplasm in the outward-facing conformation. Twenty eight key residue pairs are identified to participate in the interaction network that contributes to the conformational transmission from the NBDs to the TMDs of human P-gp. In addition, 9 key residues in each NBD are also identified. The studies have thus provided clear insight into the conformational transmission from the NBDs to the TMDs in human P-gp

The glycoproteins of Marburg and Ebola virus and their potential roles in pathogenesis.

Science.gov (United States)

Feldmann, H; Volchkov, V E; Volchkova, V A; Klenk, H D

1999-01-01

Filoviruses cause systemic infections that can lead to severe hemorrhagic fever in human and non-human primates. The primary target of the virus appears to be the mononuclear phagocytic system. As the virus spreads through the organism, the spectrum of target cells increases to include endothelial cells, fibroblasts, hepatocytes, and many other cells. There is evidence that the filovirus glycoprotein plays an important role in cell tropism, spread of infection, and pathogenicity. Biosynthesis of the glycoprotein forming the spikes on the virion surface involves cleavage by the host cell protease furin into two disulfide linked subunits GP1 and GP2. GP1 is also shed in soluble form from infected cells. Different strains of Ebola virus show variations in the cleavability of the glycoprotein, that may account for differences in pathogenicity, as has been observed with influenza viruses and paramyxoviruses. Expression of the spike glycoprotein of Ebola virus, but not of Marburg virus, requires transcriptional editing. Unedited GP mRNA yields the nonstructural glycoprotein sGP, which is secreted extensively from infected cells. Whether the soluble glycoproteins GP1 and sGP interfere with the humoral immune response and other defense mechanisms remains to be determined.

Humoral immune response to the entire human immunodeficiency virus envelope glycoprotein made in insect cells

Energy Technology Data Exchange (ETDEWEB)

Rusche, J.R.; Lynn, D.L.; Robert-Guroff, M.; Langlois, A.J.; Lyerly, H.K.; Carson, H.; Krohn, K.; Ranki, A.; Gallo, R.C.; Bolognesi, D.P.; Putney, S.D.

1987-10-01

The human immunodeficiency virus envelope gene was expressed in insect cells by using a Baculovirus expression vector. The protein has an apparent molecular mass of 160 kDa, appears on the surface of infected insect cells, and does not appear to be cleaved to glycoproteins gp120 and gp41. Goats immunized with the 160-kDa protein have high titers of antibody that neutralizes virus infection as measured by viral gene expression or cell cytolysis. In addition, immune sera can block fusion of human immunodeficiency virus-infected cells in culture. Both neutralization and fusion-blocking activities are bound to and eluted from immobilized gp120.

Humoral immune response to the entire human immunodeficiency virus envelope glycoprotein made in insect cells

International Nuclear Information System (INIS)

Rusche, J.R.; Lynn, D.L.; Robert-Guroff, M.

1987-01-01

The human immunodeficiency virus envelope gene was expressed in insect cells by using a Baculovirus expression vector. The protein has an apparent molecular mass of 160 kDa, appears on the surface of infected insect cells, and does not appear to be cleaved to glycoproteins gp120 and gp41. Goats immunized with the 160-kDa protein have high titers of antibody that neutralizes virus infection as measured by viral gene expression or cell cytolysis. In addition, immune sera can block fusion of human immunodeficiency virus-infected cells in culture. Both neutralization and fusion-blocking activities are bound to and eluted from immobilized gp120

Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners.

Science.gov (United States)

Muramatsu, Takashi

2016-05-01

Basigin, also called CD147 or EMMPRIN, is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily. Basigin has isoforms; the common form (basigin or basigin-2) has two immunoglobulin domains, and the extended form (basigin-1) has three. Basigin is the receptor for cyclophilins, S100A9 and platelet glycoprotein VI, whereas basigin-1 serves as the receptor for the rod-derived cone viability factor. Basigin tightly associates with monocarboxylate transporters and is essential for their cell surface translocation and activities. In the same membrane plane, basigin also associates with other proteins including GLUT1, CD44 and CD98. The carbohydrate portion of basigin is recognized by lectins, such as galectin-3 and E-selectin. These molecular recognitions form the basis for the role of basigin in the transport of nutrients, migration of inflammatory leukocytes and induction of matrix metalloproteinases. Basigin is important in vision, spermatogenesis and other physiological phenomena, and plays significant roles in the pathogenesis of numerous diseases, including cancer. Basigin is also the receptor for an invasive protein RH5, which is present in malaria parasites. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society.

Competitions between fibrinogen with its degradation products for interactions with the platelet-fibrinogen receptor

International Nuclear Information System (INIS)

Thorsen, L.I.; Brosstad, F.; Gogstad, G.; Sletten, K.; Solum, N.O.

1986-01-01

Direct binding of 125 -I-labelled plasmic and CNBr-derived fibrin (ogen) fragments (pre-X, X, Y, D, Degta, Efg, E1, N-DSK, N-dsk) to gel-filtered platelets was compared to their ability to support or inhibit ADP-induced aggregation, and to compete with fibrinogen for binding to ADP-stimulated platelets. Pre-X was the only fragment that supported aggregation. All fragments tested except for E derived from fibrinogen (Efg) and Degta bound specifically to the platelets and inhibited ADP-induced aggregation in the presence of fibrinogen. Competitive binding studies with fibrinogen and fragments labelled with different isotopes of iodine, or inhibition of binding of labelled fibrinogen with unlabelled fragments showed that all of the fragments except Efg and Degta were able to compete with fibrinogen for binding. When simultaneous binding of N-dsk and fibrinogen was studied, an increased binding of both ligands was observed probably due to complex formation. The results fully agree with previous findings of binding to immunoprecipitated glycoprotein IIb-IIIa after crossed immunoelectrophoresis. We conclude that the fibrinogen molecule contains at least six sequences responsible for platelet interaction, two in the E domain and two in each of the C-terminal parts of the fibrinogen molecule

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

Energy Technology Data Exchange (ETDEWEB)

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

1990-02-01

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

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

International Nuclear Information System (INIS)

Saussy, D.L. Jr.

1986-01-01

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

Strategies to overcome or circumvent P-glycoprotein mediated multidrug resistance.

Science.gov (United States)

Yuan, Hongyu; Li, Xun; Wu, Jifeng; Li, Jinpei; Qu, Xianjun; Xu, Wenfang; Tang, Wei

2008-01-01

Cancer patients who receive chemotherapy often experience intrinsic or acquired resistance to a broad spectrum of chemotherapeutic agents. The phenomenon, termed multidrug resistance (MDR), is often associated with the over-expression of P-glycoprotein, a transmembrane protein pump, which can enhance efflux of a various chemicals structurally unrelated at the expense of ATP depletion, resulting in decrease of the intracellular cytotoxic drug accumulation. The MDR has been a big threaten to the human health and the war fight for it continues. Although several other mechanisms for MDR are elucidated in recent years, considerable efforts attempting to inverse MDR are involved in exploring P-glycoprotein modulators and suppressing P-glycoprotein expression. In this review, we will report on the recent advances in various strategies for overcoming or circumventing MDR mediated by P-glycoprotein.

Platelets Proteomic Profiles of Acute Ischemic Stroke Patients.

Directory of Open Access Journals (Sweden)

Ozge Cevik

Full Text Available Platelets play a crucial role in the pathogenesis of stroke and antiplatelet agents exist for its treatment and prevention. Through the use of LC-MS based protein expression profiling, platelets from stroke patients were analyzed and then correlated with the proteomic analyses results in the context of this disease. This study was based on patients who post ischemic stroke were admitted to hospital and had venous blood drawn within 24 hrs of the incidence. Label-free protein expression analyses of the platelets' tryptic digest was performed in triplicate on a UPLC-ESI-qTOF-MS/MS system and ProteinLynx Global Server (v2.5, Waters was used for tandem mass data extraction. The peptide sequences were searched against the reviewed homo sapiens database (www.uniprot.org and the quantitation of protein variation was achieved through Progenesis LC-MS software (V4.0, Nonlinear Dynamics. These Label-free differential proteomics analysis of platelets ensured that 500 proteins were identified and 83 of these proteins were found to be statistically significant. The differentially expressed proteins are involved in various processes such as inflammatory response, cellular movement, immune cell trafficking, cell-to-cell signaling and interaction, hematological system development and function and nucleic acid metabolism. The expressions of myeloperoxidase, arachidonate 12-Lipoxygenase and histidine-rich glycoprotein are involved in cellular metabolic processes, crk-like protein and ras homolog gene family member A involved in cell signaling with vitronectin, thrombospondin 1, Integrin alpha 2b, and integrin beta 3 involved in cell adhesion. Apolipoprotein H, immunoglobulin heavy constant gamma 1 and immunoglobulin heavy constant gamma 3 are involved in structural, apolipoprotein A-I, and alpha-1-microglobulin/bikunin precursor is involved in transport, complement component 3 and clusterin is involved in immunity proteins as has been discussed. Our data provides

Peptide-Mediated Platelet Capture at Gold Micropore Arrays.

Science.gov (United States)

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

2016-11-30

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

Nonsense mutation in the glycoprotein Ibα coding sequence associated with Bernard-Soulier syndrome

International Nuclear Information System (INIS)

Ware, J.; Russell, S.R.; Vicente, V.; Scharf, R.E.; Tomer, A.; McMillian, R.; Ruggeri, Z.M.

1990-01-01

Three distinct gene products, the α and β chains of glycoprotein (GP) Ib and GP IX, constitute the platelet membrane GP Ib-IX complex, a receptor for von Willebrand factor and thrombin involved in platelet adhesion and aggregation. Defective function of the GP Ib-IX complex is the hallmark of a rare congenital bleeding disorder of still undefined pathogenesis, the Bernard-Soulier syndrome. The authors have analyzed the molecular basis of the disease in one patient in whom immunoblotting of solubilized platelets demonstrated absence of normal GP Ibα but presence of a smaller immunoreactive species. The truncated polypeptide was also present, along with normal protein, in platelets from the patient's mother and two of his four children. Genetic characterization identified a nucleotide transition changing the Trp-343 codon (TGG) to a nonsense codon (TGA). Such a mutation explains the origin of the smaller GP Ibα, which by lacking half of the sequence on the carboxyl-terminal side, including the transmembrane domain, cannot be properly inserted in the platelet membrane. Both normal and mutant codons were found in the patient, suggesting that he is a compound heterozygote with a still unidentified defect in the other GP Ibα allele. Nonsense mutation and truncated GP Ibα polypeptide were found to cosegregate in four individuals through three generations and were associated with either Bernard-Soulier syndrome or carrier state phenotype. The molecular abnormality demonstrated in this family provides evidence that defective synthesis of GP Ibα alters the membrane expression of the GP Ib-IX complex and may be responsible for Bernard-Soulier syndrome

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

International Nuclear Information System (INIS)

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

1993-01-01

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

Extending The Shelf Life Of Blood Platelets

Science.gov (United States)

Surgenor, Douglas M.

1988-01-01

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

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

LENUS (Irish Health Repository)

Power, Karen A

2009-01-01

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

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

Science.gov (United States)

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

2014-01-01

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

Indium-111 labelled platelets: experimental and clinical studies

International Nuclear Information System (INIS)

Gjerloeff Schmidt, K.

1985-10-01

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

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

Science.gov (United States)

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

2016-10-01

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

Murine and human b locus pigmentation genes encode a glycoprotein (gp75) with catalase activity

International Nuclear Information System (INIS)

Halaban, R.; Moellmann, G.

1990-01-01

Melanogenesis is regulated in large part by tyrosinase, and defective tyrosinase leads to albinism. The mechanisms for other pigmentation determinants (e.g., those operative in tyrosinase-positive albinism and in murine coat-color mutants) are not yet known. One murine pigmentation gene, the brown (b) locus, when mutated leads to a brown (b/b) or hypopigmentated (B lt /B lt ) coat versus the wild-type black (B/B). The authors show that the b locus codes for a glycoprotein with the activity of a catalase (catalase B). Only the c locus protein is a tyrosinase. Because peroxides may be by-products of melanogenic activity and hydrogen peroxide in particular is known to destroy melanin precursors and melanin, they conclude that pigmentation is controlled not only by tyrosinase but also by a hydroperoxidase. The studies indicate that catalase B is identical with gp75, a known human melanosomal glycoprotein; that the b mutation is in a heme-associated domain; and that the B lt mutation renders the protein susceptible to rapid proteolytic degradation

Ebola virus glycoprotein-mediated anoikis of primary human cardiac microvascular endothelial cells

International Nuclear Information System (INIS)

Ray, Ratna B.; Basu, Arnab; Steele, Robert; Beyene, Aster; McHowat, Jane; Meyer, Keith; Ghosh, Asish K.; Ray, Ranjit

2004-01-01

Ebola virus glycoprotein (EGP) has been implicated for the induction of cytotoxicity and injury in vascular cells. On the other hand, EGP has also been suggested to induce massive cell rounding and detachment from the plastic surface by downregulating cell adhesion molecules without causing cytotoxicity. In this study, we have examined the cytotoxic role of EGP in primary endothelial cells by transduction with a replication-deficient recombinant adenovirus expressing EGP (Ad-EGP). Primary human cardiac microvascular endothelial cells (HCMECs) transduced with Ad-EGP displayed loss of cell adhesion from the plastic surface followed by cell death. Transfer of conditioned medium from EGP-transduced HCMEC into naive cells did not induce loss of adhesion or cell death, suggesting that EGP needs to be expressed intracellularly to exert its cytotoxic effect. Subsequent studies suggested that HCMEC death occurred through apoptosis. Results from this study shed light on the EGP-induced anoikis in primary human cardiac endothelial cells, which may have significant pathological consequences

Measurement of platelet aggregation, independently of patient platelet count

DEFF Research Database (Denmark)

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

2017-01-01

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

21 CFR 864.6650 - Platelet adhesion test.

Science.gov (United States)

2010-04-01

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

Radiolabeled platelets

International Nuclear Information System (INIS)

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

1986-01-01

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

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

International Nuclear Information System (INIS)

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

1979-01-01

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

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

Science.gov (United States)

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

1980-10-15

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

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

Science.gov (United States)

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

2015-02-01

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

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

International Nuclear Information System (INIS)

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

1990-01-01

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

Glycoprotein on cell surfaces

International Nuclear Information System (INIS)

Muramatsu, T.

1975-01-01

There are conjugated polysaccharides in cell membranes and outside of animal cells, and they play important role in the control of cell behavior. In this paper, the studies on the glycoprotein on cell surfaces are reported. It was found that the glycoprotein on cell surfaces have both N-glycoside type and O-glycoside type saccharic chains. Therefore it can be concluded that the basic structure of the saccharic chains in the glycoprotein on cell surfaces is similar to that of blood serum and body fluid. The main glycoprotein in the membranes of red blood corpuscles has been studied most in detail, and it also has both types of saccharic chains. The glycoprotein in liver cell membranes was found to have only the saccharic chains of acid type and to be in different pattern from that in endoplasmic reticula and nuclear membranes, which also has the saccharic chains of neutral type. The structure of the saccharic chains of H-2 antigen, i.e. the peculiar glycoprotein on the surfaces of lymph system cells, has been studied, and it is similar to the saccharic chains of glycoprotein in blood serum. The saccharic chain structures of H-2 antigen and TL antigen are different. TL, H-2 (D), Lna and H-2 (K) are the glycoprotein on cell surfaces, and are independent molecules. The analysis of the saccharic chain patterns on cell surfaces was carried out, and it was shown that the acid type saccharic chains were similar to those of ordinary glycoprotein, because the enzyme of pneumococci hydrolyzed most of the acid type saccharic chains. The change of the saccharic chain patterns of glycoprotein on cell surfaces owing to canceration and multiplication is complex matter. (Kako, I.)

Platelet glycoprotein IIb/IIIa polymorphism HPA-3 b/b is associated with increased risk of ischemic stroke in patients under 60 years of age.

Science.gov (United States)

Duan, Hao; Cai, Yan; Sun, Xiaojiang

2012-01-01

The role of genetic risk factors in ischemic stroke is unclear. Platelet glycoprotein IIb/IIIa (GpIIb-IIIa) has been implicated in the pathogenesis of ischemic stroke. We sought to evaluate the relationship between the GpIIb/IIIa complex gene polymorphism and ischemic stroke. We investigated the association of the GpIIb/IIIa complex gene polymorphism with stroke risk in 306 patients with acute ischemic stroke and 266 control subjects by determining the GpIIb and GpIIIa genotype from leukocyte DNA by polymerase chain reaction (PCR) followed by FokI and ScrFI digestion, respectively. Compared with controls, more patients presented with coronary heart disease, hypertension, smoking history, and diabetes. In addition, the patients had higher levels of cholesterol and glucose compared with the control subjects. All donors in the GpIIIa (n=572) group expressed the GpIIIa PlA1 (HPA-1 aa) phenotype. There were no significant differences between the HPA-3 genotype (GpIIb) patient distribution (aa=39.9%, ab=41.4%, bb=28.7%) and healthy control subjects (aa=36.1%, ab=35.0%, bb=28.9%) (P=0.580). Among study participants ischemic stroke >2-fold (P=0.008). The GpIIb Ile/Ser843 gene polymorphism is associated with ischemic stroke among young and middle-aged adults (ischemic stroke.

Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo

Science.gov (United States)

von Brühl, Marie-Luise; Stark, Konstantin; Steinhart, Alexander; Chandraratne, Sue; Konrad, Ildiko; Lorenz, Michael; Khandoga, Alexander; Tirniceriu, Anca; Coletti, Raffaele; Köllnberger, Maria; Byrne, Robert A.; Laitinen, Iina; Walch, Axel; Brill, Alexander; Pfeiler, Susanne; Manukyan, Davit; Braun, Siegmund; Lange, Philipp; Riegger, Julia; Ware, Jerry; Eckart, Annekathrin; Haidari, Selgai; Rudelius, Martina; Schulz, Christian; Echtler, Katrin; Brinkmann, Volker; Schwaiger, Markus; Preissner, Klaus T.; Wagner, Denisa D.; Mackman, Nigel; Engelmann, Bernd

2012-01-01

Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features. PMID:22451716

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.

The Neutralizing Linear Epitope of Human Herpesvirus 6A Glycoprotein B Does Not Affect Virus Infectivity.

Science.gov (United States)

Wakata, Aika; Kanemoto, Satoshi; Tang, Huamin; Kawabata, Akiko; Nishimura, Mitsuhiro; Jasirwan, Chyntia; Mahmoud, Nora Fahmy; Mori, Yasuko

2018-03-01

Human herpesvirus 6A (HHV-6A) glycoprotein B (gB) is a glycoprotein consisting of 830 amino acids and is essential for the growth of the virus. Previously, we reported that a neutralizing monoclonal antibody (MAb) called 87-y-13 specifically reacts with HHV-6A gB, and we identified its epitope residue at asparagine (Asn) 347 on gB. In this study, we examined whether the epitope recognized by the neutralizing MAb is essential for HHV-6A infection. We constructed HHV-6A bacterial artificial chromosome (BAC) genomes harboring substitutions at Asn347, namely, HHV-6A BACgB(N347K) and HHV-6A BACgB(N347A). These mutant viruses could be reconstituted and propagated in the same manner as the wild type and their revertants, and MAb 87-y-13 could not inhibit infection by either mutant. In a cell-cell fusion assay, Asn at position 347 on gB was found to be nonessential for cell-cell fusion. In addition, in building an HHV-6A gB homology model, we found that the epitope of the neutralizing MAb is located on domain II of gB and is accessible to solvents. These results indicate that Asn at position 347, the linear epitope of the neutralizing MAb, does not affect HHV-6A infectivity. IMPORTANCE Glycoprotein B (gB) is one of the most conserved glycoproteins among all herpesviruses and is a key factor for virus entry. Therefore, antibodies targeted to gB may neutralize virus entry. Human herpesvirus 6A (HHV-6A) encodes gB, which is translated to a protein of about 830 amino acids (aa). Using a monoclonal antibody (MAb) for HHV-6A gB, which has a neutralizing linear epitope, we analyzed the role of its epitope residue, N347, in HHV-6A infectivity. Interestingly, this gB linear epitope residue, N347, was not essential for HHV-6A growth. By constructing a homology model of HHV-6A gB, we found that N347 was located in the region corresponding to domain II. Therefore, with regard to its neutralizing activity against HHV-6A infection, the epitope on gB might be exposed to solvents

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

Science.gov (United States)

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

2016-07-01

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

Blood platelet kinetics and platelet transfusion

OpenAIRE

Aster, Richard H.

2013-01-01

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

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

Science.gov (United States)

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

2012-05-01

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

Association of a variable number of tandem repeats (VNTR) in glycoprotein Ib alpha and HPA-2 alloantigens

NARCIS (Netherlands)

Simsek, S.; Bleeker, P. M.; van der Schoot, C. E.; von dem Borne, A. E.

1994-01-01

The human platelet alloantigen HPA-2(Koa/Kob) system is involved in two clinical syndromes, neonatal alloimmune thrombocytopenia and platelet transfusion refractoriness. We have previously described that the human platelet alloantigens HPA-2a(Kob) and HPA-2b(Koa), are caused by a Thr145Met amino

Platelet-derived growth factor receptors in the human central nervous system : autoradiographic distribution and receptor densities in multiple sclerosis

NARCIS (Netherlands)

De Keyser, J; Wilczak, N

1997-01-01

Platelet derived growth factor (PDGF) receptors were studied in postmortem adult human brain and cervical spinal cord using autoradiography with human recombinant I-125-PDGF-BB. PDGF-BB binds to the three different dimers of PDGF receptors (alpha alpha, alpha beta and beta beta) PDGF receptors were

Basic study of platelet labeling with 111In-oxine

International Nuclear Information System (INIS)

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

1981-01-01

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

Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

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Kai Xu

Full Text Available The henipaviruses, represented by Hendra (HeV and Nipah (NiV viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4 was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.

Platelet cyclooxygenase expression in normal dogs.

Science.gov (United States)

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

2011-01-01

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

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

NARCIS (Netherlands)

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

2005-01-01

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

Thyroid hormone upregulates zinc-α2-glycoprotein production in the liver but not in adipose tissue.

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Rafael Simó

Full Text Available Overproduction of zinc-α2-glycoprotein by adipose tissue is crucial in accounting for the lipolysis occurring in cancer cachexia of certain malignant tumors. The main aim of this study was to explore whether thyroid hormone could enhance zinc-α2-glycoprotein production in adipose tissue. In addition, the regulation of zinc-α2-glycoprotein by thyroid hormone in the liver was investigated. We performed in vitro (HepG2 cells and primary human adipocytes and in vivo (C57BL6/mice experiments addressed to examine the effect of thyroid hormone on zinc-α2-glycoprotein production (mRNA and protein levels in liver and visceral adipose tissue. We also measured the zinc-α2-glycoprotein serum levels in a cohort of patients before and after controlling their hyperthyroidism. Our results showed that thyroid hormone up-regulates zinc-α2-glycoprotein production in HepG2 cells in a dose-dependent manner. In addition, the zinc-α2-glycoprotein proximal promoter contains functional thyroid hormone receptor binding sites that respond to thyroid hormone treatment in luciferase reporter gene assays in HepG2 cells. Furthermore, zinc-α2-glycoprotein induced lipolysis in HepG2 in a dose-dependent manner. Our in vivo experiments in mice confirmed the up-regulation of zinc-α2-glycoprotein induced by thyroid hormone in the liver, thus leading to a significant increase in zinc-α2-glycoprotein circulating levels. However, thyroid hormone did not regulate zinc-α2-glycoprotein production in either human or mouse adipocytes. Finally, in patients with hyperthyroidism a significant reduction of zinc-α2-glycoprotein serum levels was detected after treatment but was unrelated to body weight changes. We conclude that thyroid hormone up-regulates the production of zinc-α2-glycoprotein in the liver but not in the adipose tissue. The neutral effect of thyroid hormones on zinc-α2-glycoprotein expression in adipose tissue could be the reason why zinc-α2-glycoprotein is not

Thyroid hormone upregulates zinc-α2-glycoprotein production in the liver but not in adipose tissue.

Science.gov (United States)

Simó, Rafael; Hernández, Cristina; Sáez-López, Cristina; Soldevila, Berta; Puig-Domingo, Manel; Selva, David M

2014-01-01

Overproduction of zinc-α2-glycoprotein by adipose tissue is crucial in accounting for the lipolysis occurring in cancer cachexia of certain malignant tumors. The main aim of this study was to explore whether thyroid hormone could enhance zinc-α2-glycoprotein production in adipose tissue. In addition, the regulation of zinc-α2-glycoprotein by thyroid hormone in the liver was investigated. We performed in vitro (HepG2 cells and primary human adipocytes) and in vivo (C57BL6/mice) experiments addressed to examine the effect of thyroid hormone on zinc-α2-glycoprotein production (mRNA and protein levels) in liver and visceral adipose tissue. We also measured the zinc-α2-glycoprotein serum levels in a cohort of patients before and after controlling their hyperthyroidism. Our results showed that thyroid hormone up-regulates zinc-α2-glycoprotein production in HepG2 cells in a dose-dependent manner. In addition, the zinc-α2-glycoprotein proximal promoter contains functional thyroid hormone receptor binding sites that respond to thyroid hormone treatment in luciferase reporter gene assays in HepG2 cells. Furthermore, zinc-α2-glycoprotein induced lipolysis in HepG2 in a dose-dependent manner. Our in vivo experiments in mice confirmed the up-regulation of zinc-α2-glycoprotein induced by thyroid hormone in the liver, thus leading to a significant increase in zinc-α2-glycoprotein circulating levels. However, thyroid hormone did not regulate zinc-α2-glycoprotein production in either human or mouse adipocytes. Finally, in patients with hyperthyroidism a significant reduction of zinc-α2-glycoprotein serum levels was detected after treatment but was unrelated to body weight changes. We conclude that thyroid hormone up-regulates the production of zinc-α2-glycoprotein in the liver but not in the adipose tissue. The neutral effect of thyroid hormones on zinc-α2-glycoprotein expression in adipose tissue could be the reason why zinc-α2-glycoprotein is not related to weight

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.

Stimulus-response coupling in platelets

International Nuclear Information System (INIS)

Huang, E.M.

1986-01-01

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

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

International Nuclear Information System (INIS)

Wu Guoxin; Li Jianyong; Ruan Changgeng

1991-08-01

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

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

NARCIS (Netherlands)

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

1988-01-01

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

LIM kinase-1 selectively promotes glycoprotein Ib-IX–mediated TXA2 synthesis, platelet activation, and thrombosis

OpenAIRE

Estevez, Brian; Stojanovic-Terpo, Aleksandra; Delaney, M. Keegan; O’Brien, Kelly A.; Berndt, Michael C.; Ruan, Changgeng; Du, Xiaoping

2013-01-01

Role for LIMK1 in GPIb-IX–dependent cPLA2 activation, TXA2 synthesis, and platelet activation independent of its role in actin polymerization.LIMK1 is important in arterial thrombosis in vivo but appears to be dispensable for hemostasis, suggesting a new antithrombotic target.

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

International Nuclear Information System (INIS)

Mieno, Masayuki; Isaka, Yoshinori; Kimura, Kazutumi

1990-01-01

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

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

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D. D. Zhernossekov

2017-04-01

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

Effects of drugs on platelet function.

Science.gov (United States)

Morse, E E

1977-01-01

Numerous drugs and chemicals affect the function of human blood platelets. The mechanism of action of some medications is partly understood. Aspirin is the most frequently involved drug. It appears to interfere with the platelet release reaction by acetylation of a platelet membrane protein which may be involved in the synthesis of prostaglandins. Other anti-inflammatory drugs, including indomethacin, phenylbutazone, ibuprophen (Motrin) and clonixin, also interfere with the release reaction but have a shorter acting course than aspirin. Some drugs stimulate adenylcyclase (gliclazide) or block phosphodiesterase, (dipyridamole, caffeine) both of which actions lead to an increase in adenosine cyclic 3':5' monophosphate (cAMP) and decrease aggregation by adenosine diphosphate (ADP). These interactions should be known to clinical scientists since patients using these medicaments may manifest abnormal platelet function tests in the laboratory and mild hemorrhagic syndromes in the clinic.

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

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Andreas Bayer

2017-01-01

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

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

Science.gov (United States)

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

2017-01-01

Platelet-released growth factors (PRGF) and its related clinically used formulations (e.g., Vivostat Platelet-Rich Fibrin (PRF®)) contain a variety of chemokines, cytokines, and growth factors and are therefore used to support healing of chronic, hard-to-heal, or infected wounds. Human beta-defensin-3 (hBD-3) is an antimicrobial peptide inducibly expressed in human keratinocytes especially upon wounding. The potent antimicrobial activity of hBD-3 together with its wound closure-promoting activities suggests that hBD-3 may play a crucial role in wound healing. Therefore, we analyzed the influence of PRGF on hBD-3 expression in human primary keratinocytes in vitro. In addition, we investigated the influence of Vivostat PRF on hBD-3 expression in artificially generated human skin wounds in vivo. PRGF treatment of primary keratinocytes induced a significant, concentration- and time-dependent increase in hBD-3 gene expression which was partially mediated by the epidermal growth factor receptor (EGFR). In line with these cell culture data, in vivo experiments revealed an enhanced hBD-3 expression in experimentally produced human wounds after the treatment with Vivostat PRF. Thus, the induction of hBD-3 may contribute to the beneficial effects of thrombocyte concentrate lysates in the treatment of chronic or infected wounds. PMID:28811680

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

Science.gov (United States)

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

2014-01-27

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

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...

A novel approach to optimal blood platelets logistics

NARCIS (Netherlands)

Smit Sibinga, C.; Dijk, van N.M.; Haijema, R.; Wal, van der J.

2006-01-01

The production and inventory management of blood platelets at blood establishments and hospital blood banks is a service problem of general human interest. Shortages or inadequate supplies may pur lives at risk and thus have to be managed and kept to a minimum. However, platelets have a limited

Labelled antibody techniques in glycoprotein estimation

International Nuclear Information System (INIS)

Hazra, D.K.; Ekins, R.P.; Edwards, R.; Williams, E.S.

1977-01-01

The problems in the radioimmunoassay of the glycoprotein hormones (pituitary LH, FSH and TSH and human chlorionic gonadotrophin HGG) are reviewed viz: limited specificity and sensitivity in the clinical context, interpretation of disparity between bioassay and radioimmunoassay, and interlaboratory variability. The advantages and limitations of the labelled antibody techniques - classical immonoradiometric methods and 2-site or 125 I-anti-IgG indirect labelling modifications are reviewed in general, and their theoretical potential in glycoprotein assays examined in the light of previous work. Preliminary experiments in the development of coated tube 2-site assay for glycoproteins using 125 I anti-IgG labelling are described, including conditions for maximizing solid phase extraction of the antigen, iodination of anti-IgG, and assay conditions such as effects of temperature of incubation with antigen 'hormonefree serum', heterologous serum and detergent washing. Experiments with extraction and antigen-specific antisera raised in the same or different species are described as exemplified by LH and TSH assay systems, the latter apparently promising greater sensitivity than radioimmunoassay. Proposed experimental and mathematical optimisation and validation of the method as an assay system is outlined, and the areas for further work delineated. (orig.) [de

Distribution of primaquine in human blood: Drug-binding to alpha 1-glycoprotein

International Nuclear Information System (INIS)

Kennedy, E.; Frischer, H.

1990-01-01

To clarify the distribution of the antimalarial primaquine in human blood, we measured the drug separately in the liquid, cellular, and ultrafiltrate phases. Washed red cells resuspended at a hematocrit of 0.4 were exposed to a submaximal therapeutic level of 250 ng/ml of carbon 14-labeled primaquine. The tracer was recovered quantitatively in separated plasma and red cells. Over 75% of the total labeled drug was found in red cells suspended in saline solution, but only 10% to 30% in red cells suspended in plasma. The plasma effect was not mediated by albumin. Studies with alpha 1-acid glycoprotein (AGP), tris(2-butoxyethyl)phosphate, an agent that displaces AGP-bound drugs, and cord blood known to have decreased AGP established that primaquine binds to physiologic amounts of the glycoprotein in plasma. Red cell primaquine concentration increased linearly as AGP level fell and as the free drug fraction rose. We suggest that clinical blood levels of primaquine include the red cell fraction or whole blood level because (1) erythrocytic primaquine is a sizable and highly variable component of the total drug in blood; (2) this component reflects directly the free drug in plasma, and inversely the extent of binding to AGP; (3) the amount of free primaquine may influence drug transport into specific tissues in vivo; and (4) fluctuations of AGP, an acute-phase reactant that increases greatly in patients with malaria and other infections, markedly affect the partition of primaquine in blood. Because AGP binds many basic drugs, unrecognized primaquine-drug interactions may exist

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

Energy Technology Data Exchange (ETDEWEB)

Dewanjee, M.K.; Kapadvanjwala, Mansoor; Ruzius, Kees; Serafini, A.N.; Zilleruelo, G.E.; Sfakianakis, G.N. (Miami Univ., FL (United States). School of Medicine Althin CD-Medical Inc., Miami Lakes, FL (United States))

1993-07-01

The platelet thromobogenicity of a hemodialyzer was quantified with [sup 99m]Tc- and [sup 111]In-labeled platelets. The platelets collected from blood of Beagle dogs, Yorkshire pigs and human volunteers were labeled with [sup 111]in-tropolone (detergent-free) and [sup 99m]Tc-HMPAO. Hemodialysis was performed with a hollow-fiber dialyzer (HFD) in a flow-loop, the temperature of which was maintained at 37[sup o]C, with flow-rates of 7, 150 and 270 mL/min; after dialysis, the HFD radioactivity was measured with an ionization chamber and imaged with a [gamma]-camera. The radioactivity of samples of hollow-fibers taken from the top, middle and bottom of the dialyzer was determined with a [gamma]-counter. The mean values of hemodialyzer-adherent platelet radioactivity were calculated for both radionuclides. The canine platelets were found to be more thrombogenic than porcine and human platelets. The adhesivity of porcine platelets to the biomaterial (cellulose-acetate) of the dialyzer approximated that of human platelets. The [sup 99m]Tc label underestimated the thrombus formation (P < 0.01 ). The dynamic processes of thrombosis and embolization from the hemodialyzer resulted in the large standard deviations around the mean values of the adherent thrombus. In spite of this limitation of the dynamic pathology, the quantitation of comparative throbogenicity with [sup 111]In- and [sup 99m]Tc-labeled platelets suggests that both radionuclides could be used for measurement of device-induced thrombogenicity and may provide an estimation of prosthesis-induced thrombogenicity of human platelets from animal studies. (Author).

Affinity column for purification of the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor

International Nuclear Information System (INIS)

Venton, D.L.; Arora, S.K.; Kim, S.O.; Lim, C.T.; Le Breton, G.C.

1987-01-01

The TXA 2 /PGH 2 receptor antagonist, 13-azaprostanoic acid (13-APA), was synthesized and used as the immobilized ligand in the affinity column purification of the 13-APA/U46619 binding component in human platelets. Diazo coupling of the ligand to the phenol of this tyr-gly-gly-NH-(CO)-O-Sepharose gave the affinity column material. Isolated platelet membranes were solubilized with detergent, applied directly to the affinity column and the eluate collected as 6 x 70 ml fractions. For each fraction, protein concentration and specific 3 H-13-APA/numberH-U46619 binding were determined. The majority of the applied protein (>98%) eluted in fraction number1. However, the specific 13-APA/U46619 binding per mg of protein was localized in fractions number4 and number5, representing approximately a 500-fold purification of this binding component. These results suggest that the platelet TXA 2 /PGH 2 receptor protein is retarded by this column, and that starting from crude, solubilized platelet membranes, a single pass through the column provides a 500-fold purification of the receptor

Human histologic evaluation of anorganic bovine bone mineral combined with recombinant human platelet-derived growth factor BB in maxillary sinus augmentation: case series study.

Science.gov (United States)

Nevins, Myron; Garber, David; Hanratty, James J; McAllister, Bradley S; Nevins, Marc L; Salama, Maurice; Schupbach, Peter; Wallace, Steven; Bernstein, Simon M; Kim, David M

2009-12-01

The objective of this proof-of-principle study was to examine the potential for improved bone regenerative outcomes in maxillary sinus augmentation procedures when recombinant human platelet-derived growth factor BB (0.3 mg/mL) is combined with particulate anorganic bovine bone mineral. The surgical outcomes in all treated sites were uneventful at 6 to 8 months, with sufficient regenerated bone present to allow successful placement of maxillary posterior implants. Large areas of dense, well-formed lamellar bone were seen throughout the intact core specimens in more than half of the grafted sites. Abundant numbers of osteoblasts were noted in concert with significant osteoid in all sites, indicating ongoing osteogenesis. A number of cores demonstrated efficient replacement of the normally slowly resorbing anorganic bovine bone mineral matrix particles with newly formed bone when the matrix was saturated with recombinant human platelet-derived growth factor BB.

Heterogeneity of rabbit platelets

International Nuclear Information System (INIS)

Karpatkin, S.

1978-01-01

Rabbits were injected intravenously with a cohort platelet label, 75 Se-selenomethionine. Platelet-rich plasma was separated into five different platelet density fractions on each of seven days by repetitively centrifuging the same sample of platelet-rich plasma at increasing gravitational force. The heaviest platelet sediment fraction was enriched with larger platelets. The lightest platelet sediment fraction was enriched with smaller platelets. Incorporation of isotope into the heaviest platelet fraction was considerably greater than incorporation into the lightest platelet fraction. The mean platelet survival of the lightest two fractions was significantly shorter than that of the heaviest three fractions. SDS-polyacrylamide gel electrophoresis of the platelet cell sap generally revealed 10 prominent protein bands for the heaviest platelet fractions. The lightest platelet fraction had six absent to markedly diminished platelet proteins. The data are compatible with two models, (1) heavy-large platelets are, on average, young platelets which become lighter-smaller platelets while losing platelet membranes and cell sap components with time. (2) Heavy-large platelets and light-small platelets are produced independently by specific megakarocytes. The heavy-large platelets incorporate more isotope that lighter-smaller platelets (possibly because of their megakarocyte precursor). However, they are released earlier into the circulation than lighter-smaller platelets and are therefore younger platelets. The light-smaller platelets which are released later into the circulation have a shorter survival. (author)

Platelets as autonomous drones for hemostatic and immune surveillance.

Science.gov (United States)

Li, Jackson LiangYao; Zarbock, Alexander; Hidalgo, Andrés

2017-07-18

Platelets participate in many important physiological processes, including hemostasis and immunity. However, despite their broad participation in these evolutionarily critical roles, the anucleate platelet is uniquely mammalian. In contrast with the large nucleated equivalents in lower vertebrates, we find that the design template for the evolutionary specialization of platelets shares remarkable similarities with human-engineered unmanned aerial vehicles in terms of overall autonomy, maneuverability, and expendability. Here, we review evidence illustrating how platelets are uniquely suited for surveillance and the manner in which they consequently provide various types of support to other cell types. © 2017 Li et al.

Matrix metalloproteinase content and activity in low-platelet, low-leukocyte and high-platelet, high-leukocyte platelet rich plasma (PRP) and the biologic response to PRP by human ligament fibroblasts.

Science.gov (United States)

Pifer, Matthew A; Maerz, Tristan; Baker, Kevin C; Anderson, Kyle

2014-05-01

Recent work has shown the presence of catabolic cytokines in platelet-rich plasma (PRP), but little is known about endogenous catabolic proteases such as matrix metalloproteinases (MMPs). Hypothesis/ To quantify MMP content in 2 commercially available PRP preparation systems: Arthrex Double Syringe System autologous conditioned plasma (ACP) and Biomet GPS (GPS). The hypothesis was that MMPs are actively secreted from PRP immediately after preparation. Controlled laboratory study. PRP was prepared using either ACP (low platelet, low leukocyte) or GPS (high platelet, high leukocyte). MMP-2, MMP-3, and MMP-9 concentrations were measured using multiplex enzyme-linked immunosorbent assays for up to 6 days in 2 donors, and MMP activity was measured in 3 donors using kinetic activity kits able to detect the enzymatic cleavage of a fluorogenic peptide. Human ligament fibroblasts were cultured and exposed to both ACP and GPS from 1 donor each. MMP-2, -3, and -9 concentrations were assayed in culture media at 24 and 48 hours after exposure. GPS exhibited higher total MMP-2, -3, and -9 concentrations for up to 144 hours of release, while ACP had higher platelet-normalized MMP-2 and MMP-3 concentrations. GPS had significantly higher total and endogenous MMP-2 activity (P = .004 and .014, respectively), MMP-3 activity (P = .020 and .015, respectively), and MMP-9 activity (P = .004 and .002, respectively) compared with ACP. Once normalized to platelet count, differences in MMP activity were not significant between ACP and GPS. Compared with controls, cells stimulated with interleukin-1 beta (IL-1β) and treated with ACP showed significantly higher fold changes of MMP-2 (P = .001) and MMP-3 (P = .003) concentrations at 24 hours than did cells treated with GPS. Total MMP-9 content was higher in the media of GPS-treated, IL-1β-stimulated cells compared with ACP-treated cells (P = .001). At 48 hours, IL-1β-stimulated cells treated with GPS exhibited higher fold changes of MMP-2

Platelet Arachidonic Acid Deficiency May Contribute to Abnormal Platelet Function During Parenteral Fish Oil Monotherapy in a Piglet Model.

Science.gov (United States)

Turner, Justine M; Field, Catherine J; Goruk, Sue; Wizzard, Pamela; Dicken, Bryan J; Bruce, Aisha; Wales, Paul W

2016-05-01

Fish oil monotherapy has been an advance for treating intestinal failure-associated liver disease (IFALD). However, such patients are at risk of bleeding complications from liver disease and because fish oil can inhibit thrombosis. We have previously reported abnormal platelet function in neonatal piglets given fish oil monotherapy during parenteral nutrition (PN). The purpose of this study was to determine if abnormal fatty acid composition of the platelets could explain the prior observed antiplatelet effect. Neonatal piglets were assigned to 2 treatments: PN with fish oil monotherapy (FO; n = 4) or PN with soy oil (SO; n = 5). On day 14, plasma was collected and platelets isolated by centrifuging. The fatty acid content in plasma and platelet plug were measured using gas liquid chromatography and compared with controls (CON; n = 5). The arachidonic acid (AA) content in the FO group was on average half that of the SO group, in both the platelets (FO, 3.5% vs SO, 7.6%; P = .021; CON, 4.5%-11%) and the plasma (FO, 3.8% vs SO, 9.2%; P = .002; CON, 6.1%-9.5%). No bleeding complications were observed for any piglets during PN treatment. Using platelet mapping, we have previously shown that neonatal piglets given fish oil monotherapy have abnormal platelet function in the AA pathway. This report demonstrates that such an abnormality can be explained by platelet AA deficiency. Platelet mapping and platelet fatty acid analysis should be undertaken in human infants treated with fish oil monotherapy during PN. © 2015 American Society for Parenteral and Enteral Nutrition.

Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients.