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Sample records for platelet function assay

  1. Normalization methods in time series of platelet function assays

    PubMed Central

    Van Poucke, Sven; Zhang, Zhongheng; Roest, Mark; Vukicevic, Milan; Beran, Maud; Lauwereins, Bart; Zheng, Ming-Hua; Henskens, Yvonne; Lancé, Marcus; Marcus, Abraham

    2016-01-01

    Abstract Platelet function can be quantitatively assessed by specific assays such as light-transmission aggregometry, multiple-electrode aggregometry measuring the response to adenosine diphosphate (ADP), arachidonic acid, collagen, and thrombin-receptor activating peptide and viscoelastic tests such as rotational thromboelastometry (ROTEM). The task of extracting meaningful statistical and clinical information from high-dimensional data spaces in temporal multivariate clinical data represented in multivariate time series is complex. Building insightful visualizations for multivariate time series demands adequate usage of normalization techniques. In this article, various methods for data normalization (z-transformation, range transformation, proportion transformation, and interquartile range) are presented and visualized discussing the most suited approach for platelet function data series. Normalization was calculated per assay (test) for all time points and per time point for all tests. Interquartile range, range transformation, and z-transformation demonstrated the correlation as calculated by the Spearman correlation test, when normalized per assay (test) for all time points. When normalizing per time point for all tests, no correlation could be abstracted from the charts as was the case when using all data as 1 dataset for normalization. PMID:27428217

  2. Evaluation of a BED-SIDE platelet function assay: performance and clinical utility.

    PubMed

    Lau, Wei C; Walker, C Ty; Obilby, David; Wash, Mark M; Carville, David G M; Guyer, Kirk E; Bates, Eric R

    2002-01-01

    Platelets have a pivotal role in the initial defense against insult to the vasculature and are also recognized of critical importance in the acute care settings of percutaneous coronary intervention and cardiopulmonary bypass. In these environments both platelet count and function may be markedly compromised. Unfortunately, current assays to evaluate the parameters of platelet count and function are of limited utility for bed-side testing. Moreover, it is suggested that there may be significant inter patient variation in response to antiplatelet therapy that may be exacerbated by other agents (e.g. heparin) that are routinely administered during cardiac intervention. Here we describe a practical, rapid and user-friendly whole blood platelet function assay that has been developed for use in bed-side settings. Platelet agonists were formulated with an anticoagulant and lyophilized in blood collection tubes standardised to receive a l mL fresh whole blood sample. In the presence of an agonist, platelets are activated and interact (aggregate). Using traditional cell counting principles, non-aggregated platelets are counted whereas aggregated platelets are not. The percentage (%) of functional platelets in reference to a baseline tube may then be determined. Results are available within four minutes. Platelet aggregation in whole blood demonstrated good correlation with turbidometric aggregometry for both ADP (r=0.91) and collagen (r=0.88). Moreover, in clinical settings where antiplatelet agents were administered, this rapid, bed-side, platelet function assay demonstrated utility in monitoring patient response to these therapies. This novel bed-side assay of platelet function is extremely suitable for the clinical environment with a rapid turn-around time. In addition, it provides a full haematology profile, including platelet count, and should permit enhancement of transfusion and interventional decisions. PMID:17890800

  3. High content evaluation of shear dependent platelet function in a microfluidic flow assay.

    PubMed

    Hansen, Ryan R; Wufsus, Adam R; Barton, Steven T; Onasoga, Abimbola A; Johnson-Paben, Rebecca M; Neeves, Keith B

    2013-02-01

    The high blood volume requirements and low throughput of conventional flow assays for measuring platelet function are unsuitable for drug screening and clinical applications. In this study, we describe a microfluidic flow assay that uses 50 μL of whole blood to measure platelet function on ~300 micropatterned spots of collagen over a range of physiologic shear rates (50-920 s(-1)). Patterning of collagen thin films (CTF) was achieved using a novel hydrated microcontact stamping method. CTF spots of 20, 50, and 100 μm were defined on glass substrates and consisted of a dense mat of nanoscale collagen fibers (3.74 ± 0.75 nm). We found that a spot size of greater than 20 μm was necessary to support platelet adhesion under flow, suggesting a threshold injury size is necessary for stable platelet adhesion. Integrating 50 μm CTF microspots into a multishear microfluidic device yielded a high content assay from which we extracted platelet accumulation metrics (lag time, growth rate, total accumulation) on the spots using Hoffman modulation contrast microscopy. This method has potential broad application in identifying platelet function defects and screening, monitoring, and dosing antiplatelet agents. PMID:23001359

  4. High content evaluation of shear dependent platelet function in a microfluidic flow assay.

    PubMed

    Hansen, Ryan R; Wufsus, Adam R; Barton, Steven T; Onasoga, Abimbola A; Johnson-Paben, Rebecca M; Neeves, Keith B

    2013-02-01

    The high blood volume requirements and low throughput of conventional flow assays for measuring platelet function are unsuitable for drug screening and clinical applications. In this study, we describe a microfluidic flow assay that uses 50 μL of whole blood to measure platelet function on ~300 micropatterned spots of collagen over a range of physiologic shear rates (50-920 s(-1)). Patterning of collagen thin films (CTF) was achieved using a novel hydrated microcontact stamping method. CTF spots of 20, 50, and 100 μm were defined on glass substrates and consisted of a dense mat of nanoscale collagen fibers (3.74 ± 0.75 nm). We found that a spot size of greater than 20 μm was necessary to support platelet adhesion under flow, suggesting a threshold injury size is necessary for stable platelet adhesion. Integrating 50 μm CTF microspots into a multishear microfluidic device yielded a high content assay from which we extracted platelet accumulation metrics (lag time, growth rate, total accumulation) on the spots using Hoffman modulation contrast microscopy. This method has potential broad application in identifying platelet function defects and screening, monitoring, and dosing antiplatelet agents.

  5. Radioimmune assay of human platelet prostaglandin synthetase

    SciTech Connect

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

    1982-02-01

    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/sub 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 (/sup 125/I)-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the (/sup 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/sup 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.

  6. Normalization methods in time series of platelet function assays: A SQUIRE compliant study.

    PubMed

    Van Poucke, Sven; Zhang, Zhongheng; Roest, Mark; Vukicevic, Milan; Beran, Maud; Lauwereins, Bart; Zheng, Ming-Hua; Henskens, Yvonne; Lancé, Marcus; Marcus, Abraham

    2016-07-01

    Platelet function can be quantitatively assessed by specific assays such as light-transmission aggregometry, multiple-electrode aggregometry measuring the response to adenosine diphosphate (ADP), arachidonic acid, collagen, and thrombin-receptor activating peptide and viscoelastic tests such as rotational thromboelastometry (ROTEM).The task of extracting meaningful statistical and clinical information from high-dimensional data spaces in temporal multivariate clinical data represented in multivariate time series is complex. Building insightful visualizations for multivariate time series demands adequate usage of normalization techniques.In this article, various methods for data normalization (z-transformation, range transformation, proportion transformation, and interquartile range) are presented and visualized discussing the most suited approach for platelet function data series.Normalization was calculated per assay (test) for all time points and per time point for all tests.Interquartile range, range transformation, and z-transformation demonstrated the correlation as calculated by the Spearman correlation test, when normalized per assay (test) for all time points. When normalizing per time point for all tests, no correlation could be abstracted from the charts as was the case when using all data as 1 dataset for normalization. PMID:27428217

  7. Congenital platelet function defects

    MedlinePlus

    Platelet storage pool disorder; Glanzmann's thrombasthenia; Bernard-Soulier syndrome; Platelet function defects - congenital ... disorder may also cause severe bleeding. Platelet storage pool disorder (also called platelet secretion disorder) occurs when ...

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

    PubMed

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

    2016-01-01

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

  9. Analysis of platelet function and dysfunction.

    PubMed

    Jurk, K

    2015-01-01

    Although platelets act as central players of haemostasis only their cross-talk with other blood cells, plasma factors and the vascular compartment enables the formation of a stable thrombus. Multiple activation processes and complex signalling networks are responsible for appropriate platelet function. Thus, a variety of platelet function tests are available for platelet research and diagnosis of platelet dysfunction. However, universal platelet function tests that are sensitive to all platelet function defects do not exist and therefore diagnostic algorithms for suspected platelet function disorders are still recommended in clinical practice. Based on the current knowledge of human platelet activation this review evaluates point-of-care related screening tests in comparison with specific platelet function assays and focuses on their diagnostic utility in relation to severity of platelet dysfunction. Further, systems biology-based platelet function methods that integrate global and specific analysis of platelet vessel wall interaction (advanced flow chamber devices) and post-translational modifications (platelet proteomics) are presented and their diagnostic potential is addressed.

  10. Platelet Function Tests

    MedlinePlus

    ... of the clotting process in the body ( in vivo ). A person with normal platelet function test results may still experience excessive bleeding or inappropriate clotting during and after a surgery. Most samples for platelet function testing are only stable for a very short period ...

  11. Platelet function tests: a comparative review

    PubMed Central

    Paniccia, Rita; Priora, Raffaella; Alessandrello Liotta, Agatina; Abbate, Rosanna

    2015-01-01

    In physiological hemostasis a prompt recruitment of platelets on the vessel damage prevents the bleeding by the rapid formation of a platelet plug. Qualitative and/or quantitative platelet defects promote bleeding, whereas the high residual reactivity of platelets in patients on antiplatelet therapies moves forward thromboembolic complications. The biochemical mechanisms of the different phases of platelet activation – adhesion, shape change, release reaction, and aggregation – have been well delineated, whereas their complete translation into laboratory assays has not been so fulfilled. Laboratory tests of platelet function, such as bleeding time, light transmission platelet aggregation, lumiaggregometry, impedance aggregometry on whole blood, and platelet activation investigated by flow cytometry, are traditionally utilized for diagnosing hemostatic disorders and managing patients with platelet and hemostatic defects, but their use is still limited to specialized laboratories. To date, a point-of-care testing (POCT) dedicated to platelet function, using pertinent devices much simpler to use, has now become available (ie, PFA-100, VerifyNow System, Multiplate Electrode Aggregometry [MEA]). POCT includes new methodologies which may be used in critical clinical settings and also in general laboratories because they are rapid and easy to use, employing whole blood without the necessity of sample processing. Actually, these different platelet methodologies for the evaluation of inherited and acquired bleeding disorders and/or for monitoring antiplatelet therapies are spreading and the study of platelet function is strengthening. In this review, well-tried and innovative platelet function tests and their methodological features and clinical applications are considered. PMID:25733843

  12. Laboratory testing for platelet function disorders.

    PubMed

    Israels, S J

    2015-05-01

    Platelet function testing is both complex and labor intensive. A stepwise approach to the evaluation of patients with suspected platelet disorders will optimize the use of laboratory resources, beginning with an appropriate clinical evaluation to determine whether the bleeding is consistent with a defect of primary hemostasis. Bleeding assessment tools, evaluation of platelet counts, and review of peripheral blood cell morphology can aid the initial assessment. For patients requiring further laboratory testing, platelet aggregometry, secretion assays, and von Willebrand factor assays are the most useful next steps and will direct further specialized testing including flow cytometry, electron microscopy, and molecular diagnostics. Guidelines and recommendations for standardizing platelet function testing, with a particular focus on light transmission aggregometry, are available and can provide a template for clinical laboratories in establishing procedures that will optimize diagnosis and assure quality results. This review outlines an approach to platelet function testing and reviews testing methods available to clinical laboratories.

  13. Platelet Function Tests in Bleeding Disorders.

    PubMed

    Lassila, Riitta

    2016-04-01

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

  14. The platelet serotonin-release assay.

    PubMed

    Warkentin, Theodore E; Arnold, Donald M; Nazi, Ishac; Kelton, John G

    2015-06-01

    Few laboratory tests are as clinically useful as The platelet serotonin-release assay (SRA): a positive SRA in the appropriate clinical context is virtually diagnostic of heparin-induced thrombocytopenia (HIT), a life- and limb-threatening prothrombotic disorder caused by anti-platelet factor 4 (PF4)/heparin antibodies that activate platelets, thereby triggering serotonin-release. The SRA's performance characteristics include high sensitivity and specificity, although caveats include indeterminate reaction profiles (observed in ∼4% of test sera) and potential for false-positive reactions. As only a subset of anti-PF4/heparin antibodies detectable by enzyme-immunoassay (EIA) are additionally platelet-activating, the SRA has far greater diagnostic specificity than the EIA. However, requiring a positive EIA, either as an initial screening test or as an SRA adjunct, will reduce risk of a false-positive SRA (since a negative EIA in a patient with a "positive" SRA should prompt critical evaluation of the SRA reaction profile). The SRA also provides useful information on whether a HIT serum produces strong platelet activation even in the absence of heparin: such heparin-"independent" platelet activation is a marker of unusually severe HIT, including delayed-onset HIT and severe HIT complicated by consumptive coagulopathy with risk for microvascular thrombosis. PMID:25775976

  15. Dietary manipulation of platelet function.

    PubMed

    Bachmair, E M; Ostertag, L M; Zhang, X; de Roos, B

    2014-11-01

    Activated platelets contribute to plaque formation within blood vessels in the early and late stages of atherogenesis, and therefore they have been proposed as risk factor for cardiovascular disease. Anti-platelet drugs, such as aspirin, are now the most prescribed pharmacological treatment in Europe. Certain dietary bioactives also beneficially affect platelet function, and with less side effects, albeit that effects are generally more subtle. Therefore, consumption of dietary bioactives could play a role in the prevention of atherothrombotic vascular disease. Here we review the efficacy of dietary treatment strategies, especially those involving certain dietary fatty acids and polyphenols, to modulate platelet function in healthy subjects or in patients with cardiovascular disease. Variation in study populations, small study sizes and lack of comparability between methods to assess platelet function currently limit robust evidence on the efficacy of dietary bioactives in healthy subjects or specific patient groups. Also, limited knowledge of the metabolism of dietary bioactives, and therefore of the bioavailability of bioactive ingredients, restricts our ability to identify the most effective dietary regimes to improve platelet function. Implementation of uniform point-of-care tests to assess platelet function, and enhanced knowledge of the efficacy by which specific dietary compounds and their metabolites affect platelet function, may enable the identification of functional anti-platelet ingredients that are eligible for a health claim, or combined treatment strategies, including both pharmacological anti-platelet treatment as well as dietary intervention, to tackle atherothrombotic vascular disease. PMID:24858060

  16. Assessment of platelet function in healthy sedated cats using three whole blood platelet function tests.

    PubMed

    Ho, Kimberly K; Abrams-Ogg, Anthony C G; Wood, R Darren; O'Sullivan, M Lynne; Kirby, Gordon M; Blois, Shauna L

    2015-05-01

    The objectives of this study were to establish feline references intervals for 3 commercial whole blood platelet function test analyzer systems: Multiplate analyzer (MP; Roche Diagnostics International Ltd., Rotkreuz, Switzerland), Platelet Function Analyzer-100 (PF: Siemens Canada, Mississauga, Ontario, Canada), and Plateletworks Combo-25 kit (PW; Helena Laboratories, Beaumont, TX). Venipuncture was performed on 55 healthy sedated cats, and platelet aggregation in response to adenosine diphosphate (ADP), collagen (COL), and arachidonic acid (AA; MP only) was assessed using citrated blood. For the MP analyzer, median (95% confidence intervals [CIs]) area under curve (Units) for ADP, COL, and AA agonists were 87 (11-176), 81 (32-129), and 91 (59-129), respectively. For the PF analyzer, median (95% CIs) closure time, using COL-ADP cartridges, was 69 (46-89) sec. For the PW assay, median (95% CIs) percent aggregations for ADP and COL agonists were 71 (18-92) and 49 (9-96), respectively, using impedance hematology analyzer platelet counts, and 94 (25-98) and 68 (14-119), respectively, using flow cytometry hematology analyzer platelet counts. There were low correlations between the PF analyzer (COL-ADP cartridge) and MP analyzer (COL agonist; ρ = 0.11), and between the PF analyzer (COL-ADP cartridge) and PW assay (COL agonist using impedance platelet counts; ρ = 0.14). The PW assay percent aggregations using impedance and flow cytometric platelet counts were correlated for both ADP (ρ = 0.64) and COL (ρ = 0.64) agonists. Platelet function testing using these tests are feasible in cats, but 95% CIs are wide, so single results may be difficult to interpret. Platelet counting by impedance or flow cytometry may be used for the PW assay but are not interchangeable.

  17. Platelet function alterations in dengue are associated with plasma leakage.

    PubMed

    Michels, M; Alisjahbana, B; De Groot, P G; Indrati, A R; Fijnheer, R; Puspita, M; Dewi, I M W; van de Wijer, L; de Boer, E M S; Roest, M; van der Ven, A J A M; de Mast, Q

    2014-08-01

    Severe dengue is characterised by thrombocytopenia, plasma leakage and bleeding. Platelets are important for preservation of endothelial integrity. We hypothesised that platelet activation with secondary platelet dysfunction contribute to plasma leakage. In adult Indonesian patients with acute dengue, we measured platelet activation status and the response to the platelet agonist TRAP using flow cytometer-based assays. Patients were monitored daily for plasma leakage by ultrasonography. Acute dengue was associated with platelet activation with an increased expression of the activated fibrinogen receptor (αIIbβ3), the lysosomal marker CD63 and the alpha-granule marker CD62P (P-selectin). Upon maximal platelet activation by TRAP, platelet function defects were observed with a significantly reduced maximal activated αIIbβ3 and CD63 expression and reduced platelet-monocyte and platelet-neutrophil complexes. Patients in the lowest tertile of activated αIIbβ3 and CD63 expression had an odds ratio for plasma leakage of 5.2 (95% confidence interval [CI] 1.3-22.7) and 3.9 (95% CI 1.1-13.7), respectively, compared to the highest tertile. Platelet-derived serotonin has previously been related to plasma leakage and we found increased intra-platelet serotonin concentrations in our patients. In conclusion, platelet activation with platelet function alterations can be found in patients with acute dengue and this may contribute to dengue-associated plasma leakage.

  18. Sources of variability in platelet accumulation on type 1 fibrillar collagen in microfluidic flow assays.

    PubMed

    Neeves, Keith B; Onasoga, Abimbola A; Hansen, Ryan R; Lilly, Jessica J; Venckunaite, Diana; Sumner, Meghan B; Irish, Andrew T; Brodsky, Gary; Manco-Johnson, Marilyn J; Di Paola, Jorge A

    2013-01-01

    Microfluidic flow assays (MFA) that measure shear dependent platelet function have potential clinical applications in the diagnosis and treatment of bleeding and thrombotic disorders. As a step towards clinical application, the objective of this study was to measure how phenotypic and genetic factors, as well as experimental conditions, affect the variability of platelet accumulation on type 1 collagen within a MFA. Whole blood was perfused over type 1 fibrillar collagen at wall shear rates of 150, 300, 750 and 1500 s⁻¹ through four independent channels with a height of 50 µm and a width of 500 µm. The accumulation of platelets was characterized by the lag time to 1% platelet surface coverage (Lag(T)), the rate of platelet accumulation (V(PLT)), and platelet surface coverage (SC). A cohort of normal donors was tested and the results were correlated to plasma von Willebrand factor (VWF) levels, platelet count, hematocrit, sex, and collagen receptors genotypes. VWF levels were the strongest determinant of platelet accumulation. VWF levels were positively correlated to V(PLT) and SC at all wall shear rates. A longer Lag(T) for platelet accumulation at arterial shear rates compared to venous shear rates was attributed to the time required for plasma proteins to adsorb to collagen. There was no association between platelet accumulation and hematocrit or platelet count. Individuals with the AG genotype of the GP6 gene had lower platelet accumulation than individuals with the AA genotype at 150 s⁻¹ and 300 s⁻¹. Recalcified blood collected into sodium citrate and corn trypsin inhibitor (CTI) resulted in diminished platelet accumulation compared to CTI alone, suggesting that citrate irreversibly diminishes platelet function. This study the largest association study of MFA in healthy donors (n = 104) and will likely set up the basis for the determination of the normal range of platelet responses in this type of assay. PMID:23355889

  19. Sources of Variability in Platelet Accumulation on Type 1 Fibrillar Collagen in Microfluidic Flow Assays

    PubMed Central

    Neeves, Keith B.; Onasoga, Abimbola A.; Hansen, Ryan R.; Lilly, Jessica J.; Venckunaite, Diana; Sumner, Meghan B.; Irish, Andrew T.; Brodsky, Gary; Manco-Johnson, Marilyn J.; Di Paola, Jorge A.

    2013-01-01

    Microfluidic flow assays (MFA) that measure shear dependent platelet function have potential clinical applications in the diagnosis and treatment of bleeding and thrombotic disorders. As a step towards clinical application, the objective of this study was to measure how phenotypic and genetic factors, as well as experimental conditions, affect the variability of platelet accumulation on type 1 collagen within a MFA. Whole blood was perfused over type 1 fibrillar collagen at wall shear rates of 150, 300, 750 and 1500 s−1 through four independent channels with a height of 50 µm and a width of 500 µm. The accumulation of platelets was characterized by the lag time to 1% platelet surface coverage (LagT), the rate of platelet accumulation (VPLT), and platelet surface coverage (SC). A cohort of normal donors was tested and the results were correlated to plasma von Willebrand factor (VWF) levels, platelet count, hematocrit, sex, and collagen receptors genotypes. VWF levels were the strongest determinant of platelet accumulation. VWF levels were positively correlated to VPLT and SC at all wall shear rates. A longer LagT for platelet accumulation at arterial shear rates compared to venous shear rates was attributed to the time required for plasma proteins to adsorb to collagen. There was no association between platelet accumulation and hematocrit or platelet count. Individuals with the AG genotype of the GP6 gene had lower platelet accumulation than individuals with the AA genotype at 150 s−1 and 300 s−1. Recalcified blood collected into sodium citrate and corn trypsin inhibitor (CTI) resulted in diminished platelet accumulation compared to CTI alone, suggesting that citrate irreversibly diminishes platelet function. This study the largest association study of MFA in healthy donors (n = 104) and will likely set up the basis for the determination of the normal range of platelet responses in this type of assay. PMID:23355889

  20. Monoclonal platelet antigen capture assays (MAIPA) and reagents: a statement.

    PubMed

    Kaplan, C; Freedman, J; Foxcroft, Z; Husebekk, A; Metcalfe, P; Muniz-Diaz, E; Ouwehand, W; Panzer, S; Rozman, P; Skogen, B

    2007-11-01

    This statement concerning the monoclonal-specific immobilization of platelet antigens (MAIPA) has been written on behalf of the International Society of Blood Transfusion--Working Party on Platelet Immunology. The MAIPA technique is considered as the gold standard reference technique in platelet immunology. The assay performed with reagents labelled for 'research only' is acceptable as long as it is regularly evaluated by participation of laboratories in national or international workshops held with reference laboratories. PMID:18070272

  1. [Assessment of platelet function in man].

    PubMed

    Gaussem, Pascale

    2006-01-01

    Assessment of platelet function was primarily designed to explore patients with hemostatic disorders, but is becoming important for the monitoring of anti platelet agents, mostly aspirin and clopidogrel. Beside platelet counting, morphological analysis and bleeding time, a number of dedicated platelet function instruments are now available, generally allowing a rapid evaluation of platelet function in whole blood. The other tests including aggregometry and ELISA measurement of activation markers are generally restricted to specialized laboratories. Although aggregometry is still considered as the "gold standard", the recently developed flow cytometric-based platelet function analysis provides a wide choice of tests that assess the number of surface receptors, the measure of secretion and aggregation, the quantification of microparticules and leukocyte-platelet aggregates. It also allows the measure of the function of the ADP receptor P2Y12 by the phosphorylation level of the VASP protein, method currently under evaluation to monitor the platelet response to clopidogrel treatment. PMID:17243268

  2. Platelet function defects in chronic alcoholism.

    PubMed Central

    Mikhailidis, D P; Jenkins, W J; Barradas, M A; Jeremy, J Y; Dandona, P

    1986-01-01

    Platelet function in alcoholic patients was assessed on admission and during abstinence in hospital. On admission platelets from these patients were significantly less responsive (percentage aggregation and thromboxane A2 release) to conventional in vitro aggregating agents (adrenaline, adenosine diphosphate, and collagen) than platelets from healthy, moderate drinkers. Initially, platelet counts in platelet rich plasma tended to be low and the Simplate II bleeding times frequently prolonged. Platelet aggregation and thromboxane A2 release, however, were inhibited even in patients with normal platelet counts on admission. Platelet aggregation and thromboxane A2 release returned to normal or became hyper-responsive during two to three weeks of abstinence. Platelet counts rose during this period, the largest responses occurring in those patients with the lowest counts on admission. Bleeding times reverted to normal during abstinence and correlated significantly with changes in platelet aggregation, thromboxane A2 release, and platelet count and with the estimated ethanol consumption during the week before admission. Chronic, heavy alcohol ingestion evidently exerts an inhibitory effect on platelet function even in the absence of alcohol in the blood, and this phenomenon is reversible on abstaining. The impaired platelet function, together with the reduced platelet count, may contribute to the bleeding diathesis associated with chronic alcoholism and to the increased incidence and recurrence of gastrointestinal haemorrhage associated with excessive alcohol intake. PMID:3094624

  3. Dynamic light scattering can determine platelet function

    NASA Astrophysics Data System (ADS)

    Lee, Nathan

    2011-10-01

    Platelet transfusions are life-saving procedures for patients who are bleeding or undergoing chemotherapy. The effectiveness of transfusions depends on the number of platelets transfused and the platelet function. Platelet function correlates with proportion of discoid to activated platelets, morphology response to temperature stress, and inversely correlates with microparticle content. ThromboLUX is a novel device that determines platelet function by measuring all of these characteristics using dynamic light scattering (DLS). During periods of stress, such as decreased temperature, cytoskeletal rearrangements will cause normal, discoid platelets to activate and become spiny spheres. The formation of pseudopods of various lengths facilitates the clotting cascade and also increases the apparent size of platelets. ThromboLUX uses a 37-20-37 C temperature cycle that mimics the bleeding, storage, and transfusion process. As the temperature fluctuates, DLS will measure the changing platelet hydrodynamic radius and the size of any microparticles present. ThromboLUX analysis of platelet concentrates in vitro would allow determination of high platelet function units before transfusion and would therefore improve transfusion outcomes and patient safety. This study examined how DLS is able to distinguish between discoid and activated platelets as well as measure the parameters that contribute to high platelet function.

  4. [Research Progress on Cytometric Bead Assay for Platelet Antibody Detection].

    PubMed

    Ling, Yun; Kong, Xin; Chen, Bao-An

    2015-08-01

    Anti-platelet specific antibody is one of the most important reasons leading to thrombocytopenia and megakaryocyte dysmaturity. The detection of platelet autoantibodies is an important step in the diagnosis of ITP because of the absence of specific clinic feature. The monoclonal antibody-specific immobilization of platelet antigens (MAIPA) has become a "gold standard" for determination of PLT specific antibody, which has high specificity and low sensitivity. However, this assay is time-consuming and tedious work. Routine use of this assay in hospital is difficult. Recently, some researches reporded the cytometric bead assay that has higher sensitivity than MAIPA, and so probably solves the problem of time-consuming partly, that also can use different beads for simultaneous detection. This review focuses on recent progress of the cytometric bead assay. PMID:26314475

  5. Association of MicroRNAs and YRNAs with Platelet Function

    PubMed Central

    Bender, Lukas H.; Barwari, Temo; Willeit, Peter; Pechlaner, Raimund; Sunderland, Nicholas P.; Willeit, Karin; Morton, Allison C.; Armstrong, Paul C.; Chan, Melissa V.; Lu, Ruifang; Yin, Xiaoke; Gracio, Filipe; Dudek, Katarzyna; Langley, Sarah R.; Zampetaki, Anna; de Rinaldis, Emanuele; Ye, Shu; Warner, Timothy D.; Saxena, Alka; Kiechl, Stefan; Storey, Robert F.; Mayr, Manuel

    2016-01-01

    Rationale Platelets shed microRNAs (miRNAs). Plasma miRNAs change upon platelet inhibition. It is unclear if plasma miRNA levels correlate with platelet function. Objective To link small RNAs to platelet reactivity. Methods and Results Next-generation sequencing of small RNAs in plasma revealed two peaks at 22-23 and 32-33 nucleotides corresponding to miRNAs and YRNAs, respectively. Among YRNAs, predominantly fragments of RNY4 and RNY5 were detected. Plasma miRNAs and YRNAs were measured in 125 patients with a history of ACS who had undergone detailed assessment of platelet function 30 days after the acute event. Using quantitative real-time polymerase chain reactions, 92 miRNAs were assessed in ACS patients on different anti-platelet therapies. Key platelet-related miRNAs and YRNAs were correlated with platelet function tests. MiR-223 (rp=0.28, n=121, P=0.002), miR-126 (rp=0.22, n=121, P=0.016), other abundant platelet miRNAs and YRNAs showed significant positive correlations with the vasodilator-stimulated phosphoprotein phosphorylation assay. YRNAs, miR-126 and miR-223 were also among the small RNAs showing the greatest dependency on platelets, and strongly correlated with plasma levels of P-selectin, platelet factor 4 and platelet basic protein in the population-based Bruneck study (n=669). A single nucleotide polymorphism that facilitates processing of pri-miR-126 to mature miR-126 accounted for a rise in circulating platelet activation markers. Inhibition of miR-126 in mice reduced platelet aggregation. MiR-126 directly and indirectly affects ADAM9 and P2Y12 receptor expression. Conclusions Levels of platelet-related plasma miRNAs and YRNAs correlate with platelet function tests in ACS patients and platelet activation markers in the general population. Alterations in miR-126 affect platelet reactivity. PMID:26646931

  6. State of the Art in Platelet Function Testing

    PubMed Central

    E. Kehrel, Beate; F. Brodde, Martin

    2013-01-01

    Summary Platelets perform many functions in hemostasis but also in other areas of physiology and pathology. Therefore, it is obvious that many different function tests have been developed, each one conceived and standardized for a special purpose. This review will summarize the different fields in which platelet function testing is currently in use; diagnostics of patients with bleeding disorders, monitoring patients’ response to anti-platelet therapy, monitoring in transfusion medicine (blood donors, platelet concentrates, and after transfusion), and monitoring in perioperative medicine to predict bleeding tendency. The second part of the review outlines different methods for platelet function testing, spanning bleeding time, and platelet counting as well as determining platelet adhesion, platelet secretion, platelet aggregation, platelet morphology, platelet signal transduction, platelet procoagulant activity, platelet apoptosis, platelet proteomics, and molecular biology. PMID:23653569

  7. Acidosis downregulates platelet haemostatic functions and promotes neutrophil proinflammatory responses mediated by platelets.

    PubMed

    Etulain, Julia; Negrotto, Soledad; Carestia, Agostina; Pozner, Roberto Gabriel; Romaniuk, María Albertina; D'Atri, Lina Paola; Klement, Giannoula Lakka; Schattner, Mirta

    2012-01-01

    Acidosis is one of the hallmarks of tissue injury such as trauma, infection, inflammation, and tumour growth. Although platelets participate in the pathophysiology of all these processes, the impact of acidosis on platelet biology has not been studied outside of the quality control of laboratory aggregation assays or platelet transfusion optimization. Herein, we evaluate the effect of physiologically relevant changes in extracellular acidosis on the biological function of platelets, placing particular emphasis on haemostatic and secretory functions. Platelet haemostatic responses such as adhesion, spreading, activation of αIIbβ3 integrin, ATP release, aggregation, thromboxane B2 generation, clot retraction and procoagulant activity including phosphatidilserine exposure and microparticle formation, showed a statistically significant inhibition of thrombin-induced changes at pH of 7.0 and 6.5 compared to the physiological pH (7.4). The release of alpha granule content was differentially regulated by acidosis. At low pH, thrombin or collagen-induced secretion of vascular endothelial growth factor and endostatin were dramatically reduced. The release of von Willebrand factor and stromal derived factor-1α followed a similar, albeit less dramatic pattern. In contrast, the induction of CD40L was not changed by low pH, and P-selectin exposure was significantly increased. While the generation of mixed platelet-leukocyte aggregates and the increased chemotaxis of neutrophils mediated by platelets were further augmented under acidic conditions in a P-selectin dependent manner, the increased neutrophil survival was independent of P-selectin expression. In conclusion, our results indicate that extracellular acidosis downregulates most of the haemostatic platelet functions, and promotes those involved in amplifying the neutrophil-mediated inflammatory response.

  8. Platelet function in the postprandial period

    PubMed Central

    2012-01-01

    Background Postprandial hyperlipidemia and hyperglycemia have been related to cardiovascular events. Among different underlying mechanisms platelet activation seems to be responsible too. No comparable data between various tests in normo- vs. hyperlipidemics before and at different time intervals are available after a fat meal. We aimed to compare 9 of them within the same patients at several time points in postprandial hyperlipidemia. Results For some tests baseline values between the groups were significantly different (TXB2, platelet sensitivity, sedimentation and WU-test). However, hyperlipidemia revealed a variable influence on the tests examined. Some of the available tests apparently sensitive to show platelet activation reflect the increase in triglycerides (TG), such as the sedimentation index. ADP-induced platelet aggregatory activity in count adjusted washed isolated platelet samples during postprandial hyperlipidemia indicates mildly enhanced platelet activity, but does not seem to induce significant changes in aggregation. In patients with severe hypertriglyceridemia (> 400 mg/dl fasting) changes in platelet function are more pronounced due to delayed decay and may last up to 16 hours paralleling TG reaching the prevalue. The overwhelming majority of platelet function tests do not significantly respond to postprandial hyperlipidemia. The correlation between the tests applied is poor. For standardization purpose, platelet aggregation tests, aimed to examine proaggregatory capacity in atherosclerosis, should only be performed at the same time of the day after a fasting period > 6 hours. The great variation in preanalytical work-up on comparison of various tests, large number of platelet tests available and their respective potential value are discussed. Conclusions At present, the suspicion that platelet function is significantly activated in the postprandial period cannot be supported by any of the tests used. The information provided is valuable to

  9. Pharmacological actions of nobiletin in the modulation of platelet function

    PubMed Central

    Vaiyapuri, Sakthivel; Roweth, Harvey; Ali, Marfoua S; Unsworth, Amanda J; Stainer, Alexander R; Flora, Gagan D; Crescente, Marilena; Jones, Chris I; Moraes, Leonardo A; Gibbins, Jonathan M

    2015-01-01

    Background and Purpose The discovery that flavonoids are capable of inhibiting platelet function has led to their investigation as potential antithrombotic agents. However, despite the range of studies on the antiplatelet properties of flavonoids, little is known about the mechanisms by which flavonoids inhibit platelet function. In this study, we aimed to explore the pharmacological effects of a polymethoxy flavonoid, nobiletin, in the modulation of platelet function. Experimental Approach The ability of nobiletin to modulate platelet function was explored by using a range of in vitro and in vivo experimental approaches. Aggregation, dense granule secretion and spreading assays were performed using washed platelets. Fibrinogen binding, α-granule secretion and calcium mobilization assays were performed using platelet-rich plasma and whole blood was used in impedance aggregometry and thrombus formation experiments. The effect of nobiletin in vivo was assessed by measuring tail bleeding time using C57BL/6 mice. Key Results Nobiletin was shown to suppress a range of well-established activatory mechanisms, including platelet aggregation, granule secretion, integrin modulation, calcium mobilization and thrombus formation. Nobiletin extended bleeding time in mice and reduced the phosphorylation of PKB (Akt) and PLCγ2 within the collagen receptor (glycoprotein VI)-stimulated pathway, in addition to increasing the levels of cGMP and phosphorylation of vasodilator-stimulated phosphoprotein, a protein whose activity is associated with inhibitory cyclic nucleotide signalling. Conclusions and Implications This study provides insight into the underlying molecular mechanisms through which nobiletin modulates haemostasis and thrombus formation. Therefore, nobiletin may represent a potential antithrombotic agent of dietary origins. PMID:25988959

  10. Viability and functional integrity of washed platelets

    SciTech Connect

    Pineda, A.A.; Zylstra, V.W.; Clare, D.E.; Dewanjee, M.K.; Forstrom, L.A.

    1989-07-01

    The viability and functional integrity of saline- and ACD-saline-washed platelets were compared with those of unwashed platelets. After template bleeding time (TBT) was measured, 15 healthy volunteers underwent plateletpheresis and ingested 600 mg of aspirin. Autologous /sup 111/In-labeled platelets were transfused: unwashed (n = 5), washed with 0.9 percent saline solution (SS) (n = 5), and washed with a buffered 12.6 percent solution of ACD-A in 0.9 percent saline solution (n = 5). After transfusion, we measured TBT at 1, 4, and 24 hours; platelet survival at 10 minutes and 1, 4, and 24 hours and daily for 6 days; and the percentage of uptake in liver and spleen by quantitative whole-body radionuclide scintigraphy at 24 and 190 hours. We found that saline washing affected platelet recovery, 23.47 +/- 12 percent (p less than 0.001) as compared to 52.43 +/- 17 percent (p less than 0.002) for ACD-saline and 73.17 +/- 8 percent for control; that saline washing resulted in a greater liver uptake than control and ACD-saline-washed platelets (31.9 +/- 8% (p less than 0.001) vs 17.7 +/- 4.1 and 19.3 +/- 2.1% (p greater than 0.1), respectively); that, unlike control and ACD-saline-washed platelets, saline-washed platelets did not shorten bleeding time; and that neither type of washing affected survival. Although ACD-saline washing affects recovery, it also results in intact function, normal survival, higher recovery than SS platelets, and no significant liver uptake.

  11. Coated platelets function in platelet-dependent fibrin formation via integrin αIIbβ3 and transglutaminase factor XIII

    PubMed Central

    Mattheij, Nadine J.A.; Swieringa, Frauke; Mastenbroek, Tom G.; Berny-Lang, Michelle A.; May, Frauke; Baaten, Constance C.F.M.J.; van der Meijden, Paola E.J.; Henskens, Yvonne M.C.; Beckers, Erik A.M.; Suylen, Dennis P.L.; Nolte, Marc W.; Hackeng, Tilman M.; McCarty, Owen J.T.; Heemskerk, Johan W.M.; Cosemans, Judith M.E.M.

    2016-01-01

    Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin αIIbβ3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin αIIbβ3. Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin αIIbβ3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in αIIbβ3 (Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial αIIbβ3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin αIIbβ3. PMID:26721892

  12. Effects of irradiation on platelet function

    SciTech Connect

    Rock, G.; Adams, G.A.; Labow, R.S.

    1988-09-01

    Current medical practice involves the irradiation of blood components, including platelet concentrates, before their administration to patients with severe immunosuppression. The authors studied the effect of irradiation on in vitro platelet function and the leaching of plasticizers from the bag, both immediately and after 5 days of storage. The platelet count, white cell count, pH, glucose, lactate, platelet aggregation and release reaction, and serotonin uptake were not altered by the irradiation of random-donor or apheresis units with 2000 rads carried out at 0 and 24 hours and 5 days after collection. The leaching of di(2-ethylhexyl)phthalate from the plastic bags followed by the conversion to mono(2-ethylhexyl)phthalate was not increased by irradiation. Therefore, it is possible to irradiate platelet concentrates on the day of collection and subsequently store them for at least 5 days while maintaining in vitro function. This procedure could have considerable benefit for blood banks involved in the provision of many platelet products.

  13. Low-power laser irradiation of blood inhibits platelet function: role of cyclic GMP

    NASA Astrophysics Data System (ADS)

    Brill, Alexander G.; Brill, Gregory E.; Shenkman, Boris; Tamarin, Ilya; Dardik, Rima; Varon, David; Savion, Naphtali

    1998-12-01

    The aim of the present work was to investigate effect of low power laser irradiation (LPLI) on platelet function in vitro. He-Ne laser (Optronix, USA; (lambda) - 632.8 nm, output power - 7 mW) was employed. Platelet adhesion and aggregation in whole blood (WB) under defined shear conditions were assayed by a Cone and Plate(let) Analyzer. Platelet activation was evaluated by flow cytometry. Level of platelet cGMP was estimated by immunoenzyme assay. Experiments performed showed that LPLI of WB resulted in decrease of platelet deposition on extracellular matrix at high shear rate (1300 s-1). Similar results were obtained using surfaces precoated with either collagen type I or von Willebrand factor. LPLI inhibited fibrinogen binding as well as P-selectin expression on the platelet membrane, induced by thrombin analogue. It was found out that primary acceptor of laser energy responsible for the effect on platelets was located in platelets themselves and not in blood plasma or in other blood cells. LPLI of gel- filtered platelets resulted in increase of intracellular level of cGMP both in the absence and in presence of izobutylmethylxantine (phosphodiesterase inhibitor) suggesting stimulation of synthesis rather than destruction of cGMP under the influence of LPLI. It is suggested that guanylate cyclase and/or NO-synthase might serve as primary acceptors of He-Ne laser light in platelets.

  14. Effects of methaqualone on blood platelet function.

    PubMed

    Mills, D G

    1978-06-01

    To study the mechanism whereby toxic doses of methaqualone cause a bleeding tendency in humans, the effects of methaqualone, diphenhydramine, and the combination of methaqualone plus diphenhydramine on blood platelet function were investigated. Exposure of human platelets in platelet-rich plasma in vitro to final concentrations of methaqualone ranging from 1.1 to 4.5 X 10(-4)) M resulted in nearly complete inhibition of the secondary phase and significant inhibition of the primary phase of adenosine diphosphate (ADP)--induced aggregation. Both the slope and height of collagen-induced aggregation responses were reduced significantly in vitro by the drug. When methaqualone final concentrations of 1.1, 2.3, and 4.5 X 10(-4) M were studied in the presence of diphenhydramine (1.1, 2.3, and 4.5 X 10(-5) M, respectively), the degree of inhibition of ADP-induced aggregation was only slightly greater (not significant) than that observed with methaqualone. The platelets of rabbits injected intravenously with methaqualone, 10 mg/kg, demonstrated a significantly decreased ability to aggregate with ADP and collagen 30 and 60 min after administration of the drug. These results suggest that a drug-induced defect of blood platelet function may play a role in the bleeding associated with methaqualone toxicity.

  15. Effect of sildenafil on platelet function and platelet cGMP of patients with erectile dysfunction.

    PubMed

    Akand, M; Gencer, E; Yaman, Ö; Erişgen, G; Tekin, D; Özdiler, E

    2015-12-01

    To investigate the effect of sildenafil on platelet function and cyclic guanosine monophosphate (cGMP) levels in patients with erectile dysfunction, we evaluated the association between erectile function and platelet responses after administration of 100 mg sildenafil. Erectile responses were monitored after 8 daily doses of the drug. Adenosine diphosphate (ADP) and collagen-induced platelet aggregation and simultaneous adenosine triphosphate (ATP) release and cGMP levels were determined before and after sildenafil therapy. Basal levels for platelet aggregation, ATP release and cGMP were compared with age-matched controls. There was no difference among basal levels of platelet responses between patients and controls, except for ADP-induced platelet aggregation (P = 0.04). It was significantly higher in the patient group. Analysis of the responses to sildenafil revealed that for the patients who showed a positive erectile response, there was a significant increase in platelet cGMP (P = 0.028) and a decrease in ADP-induced platelet aggregation (P = 0.04). However, for those who showed a negative or poor erectile response, there was no change in platelet cGMP levels and platelet functions. Sildenafil did not affect collagen-induced platelet responses although cGMP levels of the responders increased. It is concluded that sildenafil increases platelet cGMP in the patients with positive erectile response. Therefore, it has been speculated that platelet cGMP may be used as an index for erectile response.

  16. Platelet function in pre-eclampsia.

    PubMed

    Kazmi, Rashid S; Cooper, Alan J; Lwaleed, Bashir A

    2011-03-01

    Pronounced hemostatic changes occur during pregnancy, and the balance shifts markedly in favor of hypercoagulability. Although primarily a result of a marked rise in the levels of several procoagulants and a fall in some natural anticoagulants, platelet activation also contributes to this prothrombotic tendency. Several studies have confirmed the accentuation of platelet activation in pre-eclampsia (P-EC), which remains an important obstetric complication affecting ~2 to 4% of pregnancies. Although there is still a long way to go, significant inroads have been made in the understanding of this enigmatic condition. Whereas the pathogenesis of P-EC is protean and involves a complex interplay of placental and maternal tissues, platelet activation is likely to contribute to several clinical features. Several techniques have been used to assess platelet activation in P-EC. Detection of aberrations of platelet function and activation appear to have predictive value for its diagnosis. The findings also lend support to the use of antiplatelet agents as prophylaxis in those women with a high risk of developing the condition.

  17. Factor VIII is a positive regulator of platelet function.

    PubMed

    Obergfell, A; Sturm, A; Speer, C P; Walter, U; Grossmann, R

    2006-11-01

    FVIII is an important cofactor in the tenase coagulation factor complex, lack of FVIII causes severe bleeding, whereas high FVIII levels seem to be associated with venous and arterial thromboembolism. Resting platelets do not bind FVIII, but activated platelets bind unactivated FVIII if vWF is not present. We investigated a possible influence of platelet bound FVIII on platelet function itself as it is unclear if there is a direct effect of FVIII on platelet function. The influence of FVIII on platelet function was investigated by flow cytometric analysis of P-selectin expression (CD62P) and PAC-1 binding before and after submaximal stimulation with TRAP-6 (5 microM final concentration), by confocal microscopy and by platelet aggregometry. For flow cytometry and confocal microscopy, washed platelets were incubated with human recombinant FVIII for 5 min at 37 degrees C. Analysis of platelet surface area was measured by computerized image analysis. Treatment with FVIII only caused no changes in P-selectin expression or PAC-1 binding, respectively. Stimulation of platelets with TRAP-6 increased the expression of P-selectin (445%) and PAC-1 binding (934%) as expected. These effects were further increased when platelets were stimulated with TRAP-6 and FVIII (P-selectin 499%, difference not significant; PAC-1 1626%, P < 0.05. Values were expressed in%, related to unstimulated, buffer treated platelets). Platelet spreading on fibrinogen was significantly increased when platelets were treated with FVIII and TRAP-6 compared to TRAP-6 alone (368 vs. 307 average pixel/platelet, P<0.05). In addition platelet aggregation was enhanced when platelets were stimulated with FVIII and TRAP-6 compared to TRAP-6 alone. FVIII can act as a positive regulator of platelet function in TRAP-co-stimulated platelets. We hypothesize that FVIII induced increase in platelet activation might contribute to venous and even arterial thrombus formation in patients with high FVIII levels. PMID:17074720

  18. Platelet function tests, independent of platelet count, are associated with bleeding severity in ITP.

    PubMed

    Frelinger, Andrew L; Grace, Rachael F; Gerrits, Anja J; Berny-Lang, Michelle A; Brown, Travis; Carmichael, Sabrina L; Neufeld, Ellis J; Michelson, Alan D

    2015-08-13

    Immune thrombocytopenia (ITP) patients with similarly low platelet counts differ in their tendency to bleed. To determine if differences in platelet function in ITP patients account for this variation in bleeding tendency, we conducted a single-center, cross-sectional study of pediatric patients with ITP. Bleeding severity (assessed by standardized bleeding score) and platelet function (assessed by whole blood flow cytometry) with and without agonist stimulation was evaluated in 57 ITP patients (median age, 9.9 years). After adjustment for platelet count, higher levels of thrombin receptor activating peptide (TRAP)-stimulated percent P-selectin- and activated glycoprotein (GP)IIb-IIIa-positive platelets were significantly associated with a lower bleeding score, whereas higher levels of immature platelet fraction (IPF), TRAP-stimulated platelet surface CD42b, unstimulated platelet surface P-selectin, and platelet forward light scatter (FSC) were associated with a higher bleeding score. Thus, platelet function tests related to platelet age (IPF, FSC) and activation through the protease activated receptor 1 (PAR1) thrombin receptor (TRAP-stimulated P-selectin, activated GPIIb-IIIa, and CD42b), independent of platelet count, are associated with concurrent bleeding severity in ITP. These tests may be useful markers of future bleeding risk in ITP.

  19. Platelet function tests, independent of platelet count, are associated with bleeding severity in ITP

    PubMed Central

    Grace, Rachael F.; Gerrits, Anja J.; Berny-Lang, Michelle A.; Brown, Travis; Carmichael, Sabrina L.; Neufeld, Ellis J.; Michelson, Alan D.

    2015-01-01

    Immune thrombocytopenia (ITP) patients with similarly low platelet counts differ in their tendency to bleed. To determine if differences in platelet function in ITP patients account for this variation in bleeding tendency, we conducted a single-center, cross-sectional study of pediatric patients with ITP. Bleeding severity (assessed by standardized bleeding score) and platelet function (assessed by whole blood flow cytometry) with and without agonist stimulation was evaluated in 57 ITP patients (median age, 9.9 years). After adjustment for platelet count, higher levels of thrombin receptor activating peptide (TRAP)-stimulated percent P-selectin- and activated glycoprotein (GP)IIb-IIIa–positive platelets were significantly associated with a lower bleeding score, whereas higher levels of immature platelet fraction (IPF), TRAP-stimulated platelet surface CD42b, unstimulated platelet surface P-selectin, and platelet forward light scatter (FSC) were associated with a higher bleeding score. Thus, platelet function tests related to platelet age (IPF, FSC) and activation through the protease activated receptor 1 (PAR1) thrombin receptor (TRAP-stimulated P-selectin, activated GPIIb-IIIa, and CD42b), independent of platelet count, are associated with concurrent bleeding severity in ITP. These tests may be useful markers of future bleeding risk in ITP. PMID:26138687

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

    PubMed

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

    2012-01-01

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

  1. Resveratrol preserves the function of human platelets stored for transfusion.

    PubMed

    Lannan, Katie L; Refaai, Majed A; Ture, Sara K; Morrell, Craig N; Blumberg, Neil; Phipps, Richard P; Spinelli, Sherry L

    2016-03-01

    Stored platelets undergo biochemical, structural and functional changes that lead to decreased efficacy and safety of platelet transfusions. Not only do platelets acquire markers of activation during storage, but they also fail to respond normally to agonists post-storage. We hypothesized that resveratrol, a cardioprotective antioxidant, could act as a novel platelet storage additive to safely prevent unwanted platelet activation during storage, while simultaneously preserving normal haemostatic function. Human platelets treated with resveratrol and stored for 5 d released less thromboxane B2 and prostaglandin E2 compared to control platelets. Resveratrol preserved the ability of platelets to aggregate, spread and respond to thrombin, suggesting an improved ability to activate post-storage. Utilizing an in vitro model of transfusion and thromboelastography, clot strength was improved with resveratrol treatment compared to conventionally stored platelets. The mechanism of resveratrol's beneficial actions on stored platelets was partly mediated through decreased platelet apoptosis in storage, resulting in a longer half-life following transfusion. Lastly, an in vivo mouse model of transfusion demonstrated that stored platelets are prothrombotic and that resveratrol delayed vessel occlusion time to a level similar to transfusion with fresh platelets. We show resveratrol has a dual ability to reduce unwanted platelet activation during storage, while preserving critical haemostatic function.

  2. Platelets

    MedlinePlus

    ... are related to immunity and fighting infection. Platelet Production Platelets are produced in the bone marrow, the ... platelet destruction and also decreased bone marrow platelet production. These problems are caused by autoantibodies. Antibodies are ...

  3. Fractionation of platelets according to size: functional and biochemical characteristics

    SciTech Connect

    Carty, D.J.; Gear, A.R.

    1986-01-01

    The functional and biochemical heterogeneity of platelets has been studied using graded differential centrifugation to fractionate human platelets according to size while maintaining their morphological and functional integrity as indicated by scanning electron microscopy and content of beta-thromboglobulin. Aggregation kinetics were studied by both optical and quenched-flow methods involving single-particle counting. Large platelets were significantly more sensitive to ADP, but aggregated less rapidly than small platelets. Thrombin exerted a similar influence. Large platelets were also enriched in surface sialic acid and sulfhydryl groups and in internal glycogen, ATP, ADP, calcium, cyclic AMP, malonaldehyde, and succinate cytochrome c reductase when compared to small platelets, even when normalized per unit volume. ADP caused a more rapid breakdown of cyclic AMP in small platelets. Potential aging relationships were tested by isotope studies in rats. /sup 75/Se-selenomethionine was incorporated in vivo at a similar rate into all fractions. Large platelets labeled with /sup 51/Cr disappeared from circulation linearly and had a longer mean lifespan than small platelets, which disappeared exponentially. This behavior supports independent aging of platelet populations of differing size. The data suggest a distinct heterogeneity in platelet function and fate, which could derive from protection of large platelets against excessive activation by Ca2+-regulated events.

  4. A functional integrin ligand on the surface of platelets: intercellular adhesion molecule-2.

    PubMed Central

    Diacovo, T G; deFougerolles, A R; Bainton, D F; Springer, T A

    1994-01-01

    Activated platelets express P-selectin and release leukocyte chemoattractants; however, they have not been known to express integrin ligands important in the stabilization of leukocyte interactions with the vasculature. We now demonstrate the presence of intercellular adhesion molecular-2 (ICAM-2) (CD102), and lack of expression of other beta 2-integrin ligands, ICAM-1 (CD54) and ICAM-3 (CD50), on the surface of resting and stimulated platelets. ICAM-2 isolated from platelets migrates as a band of 59,000 M(r) in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staining of bone marrow aspirates with anti-ICAM-2 mAb demonstrates strong reactivity to megakaryocytes. Using frozen thin sections and immunogold labeling, the antigen was shown to be present on the plasma membrane and surface-connected canalicular system of resting platelets. The average number of ICAM-2 molecules per platelet is 3,000 +/- 230 and does not change after activation. In adhesion assays, resting and stimulated platelets were capable of binding through ICAM-2 to purified leukocyte function-associated antigen-1. Activation of T lymphocytes with PMA stimulated binding to platelets that was Mg2+ dependent and could be specifically inhibited by mAbs to either ICAM-2 or leukocyte function-associated antigen-1. ICAM-2 is the only known beta 2-integrin ligand present on platelets, suggesting that it may play an important role in leukocyte-platelet interactions in inflammation and thrombosis. Images PMID:8083366

  5. Platelet Function Testing-Guided Antiplatelet Therapy

    PubMed Central

    Spannagl, Michael

    2013-01-01

    Cardiovascular diseases are the leading cause of death in the Western world. Several factors have led to the increase in vascular disorders, including the aging population, unhealthy lifestyles, increasing rates of diabetes and raised lipids, and further risk factors resulting in inflammation and calcification of the vascular endothelium. Activated platelets in damaged blood vessels can trigger arterial thrombus formation, leading to vascular occlusion with subsequent organ hypoperfusion and clinical manifestation of myocardial infarction, stroke, or peripheral artery disease. Platelet inhibitors such as aspirin and clopidogrel (Plavix® and generics) are prescribed as primary or secondary prevention to attenuate chronic platelet activation. However, a significant proportion of patients do not respond adequately to uniform antiplatelet treatment. These ‘non-responders’ have an increased risk for stent thrombosis, stroke, and other ischemic complications. Platelet function (PF) tests can identify these patients thus enabling physicians to offer personalized and alternative treatment strategies. Recent alternatives to clopidogrel include prasugrel (Efient®) and ticagrelor (Brilique®) – that are both more potent than clopidogrel but also more expensive and associated with a higher risk of bleeding complications. Given these drawbacks, PF testing might help clinicians to prescribe optimal antiplatelet agent to maximize patient safety and efficacy while minimizing costs. While randomized studies using different test systems have left clinicians puzzled about the medical value of tailored antiplatelet therapy, accumulated evidence from recent studies on tailored antiplatelet therapies and the association with improved outcomes have now resulted in a consensus expert opinion for the specific adoption of PF diagnostics into clinical practice.

  6. Roll, adhere, spread and contract: structural mechanics of platelet function.

    PubMed

    Sorrentino, Simona; Studt, Jan-Dirk; Medalia, Ohad; Tanuj Sapra, K

    2015-01-01

    Platelets are involved in life-sustaining processes such as hemostasis, wound healing, atherothrombosis and angiogenesis. Mechanical trauma to blood vessels causes platelet activation resulting in their adherence and clot formation at the damaged site, culminating in clot retraction and tissue repair. Two of the major players underlying this process are the cytoskeleton, i.e., actin and microtubules, and the membrane integrin receptors. Rare congenital bleeding disorders such as Glanzmann thrombasthenia and Bernard-Soulier syndrome are associated with genetic alterations of platelet surface receptors, also affecting the platelet cytoskeletal structure. In this review, we summarize the current knowledge about platelet structure and adhesion, and delve into the mechanical aspects of platelet function. Platelets lack a nucleus, and can thus provide a minimal model of a biological cell. New biophysical tools may help to scrutinize platelets anew and to extend the existing knowledge on cell biology. PMID:25655000

  7. A simple adhesion assay for studying interactions between platelets and endothelial cells in vitro.

    PubMed

    Xu, Xian-Xiang; Gao, Xing-Hua; Pan, Rong; Lu, Dan; Dai, Yue

    2010-01-01

    Cell adhesion plays a key role during various physiological and pathological processes. Many studies have been performed to understand the interaction of platelets with endothelial cells (ECs) during the past decades. Modulation of their interaction has been shown to be therapeutically useful in thrombotic diseases. Some methods of labeling platelets such as counting and radiolabeling have been applied in the study of the platelets-ECs interaction, but these methods did not obtain full approval. A rapid, simple and sensitive assay for platelets-ECs interaction was developed in this paper. Platelets were labeled with Sudan Black B (SBB) before adding to confluent ECs monolayer. Non-adherent platelets were removed by washing with PBS. The adherent platelets were lysed with dimethylsulfoxide (DMSO) and the absorbance was recorded at 595 nm by spectrophotometer. A linear correlation was observed between the absorbance of SBB and the number of platelets. By employing the SBB method, the influence of heparin on platelets-ECs interactions was observed. Heparin (3-100 units/mL) obviously reduced platelets adhering to ECs in a concentration-dependent manner.

  8. Platelet factor V supports hemostasis in a patient with an acquired factor V inhibitor, as shown by prothrombinase and tenase assays.

    PubMed

    Perdekamp, Maria T Grosse; Rubenstein, David A; Jesty, Jolyon; Hultin, Mae B

    2006-10-01

    A woman with gross hematuria was shown to have a severe isolated factor V deficiency due to a factor V inhibitor of 200 U/ml titer. Hematuria persisted despite multiple infusions of plasma but, after one transfusion with 1 U platelets, urine red blood cells decreased by more than 98%. To evaluate the patient's platelet function we performed prothrombinase and tenase assays with platelets from the patient and from normal donors. By prothrombinase assay, ionophore-activated patient platelets showed 42% of the activity of normal platelets in their ability to support prothrombin activation by activated factor X; whereas in a 'tenase' assay, which measures the platelets' ability to support factor X activation by activated factor IX + activated factor VIII, their activity was 117% of normal. The addition of excess bovine activated factor V to the prothrombinase assay fully corrected the defect. The results demonstrate the benefit of platelet transfusion and indicate that in this case the platelets are the primary source of factor V for hemostasis.

  9. Development of a New Method for Platelet Function Test and Its Shearing Condition in Microfludic System

    NASA Astrophysics Data System (ADS)

    Lee, Hoyoon; Kim, Gyehyu; Choi, Seawhan; Shin, Sehyun; Korea University Department of Mechanical Engineering Team

    2015-11-01

    Platelet is a crucial blood cell on hemostasis. As platelet exposed to high shear stress, it can be activated showing morphological and functional changes to stop bleeding. When platelet is abnormal, there is high risk of cardiovascular diseases. Thus, quick and precise assay for platelet function is important in clinical treatment. In this study, we design a microfluidic system, which can test platelet function exposed with the stimulation of shear and agonists. The microfluidic system consists of three parts: 1) a shear mechanism with rotating stirrer; 2) multiple microchannels to flow samples and to stop; 3) camera-interfaced migration distance(MD) analyzing system. When sheared blood is driven by pressure through the microchannel, shear-activated platelets adhere to a collagen-coated surface, causing blood flow to significantly slow and eventually stop. As the micro-stirrer speed increases, MD decreases exponentially at first, but it increases beyond a critical rpm after all. These results are coincident with data measured by FACS flowcytometry. These results imply that the present system could quantitatively measure the degree of activation, aggregation and adhesion of platelets and that blood MD is potent index for measuring the shear-dependence of platelet function.

  10. Biochemical and functional abnormalities in hypercholesterolemic rabbit platelets

    SciTech Connect

    Dalal, K.B.; Ebbe, S.; Mazoyer, E.; Carpenter, D.; Yee, T. )

    1990-02-01

    This study was designed to elucidate changes in rabbit platelet lipids induced by a cholesterol rich diet and to explore the possible correlation of these lipid changes with platelet abnormalities. Pronounced biochemical alterations were observed when serum cholesterol levels of 700-1000 mg% were reached. Hypercholesterolemic (HC) platelets contained 37% more neutral lipids and 16% less phospholipids than the controls. Lysolecithin, cholesterol esters and phosphatidylinositol (PI) levels were increased in HC platelets, and the levels of phosphatidylcholine (PC) were decreased. The cholesterol/phospholipid molar ratio of lipidemic platelets increased from 0.55 +/- 0.011 to 0.89 +/- 0.016 (P less than 0.01) in eight weeks. HC platelets had 90% more arachidonic acid (AA) in the PI than normal platelets. No significant changes in AA of PC were observed. Platelet function was monitored by the uptake and release of (14C)serotonin in platelet rich plasma (PRP), using varying concentrations of collagen as an aggregating agent. The uptake of (14C)serotonin in HC and normal platelets ranged from 78-94%. The percent of (14C)serotonin released from normal and HC platelets was proportional to the concentration of collagen. However, lipidemic platelets were hyperreactive to low concentrations of collagen. Incorporation of 50 microM acetylsalicylic acid into the aggregating medium suppressed the release of (14C)serotonin in normal PRP by more than 90%, but had only a partial effect on lipidemic PRP.

  11. Effects of Physical (In)activity on Platelet Function

    PubMed Central

    Heber, Stefan; Volf, Ivo

    2015-01-01

    As platelet activation is closely related to the liberation of growth factors and inflammatory mediators, platelets play a central role in the development of CVD. Virtually all cardiovascular risk factors favor platelet hyperreactivity and, accordingly, also physical (in)activity affects platelet function. Within this paper, we will summarize and discuss the current knowledge on the impact of acute and habitual exercise on platelet function. Although there are apparent discrepancies regarding the reported effects of acute, strenuous exercise on platelet activation, a deeper analysis of the available literature reveals that the applied exercise intensity and the subjects' cardiorespiratory fitness represent critical determinants for the observed effects. Consideration of these factors leads to the summary that (i) acute, strenuous exercise can lead to platelet activation, (ii) regular physical activity and/or physical fitness diminish or prevent platelet activation in response to acute exercise, and (iii) habitual physical activity and/or physical fitness also favorably modulate platelet function at physical rest. Notably, these effects of exercise on platelet function show obvious similarities to the well-recognized relation between exercise and the risk for cardiovascular events where vigorous exercise transiently increases the risk for myocardial infarction and a physically active lifestyle dramatically reduces cardiovascular mortality. PMID:26557653

  12. Effect of serotonin on platelet function in cocaine exposed blood

    PubMed Central

    Ziu, Endrit; Hadden, Coedy; Li, Yicong; Lowery, Curtis Lee; Singh, Preeti; Ucer, Serra S.; Mercado, Charles P.; Gu, Howard H.; Kilic, Fusun

    2014-01-01

    5-hydroxytryptamine (5-HT) reuptake inhibitors counteract the pro-thrombotic effect of elevated plasma 5-HT by down-regulating the 5-HT uptake rates of platelets. Cocaine also down-regulates the platelet 5-HT uptake rates but in contrast, the platelets of cocaine-injected mice show a much higher aggregation rate than the platelets of control mice. To examine the involvement of plasma 5-HT in cocaine-mediated platelet aggregation, we studied the function of platelets isolated from wild-type and transgenic, peripheral 5-HT knock-out (TPH1-KO) mice, and cocaine-insensitive dopamine transporter knock in (DAT-KI) mice. In cocaine-injected mice compared to the control mice, the plasma 5-HT level as well as the surface level of P-selectin was elevated; in vitro platelet aggregation in the presence of type I fibrillar collagen was enhanced. However, cocaine injection lowered the 5-HT uptake rates of platelets and increased the plasma 5-HT levels of the DAT-KI mice but did not change their platelets aggregation rates further which are already hyper-reactive. Furthermore, the in vitro studies supporting these in vivo findings suggest that cocaine mimics the effect of elevated plasma 5-HT level on platelets and in 5-HT receptor- and transporter-dependent pathways in a two-step process propagates platelet aggregation by an additive effect of 5-HT and nonserotonergic catecholamine. PMID:25091505

  13. Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay

    PubMed Central

    Lordkipanidzé, Marie; Lowe, Gillian C.; Kirkby, Nicholas S.; Chan, Melissa V.; Lundberg, Martina H.; Morgan, Neil V.; Bem, Danai; Nisar, Shaista P.; Leo, Vincenzo C.; Jones, Matthew L.; Mundell, Stuart J.; Daly, Martina E.; Mumford, Andrew D.; Warner, Timothy D.; Watson, Steve P.; Watson, Steve P.; Mumford, Andrew D.; Mundell, Stuart J.; Gissen, Paul; Daly, Martina E.; Lester, Will; Clark, Justin; Williams, Mike; Motwani, Jayashree; Marshall, Dianne; Nyatanga, Priscilla; Mann, Pat; Kirwan, Julie; Wilde, Jonathan; Dunkley, Tracey; Greenway, April; Makris, Michael; Pavord, Sue; Dattani, Rashesh; Grimley, Gerry Dolan Charlotte; Stokley, Simone; Astwood, Emma; Chang, Cherry; Foros, Merri; Trower, Linda; Thachil, Jecko; Hay, Charlie; Pike, Gill; Will, Andrew; Grainger, John; Foulkes, Matt; Fareh, Mona; Talks, Kate; Biss, Tina; Kesteven, Patrick; Hanley, John; Vowles, Julie; Basey, Lesley; Barnes, Michelle; Collins, Peter; Rayment, Rachel; Alikhan, Raza; Morris, Ana Guerrero Rebecca; Mansell, Dianne; Toh, Cheng Hock; Martlew, Vanessa; Murphy, Elaine; Lachmann, Robin; Rose, Peter; Chapman, Oliver; Lokare, Anand; Marshall, Kathryn; Khan, Naseem; Keeling, David; Giangrande, Paul; Austin, Steve; Bevan, David; Alamelu, Jayanthi

    2014-01-01

    Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .0001). Normal platelet function results were particularly predictive (sensitivity, 94%; negative predictive value, 91%), whereas a positive result was not always substantiated by LTA (specificity, 67%; positive predictive value, 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This trial was registered at www.isrctn.org as #ISRCTN 77951167. PMID:24408324

  14. Platelet Consumption by Arterial Prostheses: The Effects of Endothelialization and Pharmacologic Inhibition of Platelet Function

    PubMed Central

    Harker, Laurence A.; Slichter, Sherrill J.; Sauvage, Lester R.

    1977-01-01

    The thrombogenic mechanism of arterial grafts has been studied by determining the relative utilization of platelets, fibrinogen and plasminogen by human arterial prostheses, and by direct examination of arterial grafts in a baboon model. Forty-one survival and turnover measurements of 51Crplatelets, 131I-fibrinogen and 125I-plasminogen in ten patients with aortofemoral knitted Dacron prostheses demonstrated platelet consumption after graft placement (platelet survival 4.2 days ± 0.5 and turnover 68,000 plat/ul/day ±10,000 compared with 8.2 days ± 0.3 and 35,000 plat/ul/day ± 5,000 respectively for control subjects with stable vascular disease, p < 0.01). In vitro platelet function test results were normal. Platelet consumption was interrupted by dipyridamole or a combination of dipyridamole and acetylsalicylic acid, and platelet survival normalized spontaneously during nine months postoperatively. No significantly increased consumption of fibrinogen or plasminogen was found in these patients with arterial grafts. Placement of impervious knitted Dacron velour aortic grafts in baboons reproduced platelet consumption that progressively normalized over six weeks postoperatively. Platelet survival measurements correlated directly with endothelial cell coverage of the graft luminal surface in these animals implying that endothelialization of the graft surface was also occurring postoperatively in patients. ImagesFig. 4.Fig. 5. PMID:411428

  15. Role of platelet function and platelet membrane glycoproteins in children with primary immune thrombocytopenia

    PubMed Central

    Liu, Wen-Jun; Bai, Jing; Guo, Qu-Lian; Huang, Zhe; Yang, Hong; Bai, Yong-Qi

    2016-01-01

    The aim of the present study was to examine and understand changes in platelet functions prior to and after the treatment of primary immune thrombocytopenia (ITP) in children. An automatic hematology analyzer and whole blood flow cytometry were used to detect immature platelet fraction (IPF), IPC and membrane glycoproteins (CD62p, PAC-1 and CD42b) in ITP children (ITP group), children with complete response after ITP treatment (ITP-CR group) and children with elective surgery (normal control group). The results showed that, levels of platelet count (PLT) and plateletcrit in the ITP group were lower alhtough the levels of mean platelet volume, platelet distribution width and platelet-large cell ratio (P-LCR) were higher than those in the normal control and ITP-CR groups. PLT in the ITP-CR group was lower than that in the normal controls. Additionally, IPF% was higher in the normal control and ITP-CR groups, IPC was lower in the ITP group compared to the normal control and ITP-CR groups. Furthermore, prior to ADP activation, the expression levels of CD62p, PAC-1 and CD42b in the ITP group were lower in ITP group than those in the normal control and ITP-CR groups. The expression level of PAC-1 was lower in the ITP-CR and normal control groups. No differences were identified in CD62p and CD42b expression levels. Following ATP activation, CD62p, PAC-1 and CD42b expression in the ITP group was lower than that in the normal control and ITP-CR groups. PAC-1 expression was lower while CD62p expression was higher in the ITP-CR group compared to the normal control group. In conclusion, the activation of platelets in ITP children was low. Decreased platelet function, platelet parameters and platelet glycoproteins may be used as markers for monitoring the treatment efficacy in ITP children. PMID:27431926

  16. Platelet function following trauma. A multiple electrode aggregometry study.

    PubMed

    Solomon, Cristina; Traintinger, Stefan; Ziegler, Bernhard; Hanke, Alexander; Rahe-Meyer, Niels; Voelckel, Wolfgang; Schöchl, Herbert

    2011-08-01

    Platelets play a central role in coagulation. Currently, information on platelet function following trauma is limited. We performed a retrospective analysis of patients admitted to the emergency room (ER) at the AUVA Trauma Centre, Salzburg, after sustaining traumatic injury. Immediately after admission to the ER, blood was drawn for blood cell counts, standard coagulation tests, and platelet function testing. Platelet function was assessed by multiplate electrode aggregometry (MEA) using adenosine diphosphate (ADPtest), collagen (COLtest) and thrombin receptor activating peptide-6 (TRAPtest) as activators. The thromboelastometric platelet component, measuring the contribution of platelets to the elasticity of the whole-blood clot, was assessed using the ROTEM device. The study included 163 patients, 79.7% were male, and the median age was 43 years. The median injury severity score was 18. Twenty patients (12.3%) died. Median platelet count was significantly lower among non-survivors than survivors (181,000/μl vs. 212,000/μl; p=0.01). Although platelet function defects were relatively minor, significant differences between survivors and non-survivors were observed in the ADPtest (94 vs. 79 U; p=0.0019), TRAPtest (136 vs. 115 U; p<0.0001), and platelet component (134 vs.103 MCEEXTEM - MCEFIBTEM; p=0.0012). Aggregometry values below the normal range for ADPtest and TRAPtest were significantly more frequent in non-survivors than in survivors (p=0.0017 and p=0.0002, respectively). Minor decreases in platelet function upon admission to the ER were a sign of coagulopathy accompanying increased mortality in patients with trauma. Further studies are warranted to confirm these results and investigate the role of platelet function in trauma haemostatic management. PMID:21655681

  17. Platelet function in brown bear (Ursus arctos) compared to man

    PubMed Central

    2010-01-01

    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. PMID:20525167

  18. Point-of-care platelet function tests: detection of platelet inhibition induced by nonopioid analgesic drugs.

    PubMed

    Scharbert, Gisela; Gebhardt, Kristina; Sow, Zacharia; Duris, Monika; Deusch, Engelbert; Kozek-Langenecker, Sibylle

    2007-12-01

    Detection of platelet inhibition is of clinical relevance in the preinterventional risk-benefit assessment in chronic low-back-pain patients scheduled for invasive pain therapy. We evaluated the sensitivity of various point-of-care platelet function tests for the detection of platelet inhibition induced by nonopioid analgesic drugs. After Institutional Review Board approval and informed consent, citrated whole blood from 40 patients with chronic unspecific low back pain was investigated before and 30 min after intravenous infusion of the study medication consisting of diclofenac 75 mg (plus orphenadrin 30 mg; Neodolpasse; Fresenius Kabi Austria GmbH, Austria), parecoxib 40 mg (Dynastat; Pharmacia Europe EEIG, UK), paracetamol 1 g (Perfalgan; Bieffe Medital S.P.A., Italy), or normal saline in a randomized, cross-over, double-blinded, placebo-controlled study. Platelet function was assessed using the PFA-100 platelet function analyzer and thromboelastometry, as well as impedance aggregometry (in the last 17 patients recruited after it became commercially available). Sensitivity for detecting diclofenac-induced platelet inhibition was 85% for the PFA-100 using epinephrine as agonist and 94% for arachidonic acid-induced impedance aggregometry. ADP-induced platelet function tests, as well as cytochalasin D-modified thromboelastometry were unreliable. All tests had a low incidence of false-positive test results after normal saline. Paracetamol and parecoxib had no significant platelet inhibiting effect. The PFA-100 using epinephrine as agonist and arachidonic acid-induced impedance aggregometry are recommended for the detection of cyclooxygenase-I-inhibiting effects of nonsteroidal anti-inflammatory drugs such as diclofenac. Our findings confirm that a single rescue dose of paracetamol and parecoxib has no antiplatelet effect. PMID:17982319

  19. Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation

    PubMed Central

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

    2016-01-01

    Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents. PMID:27716777

  20. Proteasome proteolysis supports stimulated platelet function and thrombosis

    PubMed Central

    Gupta, Nilaksh; Li, Wei; Willard, Belinda; Silverstein, Roy L.; McIntyre, Thomas M.

    2014-01-01

    Objective Proteasome inhibitors are in use to treat hematologic cancers, but also reduce thrombosis. Whether the proteasome participates in platelet activation or function is opaque since little is known of the proteasome in these terminally differentiated cells. Approach and Results Platelets displayed all three primary proteasome protease activities, which MG132 and bortezomib (Velcade®) inhibited. Proteasome substrates are marked by ubiquitin, and platelets contained a functional ubiquitination system that modified the proteome by mono- and poly-ubiquitination. Systemic MG132 strongly suppressed formation of occlusive, platelet-rich thrombi in FeCl3-damaged carotid arteries. Transfusion of platelets treated ex vivo with MG132 and washed prior to transfusion into thrombocytopenic mice also reduced carotid artery thrombosis. Proteasome inhibition reduced platelet aggregation by low thrombin concentrations and ristocetin-stimulated agglutination through the GPIb-IX-V complex. This receptor was not appropriately internalized after proteasome inhibition in stimulated platelets, and spreading and clot retraction after MG132 exposure also were decreased. The effects of proteasome inhibitors were not confined to a single receptor as MG132 suppressed thrombin-, ADP-, and LPS-stimulated microparticle shedding. Proteasome inhibition increased ubiquitin decoration of cytoplasmic proteins, including the cytoskeletal proteins Filamin A and Talin-1. Mass spectrometry revealed a single MG132-sensitive tryptic cleavage after R1745 in an extended Filamin A loop, which would separate its actin-binding domain from its carboxy terminal GPIbα binding domain. Conclusions Platelets contain a ubiquitin/proteasome system that marks cytoskeletal proteins for proteolytic modification to promote productive platelet-platelet and platelet-wall interactions. PMID:24177323

  1. Platelet Serotonin Transporter Function Predicts Default-Mode Network Activity

    PubMed Central

    Kasess, Christian H.; Meyer, Bernhard M.; Hofmaier, Tina; Diers, Kersten; Bartova, Lucie; Pail, Gerald; Huf, Wolfgang; Uzelac, Zeljko; Hartinger, Beate; Kalcher, Klaudius; Perkmann, Thomas; Haslacher, Helmuth; Meyer-Lindenberg, Andreas; Kasper, Siegfried; Freissmuth, Michael; Windischberger, Christian; Willeit, Matthäus; Lanzenberger, Rupert; Esterbauer, Harald; Brocke, Burkhard; Moser, Ewald; Sitte, Harald H.; Pezawas, Lukas

    2014-01-01

    Background The serotonin transporter (5-HTT) is abundantly expressed in humans by the serotonin transporter gene SLC6A4 and removes serotonin (5-HT) from extracellular space. A blood-brain relationship between platelet and synaptosomal 5-HT reuptake has been suggested, but it is unknown today, if platelet 5-HT uptake can predict neural activation of human brain networks that are known to be under serotonergic influence. Methods A functional magnetic resonance study was performed in 48 healthy subjects and maximal 5-HT uptake velocity (Vmax) was assessed in blood platelets. We used a mixed-effects multilevel analysis technique (MEMA) to test for linear relationships between whole-brain, blood-oxygen-level dependent (BOLD) activity and platelet Vmax. Results The present study demonstrates that increases in platelet Vmax significantly predict default-mode network (DMN) suppression in healthy subjects independent of genetic variation within SLC6A4. Furthermore, functional connectivity analyses indicate that platelet Vmax is related to global DMN activation and not intrinsic DMN connectivity. Conclusion This study provides evidence that platelet Vmax predicts global DMN activation changes in healthy subjects. Given previous reports on platelet-synaptosomal Vmax coupling, results further suggest an important role of neuronal 5-HT reuptake in DMN regulation. PMID:24667541

  2. Function of platelet 47K protein phosphorylation

    SciTech Connect

    Imaoka, T.

    1987-05-01

    To provide insight into the biochemical pathway of platelet activation, they purified both unphosphorylated and phosphorylated P47 to homogeneity from human platelets. This study represents the first demonstration of a change of physiological action of P47 in response to phosphorylation in platelet activation. SVI labelled unphosphorylated P47 had an ability to bind with platelet membrane fraction in the presence of phosphatidylserine. Effect of diacylglycerol was inhibitory in this PS dependent P47 binding with membrane. Unphosphorylated P47 had an inhibitory activity in platelet actin polymerization. Molar ratio to inhibit actin polymerization was 1:8 (P47:actin). These activities were Ca independent. Purified TSP-labelled P47 lost the binding ability with membrane, also the inhibitory activity in actin polymerization. Therefore, they propose the hypothesis that unphosphorylated P47 may loosely bind with the inside of plasma membrane of platelet and inhibit actin polymerization as a modulator, when stimulated, protein Kinase C rapidly phosphorylate P47 and induce the activation of cytoskeletal network and subsequently release reaction.

  3. Effects of microgravity and hypergravity on platelet functions.

    PubMed

    Dai, Kesheng; Wang, Yuedan; Yan, Rong; Shi, Quanwei; Wang, Zhicheng; Yuan, Yanhong; Cheng, Hong; Li, Suping; Fan, Yubo; Zhuang, Fengyuan

    2009-05-01

    Many serious thrombotic and haemorrhagic diseases or fatalities have been documented in human being exposed to microgravity or hypergravity environments, such as crewmen in space, roller coaster riders, and aircrew subjected to high-G training. Some possible related organs have been examined to explore the mechanisms underlying these gravity change-related diseases. However, the role of platelets which are the primary players in both thrombosis and haemostasis is unknown. Here we show that platelet aggregation induced by ristocetin or collagen and platelet adhesion to von Willebrand factor (VWF) were significantly decreased after platelets were exposed to simulated microgravity. Conversely, these platelet functions were increased after platelets were exposed to hypergravity. The tail bleeding time in vivo was significantly shortened in mice exposed to high-G force, whereas, was prolonged in hindlimb unloaded mice. Furthermore, three of 23 mice died after 15 minutes of -8 Gx stress. Platelet thrombi disseminated in the heart ventricle and blood vessels in the brain, lung, and heart from the dead mice. Finally, glycoprotein (GP) Ibalpha surface expression and its association with the cytoskeleton were significantly decreased in platelets exposed to simulated microgravity, and obviously increased in hypergravity-exposed platelets. These data indicate that the platelet functions are inhibited in microgravity environments, and activated under high-G conditions, suggesting a novel mechanism for gravity change-related haemorrhagic and thrombotic diseases. This mechanism has important implications for preventing and treating gravity change-related diseases, and also suggests that special attentions should be paid to human actions under different gravity conditions. PMID:19404544

  4. Is platelet function as measured by Thrombelastograph monitoring in whole blood affected by platelet inhibitors?

    PubMed

    Bailey, Lori A; Sistino, Joseph J; Uber, Walter E

    2005-03-01

    were comparable to control values for all parameters measured. Although statistical significance could be demonstrated with some parameters, sensitivity was only observed at increased doses and was not seen with all agents tested. In our in vitro model, the TEG monitor was unable to demonstrate clinically significant differences in platelet function and may not be reflective of platelet function in samples which have been treated with these GP IIb/IIIa inhibitors.

  5. Role of platelet function and platelet membrane glycoproteins in children with primary immune thrombocytopenia.

    PubMed

    Liu, Wen-Jun; Bai, Jing; Guo, Qu-Lian; Huang, Zhe; Yang, Hong; Bai, Yong-Qi

    2016-09-01

    The aim of the present study was to examine and understand changes in platelet functions prior to and after the treatment of primary immune thrombocytopenia (ITP) in children. An automatic hematology analyzer and whole blood flow cytometry were used to detect immature platelet fraction (IPF), IPC and membrane glycoproteins (CD62p, PAC-1 and CD42b) in ITP children (ITP group), children with complete response after ITP treatment (ITP-CR group) and children with elective surgery (normal control group). The results showed that, levels of platelet count (PLT) and plateletcrit in the ITP group were lower alhtough the levels of mean platelet volume, platelet distribution width and platelet-large cell ratio (P-LCR) were higher than those in the normal control and ITP-CR groups. PLT in the ITP-CR group was lower than that in the normal controls. Additionally, IPF% was higher in the normal control and ITP-CR groups, IPC was lower in the ITP group compared to the normal control and ITP-CR groups. Furthermore, prior to ADP activation, the expression levels of CD62p, PAC-1 and CD42b in the ITP group were lower in ITP group than those in the normal control and ITP-CR groups. The expression level of PAC-1 was lower in the ITP-CR and normal control groups. No differences were identified in CD62p and CD42b expression levels. Following ATP activation, CD62p, PAC-1 and CD42b expression in the ITP group was lower than that in the normal control and ITP-CR groups. PAC-1 expression was lower while CD62p expression was higher in the ITP-CR group compared to the normal control group. In conclusion, the activation of platelets in ITP children was low. Decreased platelet function, platelet parameters and platelet glycoproteins may be used as markers for monitoring the treatment efficacy in ITP children. PMID:27431926

  6. Multiscale prediction of patient-specific platelet function under flow.

    PubMed

    Flamm, Matthew H; Colace, Thomas V; Chatterjee, Manash S; Jing, Huiyan; Zhou, Songtao; Jaeger, Daniel; Brass, Lawrence F; Sinno, Talid; Diamond, Scott L

    2012-07-01

    During thrombotic or hemostatic episodes, platelets bind collagen and release ADP and thromboxane A(2), recruiting additional platelets to a growing deposit that distorts the flow field. Prediction of clotting function under hemodynamic conditions for a patient's platelet phenotype remains a challenge. A platelet signaling phenotype was obtained for 3 healthy donors using pairwise agonist scanning, in which calcium dye-loaded platelets were exposed to pairwise combinations of ADP, U46619, and convulxin to activate the P2Y(1)/P2Y(12), TP, and GPVI receptors, respectively, with and without the prostacyclin receptor agonist iloprost. A neural network model was trained on each donor's pairwise agonist scanning experiment and then embedded into a multiscale Monte Carlo simulation of donor-specific platelet deposition under flow. The simulations were compared directly with microfluidic experiments of whole blood flowing over collagen at 200 and 1000/s wall shear rate. The simulations predicted the ranked order of drug sensitivity for indomethacin, aspirin, MRS-2179 (a P2Y(1) inhibitor), and iloprost. Consistent with measurement and simulation, one donor displayed larger clots and another presented with indomethacin resistance (revealing a novel heterozygote TP-V241G mutation). In silico representations of a subject's platelet phenotype allowed prediction of blood function under flow, essential for identifying patient-specific risks, drug responses, and novel genotypes.

  7. Megakaryocytes and platelets express nicotinic acetylcholine receptors but nicotine does not affect megakaryopoiesis or platelet function.

    PubMed

    Schedel, Angelika; Kaiser, Kerstin; Uhlig, Stefanie; Lorenz, Florian; Sarin, Anip; Starigk, Julian; Hassmann, Dennis; Bieback, Karen; Bugert, Peter

    2016-01-01

    In our previous investigations we have shown that platelets and their precursors express nicotinic α7 acetylcholine receptors (nAChRα7) that are involved in platelet function and in vitro differentiation of the megakaryoblastic cell line MEG-01. In this study, we were interested in the expression analysis of additional nAChR and the effects of nicotine in an ex vivo model using megakaryocytic cells differentiated from cord blood derived CD34(+) cells (CBMK) and an in vivo model using blood samples from smokers. CBMK were differentiated with thrombopoietin (TPO) for up to 17 days. Quantitative real-time PCR (QRT-PCR), Western blot analysis and flow cytometry were used to investigate nAChR expression (nAChRα7, nAChRα4, nAChRβ2) and nicotine effects. In blood samples of 15 nonsmokers and 16 smokers platelet parameters (count, mean platelet volume--MPV and platelet distribution width--PDW) were determined as indicators for changes of in vivo megakaryopoiesis. Platelet function was determined by the use of whole blood aggregometry and flow cytometry. The functional role of nAChR was evaluated using specific antagonists in aggregometry. CHRNA7, CHRNA4 and CHRNB2 gene transcripts and the corresponding proteins could be identified in CBMK during all stages of differentiation. Platelets contain nAChRα7 and nAChRβ2 but not nAChRα4. Nicotine had no effect on TPO-induced differentiation of CBMK. There was no significant difference in all platelet parameters of the smokers compared to the nonsmokers. In line with this, cholinergic gene transcripts as well as the encoded proteins were equally expressed in both the study groups. Despite our observation of nAChR expression in megakaryopoiesis and platelets, we were not able to detect effects of nicotine in our ex vivo and in vivo models. Thus, the functional role of the nAChR in these cells remains open.

  8. Modification of Solid Phase Red Cell Adherence Assay for the Detection of Platelet Antibodies in Patients With Thrombocytopenia

    PubMed Central

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

    2009-01-01

    Platelet refractoriness is caused by HLA antibodies and platelet-specific antibodies. Current methods used to detect antiplatelet antibodies have limitations. Solid phase red cell adherence (SPRCA) lacks sensitivity and requires a second assay using chloroquine-treated intact platelets to specify the response due to anti-HLA. We modified SPRCA by using 2 types of antihuman platelet antibodies with different specificities toward platelet lysate and tested samples from 361 patients (69 with unexplained thrombocytopenia and 292 with poor response to platelet transfusions not explicable by alloimmunization or the clinical situation) and 50 from healthy volunteers. Our method compared favorably with platelet suspension direct immunofluorescence. All samples from healthy volunteers were negative; of the samples from the patient population, 240 were positive (147 samples had only antiplatelet and 3 samples had only anti-HLA antibodies). This modified technique had a sensitivity of 98% and a specificity of 91%. PMID:18701420

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

    PubMed

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

    2008-09-01

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

  10. Utility of the ISTH bleeding assessment tool in predicting platelet defects in participants with suspected inherited platelet function disorders

    PubMed Central

    Lowe, G C; Lordkipanidzé, M; Watson on behalf of the uk gapp study group, S P

    2013-01-01

    Background The ISTH bleeding assessment tool (ISTH-BAT) was developed to record bleeding symptoms and to aid diagnosis in patients with a possible bleeding disorder. Objectives To investigate the utility of the ISTH-BAT in predicting functional defects in platelet activation in participants with suspected inherited platelet function disorders. Patients/Methods Participants with clinical evidence of excessive bleeding and suspected inherited platelet function disorders and healthy volunteers were recruited to the Genotyping and Phenotyping of Platelets study (GAPP; ISRCTN 77951167). The ISTH-BAT questionnaire was applied by a trained investigator prior to lumiaggregometry. Results One hundred participants were included (79 with suspected inherited platelet function disorders, and 21 healthy volunteers). The ISTH-BAT score in participants with suspected inherited platelet function disorders (median 12; interquartile range [IQR] 8–16) was significantly higher than in healthy volunteers (median 0; IQR 0–0). There was no difference between participants with suspected inherited platelet function disorders with a platelet defect detected by lumiaggregometry (median 11; IQR 8–16) and those with normal platelet function (median 12; IQR 8–14) (P > 0.05). The ISTH-BAT score was not associated with a demonstrable platelet defect on platelet function testing (area under the receiver operating characteristic curve = 0.501 [95% confidence interval 0.372–0.630, P = 0.98] and odds ratio 1.01 [95% confidence interval 0.93–1.09, P = 0.91]). Conclusions The ISTH-BAT is a powerful tool for documenting lifelong bleeding history. However, the score obtained is not predictive of the presence of a platelet defect on lumiaggregometry in patients with suspected inherited platelet function disorders. PMID:23809206

  11. Platelet hexosaminidase a enzyme assay effectively detects carriers missed by targeted DNA mutation analysis.

    PubMed

    Nakagawa, Sachiko; Zhan, Jie; Sun, Wei; Ferreira, Jose Carlos; Keiles, Steven; Hambuch, Tina; Kammesheidt, Anja; Mark, Brian L; Schneider, Adele; Gross, Susan; Schreiber-Agus, Nicole

    2012-01-01

    Biochemical testing of hexosaminidase A (HexA) enzyme activity has been available for decades and has the ability to detect almost all Tay-Sachs disease (TSD) carriers, irrespective of ethnic background. This is increasingly important, as the gene pool of those who identify as Ashkenazi Jewish is diversifying. Here we describe the analysis of a cohort of 4,325 individuals arising from large carrier screening programs and tested by the serum and/or platelet HexA enzyme assays and by targeted DNA mutation analysis. Our results continue to support the platelet assay as a highly effective method for TSD carrier screening, with a low inconclusive rate and the ability to detect possible disease-causing mutation carriers that would have been missed by targeted DNA mutation analysis. Sequence analysis performed on one such platelet assay carrier, who had one non-Ashkenazi Jewish parent, identified the amino acid change Thr259Ala (A775G). Based on crystallographic modeling, this change is predicted to be deleterious, as threonine 259 is positioned proximal to the HexA alpha subunit active site and helps to stabilize key residues therein. Accordingly, if individuals are screened for TSD in broad-based programs by targeted molecular testing alone, they must be made aware that there is a more sensitive and inexpensive test available that can identify additional carriers. Alternatively, the enzyme assays can be offered as a first tier test, especially when screening individuals of mixed or non-Jewish ancestry. PMID:23430931

  12. Use of a Thrombelastograph Platelet Mapping Assay for Diagnosis of Clopidogrel Resistance: A Case Report

    PubMed Central

    Morton, John; Nelluri, Satyanarayana

    2009-01-01

    Abstract: A 62-year-old woman presented to the emergency department with sudden collapse, intractable ventricular fibrillation, and an inferior wall myocardial infarction (MI). An emergent cardiac catheterization showed a totally occluded right coronary artery (RCA). A bare-metal stent was placed in the stenosis, resulting in thrombolysis in myocardial infarction (TIMI)-III flow with 0% residual stenosis. Four days after stenting, the patient developed chest pain. A repeat cardiac catheterization showed a totally occluded stent. The patient was subsequently tested using a thrombelastograph (TEG) Platelet Mapping assay to exclude clopidogrel resistance. The assay confirmed the patient to be non-responsive to clopidogrel for the inhibition of platelet ADP receptors. In an attempt to increase ADP inhibition, the ADP antagonist was changed to ticlopidine. Further testing was confounded by the presence of abciximab; however, the patient has remained free of cardiac events. PMID:19361030

  13. Platelet function and hemolysis in centrifugal pumps: in vitro investigations.

    PubMed

    Steines, D; Westphal, D; Göbel, C; Reul, H; Rau, G

    1999-08-01

    The effects of centrifugal pumps on blood components other than erythrocytes, namely platelets and their interaction with the coagulation system, are not very well known. In a comparative study with three centrifugal pumps (BioMedicus BP-80, St. Jude Isoflow, and Sarns Delphin) and the Stockert roller pump hemolysis, platelet counts, thromboplastin and partial thromboplastin times, as well as resonance thrombography (RTG) parameters for the assessment of platelet and coagulation function were evaluated in vitro. Normalized indices of hemolysis (NIH) with ACD anticoagulation after 360 minutes were 0.008+/-0.004 (Isoflow), 0.018+/-0.017 (BP-80), 0.085+/-0.051 (Delphin), and 0.049+/-0.010 g/1001 (roller pump). Plasmatic coagulation was activated in all circuits. Platelet function was severely inhibited by the BP-80, indicated by increase in RTG platelet time to 358%+/-150% of initial values compared to 42%+/-29% (Isoflow), 40%+/-20% (Delphin), and 12%+/-10% (roller pump). Fibrin polymerization was affected similarly. The large surface area of the BP-80 leads to an extensive activation of platelets and plasminogen.

  14. No associations exist between mean platelet volume or platelet distribution width and thyroid function in Chinese

    PubMed Central

    Ren, Xiaojun; Meng, Zhaowei; Liu, Ming; Zhu, Mei; He, Qing; Zhang, Qing; Liu, Li; Song, Kun; Jia, Qiyu; Jia, Qiang; Li, Xue; Tan, Jian; Zheng, Wei; Wang, Renfei; Liu, Na; Hu, Tianpeng

    2016-01-01

    Abstract Mean platelet volume (MPV) and platelet distribution width (PDW) are morphometric indices of size distribution and variability of platelet. We aimed to explore the associations between MPV or PDW and thyroid function in a large Chinese cohort. This was a cross-sectional study with a recruitment of 13,622 self-reported healthy Chinese (8424 males, 5198 females). Clinical data of the participants comprised of anthropometric measurements, hepatic function, renal function, serum levels of lipid, glucose, C-reactive protein, erythrocyte sedimentation rate, platelet, MPV, PDW, and thyroid hormones. Database was sorted by sex, and the associations between MPV or PDW and thyroid function were analyzed by quartiles of MPV or PDW. Levels of MPV and PDW were compared in different thyroid function subgroups by 1-way analysis of variance and independent sample's t test. Receiver-operating characteristic (ROC) curve was adopted to determine diagnostic values of MPV and PDW for thyroid dysfunction. Crude and adjusted odds ratios of MPV and PDW for thyroid dysfunction with 95% confidence intervals were analyzed by binary logistic regression models. MPV, PDW, and thyroid stimulation hormone were significantly higher in females than in males. Females showed significantly higher incidence of hypothyroidism and hyperthyroidism than males. However, there were no significant differences of MPV and PDW among different thyroid function subgroups in both sexes, and no obvious correlations were revealed between MPV or PDW and thyroid function. From ROC analysis, we demonstrated no diagnostic values of MPV and PDW for thyroid dysfunction. From binary logistic regression models, no risks of different MPV and PDW quartiles were identified for thyroid dysfunction in both sexes. We could not show any association between MPV or PDW and thyroid function. Prospective studies with better defined risk groups should be performed in the future for further verification and validation. PMID

  15. A comparison of a solid phase IRC assay and the PSIFT for detection of antibodies to platelets.

    PubMed

    Häcker-Shahin, B; Giannitsis, D J

    1992-01-01

    A rapid solid phase indicator red cells assay (IRCA) for detection of platelet antibodies was developed and its sensitivity compared with PSIFT. Platelets were attached to the surface of polystyrene microtitre plate wells by means of a sodium carbonate buffer and centrifugation. Uncovered areas were blocked by a gelatin blocking buffer. After serum incubation bound platelet-specific antibodies were made visible by anti-IgG-coated indicator red cells and a brief centrifugation. A positive result, meaning the presence of an anti-platelet antibody was indicated by red cell adherence over the reaction surface. In the absence of serum antibodies to platelets the indicator red cells formed a pellet. The IRCA showed a high sensitivity; the anti-platelet antibody Thrombocyte was detectable until a dilution of 1:1,600 whereas the same antibody in the PSIFT could only be detected until a dilution of 1:400.

  16. Platelet Function During Hypothermia in Experimental Mock Circulation.

    PubMed

    Van Poucke, Sven; Stevens, Kris; Kicken, Cécile; Simons, Antoine; Marcus, Abraham; Lancé, Marcus

    2016-03-01

    Alterations in platelet function are a common finding in surgical procedures involving cardiopulmonary bypass and hypothermia. Although the combined impact of hypothermia and artificial circulation on platelets has been studied before, the ultimate strategy to safely minimize the risk for bleeding and thrombosis is yet unknown. The aim of this study was to evaluate the use of a mock circulation loop to study the impact of hypothermia for platelet-related hemostatic changes. Venous blood was collected from healthy adult humans (n = 3). Closed mock circulation loops were assembled, each consisting of a centrifugal pump, an oxygenator with integrated heat exchanger, and a hardshell venous reservoir. The experiment started with the mock circulation temperature set at 37°C (T0 [0 h]). Cooling was then initiated at T1 (+2 h), where temperature was adjusted from 37°C to 32°C. Hypothermia was maintained from T2 (+4 h) to T3 (+28 h). From that point in time, rewarming from 32°C to 37°C was initiated with similar speed as cooling. From time point T4 (+30 h), normothermia (37°C) was maintained until the experiment ended at T5 (+32 h). Blood samples were analyzed in standard hematological tests: light transmission aggregometry (LTA) (arachidonic acid [AA], adenosine diphosphate [ADP], collagen [COL], thrombin-receptor-activating-peptide-14 [TRAP]), multiple electrode aggregometry (MEA) (AA, ADP, COL, TRAP), and rotational thromboelastometry (ROTEM) (EXTEM, FIBTEM, PLTEM). Hemoglobin, hematocrit, and platelet count decrease more substantially during temperature drop (37-32°C) than during hypothermia maintenance. Hb and Hct continue to follow this trend during active rewarming (32-37°C). PC increase from the moment active rewarming was initiated. None of the values return to the initial values. LTA values demonstrate a similar decrease in aggregation after stimulation with the platelet agonists between the start of the mock circulation and the start of cooling. Except

  17. Platelet microparticles reprogram macrophage gene expression and function.

    PubMed

    Laffont, Benoit; Corduan, Aurélie; Rousseau, Matthieu; Duchez, Anne-Claire; Lee, Chan Ho C; Boilard, Eric; Provost, Patrick

    2016-01-01

    Platelet microparticles (MPs) represent the most abundant MPs subtype in the circulation, and can mediate intercellular communication through delivery of bioactives molecules, such as cytokines, proteins, lipids and RNAs. Here, we show that platelet MPs can be internalised by primary human macrophages and deliver functional miR-126-3p. The increase in macrophage miR-126-3p levels was not prevented by actinomycin D, suggesting that it was not due to de novo gene transcription. Platelet MPs dose-dependently downregulated expression of four predicted mRNA targets of miR-126-3p, two of which were confirmed also at the protein level. The mRNA downregulatory effects of platelet MPs were abrogated by expression of a neutralising miR-126-3p sponge, implying the involvement of miR-126-3p. Transcriptome-wide, microarray analyses revealed that as many as 66 microRNAs and 653 additional RNAs were significantly and differentially expressed in macrophages upon exposure to platelet MPs. More specifically, platelet MPs induced an upregulation of 34 microRNAs and a concomitant downregulation of 367 RNAs, including mRNAs encoding for cytokines/chemokines CCL4, CSF1 and TNF. These changes were associated with reduced CCL4, CSF1 and TNF cytokine/chemokine release by macrophages, and accompanied by a marked increase in their phagocytic capacity. These findings demonstrate that platelet MPs can modify the transcriptome of macrophages, and reprogram their function towards a phagocytic phenotype. PMID:26333874

  18. Platelet function in dogs with bacterial infections and leishmaniasis.

    PubMed

    Abid, Monia; Kalbantner, Kerstin; Mischke, Reinhard

    2015-01-01

    The objective of this study was to examine the influence of bacterial infections or leishmaniasis on primary haemostasis in dogs. Capillary bleeding time, automatic platelet function analysis (PFA-100), turbidimetric platelet aggregation, impedance aggregometry, platelet count and, in addition, the haematocrit were investigated in 25 dogs with bacterial infections or leishmaniasis . Results of these diseased dogs were compared to the control group and additionally classified into two subgroups based on criteria of systemic inflammatory response syndrome (SIRS) (groups "SIRS" and "Non-SIRS"). Dogs with infections had a significantly prolonged closure time of the PFA-100 using both cartridges (e. g., collagen/ADP: 83 [55-301] vs. 65 [47-99 s; median [minimum-maximum]; p < 0.0001), a significant decrease in maximal aggregation of the turbidimetric aggregometry (e. g., ADP-induced: 45.2 ± 26.8 vs. 67.3 ± 21.8%; mean ± SD; P = 0.003), a significant increase of collagen-induced impedance aggregometry and a significant suppression of arachidonic acid-induced impedance aggregometry. An enhanced collagen-induced impedance aggregation was the only significant difference between subgroups "SIRS"and "Non-SIRS". In conclusion, although individual tests indicate enhanced platelet aggregation, most of the in vitro tests revealed a normal to moderately reduced functionality. The reduced aggregabiity may partly indicate preactivation of platelets. PMID:26281441

  19. Microfluidic assessment of functional culture-derived platelets in human thrombi under flow.

    PubMed

    Kamat, Viraj; Muthard, Ryan W; Li, Ruizhi; Diamond, Scott L

    2015-10-01

    Despite their clinical significance, human platelets are not amenable to genetic manipulation, thus forcing a reliance on mouse models. Culture-derived platelets (CDPs) from human peripheral blood CD34(+) cells can be genetically altered and may eventually be used for transfusions. By use of microfluidics, the time-dependent incorporation of CD41(+)CD42(+) CDPs into clots was measured using only 54,000 CDPs doped into 27 μL of human whole blood perfused over collagen at a wall shear rate of 100 sec(-1). With the use of fluorescence-labeled human platelets (instead of CDPs) doped between 0.25% and 2% of total platelets, incorporation was highly quantitative and allowed monitoring of the anti-αIIbβ3 antagonism that occurred after collagen adhesion. CDPs were only 15% as efficient as human platelets in their incorporation into human thrombi under flow, although both cell types were equally antagonized by αIIbβ3 inhibition. Transient transfection allowed the monitoring of GFP(+) human CDP incorporation into clots. This assay quantifies genetically altered CDP function under flow. PMID:26145051

  20. Microfluidic assessment of functional culture-derived platelets in human thrombi under flow.

    PubMed

    Kamat, Viraj; Muthard, Ryan W; Li, Ruizhi; Diamond, Scott L

    2015-10-01

    Despite their clinical significance, human platelets are not amenable to genetic manipulation, thus forcing a reliance on mouse models. Culture-derived platelets (CDPs) from human peripheral blood CD34(+) cells can be genetically altered and may eventually be used for transfusions. By use of microfluidics, the time-dependent incorporation of CD41(+)CD42(+) CDPs into clots was measured using only 54,000 CDPs doped into 27 μL of human whole blood perfused over collagen at a wall shear rate of 100 sec(-1). With the use of fluorescence-labeled human platelets (instead of CDPs) doped between 0.25% and 2% of total platelets, incorporation was highly quantitative and allowed monitoring of the anti-αIIbβ3 antagonism that occurred after collagen adhesion. CDPs were only 15% as efficient as human platelets in their incorporation into human thrombi under flow, although both cell types were equally antagonized by αIIbβ3 inhibition. Transient transfection allowed the monitoring of GFP(+) human CDP incorporation into clots. This assay quantifies genetically altered CDP function under flow.

  1. p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo

    PubMed Central

    Canault, Matthias; Duerschmied, Daniel; Brill, Alexander; Stefanini, Lucia; Schatzberg, Daphne; Cifuni, Stephen M.

    2010-01-01

    Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-α and GPV. We recently demonstrated that tumor necrosis factor-α converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets. PMID:19965619

  2. p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo.

    PubMed

    Canault, Matthias; Duerschmied, Daniel; Brill, Alexander; Stefanini, Lucia; Schatzberg, Daphne; Cifuni, Stephen M; Bergmeier, Wolfgang; Wagner, Denisa D

    2010-03-01

    Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-alpha and GPV. We recently demonstrated that tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37 degrees C or 22 degrees C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

  3. Platelet Inhibitors.

    PubMed

    Shifrin, Megan M; Widmar, S Brian

    2016-03-01

    Antithrombotic medications have become standard of care for management of acute coronary syndrome. Platelet adhesion, activation, and aggregation are essential components of platelet function; platelet-inhibiting medications interfere with these components and reduce incidence of thrombosis. Active bleeding is a contraindication for administration of platelet inhibitors. There is currently no reversal agent for platelet inhibitors, although platelet transfusion may be used to correct active bleeding after administration of platelet inhibitors. PMID:26897422

  4. Antiplatelet drugs in patients with enhanced platelet turnover: biomarkers versus platelet function testing.

    PubMed

    Freynhofer, Matthias K; Gruber, Susanne C; Grove, Erik L; Weiss, Thomas W; Wojta, Johann; Huber, Kurt

    2015-08-31

    Platelets are key players in atherothrombosis. Antiplatelet therapy comprising aspirin alone or with P2Y12-inhibitors are effective for prevention of atherothrombotic complications. However, there is interindividual variability in the response to antiplatelet drugs, leaving some patients at increased risk of recurrent atherothrombotic events. Several risk factors associated with high on-treatment platelet reactivity (HTPR), including elevated platelet turnover, have been identified. Platelet turnover is adequately estimated from the fraction of reticulated platelets. Reticulated platelets are young platelets, characterised by residual messenger RNA. They are larger, haemostatically more active and there is evidence that platelet turnover is a causal and prognostic factor in atherothrombotic disease. Whether platelet turnover per se represents a key factor in pathogenesis, progression and prognosis of atherothrombotic diseases (with focus on acute coronary syndromes) or whether it merely facilitates insufficient platelet inhibition will be discussed in this state-of-the art review. PMID:26272640

  5. The impact of static work on fibrinolysis and platelet function.

    PubMed

    Vind, J; Gleerup, G; Nielsen, P T; Winther, K

    1993-12-01

    Brief stress such as dynamic work protects against thrombosis by enhancing blood fluidity. The effect of isometric work on blood fluidity, however, is not known. The aim of the present study therefore was to test the effect of isometric work on heart rate (HR), blood pressure (BP), platelet function and fibrinolytic activity. Twelve healthy male volunteers were tested before and after isometric work. Isometric work resulted in an increase in HR from 62.4 to 110.0 beats/min and in systolic BP from 118.3 to 134.5 mmHg (p < 0.01). No significant change occurred in platelet release estimated as plasma levels of B-TG and PF-4, or platelet aggregation induced by ADP. Fibrinolytic activity increased, as evidenced by a decrease in ECLT from 136.7 + 10.5 to 72.3 + 9.8 min) (p < 0.01) and an increase in t-PA of 400%. No significant change was observed in PAI. The present data suggest that isometric work increases fibrinolytic activity significantly, but leaves platelet function unchanged.

  6. Pentamethylquercetin (PMQ) reduces thrombus formation by inhibiting platelet function

    PubMed Central

    Liang, Ming-Lu; Da, Xing-Wen; He, Ao-Di; Yao, Guang-Qiang; Xie, Wen; Liu, Gang; Xiang, Ji-Zhou; Ming, Zhang-Yin

    2015-01-01

    Flavonoids exert both anti-oxidant and anti-platelet activities in vitro and in vivo. Pentamethylquercetin (PMQ), a polymethoxylated flavone derivative, has been screened for anti-carcinogenic and cardioprotective effects. However, it is unclear whether PMQ has anti-thrombotic effects. In the present study, PMQ (20 mg/kg) significantly inhibited thrombus formation in the collagen- epinephrine- induced acute pulmonary thrombosis mouse model and the ferric chloride-induced carotid injury model. To explore the mechanism, we evaluated the effects of PMQ on platelet function. We found that PMQ inhibited platelet aggregation and granule secretion induced by low dose agonists, including ADP, collagen, thrombin and U46619. Biochemical analysis revealed that PMQ inhibited collagen-, thrombin- and U46619-induced activation of Syk, PLCγ2, Akt, GSK3β and Erk1/2. Therefore, we provide the first report to show that PMQ possesses anti-thrombotic activity in vivo and inhibited platelet function in vitro, suggesting that PMQ may represent a potential therapeutic candidate for the prevention or treatment of thrombotic disorders. PMID:26059557

  7. Common variants in the human platelet PAR4 thrombin receptor alter platelet function and differ by race.

    PubMed

    Edelstein, Leonard C; Simon, Lukas M; Lindsay, Cory R; Kong, Xianguo; Teruel-Montoya, Raúl; Tourdot, Benjamin E; Chen, Edward S; Ma, Lin; Coughlin, Shaun; Nieman, Marvin; Holinstat, Michael; Shaw, Chad A; Bray, Paul F

    2014-11-27

    Human platelets express 2 thrombin receptors: protease-activated receptor (PAR)-1 and PAR4. Recently, we reported 3.7-fold increased PAR4-mediated aggregation kinetics in platelets from black subjects compared with white subjects. We now show that platelets from blacks (n = 70) express 14% more PAR4 protein than those from whites (n = 84), but this difference is not associated with platelet PAR4 function. Quantitative trait locus analysis identified 3 common single nucleotide polymorphisms in the PAR4 gene (F2RL3) associated with PAR4-induced platelet aggregation. Among these single nucleotide polymorphisms, rs773902 determines whether residue 120 in transmembrane domain 2 is an alanine (Ala) or threonine (Thr). Compared with the Ala120 variant, Thr120 was more common in black subjects than in white subjects (63% vs 19%), was associated with higher PAR4-induced human platelet aggregation and Ca2+ flux, and generated greater inositol 1,4,5-triphosphate in transfected cells. A second, less frequent F2RL3 variant, Phe296Val, was only observed in blacks and abolished the enhanced PAR4-induced platelet aggregation and 1,4,5-triphosphate generation associated with PAR4-Thr120. PAR4 genotype did not affect vorapaxar inhibition of platelet PAR1 function, but a strong pharmacogenetic effect was observed with the PAR4-specific antagonist YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole]. These findings may have an important pharmacogenetic effect on the development of new PAR antagonists.

  8. Common variants in the human platelet PAR4 thrombin receptor alter platelet function and differ by race

    PubMed Central

    Edelstein, Leonard C.; Simon, Lukas M.; Lindsay, Cory R.; Kong, Xianguo; Teruel-Montoya, Raúl; Tourdot, Benjamin E.; Chen, Edward S.; Ma, Lin; Coughlin, Shaun; Nieman, Marvin; Holinstat, Michael; Shaw, Chad A.

    2014-01-01

    Human platelets express 2 thrombin receptors: protease-activated receptor (PAR)-1 and PAR4. Recently, we reported 3.7-fold increased PAR4-mediated aggregation kinetics in platelets from black subjects compared with white subjects. We now show that platelets from blacks (n = 70) express 14% more PAR4 protein than those from whites (n = 84), but this difference is not associated with platelet PAR4 function. Quantitative trait locus analysis identified 3 common single nucleotide polymorphisms in the PAR4 gene (F2RL3) associated with PAR4-induced platelet aggregation. Among these single nucleotide polymorphisms, rs773902 determines whether residue 120 in transmembrane domain 2 is an alanine (Ala) or threonine (Thr). Compared with the Ala120 variant, Thr120 was more common in black subjects than in white subjects (63% vs 19%), was associated with higher PAR4-induced human platelet aggregation and Ca2+ flux, and generated greater inositol 1,4,5-triphosphate in transfected cells. A second, less frequent F2RL3 variant, Phe296Val, was only observed in blacks and abolished the enhanced PAR4-induced platelet aggregation and 1,4,5-triphosphate generation associated with PAR4-Thr120. PAR4 genotype did not affect vorapaxar inhibition of platelet PAR1 function, but a strong pharmacogenetic effect was observed with the PAR4-specific antagonist YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole]. These findings may have an important pharmacogenetic effect on the development of new PAR antagonists. PMID:25293779

  9. Platelet Functions and Coagulation Changes in Indian Children with Nephrotic Syndrome

    PubMed Central

    Mittal, Aliza; Aggarwal, Kailash Chandra; Saluja, Sumita; Aggarwal, Archana; Sureka, Binit

    2013-01-01

    Introduction: Only little is known on the effect of the platelet function in the paediatric nephrotic syndrome. The earlier studies which had been done on hypercoagulability have mainly featured the secondary forms of the nephrotic syndrome and the data on the minimal change type of disease is limited. We therefore, made an effort to study the platelet functions and the coagulation profile in children with the nephrotic syndrome,to find the relationship between the steroid response and the coagulation profile, and to look for the correlation between thromboembolism and the hypercoagulable states. Methodology: Twenty nine children with the steroid responsive nephrotic syndromewere studied to see the platelet aggregation and the coagulation parameters and their response to the steroid therapy. Doppler studies were done for the renal vein and the inferior vena cava (IVC) thrombus. Results: It was seen that an increased aggregability of the platelets occurred with Adenosine diphosphate (ADP) and collagen (out of the four agonists, ADP, Collagen, Ristocetin and Arachidonic acid) which were used as agonists for the assay. We also observed that the Partial thromboplastin time (PTT) had become prolonged and a significant decline in the high values of the procoagulant proteins (Protein C and Protein S) was seen after the steroid therapy and when the children went into remission. These findings were suggestive of a reversibility of the changes in the steroid responsive nephrotic syndrome with the steroid therapy. One child was found to have thrombosis of the inferior vena cava (IVC) on Doppler studies, which resolved with treatment subsequently. Conclusions: An increased platelet aggregability contributes to the hypercoagulable states, that may increase the incidence of thrombosis in such patients. Although the incidence of such complications is very low, in a given child with the hypercoagulable states, Doppler may be used to look for any evidence of a latent thrombus and

  10. The interaction of sodium nitroprusside with human endothelial cells and platelets: nitroprusside and prostacyclin synergistically inhibit platelet function

    SciTech Connect

    Levin, R.I.; Weksler, B.B.; Jaffe, E.A.

    1982-12-01

    Sodium nitroprusside (NP) is a potent vasodilator that also inhibits platelet aggregation. To test the hypothesis that NP causes both of these effects by altering the balance between prostacyclin (PGI2) produced by endothelial cells and thromboxane A2 (TXA2) produced by platelets, we incubated each of these cell types with NP for 5 minutes and assayed the PGI2 and TXA2 produced. NP at pharmacologically achieved doses (0.01--30 micrograms/ml) inhibited platelet aggregation and resultant TXA2 synthesis in a dose- and time-dependent manner (p less than 0.001). The inhibition was not dependent on cAMP production, external calcium concentration, or suppression of TXA2 synthesis. NP did not alter the production of PGI2 by cultured human endothelial cells as measured by radioimmunoassay for 6-Keto-PGF1 alpha, the stable hydrolysis product of PGI2. However, supernates of NP-treated endothelial cells containing low, noninhibitory concentrations of NP unexpectedly inhibited platelet aggregation. This inhibition of platelet aggregation was due to synergy between PGI2 (0.1--3 nM) and NP (p interaction less than 0.03). The synergistic inhibition by NP and PGI2 of platelet aggregation and TXA2 synthesis in vivo may explain some of the beneficial actions of NP in the treatment of hypertension and congestive heart failure.

  11. Solubilization of a functionally active platelet-activating factor receptor from rabbit platelets.

    PubMed Central

    Rogers, J E; Duronio, V; Wong, S I; McNeil, M; Salari, H

    1991-01-01

    Binding of platelet-activating factor (PAF) to a specific high-affinity membrane receptor has been demonstrated in numerous cell types, but very little is known about the molecular nature of this receptor. The receptor from rabbit platelets was solubilized using CHAPS, digitonin, octyl glucoside, Nonidet P-40 or sodium cholate, either with pre-bound [3H]PAF or in the absence of ligand. We have been able to demonstrate for the first time that the receptor solubilized with CHAPS, in the absence of ligand, could retain its binding activity. It migrated as a high molecular mass complex (greater than 350 kDa) on a Bio-Gel A-0.5 m gel filtration column. Binding to solubilized receptor rapidly reached an equilibrium at room temperature, but was much slower at 0 degrees C. Scatchard plots were used to calculate the number (approx. 100 per cell) and the affinity (Kd 2.5 +/- 1.4 nM) of the solubilized receptors. These values were comparable with those obtained from whole-cell binding experiments. Competition by PAF antagonists also verified that the assay was measuring PAF receptor binding activity. The presence of a protein in the receptor complex was demonstrated by heat and trypsin inactivation of binding activity. Trypsin had no effect on binding of PAF to whole cells, but was able to decrease binding activity in solubilized receptor preparations. Attempts to demonstrate the involvement of a glycoprotein by use of various lectin columns proved unsuccessful. The latter results are consistent with findings suggesting that the binding site of the PAF receptor may not be exposed at the cell surface. PMID:1654881

  12. Assays for B lymphocyte function.

    PubMed

    Bondada, Subbarao; Robertson, Darrell A

    2003-11-01

    This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first basic protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. Alternatively, the number of antibody-producing cells can be quantified by plaque-forming cell (PFC) assays presented in this unit: the Cunningham-Szenberg and the Jerne-Nordin techniques. Both methods employ specially prepared slide chambers, described here, in which the antibody-producing B cells are mixed with complement and indicator sheep red blood cells (SRBC), or with trinitrophenol-modified SRBC (TNP-SRBC), with subsequent lysis and counting of plaques. Because IgM antibodies fix complement efficiently, whereas IgG and IgA antibodies do not, unmodified PFC assays measure only IgM antibodies. The assay can be modified, however, to measure all classes of antibodies or to enumerate total immunoglobulin-secreting B cells, as described in alternate protocols. Yet another method of measuring the number of antibody-producing B cells (in a class-specific fashion) is to use the ELISPOT technique described in UNIT 7.14. The resting B cells used in these procedures are prepared as described in the final support protocols for Percoll gradient centrifugation. PMID:18432909

  13. Global analysis of the rat and human platelet proteome - the molecular blueprint for illustrating multi-functional platelets and cross-species function evolution.

    PubMed

    Yu, Yanbao; Leng, Taohua; Yun, Dong; Liu, Na; Yao, Jun; Dai, Ying; Yang, Pengyuan; Chen, Xian

    2010-07-01

    Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high-throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomic technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin-stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across-species evolutionary link that the orthologous proteins representing "core proteome", and the "evolutionary proteome" is actually a relatively static proteome.

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

    PubMed

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

    2013-06-01

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

  15. Platelet functions in relation to dietary fats in farmers from two regions of France.

    PubMed

    Renaud, S; Dumont, E; Godsey, F; Suplisson, A; Thevenon, C

    1979-02-15

    To determine whether the long-term feeding of dietary fats affect platelet functions in man, platelet aggregation (to thrombin ADP, collagen, epinephrine) and clotting activity of platelet-rich plasma (PRP), platelet-poor plasma and of washed platelets were studied in a mobile-laboratory in 44 healthy male farmers (40--45 years) from two French regions Var and Moselle, in relation to lipemia, glycemia, dietary nutriments, and platelet phospholipid composition. In the Moselle subjects, the platelet clotting activity of PRP and of washed platelets, the platelet aggregation to thrombin and ADP, were highly significantly (p less than 0.001) increased as compared to those of Var, but not the plasma cholesterol, which was identical in the two regions. In Moselle, the intake of total calories, total lipids and saturated fats was higher than in the Var. However, it was only with the saturated fat intake (mostly stearic acid) that the platelet clotting activity (p less than 0.01) and the platelet aggregation (p less than 0.001) were highly significantly correlated. The platelet clotting activity was also significantly (p less than 0.001) correlated with the fatty acid composition of the platelet phospholipid fractions phosphatidyl serine + phosphatidyl inositol.

  16. [Ultrastructural and functional assessment on platelets loaded with small molecule carbohydrates].

    PubMed

    Yang, Chao; Wang, Jie-Xi; Han, Ying; Wang, Yan; Quan, Guo-Bo; Liu, Min-Xia; Gao, Feng; Liu, An

    2008-06-01

    The aim of this study was to investigate the effect of loading some small molecule carbohydrates into human platelets on ultrastucture and function. The ultrastructure of platelets were observed by transmission electron microscope (TEM); the platelet counts and mean platelet volume (MPV) were measured by hemocytometer, the maximal platelet aggregation rate was measured optically in an aggregometer; the surface marker of platelet membranes CD62p and phosphatidyl serine were analyzed by flow cytometry. The results showed that no significant changes of the ultrastructure of platelets loaded with small molecule carbohydrates were seen. The aggregation responsiveness of platelets loaded with small molecule carbohydrates reached to 60% of the fresh control platelets. The values of platelet counts and MPV showed no significant differences. The expression level of CD62p and the binding rate with Annexin V before and after loading small molecule carbohydrates into platelets were no different. It is concluded that the platelets after loading with small molecule carbohydrates remained fine ultrastructure and function.

  17. Development of a time-resolved immunofluorometric assay for quantifying platelet-derived microparticles in human plasma.

    PubMed

    Michelsen, Annika E; Wergeland, Ragnhild; Stokke, Oddvar; Brosstad, Frank

    2006-01-01

    Platelet-derived microparticles (PMPs) are considered a marker of platelet activation. They vary considerably in size, and flow cytometry, the predominant method used to assay PMPs, is only detecting larger PMPs (>0.1 microm). We describe here a method that quantifies the amount of PMP-located GPIIb antigen in detergent-treated platelet-free plasma (PPP) by means of a one-step time-resolved immunofluorometric assay (TR-IFMA). This assay uses a streptavidin-coated microwell plate and two different monoclonal antibodies to GPIIb (CD41), one conjugated to biotin and the other labeled with europium ion. A wide linear range standard curve with low background and a high sensitivity was obtained. Pre-assay ultracentrifugation or filtration of PPP extensively reduced the fluorometric signal, indicating that the GPIIb antigen is mainly particle-located. A strong correlation between the amount of GPIIb and PMP as detected by flow cytometry was found. Consequently, the assay can be used to study PMP-related phenomena and, in contrast to flow cytometry, can be used on frozen samples and is independent of PMP size. PMID:15990159

  18. Regulation of platelet function by class B scavenger receptors in hyperlipidemia

    PubMed Central

    Zimman, Alejandro; Podrez, Eugene A.

    2010-01-01

    Platelets constitutively express class B scavenger receptors CD36 and SR-BI, two closely related pattern recognition receptors best known for their roles in lipoprotein and lipid metabolism. The biological role of scavenger receptors in platelets is poorly understood. However, in vitro and in vivo data suggest that class B scavenger receptors modulate platelet function and contribute significantly to thrombosis by sensing pathological or physiological ligands, inducing prothrombotic signaling, and increasing platelet reactivity. Platelet CD36 recognizes a novel family of endogenous oxidized choline phospholipids that accumulate in plasma of hyperlipidemic mice and in plasma of subjects with low HDL levels. This interaction leads to the activation of specific signaling pathways and promotes platelet activation and thrombosis. Platelet SR-BI, on the other hand, plays a critical role in the induction of platelet hyper-reactivity and accelerated thrombosis in conditions associated with increased platelet cholesterol content. Intriguingly, oxidized HDL, aSR-BI ligand, can suppress platelet function. These recent findings demonstrate that platelet class B scavenger receptors play roles in thrombosis in dyslipidemia and may contribute to acute cardiovascular events in vivo in hypercholesterolemia. PMID:21071700

  19. Leukaemia-associated Rho guanine nucleotide exchange factor (LARG) plays an agonist specific role in platelet function through RhoA activation.

    PubMed

    Zou, Siying; Teixeira, Alexandra M; Yin, Mingzhu; Xiang, Yaozu; Xavier-Ferruccio, Juliana; Zhang, Ping-Xia; Hwa, John; Min, Wang; Krause, Diane S

    2016-08-30

    Leukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal haemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout (KO), shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bβ3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using two different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice. PMID:27345948

  20. Functional and ultrastructural studies on In-111-Merc labeled concentrated human platelets in plasma

    SciTech Connect

    Thakur, M.L.; Sedar, A.W.; McKenney, S.L.

    1985-05-01

    Human platelets (1 billion) labeled with 200 ..mu..Ci In-111-oxine in non-plasma medium have been reported to have impaired ultrastructure and function. The ultrastructure and function of platelets labeled in plasma with In-111-Merc was examined. Human platelets (2 billion) suspended in 0.5 ml plasma were incubated with 2 ..mu..g Merc and then labeled with 390-1170 ..mu..Ci In-111. Unlabeled platelets and those incubated with Merc and decayed In-111 solution served as controls. Radiation dose received by platelets in each preparation was estimated. Aggregability studies were performed and platelets were prepared for transmission electron microscopy. Three samples from each pellet were thin sectioned and examined at 4000 to 11,000 magnification. Electron micrographs were obtained from fields containing at least 10 platelets. Assuming uniform labeling and complete decay of In-111, radiation dose per platelet ranged from 367 Gy to 1100 Gy. The aggregability of labeled platelets was unaffected (93.5 +- 3.8%) and the velocity averaged 75.5 +- 3.9% of control. Electron micrographs revealed no change in morphology of mitochondria, alpha granules, dense granules, canalicular system, microtubules and particulate glycogen; these demonstrated normal distribution. The authors conclude that despite the high radiation dose, the function and ultrastructure of platelets labeled in plasma with In-111-Merc remain unaltered.

  1. Bench-to-bedside review: Platelets and active immune functions - new clues for immunopathology?

    PubMed

    Garraud, Olivier; Hamzeh-Cognasse, Hind; Pozzetto, Bruno; Cavaillon, Jean-Marc; Cognasse, Fabrice

    2013-08-27

    Platelets display a number of properties besides the crucial function of repairing damaged vascular endothelium and stopping bleeding; these are exploited to benefit patients receiving platelet component transfusions, which might categorize them as innate immune cells. For example, platelets specialize in pro-inflammatory activities, and can secrete a large number of molecules, many of which display biological response modifier functions. Platelets also express receptors for non-self-infectious and possibly non-infectious danger signals, and can engage infectious pathogens by mechanisms barely explained beyond observation. This relationship with infectious pathogens may involve other innate immune cells, especially neutrophils. The sophisticated interplay of platelets with bacteria may culminate in sepsis, a severe pathology characterized by significant reductions in platelet count and platelet dysfunction. How this occurs is still not fully understood. Recent findings from in-depth platelet signaling studies reveal the complexity of platelets and some of the ways they evolve along the immune continuum, from beneficial functions exemplified in endothelium repair to deleterious immunopathology as in systemic inflammatory response syndrome and acute vascular diseases. This review discusses the extended role of platelets as immune cells to emphasize their interactions with infectious pathogens sensed as potentially dangerous.

  2. Dynamic, Quantitative Assays of Phagosomal Function

    PubMed Central

    Podinovskaia, Maria; VanderVen, Brian C.; Yates, Robin M.; Glennie, Sarah; Fullerton, Duncan; Mwandumba, Henry C.; Russell, David G.

    2013-01-01

    Much of the activity of the macrophage as an effector cell is performed within its phagocytic compartment. This ranges from the degradation of tissue debris as part of its homeostatic function, to the generation of the superoxide burst as part of its microbicidal response to infection. We have developed a range of real-time readouts of phagosomal function that enables these activities to be rigorously quantified. This chapter contains the description of several of these assays assessed by different methods of quantitation; including a Fluorescence Resonance Emission Transfer (FRET) assay for phagosome/lysosome fusion measured by spectrofluorometer, a fluorogenic assay for the superoxide burst measured by flow cytometry, and a fluorogenic assay for bulk proteolysis measure by confocal microscope. These assays illustrate both the range parameters that can be quantified as well as the flexibility of instrumentation that can be exploited for their quantitation. PMID:24510516

  3. Thrombocytopenia model with minimal manipulation of blood cells allowing whole blood assessment of platelet function.

    PubMed

    Tiedemann Skipper, Mette; Rubak, Peter; Halfdan Larsen, Ole; Hvas, Anne-Mette

    2016-06-01

    In vitro models of thrombocytopenia are useful research tools. Previously published models have shortcomings altering properties of platelets and other blood components. The aim of the present study was to develop a whole blood method to induce thrombocytopenia with minimal manipulation, and to describe platelet function in induced thrombocytopenia in individuals with healthy platelets. Hirudin anticoagulated blood was obtained from 20 healthy volunteers. One part of the blood was gently centrifuged at 130g for 15 minutes. The platelet-rich plasma was replaced with phosphate-buffered saline to establish thrombocytopenia. Various levels of thrombocytopenia were achieved by combining different volumes of baseline whole blood and thrombocytopenic blood. Platelet counts were measured by flow cytometry (Navios, Beckman Coulter) and routine haematological analyser (Sysmex XE-5000). Platelet function was analysed by impedance aggregometry (Multiplate® Analyzer, Roche) and by flow cytometry (Navios, Beckman Coulter) using collagen, adenosine diphosphate, thrombin receptor activating peptide-6 and ristocetin as agonists. Median baseline platelet count was 227×10(9)/l. The in vitro model yielded median platelet counts at 51×10(9)/l (range 26-93×10(9)/l). We observed minor, yet significant, changes in platelet size and maturity from baseline to modelled thrombocytopenia. In the thrombocytopenic samples, significant and positive linear associations were found between platelet count and platelet aggregation across all agonists (all p-values<0.001). Platelet function assessed by flow cytometry showed minimal alterations in the thrombocytopenic samples. A new whole blood-based model of thrombocytopenia was established and validated. This new model serves as a useful future tool, particularly to explore platelet function in patients with thrombocytopenia.

  4. Genetic regulation of platelet receptor expression and function: application in clinical practice and drug development.

    PubMed

    Williams, Marlene S; Weiss, Ethan J; Sabatine, Marc S; Simon, Daniel I; Bahou, Wadie F; Becker, Lewis C; Parise, Leslie V; Dauerman, Harold L; French, Patricia A; Smyth, Susan S; Becker, Richard C

    2010-12-01

    Understanding genetic contributions to platelet function could have profound clinical ramifications for personalizing platelet-directed pharmacotherapy, by providing insight into the risks and possible benefits associated with specific genotypes. This article represents an integrated summary of presentations related to genetic regulation of platelet receptor expression and function given at the Fifth Annual Platelet Colloquium in January 2010. It is supplemented with additional highlights from the literature covering (1) approaches to determining and evidence for the associations of genetic variants with platelet hypo- and hyperresponsive phenotypes, (2) the ramifications of these polymorphisms with regard to clinical responses to antiplatelet therapies, and (3) the role of platelet function/genetic testing in guiding antiplatelet therapy.

  5. [Recent development in platelet functions: roles beyond thrombosis].

    PubMed

    Ozaki, Yukio; Inoue, Katsue; Inoue, Osamu

    2012-01-01

    In addition to their roles in thrombosis and hemostasis, there is an increasing body of evidence to suggest that platelets have diverse functions in various biological reactions. Of these, synthesis of specific proteins in a timely manner, involvement in inflammation, roles in anti-bacteria and anti-parasite protection, and supportive roles in liver regeneration have attracted the attention of a number of scientists. We have recently found a novel platelet-activating receptor, CLEC-2, which reacts with a snake venom, Rhodocytin. CLEC-2 has an intracellular signal transduction pathway quite similar to that of GOVI, except for its hemi-Y-xx-L motif. The endogenous ligand for CLEC-2 was identified by us as podoplanin, which is present in renal podocytes, lung alveolar macrophages, and lymphatic endothelial cells, and some types of malignant tumors. We found that CLEC-2/podoplanin interaction plays an important role in the metastasis of tumor cells with podoplanin expression. We have also found that hemophilic interaction between CLEC-2 molecules contributes to thrombus formation in vivo. CLEC-2 interaction with podoplan expressed on lymphatic endothelial cells appears to play an important role in the separation between veins and lymphatic vessels during the stage of fetal development. PMID:22416457

  6. Pull-down assay for analysis of integrin-mediated activation of Rap proteins in adherent platelets.

    PubMed

    Guidetti, Gianni Francesco; Torti, Mauro

    2014-01-01

    Rap1 GTPases operate as molecular switches by cycling between a GDP-bound inactive state and a GTP-bound active state and regulate several cellular pathways in response to different stimuli. Circulating blood platelets express high levels of Rap1 proteins, mainly Rap1b, which plays a critical role in platelet adhesion and activation. Rap1 is a key element in the inside-out signaling pathway leading to the conversion of integrins into the high-affinity state for their ligands. In platelets, Rap1b regulates inside-out activation of both integrin αIIbβ3 and α2β1. In addition, Rap1b is also involved in integrin outside-in signaling. Integrin-mediated platelet adhesion leads to accumulation of GTP-bound Rap1b, which promotes integrin-mediated processes such as spreading and clot retraction. Rap1b is thus a bidirectional regulator of platelet integrin function. Here we describe a method to analyze Rap1b activation induced by platelet adhesion via integrin α2β1.

  7. Circulating primers enhance platelet function and induce resistance to antiplatelet therapy

    PubMed Central

    Blair, T A; Moore, S F; Hers, I

    2015-01-01

    Background Aspirin and P2Y12 antagonists are antiplatelet compounds that are used clinically in patients with thrombosis. However, some patients are ‘resistant’ to antiplatelet therapy, which increases their risk of developing acute coronary syndromes. These patients often present with an underlying condition that is associated with altered levels of circulating platelet primers and platelet hyperactivity. Platelet primers cannot stimulate platelet activation, but, in combination with physiologic stimuli, significantly enhance platelet function. Objectives To explore the role of platelet primers in resistance to antiplatelet therapy, and to evaluate whether phosphoinositide 3-kinase (PI3K) contributes to this process. Methods and Results We used platelet aggregation, thromboxane A2 production and ex vivo thrombus formation as functional readouts of platelet activity. Platelets were treated with the potent P2Y12 inhibitor AR-C66096, aspirin, or a combination of both, in the presence or absence of the platelet primers insulin-like growth factor-1 (IGF-1) and thrombopoietin (TPO), or the Gz-coupled receptor ligand epinephrine. We found that platelet primers largely overcame the inhibitory effects of antiplatelet compounds on platelet functional responses. IGF-1-mediated and TPO-mediated, but not epinephrine-mediated, enhancements in the presence of antiplatelet drugs were blocked by the PI3K inhibitors wortmannin and LY294002. Conclusions These results demonstrate that platelet primers can contribute to antiplatelet resistance. Furthermore, our data demonstrate that there are PI3K-dependent and PI3K-independent mechanisms driving primer-mediated resistance to antiplatelet therapy. PMID:26039631

  8. Decreased fibrinolytic activity and increased platelet function in hypertension. Possible influence of calcium antagonism.

    PubMed

    Gleerup, G; Winther, K

    1991-02-01

    Twelve patients with mild hypertension were compared, after 14 days of placebo, with an age- and gender-matched group of 12 healthy volunteers for platelet aggregability and fibrinolytic activity. Following this, 10 of the 12 hypertensives were treated with the calcium antagonist isradipine for 12 months. Blood was drawn for determinations of platelet aggregation and fibrinolytic activity after two weeks and 12 months of treatment. Platelet aggregation tended to increase in the hypertensives compared with controls, indicated by a lowering of the adenosine diphosphate (ADP) threshold value for irreversible aggregation. Tissue-plasminogen activator (t-PA) activity was significantly decreased in hypertensives compared to controls (P less than .05). During therapy, platelet aggregation decreased and t-PA activity increased (P less than .05). The present data suggest that fibrinolytic activity is decreased and platelet aggregation increased in mild hypertension. Besides the blood pressure-lowering effect, isradipine may protect against thromboembolic diseases by modifying platelet function and fibrinolytic activity.

  9. Using a Filtration Technique to Isolate Platelet Free Plasma for Assaying Pyrophosphate

    PubMed Central

    TOLOUIAN, RAMIN; CONNERY, SEAN M.; O’NEILL, W. CHARLES; GUPTA, AJAY

    2015-01-01

    SUMMARY Background Vascular calcification (VC) is a strong prognostic marker of mortality from cardiovascular disease. Extracellular inorganic pyrophosphate (PPi) is a critical inhibitor of vascular calcification and it has been reported that hemodialysis patients have reduced plasma PPi levels, suggesting that altered PPi metabolism could contribute to VC in hemodialysis patients. Platelets are rich in PPi and release of PPi from platelets during storage or processing of plasma can lead to falsely elevated plasma PPi levels. To prepare plasma samples that are suitable for measuring PPi levels, ultracentrifugation has been used to remove platelets. Consequently, plasma PPi measurements have been limited to research laboratories since the majority of clinical laboratories do not have access to an ultracentrifuge. The purpose of the present study was to test the validity of an improved method of preparing platelet free plasma that uses filtration with a 300,000 Dalton molecular weight cut-off filter to exclude platelets, while minimizing their release of PPi. Methods In 20 maintenance hemodialysis patients, PPi levels were measured in plasma samples prepared by the conventional technique of low-speed centrifugation to remove red and white blood cells versus a novel filtration technique. Results Plasma prepared by filtration had significantly lower platelet counts (0 vs. 3 – 7 103/μL) and PPi levels (1.39 ± 0.30 μM vs. 2.74 ± 1.19 μM; mean ± SD, p < 0.01). Conclusions The filtration method appears effective in excluding platelets without causing trauma to platelets and can be used by clinical laboratories to prepare platelet-depleted plasma for PPi measurement. PMID:23289181

  10. An overview of platelet indices for evaluating platelet function in children with scorpion envenomation.

    PubMed

    Konca, Capan; Tekin, Mehmet; Colak, Pinar; Uckardes, Fatih; Turgut, Mehmet

    2014-01-01

    The aim of this study was to assess the correlation between platelet indices and scorpion envenomations (SE). Medical records of 76 children who were hospitalised for scorpion stings in the paediatric intensive care unit (PICU) between February 2013 and November 2013, and 55 healthy children who were similar to the patient group in terms of age and sex, were analysed retrospectively. The leucocyte (WBC), thrombocyte (PLT), plateletcrit (PCT), platelet distribution width (PDW) and mean platelet volume (MPV) values of the 76 children with SE were recorded. These values were compared with the healthy control group. Significantly higher WBC and PDW values were noted in patients with SE in comparison to the controls. Patients with SE had significantly lower mean MPV values compared to the healthy controls (9.03 ± 1.26 compared to 10.43 ± 1.44 fL, respectively; p < 0.001). Although the mean platelet count was slightly elevated in the SE group, no statistically significant difference existed between the two groups (p = 0.097). Furthermore, the mean PCT values in the SE group compared to the control group were slightly decreased, but this decrease was not statistically significant (p = 0.141). A significant inverse correlation existed between the MPV values and the WBC (r = -0.450, p < 0.01) and PLT counts (r = -0.420, p < 0.01). The PLT values were significantly correlated with the PCT values (r = 0.687, p < 0.01). This study demonstrated that SE may lead to several alterations in platelet indices. Significantly lower values of MPV and higher values of PDW were detected in SE patients. However, the increase in the platelet counts and the decrease in the PCT values were not significant. PMID:26417303

  11. Platelet functions in relation to diet and serum lipids in British farmers.

    PubMed Central

    Renaud, S; Morazain, R; Godsey, F; Dumont, E; Symington, I S; Gillanders, E M; O'Brien, J R

    1981-01-01

    Coagulation and platelet aggregation induced by thrombin, ADP, adrenaline, and collagen were studied in three contrasted groups, each of 20 to 22 middle-aged male farmers. Serum lipids were similar in the three groups. In the west of Scotland group, however, platelet reactivity was significantly greater than in the east of Scotland. This was associated with a dietary intake, evaluated by three different techniques, higher in saturated fat but also lower in polyunsaturated fat and alcohol. Platelet function in the southern England group also correlated with dietary fats and in addition inversely with calcium intake. On an individual basis in the 63 farmers, all the platelet function tests were significantly correlated with the intake of saturated fat regulated by that of calcium and alcohol. The dietary effects on platelets appear to be mediated by the fatty acid composition of plasma lipids and of platelet phospholipids. In that fraction, the fatty acids 20:3 omega 9, 22:3 omega 9 and 20:4 were the most closely related to the platelet function tests. the trienoic acid 20:3 omega 9, identified with essential fatty acid deficiency, was also correlated with the intake of saturated fat and calcium. In this study, platelet functions were more dependent upon the dietary factors associated with coronary heart disease such as saturated fats, calcium, and alcohol than upon serum lipids. PMID:7317223

  12. A flow cytometric assay for the study of dense granule storage and release in human platelets.

    PubMed

    Ramström, A S; Fagerberg, I H; Lindahl, T L

    1999-01-01

    The clinical manifestations of platelet dense ( delta ) granule defects are easy bruising, as well as epistaxis and bleeding after delivery, tooth extractions and surgical procedures. The observed symptoms may be explained either by a decreased number of granules or by a defect in the uptake/release of granule contents. We have developed a method to study platelet dense granule storage and release. The uptake of the fluorescent marker, mepacrine, into the platelet dense granule was measured using flow cytometry. The platelet population was identified by the size and binding of a phycoerythrin-conjugated antibody against GPIb. Cells within the discrimination frame were analysed for green (mepacrine) fluorescence. Both resting platelets and platelets previously stimulated with collagen and the thrombin receptor agonist peptide SFLLRN was analysed for mepacrine uptake. By subtracting the value for mepacrine uptake after stimulation from the value for uptake without stimulation for each individual, the platelet dense granule release capacity could be estimated. Whole blood samples from 22 healthy individuals were analysed. Mepacrine incubation without previous stimulation gave mean fluorescence intensity (MFI) values of 83+/-6 (mean +/- 1 SD, range 69-91). The difference in MFI between resting and stimulated platelets was 28+/-7 (range 17-40). Six members of a family, of whom one had a known delta -storage pool disease, were analysed. The two members (mother and son) who had prolonged bleeding times also had MFI values disparate from the normal population in this analysis. The values of one daughter with mild bleeding problems but a normal bleeding time were in the lower part of the reference interval. PMID:16801086

  13. Prostaglandin E2 levels and platelet function are different in cord blood compared to adults.

    PubMed

    Schlagenhauf, Axel; Haidl, Harald; Leschnik, Bettina; Leis, Hans-Joerg; Heinemann, Akos; Muntean, Wolfgang

    2015-01-01

    Neonatal platelets support primary haemostasis and thrombin generation as well as adult platelets, despite observable hypoaggregability in vitro. High prostaglandin E2 levels at accouchement could account for inhibited platelet function via the EP4 receptor. We set out to determine prostaglandin E2 plasma levels in cord blood of healthy neonates and evaluate the impact of prostaglandin E2 on platelet function in adult and cord blood samples. Prostaglandin E2 plasma levels were measured in cord blood and venous adult blood using GC-MS. Impact of prostaglandin E2 on platelet aggregation was measured by spiking cord blood and adult samples. Contributions of EP3 and EP4 receptors were evaluated using respective antagonists. Intracellular cAMP concentrations were measured using a commercial ELISA-kit. Prostaglandin E2 plasma levels were substantially higher in cord blood than in adult samples. Spiking with prostaglandin E2 resulted in a slight but consistent reduction of platelet aggregation in adult blood, but response to PGE2 was blunted in cord blood samples. Aggregation response of spiked adult samples was still higher than with non-spiked cord blood samples. Blockage of EP4 receptors resulted in improved platelet aggregation in adult platelets upon prostaglandin E2 spiking, while aggregation in cord blood samples remained unaltered. Intracellular cAMP concentrations after preincubation with prostaglandin E2 were only increased in adult samples. In conclusion, very high prostaglandin E2 concentrations in cord blood affect platelet function. This effect may partially explain neonatal platelet hypoaggregability. Peak levels of prostaglandin E2 can potentially protect against birth stress-induced platelet activation.

  14. Lactodifucotetraose, a human milk oligosaccharide, attenuates platelet function and inflammatory cytokine release.

    PubMed

    Newburg, David S; Tanritanir, Ayse C; Chakrabarti, Subrata

    2016-07-01

    Human milk strongly quenches inflammatory processes in vitro, and breastfed infants have lower incidence of inflammatory diseases than those fed artificially. Platelets from neonates, in contrast to those from adults, are less responsive to platelet agonists such as collagen, thrombin, ADP, and epinephrine. Breastfed infants absorb oligosaccharides intact from the human milk in their gut to the circulation. This study was to determine whether these oligosaccharides can attenuate platelet function and platelet secretion of pro-inflammatory proteins, and to identify the active component. The natural mixture of oligosaccharides from human milk and pure individual human milk oligosaccharides were tested for their ability to modulate responses of platelets isolated from human blood following exposure to thrombin, ADP, and collagen. Human milk and the natural mixture of human milk oligosaccharides inhibited platelet release of inflammatory proteins. Of the purified human milk oligosaccharides tested, only lactodifucotetraose (LDFT) significantly inhibited thrombin induced release of the pro-inflammatory proteins RANTES and sCD40L. LDFT also inhibited platelet adhesion to a collagen-coated surface, as well as platelet aggregation induced by ADP or collagen. These data indicate that LDFT may help modulate hemostasis by suppressing platelet-induced inflammatory processes in breastfed infants. This activity suggests further study of LDFT for its potential as a therapeutic agent in infants and adults.

  15. Effects of zomepirac on hemostasis in healthy adults and on platelet function in vitro.

    PubMed

    Mielke, C H; Kahn, S B; Muschek, L D; Tighe, J J; Ng, K T; Minn, F L

    1980-01-01

    Zomepirac, a new nonnarcotic analgesic, was studied in 25 healthy adults for possible effects on hemostasis. Given in a single 200-mg dose or for 15 days at 300 mg/day, zomepirac prolonged template bleeding time and caused transient decreases in platelet adhesiveness, in stimulated platelet aggregation, and in the release of platelet serotonin. The short duration of these effects contrasts with the known week-long duration of the effects of aspirin. Data from in vitro platelet function studies, correlated with plasma level determinations, indicate that these effects on platelet function in man are probably dependent only on the presence of intact zomepirac and not on any metabolites. The qualitative effects of zomepirac on platelets are assumed to be the consequence of reversible inhibition of prostaglandin synthetase in these cells. Platelet concentration and the humoral clotting mechanism were not affected by zomepirac. Although no unusual bleeding has been noted in patients given zomepirc postoperatively, it should be used with the same caution as aspirin in patients with known defects in platelet function or coagulation.

  16. A shear gradient-activated microfluidic device for automated monitoring of whole blood haemostasis and platelet function

    PubMed Central

    Jain, Abhishek; Graveline, Amanda; Waterhouse, Anna; Vernet, Andyna; Flaumenhaft, Robert; Ingber, Donald E.

    2016-01-01

    Accurate assessment of blood haemostasis is essential for the management of patients who use extracorporeal devices, receive anticoagulation therapy or experience coagulopathies. However, current monitoring devices do not measure effects of haemodynamic forces that contribute significantly to platelet function and thrombus formation. Here we describe a microfluidic device that mimics a network of stenosed arteriolar vessels, permitting evaluation of blood clotting within small sample volumes under pathophysiological flow. By applying a clotting time analysis based on a phenomenological mathematical model of thrombus formation, coagulation and platelet function can be accurately measured in vitro in patient blood samples. When the device is integrated into an extracorporeal circuit in pig endotoxemia or heparin therapy models, it produces real-time readouts of alterations in coagulation ex vivo that are more reliable than standard clotting assays. Thus, this disposable device may be useful for personalized diagnostics and for real-time surveillance of antithrombotic therapy in clinic. PMID:26733371

  17. A shear gradient-activated microfluidic device for automated monitoring of whole blood haemostasis and platelet function.

    PubMed

    Jain, Abhishek; Graveline, Amanda; Waterhouse, Anna; Vernet, Andyna; Flaumenhaft, Robert; Ingber, Donald E

    2016-01-06

    Accurate assessment of blood haemostasis is essential for the management of patients who use extracorporeal devices, receive anticoagulation therapy or experience coagulopathies. However, current monitoring devices do not measure effects of haemodynamic forces that contribute significantly to platelet function and thrombus formation. Here we describe a microfluidic device that mimics a network of stenosed arteriolar vessels, permitting evaluation of blood clotting within small sample volumes under pathophysiological flow. By applying a clotting time analysis based on a phenomenological mathematical model of thrombus formation, coagulation and platelet function can be accurately measured in vitro in patient blood samples. When the device is integrated into an extracorporeal circuit in pig endotoxemia or heparin therapy models, it produces real-time readouts of alterations in coagulation ex vivo that are more reliable than standard clotting assays. Thus, this disposable device may be useful for personalized diagnostics and for real-time surveillance of antithrombotic therapy in clinic.

  18. Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells

    PubMed Central

    Grover, Amit; Sanjuan-Pla, Alejandra; Thongjuea, Supat; Carrelha, Joana; Giustacchini, Alice; Gambardella, Adriana; Macaulay, Iain; Mancini, Elena; Luis, Tiago C.; Mead, Adam; Jacobsen, Sten Eirik W.; Nerlov, Claus

    2016-01-01

    Aged haematopoietic stem cells (HSCs) generate more myeloid cells and fewer lymphoid cells compared with young HSCs, contributing to decreased adaptive immunity in aged individuals. However, it is not known how intrinsic changes to HSCs and shifts in the balance between biased HSC subsets each contribute to the altered lineage output. Here, by analysing HSC transcriptomes and HSC function at the single-cell level, we identify increased molecular platelet priming and functional platelet bias as the predominant age-dependent change to HSCs, including a significant increase in a previously unrecognized class of HSCs that exclusively produce platelets. Depletion of HSC platelet programming through loss of the FOG-1 transcription factor is accompanied by increased lymphoid output. Therefore, increased platelet bias may contribute to the age-associated decrease in lymphopoiesis. PMID:27009448

  19. Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells.

    PubMed

    Grover, Amit; Sanjuan-Pla, Alejandra; Thongjuea, Supat; Carrelha, Joana; Giustacchini, Alice; Gambardella, Adriana; Macaulay, Iain; Mancini, Elena; Luis, Tiago C; Mead, Adam; Jacobsen, Sten Eirik W; Nerlov, Claus

    2016-01-01

    Aged haematopoietic stem cells (HSCs) generate more myeloid cells and fewer lymphoid cells compared with young HSCs, contributing to decreased adaptive immunity in aged individuals. However, it is not known how intrinsic changes to HSCs and shifts in the balance between biased HSC subsets each contribute to the altered lineage output. Here, by analysing HSC transcriptomes and HSC function at the single-cell level, we identify increased molecular platelet priming and functional platelet bias as the predominant age-dependent change to HSCs, including a significant increase in a previously unrecognized class of HSCs that exclusively produce platelets. Depletion of HSC platelet programming through loss of the FOG-1 transcription factor is accompanied by increased lymphoid output. Therefore, increased platelet bias may contribute to the age-associated decrease in lymphopoiesis. PMID:27009448

  20. R1: Platelets and Megakaryocytes contain functional NF-κB

    PubMed Central

    Spinelli, Sherry L.; Casey, Ann E.; Pollock, Stephen J.; Gertz, Jacqueline M.; McMillan, David H.; Narasipura, Srinivasa D.; Mody, Nipa A.; King, Michael R.; Maggirwar, Sanjay B.; Francis, Charles W.; Taubman, Mark B.; Blumberg, Neil; Phipps, Richard P.

    2010-01-01

    The Nuclear Factor (NF)-κB transcription factor family is well-known for their role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-κB family members including the regulatory Inhibitor (I)-κB and Inhibitor Kappa Kinase (IKK) molecules. Objective Investigate the presence and role of NF-κB proteins in megakaryocytes and platelets. Methods and Results Anucleate platelets exposed to NF-κB inhibitors demonstrated impaired fundamental functions involved in repairing vascular injury and thrombus formation. Specifically, NF-κB inhibition diminished lamellapodia formation, decreased clot retraction times and reduced thrombus stability. Moreover, inhibition of I-κB-α phosphorylation (BAY-11-7082) reverts fully spread platelets back to a spheroid morphology. Addition of recombinant IKK-β or I-κB-α protein to BAY inhibitor-treated platelets partially restore platelet spreading in I-κB-α inhibited platelets, and addition of active IKK-β increased endogenous I-κB-α phosphorylation levels. Conclusions These novel findings support a crucial and non-classical role for the NF-κB family in modulating platelet function and reveal that platelets are sensitive to NF-κB inhibitors. As NF-κB inhibitors are being developed as anti-inflammatory and anti-cancer agents, they may have unintended effects on platelets. Based on these data, NF-κB is also identified as a new target to dampen unwanted platelet activation. PMID:20042710

  1. A comparison of six major platelet functional tests to assess the impact of carbon nanomaterials on platelet function: a practical guide.

    PubMed

    Laloy, Julie; Mullier, François; Alpan, Lutfiye; Mejia, Jorge; Lucas, Stéphane; Chatelain, Bernard; Toussaint, Olivier; Masereel, Bernard; Rolin, Stéphanie; Dogné, Jean-Michel

    2014-03-01

    The study of the haemocompatibility of nanomaterials that could be in contact with blood (e.g. nanoparticle (NP)-based drug-delivery system) is of major importance. The primary objective of this study was to compare the ability of six platelet functional tests to assess the impact of NPs on platelet function. The secondary objective was to determine an accurate and reliable screening test to measure the potential impact of NPs on primary haemostasis whatever their physicochemical properties. Four types of carbon NPs (carbon black, fullerenes, single-walled carbon nanotubes and multi-walled carbon nanotubes) were investigated on six platelet function tests: light transmission aggregometry, whole-blood impedance aggregometry, platelet function analyser-100 (PFA-100®) and Cone-and-Plate(let) analyser (Impact-R®), transmission- and field emission gun scanning electron microscopy (FEG-SEM). We considered that Impact-R® supported by FEG-SEM is the reference method to investigate the potential impact of NPs on platelet function.

  2. Changes in platelet morphology and function during 24 hours of storage.

    PubMed

    Braune, S; Walter, M; Schulze, F; Lendlein, A; Jung, F

    2014-01-01

    For in vitro studies assessing the interaction of platelets with implant materials, common and standardized protocols for the preparation of platelet rich plasma (PRP) are lacking, which may lead to non-matching results due to the diversity of applied protocols. Particularly, the aging of platelets during prolonged preparation and storage times is discussed to lead to an underestimation of the material thrombogenicity. Here, we study the influence of whole blood- and PRP-storage times on changes in platelet morphology and function. Blood from apparently healthy subjects was collected according to a standardized protocol and examined immediately after blood collection, four hours and twenty four hours later. The capability of platelets to adhere and form stable aggregates (PFA100, closure time) was examined in sodium citrate anticoagulated whole blood (WB) using the agonists equine type I collagen and epinephrine bitartrate (collagen/epinephrine) as well as equine type I collagen and adenosine-5'-diphosphate (collagen/ADP). Circulating platelets were quantified at each time point. Morphology of platelets and platelet aggregates were visualized microscopically and measured using an electric field multi-channel counting system (CASY). The percentage of activated platelets was assessed by means of P-selectin (CD62P) expression of circulating platelets. Furthermore, platelet factor 4 (PF4) release was measured in platelet poor plasma (PPP) at each time point. Whole blood PFA100 closure times increased after stimulation with collagen/ADP and collagen/epinephrine. Twenty four hours after blood collection, both parameters were prolonged pathologically above the upper limit of the reference range. Numbers of circulating platelets, measured in PRP, decreased after four hours, but no longer after twenty four hours. Mean platelet volumes (MPV) and platelet large cell ratios (P-LCR, 12 fL - 40 fL) decreased over time. Immediately after blood collection, no debris or platelet

  3. Platelet MAO and personality--function and dysfunction.

    PubMed

    Oreland, L; Hallman, J; Damberg, M

    2004-08-01

    Research on the association between platelet monoamine oxidase (MAO) activity and personality traits, such as sensation seeking and impulsiveness, is reviewed with an emphasis on early history and current situation. The effects of MAO-inhibiting compounds in cigarette smoke for the interpretation of this association are discussed and recent results confirming a true association between platelet MAO activity and personality and vulnerability, for e.g. type 2 alcoholism are presented. From a clinical point of view, the link between platelet MAO activity, which is highly genetically regulated and is stable in the individual, and personality traits, has had its greatest impact on the understanding of the nature of constitutional factors making individuals vulnerable, for e.g. substance abuse and other forms of sociopathic behaviour. The molecular mechanisms underlying the association between platelet MAO and behaviour are discussed and evidence that common transcriptional factors, e.g. within the AP-2 family, regulating both the expression of platelet MAO and components of the central monoaminergic systems, such as synthesising enzymes, receptors and transporters, are presented. A hypothesis is put forward, that such common transcription factors may not directly regulate platelet MAO expression, but rather mitochondrial density, or outer mitochondrial membrane surface. PMID:15279564

  4. Functional responses and molecular mechanisms involved in histone-mediated platelet activation.

    PubMed

    Carestia, A; Rivadeneyra, L; Romaniuk, M A; Fondevila, C; Negrotto, S; Schattner, M

    2013-11-01

    Histones are highly alkaline proteins found in cell nuclei and they can be released by either dying or inflammatory cells. The recent observations that histones are major components of neutrophil extracellular traps and promote platelet aggregation and platelet-dependent thrombin generation have shown that these proteins are potent prothrombotic molecules. Because the mechanism(s) of platelet activation by histones are not completely understood, we explored the ability of individual recombinant human histones H1, H2A, H2B, H3 and H4 to induce platelet activation as well as the possible molecular mechanisms involved. All histones were substrates for platelet adhesion and spreading and triggered fibrinogen binding, aggregation, von Willebrand factor release, P-selectin and phosphatidylserine (PS) exposure and the formation of platelet-leukocyte aggregates; however, H4 was the most potent. Histone-mediated fibrinogen binding, P-selectin and PS exposure and the formation of mixed aggregates were potentiated by thrombin. Histones induced the activation of ERK, Akt, p38 and NFκB. Accordingly, histone-induced platelet activation was significantly impaired by pretreatment of platelets with inhibitors of ERK (U 0126), PI3K/Akt (Ly 294002), p38 (SB 203580) and NFκB (BAY 11-7082 and Ro 106-9920). Preincubation of platelets with either aspirin or dexamethasone markedly decreased fibrinogen binding and the adhesion mediated by histones without affecting P-selectin exposure. Functional platelet responses induced by H3 and H4, but not H1, H2A and H2B, were partially mediated through interaction with Toll-like receptors -2 and -4. Our data identify histones as important triggers of haemostatic and proinflammatory platelet responses, and only haemostatic responses are partially inhibited by anti-inflammatory drugs. PMID:23965842

  5. Functional responses and molecular mechanisms involved in histone-mediated platelet activation.

    PubMed

    Carestia, A; Rivadeneyra, L; Romaniuk, M A; Fondevila, C; Negrotto, S; Schattner, M

    2013-11-01

    Histones are highly alkaline proteins found in cell nuclei and they can be released by either dying or inflammatory cells. The recent observations that histones are major components of neutrophil extracellular traps and promote platelet aggregation and platelet-dependent thrombin generation have shown that these proteins are potent prothrombotic molecules. Because the mechanism(s) of platelet activation by histones are not completely understood, we explored the ability of individual recombinant human histones H1, H2A, H2B, H3 and H4 to induce platelet activation as well as the possible molecular mechanisms involved. All histones were substrates for platelet adhesion and spreading and triggered fibrinogen binding, aggregation, von Willebrand factor release, P-selectin and phosphatidylserine (PS) exposure and the formation of platelet-leukocyte aggregates; however, H4 was the most potent. Histone-mediated fibrinogen binding, P-selectin and PS exposure and the formation of mixed aggregates were potentiated by thrombin. Histones induced the activation of ERK, Akt, p38 and NFκB. Accordingly, histone-induced platelet activation was significantly impaired by pretreatment of platelets with inhibitors of ERK (U 0126), PI3K/Akt (Ly 294002), p38 (SB 203580) and NFκB (BAY 11-7082 and Ro 106-9920). Preincubation of platelets with either aspirin or dexamethasone markedly decreased fibrinogen binding and the adhesion mediated by histones without affecting P-selectin exposure. Functional platelet responses induced by H3 and H4, but not H1, H2A and H2B, were partially mediated through interaction with Toll-like receptors -2 and -4. Our data identify histones as important triggers of haemostatic and proinflammatory platelet responses, and only haemostatic responses are partially inhibited by anti-inflammatory drugs.

  6. Cyclooxygenase Expression and Platelet Function in Healthy Dogs Receiving Low Dose Aspirin

    PubMed Central

    Dudley, Alicia; Thomason, John; Fritz, Sara; Grady, Jesse; Stokes, John; Wills, Robert; Pinchuk, Lesya; Mackin, Andrew; Lunsford, Kari

    2014-01-01

    Background Low dose aspirin is used to prevent thromboembolic complications in dogs, but some animals are non-responsive to the anti-platelet effects of aspirin (‘aspirin resistance’). Hypothesis/Objectives That low dose aspirin would inhibit platelet function, decrease thromboxane synthesis, and alter platelet cyclooxygenase (COX) expression. Animals Twenty-four healthy dogs Methods A repeated measures study. Platelet function (PFA-100® closure time, collagen/epinephrine), platelet COX-1 and COX-2 expression, and urine 11-dehydro-thromboxane B2 (11-dTXB2) was evaluated prior to and during aspirin administration (1 mg/kg Q24 hours PO, 10 days). Based on prolongation of closure times after aspirin administration, dogs were divided into categories according to aspirin responsiveness: responders, non-responders, and inconsistent responders. Results Low dose aspirin increased closure times significantly (62% by Day 10, P<0.001), with an equal distribution among aspirin responsiveness categories, 8 dogs per group. Platelet COX-1 mean fluorescent intensity (MFI) increased significantly during treatment, 13% on Day 3 (range, −29.7%–136.1%) (P=0.047) and 72% on Day 10 (range, −0.37–210.36%) (P<0.001). Platelet COX-2 MFI increased significantly by 34% (range, −29.2–270.4%) on Day 3 (P = 0.003) and 74% (range, −19.7–226.2%) on Day 10 (P<0.001). Urinary 11-dTXB2 concentrations significantly (P=0.005, P<0.001) decreased at both time points. There was no difference between aspirin responsiveness and either platelet COX expression or thromboxane production. Conclusions and Clinical Importance Low dose aspirin consistently inhibits platelet function in approximately one third of healthy dogs, despite decreased thromboxane synthesis and increased platelet COX expression in most dogs. Pre-treatment COX isoform expression did not predict aspirin resistance. PMID:23278865

  7. A novel assay for the detection of anti-human platelet antigen antibodies (HPA-1a) based on peptide aptamer technology

    PubMed Central

    Thibaut, Julien; Mérieux, Yves; Rigal, Dominique; Gillet, Germain

    2012-01-01

    Background Neonatal alloimmune thrombocytopenia is mostly due to the presence of maternal antibodies against the fetal platelet antigen HPA-1a on the platelet integrin GPIIb-IIIa. Accurate detection of anti-HPA-1a antibodies in the mother is, therefore, critical. Current diagnostic assays rely on the availability of pools of human platelets that vary according to donors and blood centers. There is still no satisfactory standardization of these assays. Design and Methods Peptide aptamer was used to detect and identify HPA-1a-specific antibodies in human serum that do not require human platelets. A peptide aptamer library was screened using an anti-HPA-1a human monoclonal antibody as a bait to isolate an aptamer that mimics the human platelet antigen HPA-1a. Results This is the first report in platelet immunology of the use of a peptide aptamer for diagnostic purposes. This assay gives better results than the MAIPA currently in use, detecting around 90% of the expected alloantibodies. Conclusions This assay could help define a standard for the quantitation of anti-HPA antibodies. This report also demonstrates that peptide aptamers can potentially detect a variety of biomarkers in body fluids; this is of particular interest for diagnostic purposes. PMID:22133781

  8. Lyn and PECAM-1 function as interdependent inhibitors of platelet aggregation.

    PubMed

    Ming, Zhangyin; Hu, Yu; Xiang, Jizhou; Polewski, Peter; Newman, Peter J; Newman, Debra K

    2011-04-01

    Inhibition of platelet responsiveness is important to control pathologic thrombus formation. Platelet-endothelial cell adhesion molecule-1 (PECAM-1) and the Src family kinase Lyn inhibit platelet activation by the glycoprotein VI (GPVI) collagen receptor; however, it is not known whether PECAM-1 and Lyn function in the same or different inhibitory pathways. In these studies, we found that, relative to wild-type platelets, platelets derived from PECAM-1-deficient, Lyn-deficient, or PECAM-1/Lyn double-deficient mice were equally hyperresponsive to stimulation with a GPVI-specific agonist, indicating that PECAM-1 and Lyn participate in the same inhibitory pathway. Lyn was required for PECAM-1 tyrosine phosphorylation and subsequent binding of the Src homology 2 domain-containing phosphatase-2, SHP-2. These results support a model in which PECAM-1/SHP-2 complexes, formed in a Lyn-dependent manner, suppress GPVI signaling. PMID:21297004

  9. Glycoprotein Ic-IIa functions as an activation-independent fibronectin receptor on human platelets.

    PubMed

    Piotrowicz, R S; Orchekowski, R P; Nugent, D J; Yamada, K Y; Kunicki, T J

    1988-04-01

    Soluble fibronectin binds specifically to glycoprotein (GP) IIb-IIIa on thrombin-activated platelets, and this binding is not observed with platelets of patients with Glanzmann's thrombasthenia (GT) which lack GPIIb-IIIa. Here we report that GT platelets retain the ability to interact with fibronectin-coated surfaces. Adhesion to fibronectin does not require platelet activation and is inhibited by soluble fibronectin, antibodies specific for fibronectin, peptides containing the sequence Arg-Gly-Asp and polyclonal antibodies specific for band 3 of the chicken embryo fibroblast fibronectin receptor (anti-band 3). Using anti-band 3, we have purified a second fibronectin receptor from human platelets, a heterodimer composed of glycoproteins previously designated GPIc and GPIIa. The GPIc-IIa complex is found on both GT and normal platelets and appears to be identical to the GP138 kD-GP160 kD complex recently immunopurified by Giancotti et al. (1986. Exp. Cell Res. 163:47-62) and by Sonnenberg et al. (1987. J. Biol. Chem. 268:10376-10383). In this report, we provide the first evidence that GPIc-IIa actually mediates adhesion of platelets to fibronectin-coated surfaces. GPIc-IIa thus represents a second functional fibronectin receptor, distinct from GPIIb-IIIa, that is largely responsible for the adhesion of nonactivated platelets to fibronectin-coated surfaces.

  10. Procoagulant and platelet-derived microvesicle absolute counts determined by flow cytometry correlates with a measurement of their functional capacity

    PubMed Central

    Ayers, Lisa; Harrison, Paul; Kohler, Malcolm; Ferry, Berne

    2014-01-01

    Background Flow cytometry is the most commonly used technology to measure microvesicles (MVs). Despite reported limitations of this technique, MV levels obtained using conventional flow cytometry have yielded many clinically relevant findings, such as associations with disease severity and ability to predict clinical outcomes. This study aims to determine if MV enumeration by flow cytometry correlates with a measurement of their functional capacity, as this may explain how flow cytometry generates clinically relevant results. Methods One hundred samples from healthy individuals and patients with obstructive sleep apnoea were analysed by conventional flow cytometry (FACSCalibur) and by three functional MV assays: Zymuphen MP-activity in which data were given as phosphatidylserine equivalent, STA® Phospholipid Procoag Assay expressed as clotting time and Endogenous Thrombin Potential (ETP) reflecting in vitro thrombin generation. Correlations were determined by Spearman correlation. Results Absolute counts of lactadherin+ procoagulant MVs generated by flow cytometry weakly correlated with the results obtained from the Zymuphen MP-activity (r=0.5370, p<0.0001); correlated with ETP (r=0.7444, p<0.0001); negatively correlated with STA® Phospholipid Procoag Assay clotting time (−0.7872, p<0.0001), reflecting a positive correlation between clotting activity and flow cytometry. Levels of Annexin V+ procoagulant and platelet-derived MVs were also associated with functional assays. Absolute counts of MVs derived from other cell types were not correlated with the functional results. Conclusions Quantitative results of procoagulant and platelet-derived MVs from conventional flow cytometry are associated with the functional capability of the MVs, as defined by three functional MV assays. Flow cytometry is a valuable technique for the quantification of MVs from different cellular origins; however, a combination of several analytical techniques may give the most comprehensive

  11. Editorial Commentary: Platelet-Rich Plasma Improves Knee Pain and Function in Patients With Knee Osteoarthritis.

    PubMed

    Lubowitz, James H

    2015-11-01

    Systematic review of overlapping meta-analyses shows that platelet-rich plasma improves knee pain and function in patients with knee osteoarthritis. Ultimately, biologics hold promise for chondroprotection in addition to symptomatic relief. PMID:26542203

  12. Regulator of G-Protein Signaling 18 Controls Both Platelet Generation and Function

    PubMed Central

    Delesque-Touchard, Nathalie; Pendaries, Caroline; Volle-Challier, Cécile; Millet, Laurence; Salel, Véronique; Hervé, Caroline; Pflieger, Anne-Marie; Berthou-Soulie, Laurence; Prades, Catherine; Sorg, Tania; Herbert, Jean-Marc; Savi, Pierre; Bono, Françoise

    2014-01-01

    RGS18 is a myeloerythroid lineage-specific regulator of G-protein signaling, highly expressed in megakaryocytes (MKs) and platelets. In the present study, we describe the first generation of a RGS18 knockout mouse model (RGS18-/-). Interesting phenotypic differences between RGS18-/- and wild-type (WT) mice were identified, and show that RGS18 plays a significant role in both platelet generation and function. RGS18 deficiency produced a gain of function phenotype in platelets. In resting platelets, the level of CD62P expression was increased in RGS18-/- mice. This increase correlated with a higher level of plasmatic serotonin concentration. RGS18-/- platelets displayed a higher sensitivity to activation in vitro. RGS18 deficiency markedly increased thrombus formation in vivo. In addition, RGS18-/- mice presented a mild thrombocytopenia, accompanied with a marked deficit in MK number in the bone marrow. Analysis of MK maturation in vitro and in vivo revealed a defective megakaryopoiesis in RGS18-/- mice, with a lower bone marrow content of only the most committed MK precursors. Finally, RGS18 deficiency was correlated to a defect of platelet recovery in vivo under acute conditions of thrombocytopenia. Thus, we highlight a role for RGS18 in platelet generation and function, and provide additional insights into the physiology of RGS18. PMID:25405900

  13. Apelin: an antithrombotic factor that inhibits platelet function.

    PubMed

    Adam, Frédéric; Khatib, Abdel-Majid; Lopez, Jose Javier; Vatier, Camille; Turpin, Sabrina; Muscat, Adeline; Soulet, Fabienne; Aries, Anne; Jardin, Isaac; Bobe, Régis; Stepanian, Alain; de Prost, Dominique; Dray, Cédric; Rosado, Juan Antonio; Valet, Philippe; Feve, Bruno; Siegfried, Geraldine

    2016-02-18

    Apelin peptide and its receptor APJ are directly implicated in various physiological processes ranging from cardiovascular homeostasis to immune signaling. Here, we show that apelin is a key player in hemostasis with an ability to inhibit thrombin- and collagen-mediated platelet activation. Mice lacking apelin displayed a shorter bleeding time and a prothrombotic profile. Their platelets exhibited increased adhesion and a reduced occlusion time in venules, and displayed a higher aggregation rate after their activation by thrombin compared with wild-type platelets. Consequently, human and mouse platelets express apelin and its receptor APJ. Apelin directly interferes with thrombin-mediated signaling pathways and platelet activation, secretion, and aggregation, but not with ADP and thromboxane A2-mediated pathways. IV apelin administration induced excessive bleeding and prevented thrombosis in mice. Taken together, these findings suggest that apelin and/or APJ agonists could potentially be useful adducts in antiplatelet therapies and may provide a promising perspective for patients who continue to display adverse thrombotic events with current antiplatelet therapies.

  14. Functionalized Nanofiber Meshes Enhance Immunosorbent Assays.

    PubMed

    Hersey, Joseph S; Meller, Amit; Grinstaff, Mark W

    2015-12-01

    Three-dimensional substrates with high surface-to-volume ratios and subsequently large protein binding capacities are of interest for advanced immunosorbent assays utilizing integrated microfluidics and nanosensing elements. A library of bioactive and antifouling electrospun nanofiber substrates, which are composed of high-molecular-weight poly(oxanorbornene) derivatives, is described. Specifically, a set of copolymers are synthesized from three 7-oxanorbornene monomers to create a set of water insoluble copolymers with both biotin (bioactive) and triethylene glycol (TEG) (antifouling) functionality. Porous three-dimensional nanofiber meshes are electrospun from these copolymers with the ability to specifically bind streptavidin while minimizing the nonspecific binding of other proteins. Fluorescently labeled streptavidin is used to quantify the streptavidin binding capacity of each mesh type through confocal microscopy. A simplified enzyme-linked immunosorbent assay (ELISA) is presented to assess the protein binding capabilities and detection limits of these nanofiber meshes under both static conditions (26 h) and flow conditions (1 h) for a model target protein (i.e., mouse IgG) using a horseradish peroxidase (HRP) colorimetric assay. Bioactive and antifouling nanofiber meshes outperform traditional streptavidin-coated polystyrene plates under flow, validating their use in future advanced immunosorbent assays and their compatibility with microfluidic-based biosensors.

  15. Cost-effectiveness of universal and platelet reactivity assay-driven antiplatelet therapy in acute coronary syndrome.

    PubMed

    Coleman, Craig I; Limone, Brendan L

    2013-08-01

    Assays monitoring P2Y12 platelet reactivity can accurately predict which patients will have a poor response to clopidogrel. We sought to determine the cost-effectiveness of using platelet reactivity assays (PRAs) to select a dual-antiplatelet regimen for patients with acute coronary syndrome. A hybrid decision tree Markov model was developed to determine the cost-effectiveness of universal clopidogrel, ticagrelor, or prasugrel (given to all patients) or PRA-driven ticagrelor or prasugrel (given to patients with high platelet reactivity, defined as >230 on the VerifyNow P2Y12 assay; the others received generic clopidogrel). We assumed a cohort of 65-year-old patients with acute coronary syndrome and an incidence of high platelet reactivity of 32% and 13% at ~24 to 48 hours after revascularization and 1 month, respectively. The 5-year costs, quality-adjusted life-years, and incremental cost-effectiveness ratios were calculated for PRA-driven ticagrelor and prasugrel compared with universal clopidogrel, ticagrelor, or prasugrel. PRA-driven ticagrelor and prasugrel were cost-effective compared with universal clopidogrel (incremental cost-effectiveness ratio $40,100 and $49,143/quality-adjusted life-year, respectively); however, universal ticagrelor and prasugrel were not (incremental cost-effectiveness ratio $61,651 and $96,261/quality-adjusted life-year, respectively). Monte Carlo simulation suggested PRA-driven ticagrelor, PRA-driven prasugrel, universal ticagrelor, and universal prasugrel would have an incremental cost-effectiveness ratio <$50,000/quality-adjusted life-year in 52%, 40%, 23%, and 2% of the iterations compared with universal clopidogrel, respectively. Universal ticagrelor and prasugrel were not cost-effective compared with their respective PRA-driven regimens (incremental cost-effectiveness ratio $68,182; $116,875/quality-adjusted life-year, respectively). Monte Carlo simulation suggested universal ticagrelor and prasugrel would have an incremental

  16. Label-free functional selectivity assays.

    PubMed

    Ferrie, Ann M; Goral, Vasiliy; Wang, Chaoming; Fang, Ye

    2015-01-01

    G protein-coupled receptors (GPCRs) represent the largest class of drug targets. Ligand-directed functional selectivity or biased agonism opens new possibility for discovering GPCR drugs with better efficacy and safety profiles. However, quantification of ligand bias is challenging. Herein, we present five different label-free dynamic mass redistribution (DMR) approaches to assess ligand bias acting at the β2-adrenergic receptor (β2AR). Multiparametric analysis of the DMR agonist profiles reveals divergent pharmacology of a panel of β2AR agonists. DMR profiling using catechol as a conformational probe detects the presence of multiple conformations of the β2AR. DMR assays under microfluidics, together with chemical biology tools, discover ligand-directed desensitization of the receptor. DMR antagonist reverse assays manifest biased antagonism. DMR profiling using distinct probe-modulated cells detects the biased agonism in the context of self-referenced pharmacological activity map. PMID:25563188

  17. Genomic landscape of megakaryopoiesis and platelet function defects

    PubMed Central

    Bianchi, Elisa; Norfo, Ruggiero; Pennucci, Valentina; Zini, Roberta

    2016-01-01

    Megakaryopoiesis is a complex, stepwise process that takes place largely in the bone marrow. At the apex of the hierarchy, hematopoietic stem cells undergo a number of lineage commitment decisions that ultimately lead to the production of polyploid megakaryocytes. On average, megakaryocytes release 1011 platelets per day into the blood that repair vascular injuries and prevent excessive bleeding. This differentiation process is tightly controlled by exogenous and endogenous factors, which have been the topics of intense research in the hematopoietic field. Indeed, a skewing of megakaryocyte commitment and differentiation may entail the onset of myeloproliferative neoplasms and other preleukemic disorders together with acute megakaryoblastic leukemia, whereas quantitative or qualitative defects in platelet production can lead to inherited platelet disorders. The recent advent of next-generation sequencing has prompted mapping of the genomic landscape of these conditions to provide an accurate view of the underlying lesions. The aims of this review are to introduce the physiological pathways of megakaryopoiesis and to present landmark studies on acquired and inherited disorders that target them. These studies have not only introduced a new era in the fields of molecular medicine and targeted therapies but may also provide us with a better understanding of the mechanisms underlying normal megakaryopoiesis and thrombopoiesis that can inform efforts to create alternative sources of megakaryocytes and platelets. PMID:26787733

  18. The effect of ageing on platelet function and fibrinolytic activity.

    PubMed

    Gleerup, G; Winther, K

    1995-08-01

    Twelve healthy male volunteers, mean age twenty-five, range twenty-one to thirty years, and 12 healthy middle-aged male volunteers mean age fifty-eight, range forty-four to seventy-two years, were tested regarding platelet aggregation induced by adenosine diphosphate and fibrinolytic activity, estimated as euglobulin clot lysis time (ECLT), tissue plasminogen activator (t-PA), and the fast-acting inhibitor against t-PA normally referred to as (PAI-1). Platelet aggregation increased significantly in the middle-aged group as compared with the young, as shown by a decrease in ADP thresholds for irreversible aggregation (P < 0.01). In healthy young volunteers, vigorous cycling exercise by itself caused platelet aggregability to decrease (P < 0.05). Such changes were not observed in the elderly. Fibrinolytic activity decreased significantly in the middle-aged group as shown by a prolongation of the ECLT (P < 0.01) and PAI-1, although not significantly, increased by approximately 100%, whereas t-PA significantly increased in the middle-aged group (P < 0.01). The present results suggest that increasing age is associated with not only increased platelet aggregability but also decreased fibrinolytic activity.

  19. Effects of condensation products of biogenic amines on human platelet function

    SciTech Connect

    Given, M.B.

    1983-01-01

    Condensation products (CP) are formed by the reaction of biogenic amines with aldehydes and alpha-keto acids. The purpose of this investigation was to examine the effects of CP on platelet function in vitro. The effect of CP on platelet aggregation was examined. Epinephrine-induced aggregation was inhibited, suggesting CP antagonistic activity on the platelet alpha/sub 2/-adrenergic receptors. Adenosine-diphosphate (ADP), collagen and arachidonic acid induced aggregation was inhibited only at high concentrations. Inhibition of epinephrine and ADP aggregation was reversible, suggesting CP are competitive inhibitors of these agonists. Binding affinities for the platelet alpha/sub 2/-adrenergic receptor were determined using (/sup 3/H)-yohimbine, a specific alpha/sup 2/-receptor antagonist. The order of potency for CP inhibition of (/sup 3/H)-yohimbine binding paralleled that determined for inhibition of epinephrine-induced aggregation. Platelet uptake of serotonin (5-HT) was competitively inhibited by CP, with the exception of salsolinol, which appears to be stimulatory. Release of 5-HT from platelets was induced by CP, with betacarbolines being more potent than tetrahydroisoquinolines. Evidence suggests that CP cause release by displacement of 5-HT from intraplatelet storage sites since this effect can be inhibited by imipramine, thus preventing accumulation of CP by platelets.

  20. Glucose ameliorates the metabolic profile and mitochondrial function of platelet concentrates during storage in autologous plasma

    PubMed Central

    Amorini, Angela M.; Tuttobene, Michele; Tomasello, Flora M.; Biazzo, Filomena; Gullotta, Stefano; De Pinto, Vito; Lazzarino, Giuseppe; Tavazzi, Barbara

    2013-01-01

    Background It is essential that the quality of platelet metabolism and function remains high during storage in order to ensure the clinical effectiveness of a platelet transfusion. New storage conditions and additives are constantly evaluated in order to achieve this. Using glucose as a substrate is controversial because of its potential connection with increased lactate production and decreased pH, both parameters triggering the platelet lesion during storage. Materials and methods In this study, we analysed the morphological status and metabolic profile of platelets stored for various periods in autologous plasma enriched with increasing glucose concentrations (13.75, 27.5 and 55 mM). After 0, 2, 4, 6 and 8 days, high energy phosphates (ATP, GTP, ADP, AMP), oxypurines (hypoxanthine, xanthine, uric acid), lactate, pH, mitochondrial function, cell lysis and morphology, were evaluated. Results The data showed a significant dose-dependent improvement of the different parameters in platelets stored with increasing glucose, compared to what detected in controls. Interestingly, this phenomenon was more marked at the highest level of glucose tested and in the period of time generally used for platelet transfusion (0–6 days). Conclusion These results indicate that the addition of glucose during platelet storage ameliorates, in a dose-dependent manner, the biochemical parameters related to energy metabolism and mitochondrial function. Since there was no correspondence between glucose addition, lactate increase and pH decrease in our experiments, it is conceivable that platelet derangement during storage is not directly caused by glucose through an increase of anaerobic glycolysis, but rather to a loss of mitochondrial functions caused by reduced substrate availability. PMID:22682337

  1. Prospective observational evaluation of the particle immunofiltration anti-platelet factor 4 rapid assay in MICU patients with thrombocytopenia

    PubMed Central

    2013-01-01

    Introduction Heparin-induced thrombocytopenia (HIT) results from antibodies to PF4/heparin complexes and clinical diagnosis is difficult. We evaluated the particle immunofiltration anti-platelet factor 4 (PIFA) rapid assay, in conjunction with a clinical risk score, in the diagnosis of HIT. Methods We performed a prospective observational study in all patients admitted to the medical intensive care unit (MICU) in a large academic medical center. Patients were screened daily for thrombocytopenia defined as either a platelet count that decreased by at least 33% or an absolute platelet count less than 150,000/μL. Patients with suspected HIT underwent PIFA and ELISA testing for anti-PF4/heparin antibodies. Available residual frozen sera were sent to a reference laboratory for serotonin release assay (SRA) testing. Results During the study period, 340 patients were admitted to the MICU, of which 143 patients met criteria for thrombocytopenia. Forty-three patients had no evidence of recent heparin exposure. PIFA and ELISA testing were performed on 100 patients, of which 92 had samples available for SRA analysis. PIFA results were negative in 62, positive in 28 and inconclusive in 2 patients. The 4Ts score showed low to intermediate risk in 57 of the PIFA negative patients. The ELISA results were negative in 86 and positive in 6 patients. SRA testing identified 3 patients with a positive SRA test and 89 patients with a negative result. All patients with a negative PIFA result also had a negative SRA result. In the one patient deemed to have clinical HIT, the pretest probability was high (4Ts score of 6) and the anti-PF4/heparin antibody testing revealed a positive SRA, inconclusive PIFA and a negative ELISA result. Conclusions While thrombocytopenia in our population is common, the prevalence of HIT is low. The combination of a low to intermediate pretest probability with a negative PIFA test can rapidly exclude the presence of platelet activating anti-PF4/heparin

  2. Glutathione peroxidase potentiates the inhibition of platelet function by S-nitrosothiols.

    PubMed Central

    Freedman, J E; Frei, B; Welch, G N; Loscalzo, J

    1995-01-01

    GSH peroxidase (Px) catalyzes the reduction of lipid hydroperoxides (LOOH), known metabolic products of platelets and vascular cells. Because interactions between these cells are modulated by nitric oxide (NO) and LOOH inactivate NO, we investigated the effect of GSH-Px on the inhibition of platelet function by the naturally occurring S-nitrosothiol, S-nitroso-glutathione (SNO-Glu). Concentrations of SNO-Glu that alone did not inhibit platelet function (subthreshold inhibitory concentrations) were added to platelet-rich plasma together with GSH-Px (0.2-20 U/ml); this led to a dose-dependent inhibition of platelet aggregation with an IC50 of 0.6 U/ml GSH-Px. In the presence of subthreshold inhibitory concentrations of SNO-Glu, the LOOH, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid, increased platelet aggregation, an effect reversed by GSH-Px. Glutathione and SNO-Glu were equally effective as cosubstrates for GSH-Px. Incubation of SNO-Glu with GSH-Px for 1 min led to a 48.5% decrease in the concentration of SNO-Glu. Incubation of SNO-Glu with serum albumin led to the formation of S-nitroso-albumin, an effect enhanced by GSH-Px. These observations suggest that GSH-Px has two functions: reduction of LOOH, thereby preventing inactivation of NO, and metabolism of SNO-Glu, thereby liberating NO and/or supporting further transnitrosation reactions. PMID:7615810

  3. The influence of herbal medicine on platelet function and coagulation: a narrative review.

    PubMed

    McEwen, Bradley J

    2015-04-01

    Cardiovascular disease (CVD) is the leading cause of death worldwide. Platelet activation and aggregation play a central role in hemostasis and thrombosis. Herbal medicines have been traditionally used in the management of CVD and can play a role in modifying CVD progression, particularly in platelet function, and have the potential of altering platelet function tests, as well as some coagulation parameters. Herbal medicines, such as feverfew, garlic, ginger, ginseng, motherwort, St John's wort, and willow bark, were found to reduce platelet aggregation. In vitro studies show promise in the reduction of platelet aggregation for Andrographis, feverfew, garlic, ginger, Ginkgo, ginseng, hawthorn, horse chestnut, and turmeric. In addition, cranberry, danshen, dong quai, Ginkgo, ginseng, green tea, and St John's wort were found to have potential interactions with warfarin. Furthermore, St John's wort interacted with clopidogrel and danshen with aspirin. Therefore, repeat testing of platelet function and coagulation studies, particularly for patients on warfarin therapy, may be required after exclusion of herbal medicines that could have possibly affected initial test results. PMID:25839871

  4. Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding

    PubMed Central

    Canault, Matthias; Ghalloussi, Dorsaf; Grosdidier, Charlotte; Guinier, Marie; Perret, Claire; Chelghoum, Nadjim; Germain, Marine; Raslova, Hana; Peiretti, Franck; Morange, Pierre E.; Saut, Noemie; Pillois, Xavier; Nurden, Alan T.; Cambien, François; Pierres, Anne; van den Berg, Timo K.; Kuijpers, Taco W.; Tregouet, David-Alexandre

    2014-01-01

    The nature of an inherited platelet disorder was investigated in three siblings affected by severe bleeding. Using whole-exome sequencing, we identified the culprit mutation (cG742T) in the RAS guanyl-releasing protein-2 (RASGRP2) gene coding for calcium- and DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). Platelets from individuals carrying the mutation present a reduced ability to activate Rap1 and to perform proper αIIbβ3 integrin inside-out signaling. Expression of CalDAG-GEFI mutant in HEK293T cells abolished Rap1 activation upon stimulation. Nevertheless, the PKC- and ADP-dependent pathways allow residual platelet activation in the absence of functional CalDAG-GEFI. The mutation impairs the platelet’s ability to form thrombi under flow and spread normally as a consequence of reduced Rac1 GTP-binding. Functional deficiencies were confined to platelets and megakaryocytes with no leukocyte alteration. This contrasts with the phenotype seen in type III leukocyte adhesion deficiency caused by the absence of kindlin-3. Heterozygous did not suffer from bleeding and have normal platelet aggregation; however, their platelets mimicked homozygous ones by failing to undergo normal adhesion under flow and spreading. Rescue experiments on cultured patient megakaryocytes corrected the functional deficiency after transfection with wild-type RASGRP2. Remarkably, the presence of a single normal allele is sufficient to prevent bleeding, making CalDAG-GEFI a novel and potentially safe therapeutic target to prevent thrombosis. PMID:24958846

  5. Correlation between the In Vitro Functionality of Stored Platelets and the Cytosolic Esterase-Induced Fluorescence Intensity with CMFDA.

    PubMed

    Wang, Jiexi; Yi, Xiaoyang; Liu, Minxia; Zhou, Qian; Ren, Suping; Wang, Yan; Yang, Chao; Zhou, Jianwei; Han, Ying

    2015-01-01

    It has been hypothesized that the cytosolic esterase-induced fluorescence intensity (CEIFI) from carboxy dimethyl fluorescein diacetate (CMFDA) in platelets may related to platelet functions. In the present study, we measured the change of CEIFI in platelets during storage, and examined the correlations of CEIFI with the in vitro functionality of stored platelets, including the ADP-induced aggregation activity, hypotonic shock response, expression of CD62P as well as platelet apoptosis. The CEIFI of fresh platelets, when tested at 10 μM CMFDA, the mean fluorescence intensity index (MFI) was 305.9 ± 49.9 (N = 80). After 1-day storage, it was 203.8 ± 34.4, the CEIFI of the stored platelets started to decline significantly, and reduced to 112.7 ±27.7 after 7-day storage. The change in CEIFI is highly correlated to all four functional parameters measured, with the correlation coefficients being 0.9813, 0.9848, -0.9945 and -0.9847 for the ADP-induced aggregation activity, hypotonic shock response (HSR), expression of CD62P and platelet apoptosis respectively. The above results show that the CEIFI measurement of platelets represents well the viability and functional state of in vitro stored platelets. This may be used as a convenient new method for quality evaluation for stored platelets if this result can be further validated by the following clinical trials.

  6. Fluorometric assay of oleate-activated phospholipase D isoenzyme in membranes of rat nervous tissue and human platelets.

    PubMed

    Krzystanek, Marek; Trzeciak, Henryk I; Krzystanek, Ewa; Małecki, Andrzej

    2010-01-01

    Phospholipase D plays a key role in the biosynthesis of phosphatidic acid, a second messenger involved in essential cellular processes. Oleate-activated phospholipase D was the first mammalian phospholipase D isoform to be discovered but is the least known. The study was aimed to test a fluorometric method of assessment of oleate-activated phospholipase D activity in different biological materials. The brain cortex of male Wistar rats, cultured rat brain astrocytes, and human platelets were processed to yield plasmatic membranes for experiments. To assess phospholipase D activity the modified fluorometric method was used. Previously, the method was used only to determine H₂O₂. In this enzyme-coupled assay phospholipase D activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine. First, phospholipase D cleaves exogenous phosphatidylcholine to yield choline and phosphatidic acid. Second, choline is oxidized by choline oxidase to betaine and H₂O₂. Finally, in the presence of horseradish peroxidase, H₂O₂ reacts with 10-acetyl-3,7-dihydroxyphenoxazine to generate the highly fluorescent product, resorufin. The concentration of resorufin was measured using excitation and emission at 560 nm and 590 nm, respectively. The proposed optimal parameters of the tested assay are 25 µg of rat brain cortex protein, 50 µg of rat brain astrocyte protein, and 50 µg of human platelet protein in a reaction volume of 200 µL, and 2 min enzymatic reaction at 37°C. The fluorometric method may be applied to assay phospholipase D in different biological materials. PMID:20835407

  7. Evaluation of a whole blood remote platelet function test for the diagnosis of mild bleeding disorders

    PubMed Central

    Dovlatova, Natalia; Lordkipanidzé, Marie; Lowe, Gillian C.; Dawood, Ban; May, Jane; Heptinstall, Stan; Watson, Steve P.; Fox, Susan C.

    2015-01-01

    Background Mild platelet function disorders (PFDs) are complex and difficult to diagnose. The current gold standard test, light transmission aggregometry (LTA), including lumi-aggregometry, is time- and labour-intensive and blood samples must be processed within a limited time after venepuncture. Furthermore, many subjects with suspected PFDs do not show a platelet abnormality on LTA. Objective To assess the diagnostic potential of an easy-to-use remote platelet function test (RPFT) as a diagnostic pre-test for suspected PFDs. Methods RPFT was compared to lumi-aggregometry in participants recruited to the Genotyping and Phenotyping of Platelets study (GAPP, ISRCTN 77951167). For RPFT, whole blood was stimulated with platelet agonists, stabilized with PAMFix and returned to the central laboratory for analysis of P-selectin and CD63 by flow cytometry. Results In the 61 study participants (42 index cases and 19 relatives) there was a good agreement between lumi-aggregometry and RPFT with diagnosis being concordant in 84% of cases (kappa=0.668, p<0.0001). According to both tests, 29 participants were identified to have a deficiency in platelet function and 22 participants appeared normal. There were 4 participants where lumi-aggregometry revealed a defect but RPFT did not, and 6 participants where RPFT detected an abnormal platelet response that was not identified by lumi-aggregometry. Conclusion This study suggests that RPFT could be an easy-to-use pre-test to select, which participants with bleeding disorders would benefit from extensive platelet phenotyping. Further development and evaluation of the test are warranted in a wider population of patients with excessive bleeding and could provide informative screening tests for PFDs. PMID:24618131

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

    NASA Astrophysics Data System (ADS)

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

    1998-04-01

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

  9. Effects of antihypertensive treatment on platelet function in essential hypertension.

    PubMed

    Gomi, T; Ikeda, T; Shibuya, Y; Nagao, R

    2000-11-01

    To evaluate the effect of antihypertensive therapy on platelet activation in essential hypertension, the plasma levels of beta-thromboglobulin (beta-TG) were examined in 45 patients with essential hypertension and 20 age-matched normotensive control subjects. Hypertensive patients were assigned to monotherapy with one of five different antihypertensive drugs for 6 months, and the change of plasma levels of beta-TG was reexamined after the completion of the monotherapy. The plasma beta-TG increased in hypertensive patients compared with levels in normotensive control subjects. Monotherapy with each drug resulted in sufficient blood pressure control in all hypertensive patients. The plasma beta-TG decreased significantly after monotherapy with an alpha-blocker or an angiotensin-converting enzyme inhibitor (ACEI). The plasma beta-TG increased with the use of a diuretic but did not change with the use of a beta-blocker or calcium antagonist. The platelet activation observed in patients with essential hypertension is reversed by monotherapy with an alpha-blocker or an ACEI. It is possible that these drugs reduce the development of hypertensive vascular complications due to suppression of platelet activation in patients with essential hypertension.

  10. Influence of different intravascular volume therapies on platelet function in patients undergoing cardiopulmonary bypass.

    PubMed

    Boldt, J; Knothe, C; Zickmann, B; Andres, P; Dapper, F; Hempelmann, G

    1993-06-01

    The influence of four different kinds of intravascular volume replacement on platelet function was investigated in 60 patients undergoing elective aortocoronary bypass grafting using cardiopulmonary bypass (CPB). In a randomized sequence, high-molecular weight hydroxyethyl starch solution (HMW-HES, mean molecular weight [Mw] 450,000 d), low-molecular weight HES (LMW-HES, Mw 200,000 d), 3.5% gelatin or 5% albumin were infused preoperatively to double reduced filling pressure (pulmonary capillary wedge pressure [PCWP] < 5 mm Hg). Fifteen untreated patients served as a control. Platelet function was assessed by aggregometry using turbidometric technique (inductors: ADP, epinephrine, collagen). Maximum aggregation, maximum gradient of aggregation, and platelet volume were measured before, during, and after CPB until the first postoperative day. HMW-HES 840 +/- 90 mL, LMW 850 +/- 100 mL, gelatin 950 +/- 110 mL, and albumin 810 +/- 100 mL were given preoperatively. Maximum platelet aggregation (ranging from -23% to -44% relative from baseline value) and maximum gradient of platelet aggregation (ranging from -26% to -45% relative from baseline values) were reduced only in the HMW-HES patients. After CPB, aggregometry also was impaired most markedly in these patients. The other volume groups showed less reduction in platelet aggregation and were similar to the untreated control. On the first postoperative day, aggregation variables had returned almost to baseline in all patients. Platelet volume was the same among the groups within the investigation period. Postbypass blood loss was highest in the HMW-HES group (890 +/- 180 mL). There was significant (P < 0.04) correlation in this group between blood loss and change in platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7684579

  11. Large-scale DNA typing for human platelet alloantigens by PCR-PHFA (preferential homoduplex formation assay).

    PubMed

    Fujiwara, K; Isa, K; Oka, T; Maekawajiri, S; Yamane, A; Akaza, T; Tadokoro, K; Juji, T; Shibata, Y; Tokunaga, K

    1996-10-01

    Alloimmunization against human platelet alloantigens (HPA) is known to be involved in disorders such as neonatal alloimmune thrombocytopenic purpura, posttransfusion purpura, and refractoriness to platelet transfusion therapy. HPA typing is essential in diagnosis and management of patients. Therefore a reliable and speedy method is necessary for HPA typing. We have successfully applied a new DNA typing method, PCR-preferential homoduplex formation assay (PHFA) method, to typing for the HPA-1, -2, -3, -4, -5 and -6 systems. This method is based on DNA strand competition during hybridization under a precisely controlled temperature gradient between a double-labelled amplicon (standard DNA), prepared from biotin- and DNP-labelled primers, and an unlabelled amplicon (sample DNA). The results obtained by PCR-PHFA typing were in good agreement with the allotypes determined by serological typing and by other DNA typing methods. The PCR-PHFA method can be easily automated, is suitable for typing both small and large numbers of samples, and thus is applicable to routine HPA typing.

  12. Detection of drug-dependent, platelet-reactive antibodies by solid-phase red cell adherence assays.

    PubMed

    Leach, M F; Cooper, L K; AuBuchon, J P

    1997-06-01

    We developed a simple modification of the solid-phase red cell adherence (SPRCA) assay system that can be used to identify drug-dependent platelet antibodies (DDPAs) reactive by either the hapten or immune complex reaction mechanisms. Between January 1994 and August 1996 we tested sera from 173 patients [123 (71%) with unexplained thrombocytopenia and 50 (29%) because of poor responses to platelet transfusions not explicable by alloimmunization or the clinical situation] for DDPAs possibly associated with the receipt of 61 different drugs. We correlated positive results with patients' clinical courses. DDPAs were identified in samples from 138 (80%) of the patients tested. Antibodies reactive only by the hapten mechanism were identified in 51 (37%) of those sera exhibiting positive reactions. The clinical courses of 108 (78%) patients were evaluable. Discontinuation of the implicated drug(s) resulted in prompt (<5 d) resolution of the thrombocytopenia or improvement in response to transfusion in all of these patients. In four cases thrombocytopenia returned upon re-exposure to the implicated drug. This adaptation of SPRCA provides a simple means of investigating the possibility of DDPAs and documents a higher frequency of these antibodies than has previously been suspected.

  13. Sulfasalazine and its metabolites inhibit platelet function in patients with inflammatory arthritis.

    PubMed

    MacMullan, Paul A; Madigan, Anne M; Paul, Nevin; Peace, Aaron J; Alagha, Ahmed; Nolan, Kevin B; McCarthy, Geraldine M; Kenny, Dermot

    2016-02-01

    The purpose of this study is to assess the effect of sulfasalazine and its metabolites on platelet function in patients with inflammatory arthritis (IA). One hundred thirty-five consecutive patients with an established diagnosis of IA were screened. Those with a history of cardiovascular disease (CVD), taking anti-platelet agents or non-steroidal anti-inflammatory drugs (NSAIDs) were excluded. A total of 32 patients were investigated, 15 taking sulfasalazine and 17 taking other disease-modifying anti-rheumatic drugs (DMARDs) and no sulfasalazine. These two cohorts were compared to 15 patients with stable CVD on long-term aspirin. The effect of sulfasalazine and its metabolites on arachidonic acid (AA)-induced platelet aggregation was also tested in vitro in samples from healthy donors (n = 18). Demographics, CVD risk factors and disease activity indices were similar in the sulfasalazine and other DMARD groups. AA-induced platelet aggregation was significantly inhibited in the sulfasalazine group (9 ± 7 %) and comparable to that in the aspirin group (10 ± 6 %). In contrast, there was no effect on AA-induced platelet aggregation in the other DMARDs group (77 ± 12 %) (p < 0.001). Furthermore, sulfasalazine therapy had no effect on platelet aggregation in response to multiple other agonists. Sulfasalazine and its metabolites (5-aminosalicylic acid and sulfapyridine) exerted an additive and dose-dependent inhibitory effect on AA-induced platelet aggregation in vitro (p < 0.001). The inhibition of AA-induced platelet aggregation by sulfasalazine is comparable to that achieved by aspirin and is dependent on both sulfasalazine and its metabolites. This represents a potential mechanism that may contribute to the known cardioprotective effect of sulfasalazine in patients with IA. PMID:25253538

  14. Desmopressin in vitro effects on platelet function, monitored with Multiplate, ROTEM and Sonoclot.

    PubMed

    Pearson, Kevin; Jensen, Hanna; Kander, Thomas; Schött, Ulf

    2016-07-01

    Background and aims The vasopressin analogue desmopressin has demonstrated efficacy in decreasing bleeding time by increasing the circulating levels of coagulation factor VIII and von Willebrand factor, but also by direct effects on platelets. Previous studies have demonstrated contrasting results regarding the effect of desmopressin on platelets in vitro. The aim of this study was to investigate the dose-response effects of in vitro desmopressin in whole blood. Our hypothesis was that desmopressin could increase platelet function in anticoagulated whole blood being stored up to 4 hours. Methods Desmopressin was administered with up to four different concentrations to venous whole blood, sampled with standard vacutainer tubes from 10 healthy volunteers after consent. Platelet function was analyzed with three different point-of-care techniques: Multiplate platelet aggregometry with adenosine diphosphate, collagen, thrombin receptor activating peptide-6, ristocetin and arachidonic acid agonists, tissue factor-activated thromboelastometry and Sonoclot glass bead viscoelastic coagulation tests at baseline and 4 hours later using different activator reagents. Results Thromboelastometry and Sonoclot did not show any significant change between baseline and 4 h later. A significant decrease in area under curve (AUC) could be seen with the Multiplate between baseline and after 4 h. Desmopressin did not improve any of these tests at baseline or during a 4 h storage and incubation period. Conclusion In vitro administered desmopressin could not increase normal platelet function or coagulation being measured with thromboelastometry and Sonoclot. Multiplate indicated decreased platelet aggregation over time, without any effect of in vitro added desmopressin. PMID:26923171

  15. Comparison between urinary 11-dehydrothromboxane B2 detection and platelet Light Transmission Aggregometry (LTA) assays for evaluating aspirin response in elderly patients with coronary artery disease.

    PubMed

    Liu, Tengfei; Zhang, Jingwei; Chen, Xiahuan; Feng, Xueru; Fu, Sidney W; McCaffrey, Timothy A; Liu, Meilin

    2015-10-15

    Aspirin is widely used in the primary and secondary prevention of cardiovascular diseases. The aim of our study was to compare between two established methods of aspirin response, urinary 11-dehydrothromboxane B2 (11dhTXB2) and platelet Light Transmission Aggregometry (LTA) assays in elderly Chinese patients with coronary artery disease (CAD), and to investigate the clinical significance of both methods in predicting cardiovascular events. Urinary 11dhTxB2 assay and arachidonic acid-induced (AA, 0.5mg/ml) platelet aggregation by Light Transmission Aggregometry (LTAAA) assay were measured to evaluate aspirin responses. High-on aspirin platelet reactivity (HAPR) was defined as urinary 11dhTxB2>1500pg/mg or AA-induced platelet aggregation≥15.22%-the upper quartile of our enrolled population. The two tests showed a poor correlation for aspirin inhibition (r=0.063) and a poor agreement in classifying HAPR (kappa=0.053). With a mean follow-up time of 12months, cardiovascular events occurred more frequently in HAPR patients who were diagnosed by LTA assay as compared with no-HAPR patients (22.5% versus 10.6%, P=0.039, OR=2.45, 95% CI=1.06-5.63). However, the HAPR status, as determined by urinary 11dTXB2 measurement, did not show a significant correlation with outcomes. PMID:26095809

  16. Comparison between urinary 11-dehydrothromboxane B2 detection and platelet Light Transmission Aggregometry (LTA) assays for evaluating aspirin response in elderly patients with coronary artery disease.

    PubMed

    Liu, Tengfei; Zhang, Jingwei; Chen, Xiahuan; Feng, Xueru; Fu, Sidney W; McCaffrey, Timothy A; Liu, Meilin

    2015-10-15

    Aspirin is widely used in the primary and secondary prevention of cardiovascular diseases. The aim of our study was to compare between two established methods of aspirin response, urinary 11-dehydrothromboxane B2 (11dhTXB2) and platelet Light Transmission Aggregometry (LTA) assays in elderly Chinese patients with coronary artery disease (CAD), and to investigate the clinical significance of both methods in predicting cardiovascular events. Urinary 11dhTxB2 assay and arachidonic acid-induced (AA, 0.5mg/ml) platelet aggregation by Light Transmission Aggregometry (LTAAA) assay were measured to evaluate aspirin responses. High-on aspirin platelet reactivity (HAPR) was defined as urinary 11dhTxB2>1500pg/mg or AA-induced platelet aggregation≥15.22%-the upper quartile of our enrolled population. The two tests showed a poor correlation for aspirin inhibition (r=0.063) and a poor agreement in classifying HAPR (kappa=0.053). With a mean follow-up time of 12months, cardiovascular events occurred more frequently in HAPR patients who were diagnosed by LTA assay as compared with no-HAPR patients (22.5% versus 10.6%, P=0.039, OR=2.45, 95% CI=1.06-5.63). However, the HAPR status, as determined by urinary 11dTXB2 measurement, did not show a significant correlation with outcomes.

  17. Minimal amounts of kindlin-3 suffice for basal platelet and leukocyte functions in mice.

    PubMed

    Klapproth, Sarah; Moretti, Federico A; Zeiler, Marlis; Ruppert, Raphael; Breithaupt, Ute; Mueller, Susanna; Haas, Rainer; Mann, Matthias; Sperandio, Markus; Fässler, Reinhard; Moser, Markus

    2015-12-10

    Hematopoietic cells depend on integrin-mediated adhesion and signaling, which is induced by kindlin-3 and talin-1. To determine whether platelet and polymorphonuclear neutrophil (PMN) functions require specific thresholds of kindlin-3, we generated mouse strains expressing 50%, 10%, or 5% of normal kindlin-3 levels. We report that in contrast to kindlin-3-null mice, which die perinatally of severe bleeding and leukocyte adhesion deficiency, mice expressing as little as 5% of kindlin-3 were viable and protected from spontaneous bleeding and infections. However, platelet adhesion and aggregation were reduced in vitro and bleeding times extended. Similarly, leukocyte adhesion, extravasation, and bacterial clearance were diminished. Quantification of protein copy numbers revealed stoichiometric quantities of kindlin-3 and talin-1 in platelets and neutrophils, indicating that reduction of kindlin-3 in our mouse strains progressively impairs the cooperation with talin-1. Our findings show that very low levels of kindlin-3 enable basal platelet and neutrophil functions, whereas in stress situations such as injury and infection, platelets and neutrophils require a maximum of functional integrins that is achieved with high and stoichiometric quantities of kindlin-3 and talin-1.

  18. Effects of high flavanol dark chocolate on cardiovascular function and platelet aggregation.

    PubMed

    Rull, Gurvinder; Mohd-Zain, Zetty N; Shiel, Julian; Lundberg, Martina H; Collier, David J; Johnston, Atholl; Warner, Timothy D; Corder, Roger

    2015-08-01

    Regular consumption of chocolate and cocoa products has been linked to reduced cardiovascular mortality. This study compared the effects of high flavanol dark chocolate (HFDC; 1064mg flavanols/day for 6weeks) and low flavanol dark chocolate (LFDC; 88mg flavanols/day for 6weeks) on blood pressure, heart rate, vascular function and platelet aggregation in men with pre-hypertension or mild hypertension. Vascular function was assessed by pulse wave analysis using radial artery applanation tonometry in combination with inhaled salbutamol (0.4mg) to assess changes due to endothelium-dependent vasodilatation. HFDC did not significantly reduce blood pressure compared to baseline or LFDC. Heart rate was increased by LFDC compared to baseline, but not by HFDC. Vascular responses to salbutamol tended to be greater after HFDC. Platelet aggregation induced by collagen or the thromboxane analogue U46619 was unchanged after LFDC or HFDC, whereas both chocolates reduced responses to ADP and the thrombin receptor activator peptide, SFLLRNamide (TRAP6), relative to baseline. Pre-incubation of platelets with theobromine also attenuated platelet aggregation induced by ADP or TRAP6. We conclude that consumption of HFDC confers modest improvements in cardiovascular function. Platelet aggregation is modulated by a flavanol-independent mechanism that is likely due to theobromine.

  19. Endothelial and platelet function alterations in HIV-infected patients.

    PubMed

    Gresele, P; Falcinelli, E; Sebastiano, M; Baldelli, F

    2012-03-01

    The HIV epidemic has huge dimensions: in 2009, 33.3million people worldwide, including 2.5million children, were affected by human immunodeficiency virus (HIV) infection. The introduction of Highly Active Anti-Retroviral Therapy (HAART) has significantly modified the course of HIV disease, with longer survival and improved quality of life, but it has simultaneously lead to the appearance of previously unrecognized complications, such as ischemic cardiovascular events. Many studies have shown a higher rate of premature atherosclerosis in patients with HIV infection, leading to coronary, cerebrovascular, or peripheral arterial disease. However, it is still debated whether cardiovascular complications are a consequence of HIV infection itself or of the long-term use of HAART. In particular, myocardial infarction has been suggested to be associated with the use of abacavir. Endothelial dysfunction and platelet activation are markers of atherosclerosis and of increased cardiovascular risk. Here we review the evidence that endothelial dysfunction and platelet alterations are associated with chronic HIV infection, the possible role of different HAARTs, and the possible pathophysiologic mechanisms. Potential therapeutic implications are also discussed.

  20. Role of glycoprotein IIa (beta 1 subunit of very late activation antigens) in platelet functions.

    PubMed

    Parmentier, S; Catimel, B; McGregor, L; Leung, L L; McGregor, J L

    1991-10-15

    Very late activation antigens (VLAs) are glycoproteins (GPs) that play a major role in platelet adhesion to extracellular matrix. These GPs, members of the integrin family, are heterodimer complexes with different alpha subunits noncovalently associated with a common beta 1 subunit known as GPIIa. GPIa-IIa (also known as VLA2), GPIc-IIa (VLA5), and GPIc*-IIa (VLA6) are involved, respectively, in platelet adhesion to collagen, fibronectin, and laminin. At this stage, very little is known about the role of GPIIa in platelet adhesive functions. In this study, we have generated a monoclonal antibody (MoAb) (LYP22) directed against GPIIa. Immunoaffinity chromatography using LYP22 combined with two-dimensional nonreduced-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the antibody brings down all VLA subunits. Western blots indicate that the binding site of LYP22 on GPIIa is disulfide bridge-dependent. The number of LYP22 binding sites is not increased on stimulation with thrombin and is in the range of what is observed with another anti-GPIIa MoAb (A-1A5). LYP22 is the first anti-GPIIa MoAb to inhibit aggregation and secretion of washed platelets stimulated with collagen, thrombin, or arachidonic acid. Moreover, the lag-phase usually observed on collagen stimulation is significantly prolonged (by 60 seconds) in the presence of LYP22. This lag-phase, mediated by LYP22, is also observed in the presence of plasma proteins and is coupled with a reduced effect on collagen-induced platelet aggregation. In addition, LYP22 affects the adhesion of resting platelets to type III collagen, but not to fibronectin, laminin, or type I collagen. These results strongly indicate that the site on GPIIa, bearing the LYP22 epitope, is an active participant in signal transduction controlling platelet functions.

  1. Platelet proteomics.

    PubMed

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

    2012-01-01

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

  2. [Effects of silkworm pupa oil on serum lipids level and platelet function in rats].

    PubMed

    Yang, Xuefeng; Huang, Lianzhen; Hu, Jianping; Li, Tao

    2002-08-01

    To observe the effects of silkworm pupa oil on serum lipids level and platelet function in rats, according to serum TG, TC level, 40 male Wistar rats are divided into four groups (normal control group, high fat control group, silkworm pupa oil group and silkworm pupa oil + VE group). The rats are fed different diets and six weeks later, serum lipids level and platelet function are measured. The results show that (1) Compared with high fat control group, serum TC, TG, LDL-C level, AI value, Platelet aggregability, plasma TXB2 level and T/P ratio decrease significantly while HDL-C level and 6-k-PGF1 level increase in silkworm pupa oil group; (2) Serum TC, LDL-C level, T/P ratio and platelet aggregability are significantly lower in silkworm pupa oil + VE group than in silkworm pupa oil group. It is suggested that silkworm pupa oil rich in alpha-linolenic acid can reduce serum lipids level and inhibit platelet aggregation, which is more effective with the supplementation with VE.

  3. Thrombin-linked aptamer assay for detection of platelet derived growth factor BB on magnetic beads in a sandwich format.

    PubMed

    Guo, Limin; Zhao, Qiang

    2016-09-01

    Here we describe a thrombin-linked aptamer assay (TLAA) for protein by using thrombin as an enzyme label, harnessing enzyme activity of thrombin and aptamer affinity binding. TLAA converts detection of specific target proteins to the detection of thrombin by using a DNA sequence that consists of two aptamers with the first aptamer binding to the specific target protein and the second aptamer binding to thrombin. Through the affinity binding, the thrombin enzyme is labeled on the protein target, and thrombin catalyzes the hydrolysis of small peptide substrate into product, generating signals for quantification. As a proof of principle, we show a sandwich TLAA for platelet derived growth factor BB (PDGF-BB) by using anti-PDGF-BB antibody coated on magnetic beads and an oligonucleotide containing the aptamer for PDGF-BB and the aptamer for thrombin. The binding of PDGF-BB to both the antibody and the aptamer results in labeling the complex with thrombin. We achieved detection of PDGF-BB at 16 pM. This TLAA contributes a new application of thrombin and its aptamer in bioanalysis, and shows potentials in assay developments. PMID:27343590

  4. Inherited platelet disorders.

    PubMed

    Franchini, Massimo; Lippi, Giuseppe; Veneri, Dino; Targher, Giovanni; Zaffanello, Marco; Guidi, Gian Cesare

    2008-01-01

    Inherited platelet disorders are a rare, but probably underdiagnosed, cause of symptomatic bleeding. They are characterized by abnormalities of platelet number (inherited thrombocytopenias), function (inherited disorders of platelet function) or both. This review briefly discusses the inherited platelet disorders with respect to molecular defects, diagnostic evaluation and treatment strategies.

  5. [Cyclooxygenase inhibitors in some dietary vegetables inhibit platelet aggregation function induced by arachidonic acid].

    PubMed

    Wang, Xin-Hua; Shao, Dong-Hua; Liang, Guo-Wei; Zhang, Ru; Xin, Qin; Zhang, Tao; Cao, Qing-Yun

    2011-10-01

    The study was purposed to investigate whether the cyclooxygenase inhibitors from some dietary vegetables can inhibit platelet aggregation function by the arachidonic acid (AA). The vegetable juice was mixed with platelet rich plasma (PRP), and asprin was used as positive control. The maximum ratio of platelet aggregation induced by AA was measured on the aggregometer; heme and cyclooxygenase-1 (COX(1)) or cyclooxygenase-2 (COX(2)) were added to test tubes containing COX reaction buffer, the mixture was vortex-mixed and exposed to aspirin or vegetable juice, followed by addition of AA and then hydrochloric acid (1 mol/L) was added to stop the COX reaction, followed by chemical reduction with stannous chloride solution. The concentration of COX inhibitors was detected by the enzyme immunoassay kit; vegetable juice (aspirin as positive control) was mixed with whole blood, which was followed by the addition of AA, and then the reaction was stopped by adding indomethacin, centrifuged, then the supernatant was collected, and the plasma thromboxane B(2) (TXB(2)) was measured by radioimmunoassay. The results showed that spinach juice, garlic bolt juice, blanched garlic leave juice and Chinese leek juice could inhibit by 80% human platelet aggregation induced by AA. 4 kinds of vegetables were all found a certain amount of cyclooxygenase inhibitors, which COX(1) and COX(2) inhibitor concentrations of spinach were higher than that of aspirin; 4 vegetable juice could significantly reduce the human plasma concentrations of TXB(2) induced by AA (p < 0.05). It is concluded that 4 kinds of raw vegetables containing cyclooxygenase inhibitors inhibit the production of TXA(2) and thus hinder platelet aggregation. Raw spinach, garlic bolt, blanched garlic and chinese leek inhibit significantly AA-induced human platelet aggregation in vitro. 4 kinds of vegetables may have a good potential perspective of anti-platelet aggregation therapy or prevention of thrombosis.

  6. Poly (I:C) downregulates platelet production and function through type I interferon.

    PubMed

    Rivadeneyra, Leonardo; Pozner, Roberto Gabriel; Meiss, Roberto; Fondevila, Carlos; Gómez, Ricardo Martin; Schattner, Mirta

    2015-11-01

    Thrombocytopenia is a frequent complication of viral infections; the underlying mechanisms appear to depend on the identity of the virus involved. Previous research, including reports from our group, indicates that as well as having antiviral activity type I interferons (IFN I) selectively downregulate platelet production. In this study we extended understanding of the role of endogenous IFN I in megakaryo/thrombopoiesis by evaluating platelet and megakaryocyte physiology in mice treated with polyinosinic:polycytidylic acid [poly (I:C)], a synthetic analogue of double-stranded RNA, Toll-like receptor-3 ligand and strong IFNβ inducer. Mice-treated with poly (I:C) showed thrombocytopaenia, an increase in mean platelet volume and abnormal haemostatic and inflammatory platelet-mediated functionality, indicated by decreased fibrinogen binding and platelet adhesion, prolonged tail bleeding times and impaired P-Selectin externalisation, RANTES release and thrombin-induced platelet-neutrophil aggregate formation. These changes were associated with an increase in size and an abnormal distribution of bone marrow megakaryocytes within the vascular niche and were directly correlated with the plasmatic and bone marrow IFNβ levels. All these effects were absent in genetically modified mice lacking the IFN I receptor. Our results suggest that IFN I is the central mediator of poly (I:C)-induced thrombocytopenia and platelet dysfunction and indicate that these abnormalities are due to changes in the last stages of megakaryocyte development. These data provide new evidence for the role of IFN I in megakaryocyte distribution in the bone marrow niches and its influence on thrombopoiesis and haemostasis. PMID:26134179

  7. [Effects of lysine clonixinate on platelet function. Comparison with other non-steroidal anti-inflammatory agents].

    PubMed

    Kramer, E H; Sassetti, B; Kaminker, A J; De Los Santos, A R; Martí, M L; Di Girolamo, G

    2001-01-01

    One of the mechanisms of action of non steroid antiinflammatory drugs (NSAIDs) consists of inhibition of prostaglandin synthesis. This explains many of the pharmacological effects and adverse events observed in medical practice. Administration of NSAIDs to patients with hemostatic disorders or perioperative conditions entails the risk of bleeding due to inhibition of platelet function. This study deals with platelet changes induced by lysine clonixinate vs diclofenac, ibuprofen and aspirin in classical tests such as platelet count, platelet factor 3 (PF3) activity and platelet aggregation with various inductors and more recent procedures such as P-selectin measurement by flow cytometry. Unlike control drugs, lysine clonixinate did not induce changes in platelet count or function when administered to healthy volunteers at the commonly used therapeutic doses.

  8. The association of thromboxane A2 receptor with lipid rafts is a determinant for platelet functional responses.

    PubMed

    Moscardó, A; Vallés, J; Latorre, A; Santos, M T

    2014-08-25

    We have investigated the presence of thromboxane A2 (TXA2) receptor associated with lipid rafts in human platelets and the regulation of platelet function in response to TXA2 receptor agonists when lipid rafts are disrupted by cholesterol extraction. Platelet aggregation with TXA2 analogs U46619 and IBOP was almost blunted in cholesterol-depleted platelets, as well as αIIbβ3 integrin activation and P-selectin exposure. Raft disruption also inhibited TXA2-induced cytosolic calcium increase and nucleotide release, ruling out an implication of P2Y12 receptor. An important proportion of TXA2 receptor (40%) was colocalized at lipid rafts. The presence of the TXA2 receptor associated with lipid rafts in platelets is important for functional platelet responses to TXA2.

  9. Fibronectin unfolded by adherent but not suspended platelets: An in vitro explanation for its dual role in haemostasis.

    PubMed

    Huynh, Khon; Gyenes, Marianna; Hollenberg, Cornelis P; Nguyen, Thi-Hiep; Van Vo, Toi; Stoldt, Volker R

    2015-10-01

    Fibronectin (FN), a dimeric adhesive glycoprotein, which is present both in plasma and the extracellular matrix can interact with platelets and thus contribute to platelet adhesion and aggregation. It has been shown that FN can decrease platelet aggregation but enhance platelet adhesion, suggesting a dual role of FN in haemostasis. The prevalent function(s) of FN may be determined by its fibril form. To explore the suggested dual role of this adhesive protein for haemostasis in further detail, we now tested for any differences of adherent and suspended platelets with regard to their effect to unfold and assemble FN upon interaction. Platelet aggregation and adhesion assays were performed using washed platelets in the presence of exogenous FN. Addition of plasma FN reduced platelet aggregation in response to collagen or PMA by 50% or 25% but enhanced platelet adhesion onto immobilized collagen, as compared to control experiments. Analyses by fluorescence resonance energy transfer (FRET) demonstrated that adherent platelets but not suspended platelets were capable of unfolding FN during 3h incubation. Fluorescence microscopy and deoxycholate (DOC) solubility assays demonstrated that FN fibrils formed only on the surfaces of adherent platelets. In addition, platelets adherent onto FN revealed a significantly higher activity of specific Src phosphorylation (pY418) than platelets in suspension. These data suggest (1) that the function of FN in haemostasis is prevalent to its assembly, unfolding and subsequent fibril formation on the surface of adherent platelets and (2) that outside-in signaling contributes to the interaction of platelets and FN.

  10. Effect of Haemophilus influenzae infection and moxalactam on platelet function in children.

    PubMed Central

    Kaplan, S L; Courtney, J T; Kenal, K A

    1987-01-01

    In a prospective randomized study, children with Haemophilus influenzae type b meningitis received moxalactam or ampicillin or chloramphenicol. Of 41 children, 6 had prolonged bleeding times (greater than 6 min), and 7 of 9 tested had abnormal platelet aggregation at hospital admission. At the end of therapy, no children in the ampicillin-chloramphenicol group, compared with 5 of 22 moxalactam-treated children (23%) (P = 0.08), had prolonged bleeding times (6.5 to 7.5 min). Our data suggest that H. influenzae meningitis and treatment with moxalactam may each have an effect on platelet function in children. PMID:3579263

  11. Blood rheology and platelet function in untreated early-stage essential hypertensives complicated with metabolic syndrome.

    PubMed

    Sugimori, Hiroko; Tomoda, Fumihiro; Koike, Tsutomu; Kinuno, Hiroyuki; Kurosaki, Hiroko; Masutani, Toshitaka; Inoue, Hiroshi

    2012-01-01

    We examined whether hemorheology and platelet function are affected in essential hypertensives (EHTs) of the World Health Organization stage I when complicated with metabolic syndrome (Mets). In 156 untreated EHTs, blood viscosity and platelet surface markers were determined. Blood viscosity was significantly elevated in 54 subjects with Mets compared with 102 subjects without Mets. Hematocrit and plasma viscosity increased in the group with Mets, although red blood cell rigidity index "k" did not differ between groups. As a whole group, blood viscosity correlated positively with hematocrit and plasma viscosity. Additionally, plasma viscosity correlated positively with plasma leptin, triglyceride, homeostasis model assessment index, C-reactive protein, and plasma fibrinogen, but negatively with high-density lipoprotein cholesterol. In contrast, no differences were seen in platelet surface markers between groups. In conclusion, EHTs of the early stage complicated with Mets are characterized by increased blood viscosity due to hemoconcentration and increased plasma viscosity. PMID:22570768

  12. New families of adhesion molecules play a vital role in platelet functions.

    PubMed

    Parmentier, S; Kaplan, C; Catimel, B; McGregor, J L

    1990-07-01

    Adhesion molecules play a crucial part in cell-matrix and in cell-cell interactions. These interactions, which are essential to the body's defense processes, involve adhesion molecules belonging to different families: integrins, immunoglobulins and selectins. Integrins are expressed by a large number of tissues, whereas other adhesion molecule families are restricted to a small number of cell types. A recent symposium dealt with the recruitment of circulating platelets at specific sites, their adhesion to extracellular matrix components and their activation by agonists leading to aggregation or attachment to other cells. These events, supporting hemostasis and thrombosis, involve integrins, selectins and other adhesion molecules. This report focuses on newly reported integrins (GPIa, GPIc, GPIIa), selectins (GMP-140) and GPIIIb, previously known as 'minor' surface oriented platelet glycoproteins. Major membrane glycoproteins such as GPIIb-IIIa (an integrin) and GPIb, which also play a vital role in platelet functions, have been extensively reviewed elsewhere.

  13. Inhibitory effects of Cyperus digitatus extract on human platelet function in vitro.

    PubMed

    Fuentes, Eduardo; Forero-Doria, Oscar; Alarcón, Marcelo; Palomo, Iván

    2015-01-01

    The purpose of this research was to investigate the mechanisms of antiplatelet action of Cyperus digitatus. The antiplatelet action of C. digitatus was studied on platelet function: secretion, adhesion, aggregation, and sCD40L release. The platelet ATP secretion and aggregation were significantly inhibited by CDA (ethyl acetate extract) at 0.1 mg/ml and after the incubation of whole blood with CDA, the platelet coverage was inhibited by 96 ± 3% (p < 0.001). At the same concentration, CDA significantly decreased sCD40L levels. The mechanism of antiplatelet action of CDA could be by NF-κB inhibition and that is cAMP independent. In conclusion, C. digitatus extract may serve as a new source of antiplatelet agents for food and nutraceutical applications. PMID:25548968

  14. Towards Personalized Medicine Based on Platelet Function Testing for Stent Thrombosis Patients

    PubMed Central

    Godschalk, Thea Cornelia; Hackeng, Christian Marcus; ten Berg, Jurriën Maria

    2012-01-01

    Stent thrombosis (ST) is a severe and feared complication of coronary stenting. Patients who have suffered from ST are usually treated according to the “one-size-fits-all” dosing regimen of aspirin and clopidogrel. Many ST patients show high on-treatment platelet reactivity (HPR) despite this antiplatelet therapy (APT). It has been shown that HPR is a risk factor for major adverse cardiac events. Therefore, ST patients with HPR are at a high risk for recurrent atherothrombotic events. New insights into the variable response to clopidogrel and the advent of stronger P2Y12 inhibitors prasugrel and ticagrelor have changed the attention from a fixed APT treatment strategy towards “personalized APT strategies.” Strategies can be based on platelet function testing, which gives insight into the overall response of a patient to APT. At our outpatient ST clinic, we practice personalized APT based on platelet function testing to guide the cardiologist to a presumed optimal antiplatelet treatment of ST patients. Beside results of platelet function testing, comedication, clinical characteristics, and genetics have to be considered to decide on personalized APT. Ongoing studies have yet to reveal the optimal personalized APT strategy for cardiologists to prevent their patients from atherothrombotic and bleeding events. PMID:23320159

  15. Platelet lipidomic.

    PubMed

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

    2012-01-01

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

  16. Cocoa flavanols and platelet and leukocyte function: recent in vitro and ex vivo studies in healthy adults.

    PubMed

    Heptinstall, Stan; May, Jane; Fox, Sue; Kwik-Uribe, Catherine; Zhao, Lian

    2006-01-01

    There is growing interest in possible beneficial effects of specific dietary components on cardiovascular health. Platelets and leukocytes contribute to arterial thrombosis and to inflammatory processes. Previous studies performed in vitro have demonstrated inhibition of platelet function by (-)-epicatechin and (+)-catechin, flavan-3-ols (flavanols) that are present in several foods including some cocoas. Also, some modest inhibition of platelet function has been observed ex vivo after the consumption of flavanol-containing cocoa products by healthy adults. So far there are no reports of effects of cocoa flavanols on leukocytes. This paper summarizes 2 recent investigations. The first was a study of the effects of cocoa flavanols on platelet and leukocyte function in vitro. The second was a study of the effects of consumption of a flavanol-rich cocoa beverage by healthy adults on platelet and leukocyte function ex vivo. Measurements were made of platelet aggregation, platelet-monocyte conjugate formation (P/M), platelet-neutrophil conjugate formation (P/N), platelet activation (CD62P on monocytes and neutrophils), and leukocyte activation (CD11b on monocytes and neutrophils) in response to collagen and/or arachidonic acid. In the in vitro study several cocoa flavanols and their metabolites were shown to inhibit platelet aggregation, P/M, P/N, and platelet activation. Their effects were similar to those of aspirin and the effects of a cocoa flavanol and aspirin did not seem to be additive. There was also inhibition of monocyte and neutrophil activation by flavanols, but this was not replicated by aspirin. 4'-O-methyl-epicatechin, 1 of the known metabolites of the cocoa flavanol (-)-epicatechin, was consistently effective as an inhibitor of platelet and leukocyte activation. The consumption of a flavanol-rich cocoa beverage also resulted in significant inhibition of platelet aggregation, P/M and P/N, and platelet activation induced by collagen. The inhibitory effects

  17. Platelet function and fibrinolytic activity during rest and exercise in borderline hypertensive patients.

    PubMed

    Gleerup, G; Vind, J; Winther, K

    1995-04-01

    In this study we examined whether the reduced fibrinolysis and increased platelet activity that are known to occur in hypertension are already present in borderline hypertension. Twelve patients with 'borderline' hypertension (diastolic blood pressure 90-95 mmHg) were found to have substantially reduced fibrinolytic activity, both at rest and during exercise, compared with 12 normotensive controls. Euglobulin clot lysis time (ECLT) was significantly higher in hypertensive subjects (218 min vs. 145 min; P < 0.05), and this difference persisted during exercise. Resting tissue plasminogen activator activity (t-PA) did not differ in the two groups, but the brisk increase in t-PA in controls during exercise (0.64 rising to 1.44 IU mL-1; P < 0.01) did not occur to the same extent in the borderline hypertensive subjects. Levels of the fast-acting t-PA inhibitor, normally referred to as PAI-1, were considerably higher in hypertensives (9.22 vs. 4.41 IU mL-1; P < 0.02), and this difference persisted in the upright posture, indicating a decrease in fibrinolytic activity. Platelet aggregability induced by ADP in vitro was not significantly higher in the hypertensive subjects, but indices of platelet activity in vivo (B-TG and PF-4 levels) revealed enhanced platelet function in the hypertensives. These results indicate that the indicators of altered haemostatic function known to occur in hypertension, namely diminished fibrinolytic activity and increased platelet function, are already detectable during the very earliest stage of the disease.

  18. EphB2 regulates contact-dependent and contact-independent signaling to control platelet function

    PubMed Central

    Vaiyapuri, Sakthivel; Sage, Tanya; Rana, Rekha H.; Schenk, Michael P.; Ali, Marfoua S.; Unsworth, Amanda J.; Jones, Chris I.; Stainer, Alexander R.; Kriek, Neline; Moraes, Leonardo A.

    2015-01-01

    The Eph kinases, EphA4 and EphB1, and their ligand, ephrinB1, have been previously reported to be present in platelets where they contribute to thrombus stability. Although thrombus formation allows for Eph-ephrin engagement and bidirectional signaling, the importance specifically of Eph kinase or ephrin signaling in regulating platelet function remained unidentified. In the present study, a genetic approach was used in mice to establish the contribution of signaling orchestrated by the cytoplasmic domain of EphB2 (a newly discovered Eph kinase in platelets) in platelet activation and thrombus formation. We conclude that EphB2 signaling is involved in the regulation of thrombus formation and clot retraction. Furthermore, the cytoplasmic tail of this Eph kinase regulates initial platelet activation in a contact-independent manner in the absence of Eph-ephrin ligation between platelets. Together, these data demonstrate that EphB2 signaling not only modulates platelet function within a thrombus but is also involved in the regulation of the function of isolated platelets in a contact-independent manner. PMID:25370417

  19. A protein with characteristics of factor H is present on rodent platelets and functions as the immune adherence receptor.

    PubMed

    Alexander, J J; Hack, B K; Cunningham, P N; Quigg, R J

    2001-08-24

    Complement-coated particles interact with specific immune adherence receptors (IAR). In primates, this function is served by complement receptor 1 (CR1) on erythrocytes. In contrast, rodent platelets bear IAR distinct from CR1, the identity of which was studied here. A 150-kDa C3b-binding protein was isolated from rat platelets, which had immunochemical and biochemical identity to plasma factor H. Immunofluorescence microscopy and flow cytometry demonstrated that factor H was present on the surface of rat and mouse platelets, which could be removed by treatment with neuraminidase. Sheep erythrocytes bearing C3b underwent immune adherence with rat and mouse platelets, which was blocked with anti-factor H F(ab')(2) antibodies, but not with antibodies binding to the complement regulator, Crry, on the platelet surface. By reverse transcription-polymerase chain reaction using rat platelet RNA and primers designed from mouse factor H, a 472-base pair product was generated that was identical in sequence to that produced from rat liver RNA. The translated protein product was 85% similar to mouse liver factor H. The 3'-nucleotide sequence from platelets predicted a soluble factor H protein. By Northern analysis, liver and platelets had identically sized factor H mRNA. Thus, rat and mouse platelets have a membrane protein with characteristics of factor H that is linked via sialic acid residues and functions as the IAR. Whether platelet factor H is acquired by passive adsorption from sera and/or is produced by platelets remains to be determined.

  20. Grafting of phosphorylcholine functional groups on polycarbonate urethane surface for resisting platelet adhesion.

    PubMed

    Gao, Bin; Feng, Yakai; Lu, Jian; Zhang, Li; Zhao, Miao; Shi, Changcan; Khan, Musammir; Guo, Jintang

    2013-07-01

    In order to improve the resistance of platelet adhesion on material surface, 2-methacryloyloxyethyl phosphorylcholine (MPC) was grafted onto polycarbonate urethane (PCU) surface via Michael reaction to create biomimetic structure. After introducing primary amine groups via coupling tris(2-aminoethyl)amine (TAEA) onto the polymer surface, the double bond of MPC reacted with the amino group to obtain MPC modified PCU. The modified surface was characterized by Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The results verified that MPC was grafted onto PCU surface by Michael reaction method. The MPC grafted PCU surface had a low water contact angle and a high water uptake. This means that the hydrophilic PC functional groups improved the surface hydrophilicity significantly. In addition, surface morphology of MPC grafted PCU film was imaged by atomic force microscope (AFM). The results showed that the grafted surface was rougher than the blank PCU surface. In addition, platelet adhesion study was evaluated by scanning electron microscopy (SEM) observation. The PCU films after treated with platelet-rich plasma demonstrated that much fewer platelets adhered to the MPC-grafted PCU surface than to the blank PCU surface. The antithrombogenicity of the MPC-grafted PCU surface was determined by the activated partial thromboplastin time (APTT). The result suggested that the MPC modified PCU may have potential application as biomaterials in blood-contacting and some subcutaneously implanted devices.

  1. 14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function

    PubMed Central

    Schoenwaelder, Simone M.; Darbousset, Roxane; Cranmer, Susan L.; Ramshaw, Hayley S.; Orive, Stephanie L.; Sturgeon, Sharelle; Yuan, Yuping; Yao, Yu; Krycer, James R.; Woodcock, Joanna; Maclean, Jessica; Pitson, Stuart; Zheng, Zhaohua; Henstridge, Darren C.; van der Wal, Dianne; Gardiner, Elizabeth E.; Berndt, Michael C.; Andrews, Robert K.; James, David E.; Lopez, Angel F.; Jackson, Shaun P.

    2016-01-01

    The 14-3-3 family of adaptor proteins regulate diverse cellular functions including cell proliferation, metabolism, adhesion and apoptosis. Platelets express numerous 14-3-3 isoforms, including 14-3-3ζ, which has previously been implicated in regulating GPIbα function. Here we show an important role for 14-3-3ζ in regulating arterial thrombosis. Interestingly, this thrombosis defect is not related to alterations in von Willebrand factor (VWF)–GPIb adhesive function or platelet activation, but instead associated with reduced platelet phosphatidylserine (PS) exposure and procoagulant function. Decreased PS exposure in 14-3-3ζ-deficient platelets is associated with more sustained levels of metabolic ATP and increased mitochondrial respiratory reserve, independent of alterations in cytosolic calcium flux. Reduced platelet PS exposure in 14-3-3ζ-deficient mice does not increase bleeding risk, but results in decreased thrombin generation and protection from pulmonary embolism, leading to prolonged survival. Our studies define an important role for 14-3-3ζ in regulating platelet bioenergetics, leading to decreased platelet PS exposure and procoagulant function. PMID:27670677

  2. Platelet and other hemostatic characteristics in patients with chronic urticaria.

    PubMed

    Isiksacan, Nilgun; Koser, Murat; Cemsitoglu, Ferhan; Kucuksezer, Umut C; Gurdol, Figen

    2015-04-01

    Several publications have pointed out the importance of coagulation and fibrinolysis in the occurrence of chronic urticaria (CU), but only a few indicated the direct role of platelets. We assessed platelet aggregation and evaluated parameters of coagulation and fibrinolysis in patients with CU. Patients (n = 34) diagnosed as having CU and 36 healthy controls were enrolled. Platelet aggregation was assayed using an impedance aggregometer and adenosine diphosphate, arachidonic acid, thrombin receptor-activating peptide (TRAP), and ristocetin as agonists. In patients with CU, significantly decreased platelet aggregation to some agonists (ristocetin and TRAP) was observed. The D-dimer levels were elevated, mean platelet volume was decreased, but no alteration was observed in other coagulation assays. Elevated D-dimer levels indicated that coagulation and fibrinolysis are activated in the patients with CU. Evaluation of platelet function may contribute to identify the role of these cells in the pathogenesis of CU.

  3. The effects of Lonomin V, a toxin from the caterpillar (Lonomia achelous), on hemostasis parameters as measured by platelet function.

    PubMed

    Guerrero, Belsy; Arocha-Piñango, Carmen L; Salazar, Ana M; Gil, Amparo; Sánchez, Elda E; Rodríguez-Acosta, Alexis; Lucena, Sara

    2011-09-15

    Platelets play a central role in hemostasis during vascular injury. Patients affected with the hemorrhagic syndrome caused by contact with Lonomia achelous caterpillars (Lac) Lepidoptera distributed in various South American countries, show digestive, pulmonary and intraperitoneal bleeding in combination with hematomas and echymosis. In the present study, we have evaluated the effects of Lonomin V (serine protease isolated from Lac hemolymph) on some functional properties of platelets, evaluating its importance in primary hemostasis. Platelet adhesion to fibrinogen was reduced by 19, 20, 36, and 37% after pre-treated with 0.2, 2, 20 and 40 nM of Lonomin V, respectively. Pre-incubation of the platelets with 408 nM of Lonomin V, for 4 min at 37 °C, resulted in complete inhibition of the collagen-induced platelet aggregation, in contrast to 56% inhibition of the ADP - induced platelet aggregation. Lonomin V also inhibited anti-α(IIb)β(3) integrin binding to platelets by 56, 57, 52 and 54% at concentrations of 0.2, 2, 20 and 40 nM respectively. Additionally, Lonomin V inhibited anti-P-selectin binding to platelets by 28, 37, 33 and 33% at the same concentrations. The platelets tested with Lonomin V did not modify their viability. In summary, Lonomin V inhibited platelet aggregation, probably caused by the degradation of collagen. The anti-platelet activity of Lonomin V has been shown to be unique and a potentially useful tool for investigating cell-matrix and cell-cell interactions and for the development of antithrombotic agents in terms of their anti-adhesive activities.

  4. A comparison of platelet function tests and thromboxane metabolites to evaluate aspirin response in healthy individuals and patients with coronary artery disease.

    PubMed

    Grove, Erik Lerkevang; Hvas, Anne-Mette; Johnsen, Helle Ladefoged; Hedegaard, Sofie Sommer; Pedersen, Susanne Bendesgaard; Mortensen, Jette; Kristensen, Steen Dalby

    2010-06-01

    Individualised antiplatelet therapy and platelet function testing have attracted considerable clinical interest, but several aspects of test performance have not been thoroughly evaluated. We investigated repeatability and concordance of light transmission aggregometry (LTA) induced with arachidonic acid (AA) 1.0 mM, PFA-100 induced with collagen/epinephrine, multiple electrode aggregometry (MEA) induced with AA 0.5 or 0.75 mM and VerifyNow Aspirin. Patients with stable coronary artery disease (n=43) and healthy individuals (n=21) were included. All tests were performed in duplicate at baseline in healthy individuals and in duplicate for four days in all study participants during aspirin treatment. Serum and urinary thromboxane metabolites were measured several times to evaluate cyclooxygenase-1 inhibition by aspirin. MEA was most sensitive for aspirin as treatment induced a 12-fold difference in AA-induced platelet aggregation. Coefficients of variation for duplicate measurements at baseline (0.4-12%), during aspirin treatment (3-46%) and for day-to-day variability (3-37%) differed markedly between tests and were lowest for VerifyNow. The prevalence of aspirin low-responsiveness also differed between tests (0-9%) and the agreement was low: kappaplatelet function test, whereas serum thromboxane correlated with VerifyNow Aspirin (r=0.41, p=0.001). Overall, repeatability was moderate and the correlation between tests was low. VerifyNow Aspirin proved most reproducible, and this was the only assay showing a significant positive correlation with serum thromboxane. This study demonstrated that conclusions based on platelet function testing strongly depend on the assay used. PMID:20352155

  5. Proplatelets and stress platelets.

    PubMed

    Tong, M; Seth, P; Penington, D G

    1987-02-01

    The process of platelet formation by the fragmentation of megakaryocyte pseudopodia, termed proplatelets, demonstrable in the marrow sinusoids is poorly understood. "Stress" platelets produced under conditions of stimulated platelet production differ from normal circulating platelets with respect to volume and a number of functional characteristics. To clarify the relationship of stress platelets to proplatelets, rats were injected with heterologous platelet antiserum. Nondiscoid platelet forms, some characteristically beaded in appearance, strongly resembling bone marrow proplatelets, can be recovered in the circulation of normal rats. During the early period of recovery from acute thrombocytopenia, there was a substantial increase in the proportion of these elongated platelets in the citrated platelet rich plasma. Exposure to EDTA rendered them spherical. Circulating proplatelets may contribute significantly to the prompt increase in platelet volume during recovery from acute thrombocytopenia at a time prior to significant increase in megakaryocyte size and ploidy. PMID:3801667

  6. Small-size platelet microparticles trigger platelet and monocyte functionality and modulate thrombogenesis via P-selectin.

    PubMed

    Montoro-García, Silvia; Shantsila, Eduard; Hernández-Romero, Diana; Jover, Eva; Valdés, Mariano; Marín, Francisco; Lip, Gregory Y H

    2014-08-01

    This study aimed to examine the mechanisms of cellular activation by small-size platelet microparticles (sPMP) and to present the performance of high-resolution flow cytometry for the analysis of subcellular entities from different origins. Plasma counts of sPMP were analysed in coronary artery disease patients (n = 40) and healthy controls (n = 40). The effect of sPMP and platelet debris (PD) in pathophysiologically relevant doses on platelet and monocyte activation parameters and thrombogenesis was investigated via flow cytometry and thromboelastometry. New generation flow cytometry identifies differences in size, levels and surface molecules of sPMP derived in the absence of stimulus, thrombin activation and platelet disruption. Addition of sPMP resulted in platelet degranulation and P-selectin redistribution to the membrane (P = 0·019) in a dose and time-dependent manner. Blood clotting time decreased after addition of sPMP (P = 0·005), but was not affected by PD. Blocking P-selectin (CD62P) in sPMP markedly reverted the effect on thrombus kinetics (P = 0·035). Exposure to sPMP stimulated monocyte expression of intercellular adhesion molecule-1 (P < 0·03) and decreased monocyte interleukin-6 receptor density (P < 0·01). These results implicate sPMP as a direct source of downstream platelet and monocyte activation. In pathological coronary artery disease conditions, higher levels of sPMP favour a prothrombotic state, partly through P-selectin expression.

  7. Variability of residual platelet function despite clopidogrel treatment in patients with peripheral arterial occlusive disease.

    PubMed

    Linnemann, Birgit; Schwonberg, Jan; Toennes, Stefan W; Mani, Helen; Lindhoff-Last, Edelgard

    2010-04-01

    Residual platelet function despite treatment with clopidogrel may predict an unfavourable cardiovascular outcome. The majority of studies have investigated the effects of clopidogrel administration in conjunction with aspirin in patients undergoing percutaneous coronary intervention. The primary objective of the present study was to assess the platelet response to clopidogrel in the absence of aspirin in patients with peripheral arterial occlusive disease (PAOD) and to investigate whether non-responsiveness to clopidogrel is reproducible during long-term follow-up. Fifty-four clinically stable PAOD patients on a maintenance dose of 75 mg/d clopidogrel were enrolled in this study. Platelet function was assessed at baseline and after a median follow-up of 18 months using light transmittance aggregometry (LTA) with 2 microM ADP as an agonist. HPLC-coupled mass spectrometry was used to detect clopidogrel and clopidogrel carboxylic acid, the main metabolite of clopidogrel. Residual platelet function, as defined by late aggregation values within the reference range (i.e., >43%), was observed in 35.2% of patients at baseline and 17.6% during follow-up. During the observation period, 26.5% had switched from responder to non-responder status or vice versa. Among non-responders, either clopidogrel or its metabolite was detected in 89.5% and 83.3% of patients at baseline and at follow-up, respectively. We conclude that non-responsiveness to clopidogrel as determined by ADP-induced LTA is not stable over time. This phenomenon cannot be attributed to non-compliance alone. PMID:20153859

  8. Anucleate platelets generate progeny

    PubMed Central

    Schwertz, Hansjörg; Köster, Sarah; Kahr, Walter H. A.; Michetti, Noemi; Kraemer, Bjoern F.; Weitz, David A.; Blaylock, Robert C.; Kraiss, Larry W.; Greinacher, Andreas; Zimmerman, Guy A.

    2010-01-01

    Platelets are classified as terminally differentiated cells that are incapable of cellular division. However, we observe that anucleate human platelets, either maintained in suspension culture or captured in microdrops, give rise to new cell bodies packed with respiring mitochondria and α-granules. Platelet progeny formation also occurs in whole blood cultures. Newly formed platelets are structurally indistinguishable from normal platelets, are able to adhere and spread on extracellular matrix, and display normal signal-dependent expression of surface P-selectin and annexin V. Platelet progeny formation is accompanied by increases in biomass, cellular protein levels, and protein synthesis in expanding populations. Platelet numbers also increase during ex vivo storage. These observations indicate that platelets have a previously unrecognized capacity for producing functional progeny, which involves a form of cell division that does not require a nucleus. Because this new function of platelets occurs outside of the bone marrow milieu, it raises the possibility that thrombopoiesis continues in the bloodstream. PMID:20086251

  9. Luminal platelet aggregates in functional deficits in parenchymal vessels after subarachnoid hemorrhage

    PubMed Central

    Friedrich, Victor; Flores, Rowena; Muller, Artur; Sehba, Fatima A.

    2010-01-01

    The pathophysiology of early ischemic injury after aneurysmal subarachnoid hemorrhage (SAH) is not understood. This study examined the acute effect of endovascular puncture-induced SAH on parenchymal vessel function in rat, using intravascular fluorescent tracers to assess flow and vascular permeability and immunostaining to assess structural integrity and to visualize platelet aggregates. In sham-operated animals, vessels were well filled with tracer administered 10 seconds before sacrifice, and parenchymal escape of tracer was rare. At ten minutes and 3 hours after hemorrhage, patches of poor vascular filling were distributed throughout the forebrain. Close examination of these regions revealed short segments of narrowed diameter along many profiles. Most vascular profiles with reduced perfusion contained platelet aggregates and in addition showed focal loss of collagen IV, a principal component of basal lamina. In contrast, vessels were well filled at 24 hours post-hemorrhage, indicating that vascular perfusion had recovered. Parenchymal escape of intravascular tracer was detected at 10 minutes post-hemorrhage and later as plumes of fluorescence emanating into parenchyma from restricted microvascular foci. These data demonstrate that parenchymal microvessels are compromised in function by 10 minutes after SAH and identify focal microvascular constriction and local accumulation of luminal platelet aggregates as potential initiators of that compromise. PMID:20654597

  10. Can the Platelet Function Analyzer (PFA)-100 test substitute for the template bleeding time in routine clinical practice?

    PubMed

    Francis, J; Francis, D; Larson, L; Helms, E; Garcia, M

    1999-01-01

    The bleeding time (BT) is widely used in clinical medicine as a screening test of platelet function, although its deficiencies in such a role are well recognized. The Platelet Function Analyzer (PFA)-100 measures the ability of platelets activated in a high-shear environment to occlude an aperture in a membrane treated with collagen and epinephrine (CEPI) or collagen and ADP (CADP). The time taken for flow across the membrane to stop (closure time) is recorded. This study compared the PFA-100 with the BT as a screening test of platelet dysfunction in 113 hospital inpatients. The PFA-100 test was performed initially using the CEPI cartridge; CADP tests were performed on those with abnormal (> 163 s) CEPI closure times. Whole blood platelet aggregation studies and chart review were performed on patients in whom the BT and PFA-100 results did not agree.Abnormal bleeding times and PFA-100 results were obtained in 20.4% and 35.4% of patients, respectively. The results of BT and PFA-100 agreed in 74.3% of patients. Of the 29 patients in whom the BT and PFA-100 results were discordant, whole blood platelet aggregation studies supported the PFA-100 result in 25 (86.2%). The PFA-100 was more sensitive to aspirin-induced platelet dysfunction and was more rapidly and cheaply performed than the BT. Since the PFA-100 test reflects platelet function better than the BT, we conclude that this test could replace the BT as a first-line screening test for platelet dysfunction in clinical practice.

  11. Can the Platelet Function Analyzer (PFA)-100 test substitute for the template bleeding time in routine clinical practice?

    PubMed

    Francis, J; Francis, D; Larson, L; Helms, E; Garcia, M

    1999-01-01

    The bleeding time (BT) is widely used in clinical medicine as a screening test of platelet function, although its deficiencies in such a role are well recognized. The Platelet Function Analyzer (PFA)-100 measures the ability of platelets activated in a high-shear environment to occlude an aperture in a membrane treated with collagen and epinephrine (CEPI) or collagen and ADP (CADP). The time taken for flow across the membrane to stop (closure time) is recorded. This study compared the PFA-100 with the BT as a screening test of platelet dysfunction in 113 hospital inpatients. The PFA-100 test was performed initially using the CEPI cartridge; CADP tests were performed on those with abnormal (> 163 s) CEPI closure times. Whole blood platelet aggregation studies and chart review were performed on patients in whom the BT and PFA-100 results did not agree.Abnormal bleeding times and PFA-100 results were obtained in 20.4% and 35.4% of patients, respectively. The results of BT and PFA-100 agreed in 74.3% of patients. Of the 29 patients in whom the BT and PFA-100 results were discordant, whole blood platelet aggregation studies supported the PFA-100 result in 25 (86.2%). The PFA-100 was more sensitive to aspirin-induced platelet dysfunction and was more rapidly and cheaply performed than the BT. Since the PFA-100 test reflects platelet function better than the BT, we conclude that this test could replace the BT as a first-line screening test for platelet dysfunction in clinical practice. PMID:16801082

  12. Effect of troxerutin on laser-induced thrombus formation in rat mesenteric vessels, coagulation parameters and platelet function.

    PubMed

    Krupiński, K; Giedrojć, J; Bielawiec, M

    1996-01-01

    The antithrombotic effect of Troxerutin have been studied in an experimental model of thrombosis in which rat mesenteric vessels (arterioles and venules) 25-30 microns in diameter were injured by well defined argon laser lesions. Furthermore in vitro effect of this agent on coagulation parameters (IIa, Xa inhibition, TT, heptest), and platelet function (platelet adhesion to the siliconised glass and extracellular matrix, platelet spreading) has been investigated 2 h after oral drug administration. Troxerutin at a dose of 10 mg/kg markedly inhibited thrombus formation in venules. Higher dose (50 mg/kg) was needed to obtain the same antithrombotic effect when arterioles were studied. After application of a single dose of Troxerutin (100 mg/kg) antithrombotic effect lasted for 6 h to 7.5 h when venules were studied, and for 4.5 h to 6 h when arterioles were investigated. In in vitro study we did not observe any effect of Troxerutin on coagulation parameters. In concentrations of 100 micrograms/ml in platelet rich plasma Troxerutin significantly inhibited platelet adhesion to the extracellular matrix and siliconised glass as well as platelet spreading. It is likely that this drug possesses antithrombotic effect evaluated by inhibition of platelet function and protection of endothelial cells.

  13. The Hydraulic Permeability of Blood Clots as a Function of Fibrin and Platelet Density

    PubMed Central

    Wufsus, A.R.; Macera, N.E.; Neeves, K.B.

    2013-01-01

    Interstitial fluid flow within blood clots is a biophysical mechanism that regulates clot growth and dissolution. Assuming that a clot can be modeled as a porous medium, the physical property that dictates interstitial fluid flow is the hydraulic permeability. The objective of this study was to bound the possible values of the hydraulic permeability in clots formed in vivo and present relationships that can be used to estimate clot permeability as a function of composition. A series of clots with known densities of fibrin and platelets, the two major components of a clot, were formed under static conditions. The permeability was calculated by measuring the interstitial fluid velocity through the clots at a constant pressure gradient. Fibrin gels formed with a fiber volume fraction of 0.02–0.54 had permeabilities of 1.2 × 10−1–1.5 × 10−4μm2. Platelet-rich clots with a platelet volume fraction of 0.01–0.61 and a fibrin volume fraction of 0.03 had permeabilities over a range of 1.1 × 10−2–1.5 × 10−5μm2. The permeability of fibrin gels and of clots with platelet volume fraction of <0.2 were modeled as an array of disordered cylinders with uniform diameters. Clots with a platelet volume fraction of >0.2 were modeled as a Brinkman medium of coarse solids (platelets) embedded in a mesh of fine fibers (fibrin). Our data suggest that the permeability of clots formed in vivo can vary by up to five orders of magnitude, with pore sizes that range from 4 to 350 nm. These findings have important implications for the transport of coagulation zymogens/enzymes in the interstitial spaces during clot formation, as well as the design of fibrinolytic drug delivery strategies. PMID:23601328

  14. Platelet function: aggregation by PAF or sequestration in lung is not modified during immediate or late allergen-induced bronchospasm in man.

    PubMed

    Hemmendinger, S; Pauli, G; Tenabene, A; Pujol, J L; Bessot, J C; Eber, M; Cazenave, J P

    1989-05-01

    Among the mediators involved in the pathophysiologic mechanisms that underly the reactions of the acute and delayed phases of bronchospasm induced by allergens in man, platelet-activating factor (PAF) could play an important role, in particular by its effects on platelets. In animals, inhalation or injection of PAF causes a platelet-dependent bronchoconstriction that is blocked by prior administration of an antiplatelet antiserum and accompanied by platelet accumulation in the pulmonary vessels. In man, inhalation of PAF causes a bronchospasm and induces a bronchial hyperreactivity. Abnormalities of platelet aggregation and the secretion into plasma of platelet factor 4 and beta-thromboglobulin have been described in patients with asthma during induced bronchospasm. Platelet functions have been studied in 15 patients with asthma before and after allergen bronchial provocation tests. There was no difference between platelet counts, plasma concentrations of platelet factor 4 and beta-thromboglobulin, and platelet aggregation induced by several agonists (adrenaline, arachidonic acid, or PAF) before and immediately after the allergen bronchial provocation test. There was no platelet pulmonary sequestration as studied with 111Indium-labeled platelets during 24 hours after the antigen challenge, and the life span of circulating platelets was normal. Our results do not support an important direct role for PAF in the pathophysiology of asthma. It is still possible that the current methodology is too insensitive to detect amounts of PAF in the circulation or that PAF is acting locally. PMID:2523922

  15. Heat shock protein 70 regulates platelet integrin activation, granule secretion and aggregation.

    PubMed

    Rigg, Rachel A; Healy, Laura D; Nowak, Marie S; Mallet, Jérémy; Thierheimer, Marisa L D; Pang, Jiaqing; McCarty, Owen J T; Aslan, Joseph E

    2016-04-01

    Molecular chaperones that support protein quality control, including heat shock protein 70 (Hsp70), participate in diverse aspects of cellular and physiological function. Recent studies have reported roles for specific chaperone activities in blood platelets in maintaining hemostasis; however, the functions of Hsp70 in platelet physiology remain uninvestigated. Here we characterize roles for Hsp70 activity in platelet activation and function. In vitro biochemical, microscopy, flow cytometry, and aggregometry assays of platelet function, as well as ex vivo analyses of platelet aggregate formation in whole blood under shear, were carried out under Hsp70-inhibited conditions. Inhibition of platelet Hsp70 blocked platelet aggregation and granule secretion in response to collagen-related peptide (CRP), which engages the immunoreceptor tyrosine-based activation motif-bearing collagen receptor glycoprotein VI (GPVI)-Fc receptor-γ chain complex. Hsp70 inhibition also reduced platelet integrin-αIIbβ3 activation downstream of GPVI, as Hsp70-inhibited platelets showed reduced PAC-1 and fibrinogen binding. Ex vivo, pharmacological inhibition of Hsp70 in human whole blood prevented the formation of platelet aggregates on collagen under shear. Biochemical studies supported a role for Hsp70 in maintaining the assembly of the linker for activation of T cells signalosome, which couples GPVI-initiated signaling to integrin activation, secretion, and platelet function. Together, our results suggest that Hsp70 regulates platelet activation and function by supporting linker for activation of T cells-associated signaling events downstream of platelet GPVI engagement, suggesting a role for Hsp70 in the intracellular organization of signaling systems that mediate platelet secretion, "inside-out" activation of platelet integrin-αIIbβ3, platelet-platelet aggregation, and, ultimately, hemostatic plug and thrombus formation.

  16. Different protein expression in normal and dysfunctional platelets from uremic patients.

    PubMed

    Marques, María; Sacristán, Daniel; Mateos-Cáceres, Petra J; Herrero, José; Arribas, María J; González-Armengol, Juan J; Villegas, Ana; Macaya, Carlos; Barrientos, Alberto; López-Farré, Antonio J

    2010-01-01

    Although many uremic patients show platelet dysfunctionality, there are others with normal platelet functionality and even with thrombotic tendencies. Our aim was to evaluate changes in the expression of proteins in functional and dysfunctional uremic platelets. Using the platelet function analyzer (PFA-100) assay, uremic patients were divided according to their platelet functionality into normal (n=7) and dysfunctional (n=8). There were no significant differences in the number of circulating platelets and hematocrit and hemoglobin levels. Two-dimensional electrophoresis and mass spectrometry were used to determine and identify changes in protein expression. The closure time (CT) in the PFA-100 assay was significantly prolonged in the dysfunctional uremic platelets. In the dysfunctional platelets, actin-interacting protein-1 isotype 1 was down-regulated, while integrin IIb was up-regulated. Glutathione-S-transferase isotypes 1 and 2 and peroxiredoxin VI were up-regulated in the dysfunctional platelets. Pearson analysis showed a negative correlation between the platelet expression of integrin IIb and creatinine clearance. A positive correlation was found between creatinine clearance and glutathione-S-transferase isotype 2. Serum uric acid concentration was positively correlated with CT values and glutathione-S-transferase isotype 1. In conclusion, the analysis of the protein expression in uremic platelets with normal and dysfunctional activity revealed differences which may occur at the megakaryocyte level.

  17. Factor VIIa analog has marked effects on platelet function and clot kinetics in blood from patients with hemophilia A.

    PubMed

    Brophy, Donald F; Martin, Erika J; Nolte, Melinda E; Kuhn, Janice G; Barrett, J Christian; Ezban, Mirella

    2010-09-01

    To evaluate the hemostatic effects of NN1731 and rFVIIa, an ex-vivo study in hemophilia patients used the Hemodyne Hemostasis Analysis System (HAS) to measure platelet contractile force (PCF), clot elastic modulus (CEM), and force onset time (FOT), and the Haemoscope Thrombelastograph (TEG) to measure reaction time (R), kinetics time (K), and maximum amplitude (MA). Blood samples from 10 healthy volunteers and 10 Factor VIII-deficient patients of varying severity (mild, moderate, severe), were spiked with rFVIIa and NN1731 (both 0.64 and 1.28 microg/ml, respectively) and analyzed to characterize platelet function and clot kinetics. There was wide variability in the rFVIIa response. NN1731 had greater and more consistent effects on PCF, CEM, FOT, R, and K relative to rFVIIa, in all hemophilia groups. The lowest NN1731 concentration (0.64 microg/ml) shortened R and FOT, and increased CEM and PCF more than rFVIIa 1.28 microg/ml. NN1731 normalized clotting parameters equivalent to values obtained in healthy volunteers. FOT and R were highly correlated (r = 0.96). No correlation was observed between CEM and MA. NN1731 produced less variable, more pronounced and predictable ex-vivo hemostatic effects on PCF, CEM, FOT, R and K than rFVIIa in all hemophilia groups. HAS and TEG assays provided similar estimates of FOT and R, however CEM appeared to be more sensitive than MA to changes in clot firmness.

  18. Examining Endothelial Function and Platelet Reactivity in Patients with Depression before and after SSRI Therapy.

    PubMed

    Dawood, Tye; Barton, David A; Lambert, Elisabeth A; Eikelis, Nina; Lambert, Gavin W

    2016-01-01

    Although it is recognized that patients with major depressive disorder (MDD) are at increased risk of developing cardiovascular disease (CVD) the mechanisms responsible remain unknown. Endothelial dysfunction is one of the first signs of CVD. Using two techniques, flow-mediated dilatation in response to reactive hyperemia and laser Doppler velocimetry with iontophoresis, we examined endothelial function in the forearm before and after serotonin-specific reuptake inhibitor (SSRI) treatment in 31 patients with MDD. Measurement of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, soluble P-selectin, and noradrenaline in plasma was also performed. Prior to treatment, markers of endothelial and vascular function and platelet reactivity were within the normal range. Following SSRI therapy (95 ± 5 days) symptoms of depression were reduced (paired difference between pre- and post-treatment Hamilton rating -18 ± 1, P < 0.001) with 19 patients recovered and 4 remitted. There occurred no significant change in markers of endothelial or vascular function following SSRI therapy. The improvement in Hamilton depression rating in response to therapy could be independently predicted by the baseline arterial plasma noradrenaline concentration (r (2) = 0.36, P = 0.003). In this cohort of patients with MDD, SSRI therapy did not influence endothelial function or markers of vascular or platelet reactivity. Patient response to SSRI therapy could be predicted by the initial circulating level of noradrenaline, with noradrenaline levels being lower in responders. PMID:26924994

  19. Examining Endothelial Function and Platelet Reactivity in Patients with Depression before and after SSRI Therapy

    PubMed Central

    Dawood, Tye; Barton, David A.; Lambert, Elisabeth A.; Eikelis, Nina; Lambert, Gavin W.

    2016-01-01

    Although it is recognized that patients with major depressive disorder (MDD) are at increased risk of developing cardiovascular disease (CVD) the mechanisms responsible remain unknown. Endothelial dysfunction is one of the first signs of CVD. Using two techniques, flow-mediated dilatation in response to reactive hyperemia and laser Doppler velocimetry with iontophoresis, we examined endothelial function in the forearm before and after serotonin-specific reuptake inhibitor (SSRI) treatment in 31 patients with MDD. Measurement of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, soluble P-selectin, and noradrenaline in plasma was also performed. Prior to treatment, markers of endothelial and vascular function and platelet reactivity were within the normal range. Following SSRI therapy (95 ± 5 days) symptoms of depression were reduced (paired difference between pre- and post-treatment Hamilton rating −18 ± 1, P < 0.001) with 19 patients recovered and 4 remitted. There occurred no significant change in markers of endothelial or vascular function following SSRI therapy. The improvement in Hamilton depression rating in response to therapy could be independently predicted by the baseline arterial plasma noradrenaline concentration (r2 = 0.36, P = 0.003). In this cohort of patients with MDD, SSRI therapy did not influence endothelial function or markers of vascular or platelet reactivity. Patient response to SSRI therapy could be predicted by the initial circulating level of noradrenaline, with noradrenaline levels being lower in responders. PMID:26924994

  20. Sulphate conjugation of p-hydroxytriamterene by platelet phenol sulphotransferase: assay conditions and correlation with metabolism in man.

    PubMed Central

    Reiter, C; Werness, P G; Van Loon, J; Smith, L H; Weinshilboum, R M

    1983-01-01

    1 Sulphate conjugation catalyzed by phenol sulphotransferase (PST) is an important pathway in the metabolism of many drugs including triamterene. Variations in PST activity in an easily obtained tissue such as the platelet might reflect individual differences in the sulphate conjugation in other organs and tissues. Human platelets contain at least two forms of PST, a thermolabile (TL) form for which dopamine is a substrate and a thermostable (TS) form for which low concentrations of p-nitrophenol serve as a substrate. 2 p-OH-triamterene, the major metabolite of triamterene, is conjugated with sulphate in vivo. p-OH-triamterene was a substrate for platelet PST with an apparent Michaelis-Menten value of 26 microM. Thermal stability studies indicated that p-OH-triamterene was a substrate for only the TS form of platelet PST. 3 When platelet homogenates from 29 individual subjects were tested, there was a significant correlation between PST activities measured with 4 microM p-nitrophenol and with p-OH-triamterene (r = 0.985, P less than 0.0001) but not between activities measured with dopamine and with p-OH-triamterene (r = 0.023, P greater than 0.2). These results confirmed that p-OH-triamterene was a substrate for only the TS form of human platelet PST. 4 The same 29 subjects were treated with 1 mg/kg of triamterene orally. 24-h urinary excretions of triamterene, p-OH-triamterene and p-OH-triamterene sulphate averaged 15.3%, 6.3% and 78.4%, respectively, of the total of triamterene plus measured metabolites excreted. The excretion of triamterene plus the two metabolites averaged 43.1 +/- 2.6% (mean +/- s.e. mean) of the ingested dose. There was not a significant correlation between the proportion of p-OH-triamterene excreted as sulphate conjugate and the activities of either the TS or TL forms of platelet PST activity. PMID:6573903

  1. Structural functional and folding scenario of an anti platelet and thrombolytic enzyme crinumin.

    PubMed

    Singh, Kunwar Awaneesh; Singh, Sanjay; Jagannadham, Medicherla V

    2014-07-01

    A folding pattern, conformational stability and therapeutic role of a protein helps in developing a suitable drug. Crinumin, a thrombolytic and anti platelet agent, has been studied for its functional and conformational properties by equilibrium unfolding methods. The crinumin belongs to α+β class of protein and exhibits a non native structure and two molten globule states at different conditions. Two domains in the molecular structure of the protein with altered stability are present that unfold sequentially. The enzyme maintains activity as well as structural integrity even in adverse conditions. These observations provide an understanding of protein folding as well as facilitate the development of a potential drug. PMID:24726528

  2. Platelet antibody screening by flow cytometry is more sensitive than solid phase red cell adherence assay and lymphocytotoxicity technique: a comparative study in Thai patients.

    PubMed

    Buakaew, Jarin; Promwong, Charuporn

    2010-01-01

    The objective of this study was to compare the sensitivity and specificity of lymphocytotoxicity test (LCT), solid phase red cell adherence assay (SPRCA) and flow cytometry in detecting platelet reactive antibodies against human leukocyte antigens (HLA) class I and human platelet antigens (HPA). Sera from 38 thrombocytopenic patients and 5 mothers of thrombocytopenic newborns were screened for platelet reactive antibodies by these three methods using screening platelets and/or lymphocytes panels derived from six subjects. The sensitivity and specificity of each method and levels of agreement were analysed. HLA antibodies were found in 18, 17 and 19 out of 43 patients' sera tested by LCT, SPRCA and flow cytometry, respectively. Four out of 43 patients' sera were reactive against HPA by flow cytometry, but were reactive to only 2 sera by SPRCA. Using flow cytometry as the reference method, the sensitivities/specificities of SPRCA and LCT in HLA antibody detection were 84.21/95.83% and 94.73/100%, respectively, with a good strength of agreement. SPRCA had 50% sensitivity and 100% specificity in HPA antibody detection compare to flow cytometry. Flow cytometry appeared to be the most sensitive technique compared with SPRCA and LCT for both HPA and HLA antibody screening. SPRCA sensitivity was too low for HPA antibody detection, but this might be because of the small number of samples. There was one serum from the mother of a baby suffering neonatal alloimmune thrombocytopenia (NAIT), in whom SPRCA could not detect HPA antibodies, while flow cytometry came out positive. Therefore, SPRCA should not be used in NAIT investigation and flow cytometry should be employed instead.

  3. Helenalin and 11 alpha,13-dihydrohelenalin, two constituents from Arnica montana L., inhibit human platelet function via thiol-dependent pathways.

    PubMed

    Schröder, H; Lösche, W; Strobach, H; Leven, W; Willuhn, G; Till, U; Schrör, K

    1990-03-15

    This study investigates the effect on human platelet function of two sesquiterpene lactones from Arnica montana L., helenalin (H) and 11 alpha,13-dihydrohelenalin (DH). Both compounds inhibited collagen-induced platelet aggregation, thromboxane formation and 5-hydroxytryptamine secretion in a concentration-dependent manner at 3-300 microM. When arachidonic acid was used as stimulus, thromboxane formation remained unaffected despite of inhibition of platelet aggregation. Both H and DH reduced the number of acid-soluble sulfhydryl groups in platelets, by up to 78% at anti-aggregatory concentrations. Moreover, H- and DH-induced platelet inhibition could be prevented by the thiol containing amino acid cysteine. It is concluded that H and DH inhibit platelet function via interaction with platelet sulfhydryl groups, probably associated with reduced phospholipase A2 activity.

  4. Dose-dependent platelet stimulation and inhibition induced by anti-PIA1 IgG

    SciTech Connect

    Ryu, T.; Davis, J.M.; Schwartz, K.A. )

    1990-07-01

    The PIA1 antibody produces several clinically distinct and severe thrombocytopenias. Investigations have demonstrated divergent effects on platelet function; prior reports demonstrated inhibition, while a conflicting publication showed platelet activation. We have resolved this conflict using anti-PIA1 IgG produced by a patient with posttransfusion purpura. Relatively low concentrations stimulated platelet aggregation and release of adenosine triphosphate (ATP) whereas high concentrations inhibited platelet function, producing a thrombasthenia-like state. The number of molecules of platelet-associated IgG necessary to initiate aggregation and ATP release (2,086 +/- 556) or produce maximum aggregation (23,420 +/- 3,706) or complete inhibition (63,582 +/- 2654) were measured with a quantitative radiometric assay for bound anti-PIA1. Preincubation of platelets with high concentrations of PIA1 antibody inhibited platelet aggregation with 10 mumol/L adenosine diphosphate and blocked 125I-labeled fibrinogen platelet binding. Platelet activation with nonfibrinogen dependent agonist, 1 U/ml thrombin, was not inhibited by this high concentration of PIA1 IgG. In conclusion, anti-PIAI IgG produces (1) stimulation of platelet aggregation and ATP release that is initiated with 2000 molecules IgG per platelet and is associated with an increase of 125I-fibrinogen binding; (2) conversely, inhibition of platelet aggregation is observed with maximum antibody binding, 63,000 molecules IgG per platelet, and is mediated via a blockade of fibrinogen binding.

  5. The importance of platelets in the expression of monocyte tissue factor antigen measured by a new whole blood flow cytometric assay.

    PubMed

    Amirkhosravi, A; Alexander, M; May, K; Francis, D A; Warnes, G; Biggerstaff, J; Francis, J L

    1996-01-01

    Previous methods for the determination of monocyte tissue factor (TF) have been technically complex, difficult to standardize, prone to spuriously elevated results and difficult to implement in a clinical laboratory environment. We report the development of a two-color whole blood cytometric technique that overcomes many of these disadvantages. The assay uses small volumes of citrated blood (1.0 ml), can be performed in under one hour (if endotoxin stimulation is not performed), is reproducible (CV = 5%) and uses methodology commonly available in clinical laboratories. Baseline (mean +/- SD) expression of monocyte TF in normal subjects was very low (1.1 +/- 0.95%, Mean Fluorescence [Mean FL] 0.20 +/- 0.01) making relatively small increases easy to detect. Monocyte TF expression following endotoxin (LPS) stimulation for 1 h was 34.6 +/- 11.2% (Mean FL 0.32 +/- 0.04). LPS-stimulated activity varied between subjects (21-68%) but was remarkably consistent for individual subjects (CV = 5.4%). Stimulated monocyte TF expression was directly proportional to the platelet count and was reduced by platelet protective anticoagulants and by ingestion of aspirin. Non LPS-stimulated monocyte TF was markedly increased, in a dose-dependent manner, by adding collagen to whole blood. This was apparently associated with platelet-monocyte binding and could be abolished by anti-P-Selectin. We conclude that the whole blood flow cytometric assay of monocyte TF may be a valuable tool for clinical use and a useful model system for evaluating the humoral and cellular factors governing monocyte TF expression in a natural environment.

  6. The importance of platelets in the expression of monocyte tissue factor antigen measured by a new whole blood flow cytometric assay.

    PubMed

    Amirkhosravi, A; Alexander, M; May, K; Francis, D A; Warnes, G; Biggerstaff, J; Francis, J L

    1996-01-01

    Previous methods for the determination of monocyte tissue factor (TF) have been technically complex, difficult to standardize, prone to spuriously elevated results and difficult to implement in a clinical laboratory environment. We report the development of a two-color whole blood cytometric technique that overcomes many of these disadvantages. The assay uses small volumes of citrated blood (1.0 ml), can be performed in under one hour (if endotoxin stimulation is not performed), is reproducible (CV = 5%) and uses methodology commonly available in clinical laboratories. Baseline (mean +/- SD) expression of monocyte TF in normal subjects was very low (1.1 +/- 0.95%, Mean Fluorescence [Mean FL] 0.20 +/- 0.01) making relatively small increases easy to detect. Monocyte TF expression following endotoxin (LPS) stimulation for 1 h was 34.6 +/- 11.2% (Mean FL 0.32 +/- 0.04). LPS-stimulated activity varied between subjects (21-68%) but was remarkably consistent for individual subjects (CV = 5.4%). Stimulated monocyte TF expression was directly proportional to the platelet count and was reduced by platelet protective anticoagulants and by ingestion of aspirin. Non LPS-stimulated monocyte TF was markedly increased, in a dose-dependent manner, by adding collagen to whole blood. This was apparently associated with platelet-monocyte binding and could be abolished by anti-P-Selectin. We conclude that the whole blood flow cytometric assay of monocyte TF may be a valuable tool for clinical use and a useful model system for evaluating the humoral and cellular factors governing monocyte TF expression in a natural environment. PMID:8713785

  7. An association of platelet indices with blood pressure in Beijing adults: Applying quadratic inference function for a longitudinal study.

    PubMed

    Yang, Kun; Tao, Lixin; Mahara, Gehendra; Yan, Yan; Cao, Kai; Liu, Xiangtong; Chen, Sipeng; Xu, Qin; Liu, Long; Wang, Chao; Huang, Fangfang; Zhang, Jie; Yan, Aoshuang; Ping, Zhao; Guo, Xiuhua

    2016-09-01

    The quadratic inference function (QIF) method becomes more acceptable for correlated data because of its advantages over generalized estimating equations (GEE). This study aimed to evaluate the relationship between platelet indices and blood pressure using QIF method, which has not been studied extensively in real data settings.A population-based longitudinal study was conducted in Beijing from 2007 to 2012, and the median of follow-up was 6 years. A total of 6515 cases, who were aged between 20 and 65 years at baseline and underwent routine physical examinations every year from 3 Beijing hospitals were enrolled to explore the association between platelet indices and blood pressure by QIF method. The original continuous platelet indices were categorized into 4 levels (Q1-Q4) using the 3 quartiles of P25, P50, and P75 as a critical value. GEE was performed to make a comparison with QIF.After adjusting for age, usage of drugs, and other confounding factors, mean platelet volume was negatively associated with diastolic blood pressure (DBP) (Equation is included in full-text article.)in males and positively linked with systolic blood pressure (SBP) (Equation is included in full-text article.). Platelet distribution width was negatively associated with SBP (Equation is included in full-text article.). Blood platelet count was associated with DBP (Equation is included in full-text article.)in males.Adults in Beijing with prolonged exposure to extreme value of platelet indices have elevated risk for future hypertension and evidence suggesting using some platelet indices for early diagnosis of high blood pressure was provided. PMID:27684843

  8. Rhesus monkey platelets

    SciTech Connect

    Harbury, C.B.

    1986-03-01

    The purpose of this abstract is to describe the adenine nucleotide metabolism of Rhesus monkey platelets. Nucleotides are labelled with /sup 14/C-adenine and extracted with EDTA-ethanol (EE) and perchlorate (P). Total platelet ATP and ADP (TATP, TADP) is measured in the Holmsen Luciferase assay, and expressed in nanomoles/10/sup 8/ platelets. TR=TATP/TADP. Human platelets release 70% of their TADP, with a ratio of released ATP/ADP of 0.7. Rhesus platelets release 82% of their TADP, with a ratio of released ATP/ADP of 0.33. Thus, monkey platelets contain more ADP than human platelets. Thin layer chromatography of EE gives a metabolic ratio of 11 in human platelets and 10.5 in monkey platelets. Perchlorate extracts metabolic and actin bound ADP. The human and monkey platelets ratios were 5, indicating they contain the same proportion of actin. Thus, the extra ADP contained in monkey platelets is located in the secretory granules.

  9. Scalable Functionalized Graphene Nano-platelets as Tunable Cathodes for High-performance Lithium Rechargeable Batteries

    PubMed Central

    Kim, Haegyeom; Lim, Hee-Dae; Kim, Sung-Wook; Hong, Jihyun; Seo, Dong-Hwa; Kim, Dae-chul; Jeon, Seokwoo; Park, Sungjin; Kang, Kisuk

    2013-01-01

    High-performance and cost-effective rechargeable batteries are key to the success of electric vehicles and large-scale energy storage systems. Extensive research has focused on the development of (i) new high-energy electrodes that can store more lithium or (ii) high-power nano-structured electrodes hybridized with carbonaceous materials. However, the current status of lithium batteries based on redox reactions of heavy transition metals still remains far below the demands required for the proposed applications. Herein, we present a novel approach using tunable functional groups on graphene nano-platelets as redox centers. The electrode can deliver high capacity of ~250 mAh g−1, power of ~20 kW kg−1 in an acceptable cathode voltage range, and provide excellent cyclability up to thousands of repeated charge/discharge cycles. The simple, mass-scalable synthetic route for the functionalized graphene nano-platelets proposed in this work suggests that the graphene cathode can be a promising new class of electrode. PMID:23514953

  10. Platelet function and fibrinolytic activity in hypertensive and normotensive sleep apnea patients.

    PubMed

    Rångemark, C; Hedner, J A; Carlson, J T; Gleerup, G; Winther, K

    1995-04-01

    Platelet function and fibrinolytic activity was studied during rest and after ergometric exercise in 13 hypertensive or normotensive patients with obstructive sleep apnea (OSA) and in 10 sex- and weight-matched controls. All patients had undergone a complete polysomnography for the diagnosis of OSA. The controls did not undergo any sleep investigation but had no history of snoring or witnessed apneas during sleep. On antihypertensive drug wash-out, two of the patients were normotensive, whereas 11 had mild to moderate hypertension. Platelet aggregation measured by adenosine 5'-diphosphate- or adrenaline-induced aggregation, platelet factor-4 or beta-thromboglobulin did not differ between patients and controls. During exercise beta-thromboglobulin decreased significantly in both OSA patients and controls. Plasma tissue plasminogen activator activity was similar in OSA patients and controls and increased significantly in both groups after exercise. Plasminogen activator inhibitor type 1 (PAI-1) was 18.4 +/- 3.6 IU/ml in OSA patients compared with 8.2 +/- 1.7 IU/ml in controls (p < 0.029) during rest, indicating decreased fibrinolytic activity. The difference between groups remained after exercise (p < 0.017). Blood pressure elevation was more common and body mass index (BMI) was higher in patients with OSA, but there was no direct relation between blood pressure level or BMI and PAI-1. Nevertheless, differences between groups were smaller when blood pressure and obesity were accounted for. It is concluded that patients with OSA may exhibit decreased fibrinolytic activity. Low fibrinolytic activity may represent a confounding pathophysiological mechanism behind the high incidence of myocardial infarction and stroke in patients with OSA.

  11. Platelet function and fibrinolytic activity in hypertension: differential effects of calcium antagonists and beta-adrenergic receptor blockers.

    PubMed

    Winther, K; Gleerup, G; Hedner, T

    1991-01-01

    Platelet function was investigated in healthy volunteers and patients with essential hypertension by measurement of thresholds for ADP and adrenaline-induced aggregation and plasma concentrations of platelet factor 4 (PF-4) and beta-thromboglobulin (beta-TG) after administration of antihypertensive drugs. Fibrinolytic activity was investigated by the euglobulin clot lysis time (ECLT) and tissue plasminogen activator (t-PA) activity. Compared to normotensive controls, patients with essential hypertension showed increased aggregation as evidenced by a decrease in ADP thresholds for ex vivo platelet aggregation. ECLT was significantly prolonged and t-PA significantly lowered, indicating impaired fibrinolytic activity in mild hypertension. In different studies, we have shown that various antihypertensive drug regimens differ in their effects on platelet function and fibrinolytic activity when given to healthy volunteers or patients with mild-to-moderate essential hypertension. In normal volunteers, treatment with the calcium antagonists verapamil, nifedipine, and felodipine lowered plasma concentrations of PF-4 and beta-TG, indicating a reduced platelet activity in vivo. Fibrinolytic activity was not influenced by calcium antagonist treatment in the normal volunteers. Interestingly, however, t-PA increased significantly in the hypertensive group. When compared to placebo or beta 1-selective blockers, propranolol, a non-selective beta-adrenergic blocker without partial agonist activity, reduced ADP and adrenaline threshold values for ex vivo platelet aggregation in hypertensive subjects and impaired fibrinolytic activity in the normal volunteers as well as in the hypertensive groups by increasing ECLT and reducing t-PA. Hypothetically, the effects of antihypertensive drugs on platelet function and fibrinolytic activity could be of importance for their proposed actions on cardiovascular morbidity and mortality.

  12. The role of peroxides as modulators of human blood platelet function

    SciTech Connect

    Jesse, R.L.; Hess, M.L. )

    1990-02-26

    The ability to peroxidize lipid in the vicinity of arterial walls has been demonstrated through oxidation of the LDL-particle by monocytes/macrophages, smooth muscle cells, and endothelial cells. The authors questioned what effect increased levels of peroxides might have on platelet function. Platelet aggregation, studied trubidometrically, was initiated by arachidonic acid (AA) alone or in the presence of a low concentration of H{sub 2}O{sub 2}, (which by itself could not initiate aggregation). Aggregation was analyzed quantitatively by measuring the time it took to reach 1/2 the maximal extent of aggregation (T1/2Max). Using the minimal concentration of AA still able to initiate aggregation (0.2-0.45 mM) the T1/2Max was 112{+-}35 (n=10). With 50% of the respective AA conc. plus 17 {mu}M H{sub 2}O{sub 2}, the T1/2Max was 52{+-}21. Under similar circumstances, the amount of AA able to initiate aggregation could be reduced by 80% with the addition of H{sub 2}O{sub 2}. These effects could be duplicated by glucose oxidase, 0.015-0.15 IU, and could be eliminated by the addition of catalase (<25 U/ml) at any time prior to the start of aggregation. With non-aggregating concentrations of AA alone, synthesis of Thromboxane B{sub 2} was negligible; with aggregation by the same AA plus H{sub 2}O{sub 2} it was 550{+-}79 pg/10{sup 3} platelets. Both aggregation and TxB{sub 2} synthesis were completely inhibited by aspirin or indomethacin, and by the antioxidants phenol and nor-dihydroguaretic acid, at concentrations known to inhibit cyclooxygenase. In platelets with a defect in the second wave of ADP induced aggregation, but having normal aggregation with added AA (indicating the cyclooxygenase-thromboxane axis was intact), sub-aggregating concentrations of AA, plus H{sub 2}O{sub 2} resulted in brisk aggregation.

  13. Platelet interactions with titanium: modulation of platelet activity by surface topography.

    PubMed

    Park, J Y; Gemmell, C H; Davies, J E

    2001-10-01

    Endosseous implants initially come into contact with blood. Thus, the nature of the interactions between blood and implanted endosseous implants may influence subsequent bone healing events in the peri-implant healing compartment. We conducted studies to address the following question: Does implant surface microtexture modulate platelet activity? We used commercially pure Ti (cpTi) disks with four different surface finishes: dual acid-etched (DAE), 320 grit (320G) abraded, machined, and p1200 polished cpTi. Surfaces were characterized by scanning electron microscopy (SEM) and optical profilometry. The DAE and 320G surfaces presented more complex microtextures than the machined or polished surfaces. Platelet activities were measured by quantifying platelet adherence, platelet-derived microparticle (MP) formation, and P-selectin expression as function of surface type. Platelet adhesion, measured using a lactate dehydrogenase (LDH) assay. was increased on DAE and 320G surfaces compared to machined and polished surfaces (p < 0.05). M P formation and P-selectin expression, assayed by flow cytometry, also showed increased activation of platelets on DAE and 320G surfaces. Because increased activation of platelets may lead to up-regulation of osteogenic responses during bone healing, these results may explain the enhanced osteoconductivity known to occur with DAE cpTi surfaces in comparison with machined cpTi surfaces. PMID:11519787

  14. Targeted drug delivery to circulating tumor cells via platelet membrane-functionalized particles.

    PubMed

    Li, Jiahe; Ai, Yiwei; Wang, Lihua; Bu, Pengcheng; Sharkey, Charles C; Wu, Qianhui; Wun, Brittany; Roy, Sweta; Shen, Xiling; King, Michael R

    2016-01-01

    Circulating tumor cells (CTCs) are responsible for metastases in distant organs via hematogenous dissemination. Fundamental studies in the past decade have suggested that neutralization of CTCs in circulation could represent an effective strategy to prevent metastasis. Current paradigms of targeted drug delivery into a solid tumor largely fall into two main categories: unique cancer markers (e.g. overexpression of surface receptors) and tumor-specific microenvironment (e.g. low pH, hypoxia, etc.). While relying on a surface receptor to target CTCs can be greatly challenged by cancer heterogeneity, targeting of tumor microenvironments has the advantage of recognizing a broader spectrum of cancer cells regardless of genetic differences or tumor types. The blood circulation, however, where CTCs transit through, lacks the same tumor microenvironment as that found in a solid tumor. In this study, a unique "microenvironment" was confirmed upon introduction of cancer cells of different types into circulation where activated platelets and fibrin were physically associated with blood-borne cancer cells. Inspired by this observation, synthetic silica particles were functionalized with activated platelet membrane along with surface conjugation of tumor-specific apoptosis-inducing ligand cytokine, TRAIL. Biomimetic synthetic particles incorporated into CTC-associated micro-thrombi in lung vasculature and dramatically decreased lung metastases in a mouse breast cancer metastasis model. Our results demonstrate a "Trojan Horse" strategy of neutralizing CTCs to attenuate metastasis.

  15. What’s new in using platelet research? To unravel thrombopathies and other human disorders

    PubMed Central

    Labarque, Veerle; Thys, Chantal; Wittevrongel, Christine; Geet, Chris Van

    2007-01-01

    This review on platelet research focuses on defects of adhesion, cytoskeletal organisation, signal transduction and secretion. Platelet defects can be studied by different laboratory platelet functional assays and morphological studies. Easy bruising or a suspected platelet-based bleeding disorder is of course the most obvious reason to test the platelet function in a patient. However, nowadays platelet research also contributes to our understanding of human pathology in other disciplines such as neurology, nephrology, endocrinology and metabolic diseases. Apart from a discussion on classical thrombopathies, this review will also deal with the less commonly known relation between platelet research and disorders with a broader clinical phenotype. Classical thrombopathies involve disorders of platelet adhesion such as Glanzmann thrombastenia and Bernard-Soulier syndrome, defective G protein signalling diseases with impaired phospholipase C activation, and abnormal platelet granule secretion disorders such as gray platelet disorder and delta-storage pool disease. Other clinical symptoms besides a bleeding tendency have been described in MYH9-related disorders and Duchenne muscular dystrophy due to adhesion defects, and also in disorders of impaired Gs signalling, in Hermansky Pudlack disease and Chediak Higashi disease with abnormal secretion. Finally, platelet research can also be used to unravel novel mechanisms involved in many neurological disorders such as depression and autism with only a subclinical platelet defect. PMID:17619901

  16. Inhibition of platelet function with clopidogrel, as measured with a novel whole blood impedance aggregometer in horses.

    PubMed

    Roscher, Katja A; Failing, Klaus; Moritz, Andreas

    2015-03-01

    This study aimed to validate a loading and maintenance clopidogrel dosing scheme for the inhibition of platelet function, measured by whole blood impedance aggregometry in healthy adult horses. Ten Warmblood horses received oral clopidogrel once daily. Doses were based on 50 kg weight categories and resulted in one loading dose of 6-6.5 mg/kg bodyweight and maintenance doses of 1.2-1.4 mg/kg over the next 4 days. Platelet function was measured via whole blood multiple electrode impedance aggregometry prior to (T0) and at 6, 12, 24, 48, 72, 96, 144, 192 and 240 h following the loading dose. Aggregometries for collagen (COLtest), arachidonic acid (ASPItest), adenosine diphosphate (ADPtest) and ADP with prostaglandin E1 (ADPtestHS) were performed. Statistical analyses included one way repeated measures ANOVAs and subsequent Dunnett's tests. Platelet aggregation induced by collagen remained unchanged. There were significant inhibitions in the ASPItest (P <0.01 at 192 h, and P <0.05 at 240 h) and the ADPtest and ADPtestHS (P < 0.01, with the exception of 240 h). The loading dose of clopidogrel induced rapid inhibition of platelet function within hours, and the low dose was suitable for maintaining the inhibition over the 4 days of therapy. Recovery of platelet function was restored 6 days after the cessation of medication, determined with the ADPtest and ADPtestHS, but remained inhibited with the ASPItest. The prolonged effect of clopidogrel may indicate differences in the activation of platelets between horses and humans that were previously unknown.

  17. A noble function of BAY 11-7082: Inhibition of platelet aggregation mediated by an elevated cAMP-induced VASP, and decreased ERK2/JNK1 phosphorylations.

    PubMed

    Lee, Hyun-Sub; Kim, Sung Dae; Lee, Whi Min; Endale, Mehari; Kamruzzaman, S M; Oh, Won Jun; Cho, Jae Youl; Kim, Sang Keun; Cho, Hyun-Jeong; Park, Hwa-Jin; Rhee, Man Hee

    2010-02-10

    Platelets, though anucleated, possess several transcription factors, including NF-kappaB, that exert non-genomic functions regulating platelet activation. Since platelets have not only been recognized as central players of homeostasis, but also participated in pathological conditions such as thrombosis, atherosclerosis, and inflammation, we examined rat platelet NF-kappaB expression and evaluated the effects of anti-inflammatory drug BAY 11-7082, an inhibitor of NF-kappaB activation, in platelet physiology. Western blotting revealed that rat platelets express NF-kappaB. BAY 11-7082, dose dependently, inhibited collagen- or thrombin-induced-platelet aggregation. ATP release, TXB(2) formation, P-selectin expression, and intercellular Ca(2+) concentration activated by collagen were reduced in BAY 11-7082-treated platelets. BAY 11-7082 elevated intracellular levels of cAMP, but not cGMP, and its co-incubation with cAMP-activating agent (forskolin) or its hydrolyzing enzyme inhibitor (3-isobutyl-1-methylxanthine, IBMX), synergistically inhibited collagen-induced-platelet aggregation. In addition, vasodilator-stimulated-phosphoprotein (VASP) phosphorylation was enhanced in BAY 11-7082-treated platelets, which was partially inhibited by a protein kinase A (PKA) inhibitor, H-89. Moreover, while p38 mitogen-activated protein kinase (MAPK) was not affected, BAY 11-7082 attenuated c-Jun N-terminal kinase 1 (JNK1) and extracellular-signal-regulated protein kinase 2 (ERK2) phosphorylations. In conclusion, BAY 11-7082 inhibits platelet activation, granule secretion, and aggregation, and that this effect is mediated by inhibition of JNK1 and ERK2 phosphorylations, and partially by stimulation of cAMP-dependent PKA VASP phosphorylation. The ability of BAY 11-7082 to inhibit platelet function might be relevant in cases involving aberrant platelet activation where the drug is considered as anti-atherothrombosis, and anti-inflammatory therapy.

  18. The effects of haemodilution with hydroxyethyl starch 130/0.4 solution on coagulation as assessed by thromboelastography and platelet receptor function studies in vitro.

    PubMed

    Williams, P; Yang, K; Kershaw, G; Wong, G; Dunkley, S; Kam, P C A

    2015-11-01

    This study evaluated the effects of haemodilution with either 6% hydroxyethyl starch (HES) 130/0.4 (Voluven(®)) or 0.9% normal saline (NS) on blood coagulation in vitro. Haemodilution with 6% HES 130/0.4 impaired coagulation, as indicated by the changes in thromboelastographic parameters k-time, α-angle and maximum amplitude. Light transmission aggregometry and multiple electrode aggregometry demonstrated that impaired platelet receptor function occurred only at high levels of haemodilution (40%) with both fluids, but there was no significant difference between the two fluids (P=0.05). The thromboelastographic functional fibrinogen assay showed that the fibrinogen component of clot strength was significantly impaired with haemodilution with HES 130/0.4 compared with haemodilution with NS (whole blood [14.4 ± 4.6 mm] versus 40% HES dilution [3.7 ± 1.9], [P=0.001]; versus 40% NS dilution [10.4 ± 4.6], [P=0.129]). These findings suggest that there is little difference between HES or NS in relation to coagulation or platelet function during minor or moderate haemodilution, but at high levels of haemodilution with HES, fibrinogen activity is more impaired compared with NS. PMID:26603798

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

    SciTech Connect

    Laduca, F.M.; Bell, W.R.; Bettigole, R.E. State Univ. of New York, Buffalo )

    1987-11-01

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

  20. Highly active antiretroviral therapy-related mechanisms of endothelial and platelet function alterations.

    PubMed

    Gresele, Paolo; Falcinelli, Emanuela; Momi, Stefania; Francisci, Daniela; Baldelli, Franco

    2014-01-01

    Highly active antiretroviral therapy (HAART) has transformed human immunodeficiency virus (HIV) infection into a chronic condition, which has allowed the infected population to age and become prone to chronic degenerative diseases common to the general population, including atherosclerotic cardiovascular disease, and coronary artery disease (CAD). Possible causative mechanisms of HIV-associated CAD are related to classic cardiovascular risk factors, such as dyslipidemia, insulin resistance, and fat redistribution, which may be due to either HIV infection or to HAART-associated toxicity. However, other mechanisms are emerging as crucial for the cardiovascular complication of HIV and HAART. This article analyzes the effects of HIV and HAART on endothelial function, endothelium-leukocyte interactions, and platelets as possible mechanisms of enhanced cardiovascular risk.

  1. Effects of hormones on platelet aggregation.

    PubMed

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

    2014-04-01

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

  2. Platelet-derived CXCL12 regulates monocyte function, survival, differentiation into macrophages and foam cells through differential involvement of CXCR4–CXCR7

    PubMed Central

    Chatterjee, M; von Ungern-Sternberg, S N I; Seizer, P; Schlegel, F; Büttcher, M; Sindhu, N A; Müller, S; Mack, A; Gawaz, M

    2015-01-01

    Platelets store and release CXCL12 (SDF-1), which governs differentiation of hematopoietic progenitors into either endothelial or macrophage-foam cells. CXCL12 ligates CXCR4 and CXCR7 and regulates monocyte/macrophage functions. This study deciphers the relative contribution of CXCR4–CXCR7 in mediating the effects of platelet-derived CXCL12 on monocyte function, survival, and differentiation. CXCL12 and macrophage migration inhibitory factor (MIF) that ligate CXCR4–CXCR7 induced a dynamic bidirectional trafficking of the receptors, causing CXCR4 internalization and CXCR7 externalization during chemotaxis, thereby influencing relative receptor availability, unlike MCP-1. In vivo we found enhanced accumulation of platelets and platelet-macrophage co-aggregates in peritoneal fluid following induction of peritonitis in mice. The relative surface expression of CXCL12, CXCR4, and CXCR7 among infiltrated monocytes was also enhanced as compared with peripheral blood. Platelet-derived CXCL12 from collagen-adherent platelets and recombinant CXCL12 induced monocyte chemotaxis specifically through CXCR4 engagement. Adhesion of monocytes to immobilized CXCL12 and CXCL12-enriched activated platelet surface under static and dynamic arterial flow conditions were mediated primarily through CXCR7 and were counter-regulated by neutralizing platelet-derived CXCL12. Monocytes and culture-derived-M1–M2 macrophages phagocytosed platelets, with the phagocytic potential of culture-derived-M1 macrophages higher than M2 involving CXCR4–CXCR7 participation. CXCR7 was the primary receptor in promoting monocyte survival as exerted by platelet-derived CXCL12 against BH3-mimetic induced apoptosis (phosphatidylserine exposure, caspase-3 activation, loss of mitochondrial transmembrane potential). In co-culture experiments with platelets, monocytes predominantly differentiated into CD163+ macrophages, which was attenuated upon CXCL12 neutralization and CXCR4/CXCR7 blocking antibodies

  3. Platelet Disorders

    MedlinePlus

    ... higher risk of blood clots. With other platelet disorders, the platelets do not work as they should. For example, in von Willebrand Disease, the platelets cannot stick together or cannot attach ...

  4. Does calcium channel blockade and beta-adrenergic blockade affect platelet function and fibrinolysis to a varying degree?

    PubMed

    Gleerup, G; Mehlsen, J; Winther, K

    1995-01-01

    The effects of isradipine and atenolol on platelet function and fibrinolytic activity were studied in 10 male patients with mild untreated hypertension. After a 2-week placebo run-in period, the volunteers were randomized to either isradipine 2.5 mg twice daily or atenolol 100 mg daily for a 6-month period. Those initially receiving isradipine then received atenolol and vice versa. After each therapy regimen, blood was drawn at rest and 1 h after exercise during a maximum exercise test. Platelet activity in vivo was estimated as release of B-TG and PF-4. Fibrinolytic activity was estimated as the fast-acting inhibitor against tissue plasminogen activator usually termed PAI-1. During atenolol and isradipine therapy, blood pressure (BP) was equally reduced (p < 0.05). Heart rate (HR) decreased during atenolol treatment but was not changed by isradipine. Platelet activity in vivo estimated as B-TG and PF-4 decreased irrespective of therapy (p < 0.02). During atenolol, as during placebo therapy, exercise resulted in a significant increase in platelet activity, as shown by an increase in B-TG (p < 0.02) and in PF-4 (p < 0.01). Such increase was not observed during isradipine treatment. Both treatments tended to improve fibrinolysis, as shown by a decrease in PAI, 1 h after exercise. Reducing BP with isradipine or atenolol results in a similar decrease in platelet activity and PAI-level, tested at rest and 1 h after rest, respectively. During exercise, platelet activity increased during atenolol treatment; such change did not occur during isradipine treatment.

  5. Effect of spirapril and hydrochlorothiazide on platelet function and euglobulin clot lysis time in patients with mild hypertension.

    PubMed

    Gleerup, G; Petersen, J R; Mehlsen, J; Winther, K

    1996-10-01

    Thirteen patients with mild hypertension (untreated diastolic blood pressure of 95 to 114 mmHg) received, in random order, three successive treatments of four weeks with placebo, spirapril (6 mg daily), or hydrochlorothiazide (HCT2) (24 mg daily). At the end of each treatment, blood samples for assessment of platelet aggregation and platelet release of platelet factor 4 (PF4) and for assessment of fibrinolysis, estimated by tissue plasminogen activator (t-PA), plasminogen activator inhibitor-type 1 (PAI-1), and euglobulin clot lysis time (ECLT), were taken, first at rest, then immediately after five to ten minutes of vigorous exercise, and finally after the subsequent hour of recovery rest. Platelet aggregation induced in vitro by adrenaline significantly decreased during treatment with HCT2, the threshold rising to 10 microM as compared with 1.0 with placebo (P < 0.05) at rest, and the threshold for adenosine diphosphate (ADP) aggregation also rose, from 2 microM to 4 (NS). The resting plasma PF4 value fell, although not significantly, during HCT2 treatment from the placebo value of 3.28 to 2.56 ng/mL. During spirapril treatment there was no change in the threshold of either adrenaline or ADP for aggregation of platelets sampled at rest, and the PF4 plasma levels showed no significant reductions at rest. However, during exercise PF4 showed an approximate doubling of the resting value irrespective of therapy. This exercise-induced increase in PF4 was significantly reduced by spirapril as compared with placebo (P < 0.05). ECLT and t-PA did not shift significantly from the placebo level during either therapy. PAI-1 did not change during spirapril therapy, but during HCT2 treatment it fell, although not significantly, to 9.36 IU/mL from 15.91 with placebo (NS). Spirapril and HCT2 did not produce any unwanted side effect on platelet function or fibrinolysis. HCT2 seems to decrease platelet activity at rest, whereas spirapril seems to some extent to decrease platelet

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

    PubMed

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

    2015-04-01

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

  7. Development of Poly Unsaturated Fatty Acid Derivatives of Aspirin for Inhibition of Platelet Function.

    PubMed

    Roy, Jahnabi; Adili, Reheman; Kulmacz, Richard; Holinstat, Michael; Das, Aditi

    2016-10-01

    The inhibition of platelet aggregation is key to preventing conditions such as myocardial infarction and ischemic stroke. Aspirin is the most widely used drug to inhibit platelet aggregation. Aspirin absorption can be improved further to increase its permeability across biologic membranes via esterification or converting the carboxylic acid to an anhydride. There are several reports indicating that ω-3 and ω-6 fatty acids such as linoleic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) separately inhibit platelet aggregation. Herein, we synthesize anhydride conjugates of aspirin with linoleic acid, EPA, and DHA to form aspirin anhydrides that are expected to have higher permeability across cellular membranes. These aspirin-fatty acid anhydrides inhibited platelet aggregation in washed human platelets and platelet-rich plasma in a dose-dependent manner. In particular, the aspirin-DHA anhydride displayed similar effectiveness to aspirin. Platelet aggregation studies conducted in the presence of various platelet agonists indicated that the aspirin-lipid conjugates act through inhibition of the cyclooxygenase (COX)-thromboxane synthase (TXAS) pathway. Hence, we performed detailed biochemical studies using purified COX-1 as well as TXAS stabilized in nanoscale lipid bilayers of nanodiscs to confirm results from the platelet aggregation studies. We show that although all of the aspirin conjugates act through the COX-TXAS pathway by inhibiting COX-1, the parent fatty acids do not act via this pathway. Finally, we studied the hydrolysis of these compounds in buffer and human plasma, and we demonstrate that all of the aspirin-fatty acid conjugates hydrolyze to the parent molecules aspirin and fatty acid in a controlled manner. PMID:27488919

  8. Optical Assay of Erythrocyte Function in Banked Blood

    PubMed Central

    Bhaduri, Basanta; Kandel, Mikhail; Brugnara, Carlo; Tangella, Krishna; Popescu, Gabriel

    2014-01-01

    Stored red blood cells undergo numerous biochemical, structural, and functional changes, commonly referred to as storage lesion. How much these changes impede the ability of erythrocytes to perform their function and, as result, impact clinical outcomes in transfusion patients is unknown. In this study we investigate the effect of the storage on the erythrocyte membrane deformability and morphology. Using optical interferometry we imaged red blood cell (RBC) topography with nanometer sensitivity. Our time-lapse imaging quantifies membrane fluctuations at the nanometer scale, which in turn report on cell stiffness. This property directly impacts the cell's ability to transport oxygen in microvasculature. Interestingly, we found that cells which apparently maintain their normal shape (discocyte) throughout the storage period, stiffen progressively with storage time. By contrast, static parameters, such as mean cell hemoglobin content and morphology do not change during the same period. We propose that our method can be used as an effective assay for monitoring erythrocyte functionality during storage time. PMID:25189281

  9. Optical Assay of Erythrocyte Function in Banked Blood

    NASA Astrophysics Data System (ADS)

    Bhaduri, Basanta; Kandel, Mikhail; Brugnara, Carlo; Tangella, Krishna; Popescu, Gabriel

    2014-09-01

    Stored red blood cells undergo numerous biochemical, structural, and functional changes, commonly referred to as storage lesion. How much these changes impede the ability of erythrocytes to perform their function and, as result, impact clinical outcomes in transfusion patients is unknown. In this study we investigate the effect of the storage on the erythrocyte membrane deformability and morphology. Using optical interferometry we imaged red blood cell (RBC) topography with nanometer sensitivity. Our time-lapse imaging quantifies membrane fluctuations at the nanometer scale, which in turn report on cell stiffness. This property directly impacts the cell's ability to transport oxygen in microvasculature. Interestingly, we found that cells which apparently maintain their normal shape (discocyte) throughout the storage period, stiffen progressively with storage time. By contrast, static parameters, such as mean cell hemoglobin content and morphology do not change during the same period. We propose that our method can be used as an effective assay for monitoring erythrocyte functionality during storage time.

  10. Effect of hydrogen peroxide and catalase derivatives on functional activity of platelets.

    PubMed

    Vavaev, A V; Buryachkovskaya, L I; Uchitel, I A; Tishchenko, E G; Maksimenko, A V

    2012-01-01

    Effects of H(2)O(2) on platelet aggregation were estimated in vitro in the presence and absence of inductors (ADP, serotonin, TRAP) and native and modified catalase. Dose-dependent effect of H(2)O(2) (50 μM or more) was investigated in a pathophysiological concentration of 300 μM inducing platelet aggregation. H(2)O(2) modulated aggregation induced by ADP, serotonin, and TRAP significantly increasing the initial platelet aggregation followed by disaggregation, which was always more pronounced than in control. Catalase derivatives (native and modified forms) dose-dependently reduced the effect of H(2)O(2) and completely abolished it in a dose of 9000 U catalase activity per 1 ml of solution for native catalase and 1200 U/ml for modified one. Modified catalase, in contrast to native one, produced an independent inhibitory effect on induced platelet aggregation. Components of modified catalase (individual substance and simple mixture) had no antiaggregant effect.

  11. Identification of anti-HPA-1a allo-antibodies using IgG platelet antibody detection and crossmatch system assay with Galileo Echo.

    PubMed

    Di Cristofaro, Julie; Frassati, Coralie; Montagnie, Rolande; Basire, Agnes; Merieux, Yves; Picard, Christophe

    2015-01-01

    Fetal/neonatal allo-immune thrombocytopenia is the most frequent and the most dangerous clinical condition involving anti-human platelet antigens (HPA)-1a allo-antibodies. Anti-HPA-1a allo-immunization requires rapid and accurate diagnosis to determine appropriate treatment. The Capture-P Ready-Screen assay (C-PRS) is a new qualitative immunoassay to detect IgG anti-human leukocyte antigen (HLA) and anti-HPA allo-antibodies. The aim of this study is to assess the identification of anti-HPA-1a allo-antibodies using the C-PRS assay, associated with HLA class I stripping reagents, on the automated benchtop analyzer Galileo Echo. Forty-nine sera were analyzed: without anti-HLA class I or anti-HPA allo-antibodies, with anti-HLA class I allo-antibodies, with anti-HPA-1a allo-antibodies, among which with anti-HLA class I allo-antibodies. None of the samples without allo-antibodies were reactive. Only anti-HLA antibodies, detected by cytotoxicity-dependent complement and not by Luminex, remained positive before and after stripping reagents. Of the 13 samples, anti-HPA-1a allo-antibodies that were correctly identified before and after incubation with HLA assassin reagent were 70% and 85%, respectively. Anti-glycoprotein auto-antibodies and anti-HLA allo-antibodies do not interfere with the detection of anti-HPA-1a antibodies. This preliminary study indicates that further improvement of the test will be helpful in developing a clinically useful assay in the future. PMID:25101933

  12. Identification of anti-HPA-1a allo-antibodies using IgG platelet antibody detection and crossmatch system assay with Galileo Echo.

    PubMed

    Di Cristofaro, Julie; Frassati, Coralie; Montagnie, Rolande; Basire, Agnes; Merieux, Yves; Picard, Christophe

    2015-01-01

    Fetal/neonatal allo-immune thrombocytopenia is the most frequent and the most dangerous clinical condition involving anti-human platelet antigens (HPA)-1a allo-antibodies. Anti-HPA-1a allo-immunization requires rapid and accurate diagnosis to determine appropriate treatment. The Capture-P Ready-Screen assay (C-PRS) is a new qualitative immunoassay to detect IgG anti-human leukocyte antigen (HLA) and anti-HPA allo-antibodies. The aim of this study is to assess the identification of anti-HPA-1a allo-antibodies using the C-PRS assay, associated with HLA class I stripping reagents, on the automated benchtop analyzer Galileo Echo. Forty-nine sera were analyzed: without anti-HLA class I or anti-HPA allo-antibodies, with anti-HLA class I allo-antibodies, with anti-HPA-1a allo-antibodies, among which with anti-HLA class I allo-antibodies. None of the samples without allo-antibodies were reactive. Only anti-HLA antibodies, detected by cytotoxicity-dependent complement and not by Luminex, remained positive before and after stripping reagents. Of the 13 samples, anti-HPA-1a allo-antibodies that were correctly identified before and after incubation with HLA assassin reagent were 70% and 85%, respectively. Anti-glycoprotein auto-antibodies and anti-HLA allo-antibodies do not interfere with the detection of anti-HPA-1a antibodies. This preliminary study indicates that further improvement of the test will be helpful in developing a clinically useful assay in the future.

  13. Increased Platelet Concentration does not Improve Functional Graft Healing in Bio-Enhanced ACL Reconstruction

    PubMed Central

    Fleming, Braden C.; Proffen, Benedikt L.; Vavken, Patrick; Shalvoy, Matthew R.; Machan, Jason T.; Murray, Martha M.

    2014-01-01

    Purpose The use of an extra-cellular matrix scaffold (ECM) combined with platelets to enhance healing of an ACL graft (“bio-enhanced ACL reconstruction”) has shown promise in animal models. However, the effects of platelet concentration on graft healing remains unknown. The objectives of this study were to determine if increasing the platelet concentration in the ECM scaffold would; 1) improve the graft biomechanical properties, and 2) decrease cartilage damage after surgery. Methods Fifty-five adolescent minipigs were randomized to 5 treatment groups; untreated ACL transection (n=10), conventional ACL reconstruction (n=15), and bio-enhanced ACL reconstruction using 1X (n=10), 3X (n=10) or 5X (n=10) platelet-rich plasma. The graft biomechanical properties, anteroposterior (AP) knee laxity, graft histology and macroscopic cartilage integrity were measured at 15 weeks. Results The mean linear stiffness of the bio-enhanced ACL reconstruction procedure using the 1X preparation was significantly greater than traditional reconstruction while the 3X and 5X preparations were not. The failure loads of all the ACL reconstructed groups were equivalent but significantly greater than untreated ACL transection. There were no significant differences in the ligament maturity index or AP laxity between reconstructed knees. Macroscopic cartilage damage was relatively minor, though significantly less when the ECM-platelet composite was used. Conclusions Only the 1X platelet concentration improved healing over traditional ACL reconstruction. Increasing the platelet concentration from 1X to 5X in the ECM scaffold did not further improve the graft mechanical properties. The use of an ECM-platelet composite decreased the amount of cartilage damage seen after ACL surgery. PMID:24633008

  14. Taurine transporter in fetal T lymphocytes and platelets: differential expression and functional activity.

    PubMed

    Iruloh, C G; D'Souza, S W; Speake, P F; Crocker, I; Fergusson, W; Baker, P N; Sibley, C P; Glazier, J D

    2007-01-01

    Transplacental transfer of taurine, a beta-amino acid essential for fetal and neonatal development, constitutes the primary source of taurine for the fetus. Placental transport of taurine is compromised in pregnancies complicated by intrauterine growth restriction, resulting in a reduced concentration of taurine in cord plasma. This could impact on fetal cellular metabolism as taurine represents the most abundant intracellular amino acid in many fetal cell types. In the present study, we have used pure isolates of fetal platelets and T lymphocytes from cord blood of placentas, from normal, term pregnancies, as fetal cell types to examine the cellular uptake mechanisms for taurine by the system beta transporter and have compared gene and protein expression for the taurine transporter protein (TAUT) in these two cell types. System beta activity in fetal platelets was 15-fold higher compared with fetal T lymphocytes (P < 0.005), mirroring greater TAUT mRNA expression in platelets than T lymphocytes (P < 0.005). Cell-specific differences in TAUT protein moieties were detected with a doublet of 75 and 80 kDa in fetal platelets compared with 114 and 120 kDa in fetal T lymphocytes, with relatively higher expression in platelets. We conclude that greater system beta activity in fetal platelets compared with T lymphocytes is the result of relatively greater TAUT mRNA and protein expression. This study represents the first characterization of amino acid transporters in fetal T lymphocytes.

  15. Evaluation of Pediatric Bleeding Questionnaire in Turkish Children With Von Willebrand Disease and Platelet Function Disorders.

    PubMed

    Belen, Burcu; Kocak, Ulker; Isik, Melek; Keskin, Ebru Yilmaz; Oner, Nergiz; Sal, Ertan; Kaya, Zuhre; Yenicesu, Idil; Gursel, Turkiz

    2015-09-01

    The diagnosis of mild bleeding disorders is not easy as most of the "healthy" individuals also report bleeding symptoms. In order to get a precise bleeding history, Pediatric Bleeding Questionnaire (PBQ) has been developed. In our study, Turkish children diagnosed with Von Willebrand disease (VWD), platelet function defect (PFD), and healthy children without any symptoms (control group 1) and healthy children with symptoms but found hemostatically normal (control group 2) were analyzed with PBQ. The cut off level for "positive bleeding score" was found to be ≥2 (area under the curve [AUC]: 0.785, 95% confidence interval [CI]: 0.718-0.852). The sensitivity, specificity, positive predictive value, and negative predictive value of PBQ to define VWD versus control group 1 was 100%, 97.4%, 96.4%, and 100%; VWD versus control group 2 was 100%, 53.1%, 64.3%, and 100%; PFD versus control group 1 was 93.3%, 53.1%, 73.7%, and 85%; and PFD versus control group 2 was 93.3%, 53.1%, 73.7%, and 85%, respectively.

  16. [Effects of intravenous nicardipine on blood pressure, hemorheology platelet function in arterial hypertension. Dose-effect relations].

    PubMed

    Zannad, F; Voisin, P; Sadoul, N; Marchal, C; Donetti, C; Stoltz, J F; Gilgenkrantz, J M

    1987-06-01

    In arterial hypertension, hyperviscosity with hemorheological disturbances and platelet dysfunction may play a role in the prognosis and complications of the disease. We studied the effects of Nicardipine (NIC) on these blood disturbances in a group of 21 untreated patients with essential hypertension, aged 25 to 70 years (SBP/DBP = 185 +/- 28/105 +/- 17 mmHg). During one hour before and 4 hours after the IV injection of single doses of 5, 7.5 or 10 mg NIC over 5 min, blood pressure was recorded automatically (Dinamap). Hemorheological variables and platelet function were studied before and 30 min, 3 h and 24 hours after the injection. NIC lowered blood pressure and increased heart rate significantly (At 5 min, SBP = -24 mmHg; DBP = -18 mmHg; HR = +22 b/min). These effects were dose-dependent with rapid onset and short duration (less than 2 hrs). NIC decreased plasma viscosity from 1.36 +/- 0.08 to 1.30 +/- 0.07 Cst; p less than 0.01, whole blood viscosity from 22.4 +/- 2.8 to 20.7 +/- 1.5 mPas; p less than 0.05 for gamma = 0.512 s-1, and erythrocyte filterability with the Ca++ ionophore A 23187 from 16.3 +/- 3.8 to 13.5 +/- 3.1; p less than 0.01. Platelet aggregation with ADP was unchanged, but aggregation with A 23187 decreased from 46.9 +/- 21.2 to 31.3 +/- 25.6; p less than 0.05, as well as plasma levels of beta-thromboglobulin (71.2 +/- 29.8 to 55.4 +/- 24.3 ng/ml; p less than 0.02) and platelet generated malonaldehyde (7.2 +/- 1.8 to 6.7 +/- 1.4 nM/10(9) platelets; NS).(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Functional groups grafted nonwoven fabrics for blood filtration-The effects of functional groups and wettability on the adhesion of leukocyte and platelet

    NASA Astrophysics Data System (ADS)

    Yang, Chao; Cao, Ye; Sun, Kang; Liu, Jiaxin; Wang, Hong

    2011-01-01

    In this work, the effects of grafted functional groups and surface wettability on the adhesion of leukocyte and platelet were investigated by the method of blood filtration. The filter materials, poly(butylene terephthalate) nonwoven fabrics bearing different functional groups including hydroxyl (OH), carboxyl (COOH), sulfonic acid group (SO3H) and zwitterionic sulfobetaine group (⊕N((CH3)2)(CH2)3SO3⊖) with controllable wettability were prepared by UV radiation grafting vinyl monomers with these functional groups. Our results emphasized that both surface functional groups and surface wettability had significant effects on the adhesion of leukocyte and platelet. In the case of filter materials with the same wettability, leukocytes adhering to filter materials decreased in the order: the surface bearing OH only > the surface bearing both OH and COOH > the surface bearing sulfobetaine group > the surface bearing SO3H, while platelets adhering to filter materials decreased as the following order: the surface bearing SO3H > the surface bearing both OH and COOH > the surface bearing OH only > the surface bearing sulfobetaine group. As the wettability of filter materials increased, both leukocyte and platelet adhesion to filter materials declined, except that leukocyte adhesion to the surface bearing OH only remained unchanged.

  18. Platelets and platelet-like particles mediate intercellular RNA transfer

    PubMed Central

    Risitano, Antonina; Beaulieu, Lea M.; Vitseva, Olga

    2012-01-01

    The role of platelets in hemostasis and thrombosis is clearly established; however, the mechanisms by which platelets mediate inflammatory and immune pathways are less well understood. Platelets interact and modulate the function of blood and vascular cells by releasing bioactive molecules. Although the platelet is anucleate, it contains transcripts that may mirror disease. Platelet mRNA is only associated with low-level protein translation; however, platelets have a unique membrane structure allowing for the passage of small molecules, leading to the possibility that its cytoplasmic RNA may be passed to nucleated cells. To examine this question, platelet-like particles with labeled RNA were cocultured with vascular cells. Coculture of platelet-like particles with activated THP-1, monocytic, and endothelial cells led to visual and functional RNA transfer. Posttransfer microarray gene expression analysis of THP-1 cells showed an increase in HBG1/HBG2 and HBA1/HBA2 expression that was directly related to the transfer. Infusion of wild-type platelets into a TLR2-deficient mouse model established in vivo confirmation of select platelet RNA transfer to leukocytes. By specifically transferring green fluorescent protein, we also observed external RNA was functional in the recipient cells. The observation that platelets possess the capacity to transfer cytosolic RNA suggests a new function for platelets in the regulation of vascular homeostasis. PMID:22596260

  19. Effects of isoflurane, sevoflurane, and desflurane on platelet function: A prospective, randomized, single-blind, in vivo study

    PubMed Central

    Bozdogan, Nesrin; Madenoglu, Halit; Dogru, Kudret; Yildiz, Karamehmet; Kotanoglu, Mustafa S.; Cetin, Mustafa; Boyaci, Adem

    2005-01-01

    Background: The primary physiologic function of platelets is to facilitate hemostasisby aggregation. Volatile anesthetics have been reported to decrease platelet aggregation in vivo and in vitro. Objective: The aim of this study was to investigate the hematologic effectsof the anesthetics isoflurane, sevoflurane, and desflurane on hemoglobin (Hb), hematocrit (Hct), platelet count, activated partial thromboplastin time (aPTT), prothrombin time (PT), international normalized ratio (INR), and platelet aggregation after minor surgery. Methods: Patients aged 20 to 60 years who were scheduled to undergominor surgery and American Society of Anesthesiologists physical status P1 or P2 (healthy or mild systemic disease) were randomized to 1 of 3 groups: 1 minimum alveolar concentration (MAC) of isoflurane, sevoflurane, or desflurane. None of the patients received premedication. Anesthesia was induced using IV thiopental 5 to 6 mg/kg, fentanyl 1 to 2 μg/kg, and vecuronium 0.1 mg/kg, and maintained with 1 MAC of isoflurane, sevoflurane, or desflurane in 66% nitrous oxide and 33% oxygen. Vecuronium 0.03 mg/kg was given when necessary for muscle relaxation. All patients were monitored throughout surgery; isotonic saline was given at a rate of 5 mL/kg · h. Hematologic studies were performed preoperatively, 15 minutes after intubation, and 1 hour after the end of surgery. Platelet aggregation tests were performed in a laboratory using a platelet function analyzer (PFA), collagen/epinephrine PFA test cartridges, collagen/adenosine diphosphate PFA test cartridges, and PFA trigger solution. Results: This prospective, randomized, single-blind, in vivo study was conducted at Gevher Nesibe Teaching Hospital, Erciyes University, Kayseri, Turkey. Thirty patients (15 men, 15 women) were randomized to the 3 treatment groups (each, n = 10). Hb, Hct, platelet count, aPTT, PT, and INR were statistically similar between all 3 groups. The measured parameters were not significantly different

  20. A functional assay-based strategy for nanomaterial risk forecasting.

    PubMed

    Hendren, Christine Ogilvie; Lowry, Gregory V; Unrine, Jason M; Wiesner, Mark R

    2015-12-01

    The study of nanomaterial impacts on environment, health and safety (nanoEHS) has been largely predicated on the assumption that exposure and hazard can be predicted from physical-chemical properties of nanomaterials. This approach is rooted in the view that nanoöbjects essentially resemble chemicals with additional particle-based attributes that must be included among their intrinsic physical-chemical descriptors. With the exception of the trivial case of nanomaterials made from toxic or highly reactive materials, this approach has yielded few actionable guidelines for predicting nanomaterial risk. This article addresses inherent problems in structuring a nanoEHS research strategy based on the goal of predicting outcomes directly from nanomaterial properties, and proposes a framework for organizing data and designing integrated experiments based on functional assays (FAs). FAs are intermediary, semi-empirical measures of processes or functions within a specified system that bridge the gap between nanomaterial properties and potential outcomes in complex systems. The three components of a functional assay are standardized protocols for parameter determination and reporting, a theoretical context for parameter application and reference systems. We propose the identification and adoption of reference systems where FAs may be applied to provide parameter estimates for environmental fate and effects models, as well as benchmarks for comparing the results of FAs and experiments conducted in more complex and varied systems. Surface affinity and dissolution rate are identified as two critical FAs for characterizing nanomaterial behavior in a variety of important systems. The use of these FAs to predict bioaccumulation and toxicity for initial and aged nanomaterials is illustrated for the case of silver nanoparticles and Caenorhabditis elegans.

  1. A functional assay-based strategy for nanomaterial risk forecasting.

    PubMed

    Hendren, Christine Ogilvie; Lowry, Gregory V; Unrine, Jason M; Wiesner, Mark R

    2015-12-01

    The study of nanomaterial impacts on environment, health and safety (nanoEHS) has been largely predicated on the assumption that exposure and hazard can be predicted from physical-chemical properties of nanomaterials. This approach is rooted in the view that nanoöbjects essentially resemble chemicals with additional particle-based attributes that must be included among their intrinsic physical-chemical descriptors. With the exception of the trivial case of nanomaterials made from toxic or highly reactive materials, this approach has yielded few actionable guidelines for predicting nanomaterial risk. This article addresses inherent problems in structuring a nanoEHS research strategy based on the goal of predicting outcomes directly from nanomaterial properties, and proposes a framework for organizing data and designing integrated experiments based on functional assays (FAs). FAs are intermediary, semi-empirical measures of processes or functions within a specified system that bridge the gap between nanomaterial properties and potential outcomes in complex systems. The three components of a functional assay are standardized protocols for parameter determination and reporting, a theoretical context for parameter application and reference systems. We propose the identification and adoption of reference systems where FAs may be applied to provide parameter estimates for environmental fate and effects models, as well as benchmarks for comparing the results of FAs and experiments conducted in more complex and varied systems. Surface affinity and dissolution rate are identified as two critical FAs for characterizing nanomaterial behavior in a variety of important systems. The use of these FAs to predict bioaccumulation and toxicity for initial and aged nanomaterials is illustrated for the case of silver nanoparticles and Caenorhabditis elegans. PMID:26188653

  2. Subpopulations in purified platelets adhering on glass.

    PubMed

    Donati, Alessia; Gupta, Swati; Reviakine, Ilya

    2016-01-01

    Understanding how platelet activation is regulated is important in the context of cardiovascular disorders and their management with antiplatelet therapy. Recent evidence points to different platelet subpopulations performing different functions. In particular, procoagulant and aggregating subpopulations have been reported in the literature in platelets treated with the GPVI agonists. How the formation of platelet subpopulations upon activation is regulated remains unclear. Here, it is shown that procoagulant and aggregating platelet subpopulations arise spontaneously upon adhesion of purified platelets on clean glass surfaces. Calcium ionophore treatment of the adhering platelets resulted in one platelet population expressing both the procoagulant and the adherent population markers phosphatidylserine and the activated form of GPIIb/IIIa, while all of the platelets expressed CD62P independently of the ionophore treatment. Therefore, all platelets have the capacity to express all three activation markers. It is concluded that platelet subpopulations observed in various studies reflect the dynamics of the platelet activation process. PMID:27338300

  3. Use of 8-methoxypsoralen and long-wavelength ultraviolet radiation for decontamination of platelet concentrates

    SciTech Connect

    Lin, L.; Wiesehahn, G.P.; Morel, P.A.; Corash, L. )

    1989-07-01

    Transmission of viral diseases through blood products remains an unsolved problem in transfusion medicine. We have developed a psoralen photochemical system for decontamination of platelet concentrates in which platelets are treated with long wavelength ultraviolet radiation (UVA, 320-400 nm) in the presence of 8-methoxypsoralen (8-MOP). Bacteria, RNA viruses, and DNA viruses ranging in genome size from 1.2 x 10(6) daltons, encompassing the size range of human pathogens, were inoculated into platelet concentrates and subjected to treatment. This system inactivated 25 to 30 logs/h of bacteria Escherichia coli or Staphylococcus aureus, 6 logs/h of bacteriophage fd, 0.9 log/h of bacteriophage R17 and 1.1 logs/h of feline leukemia virus (FeLV) in platelet concentrates maintained in standard storage bags. Platelet integrity and in vitro function before, immediately following photochemical treatment, and during prolonged storage after treatment, were evaluated by measuring: (1) extracellular pH; (2) platelet yields; (3) extracellular lactate dehydrogenase (LDH) levels; (4) platelet morphology; (5) platelet aggregation responsiveness; (6) thromboxane beta-2 (TXB-2) production; (7) dense body secretion; and (8) alpha granule secretion. These assays demonstrated that this photochemical inactivation system inactivated bacteria and viruses in platelet concentrates with minimal adverse effects on the in vitro function of platelets in comparison to untreated control concentrates maintained under current, standard blood bank conditions.

  4. Effect of disulfiram on the platelet function and fibrinolysis in healthy volunteers.

    PubMed

    Gleerup, G; Boström, S; Hansson, G; Teger-Nilsson, A C; Sjöquist, P O; Winther, K

    1990-01-01

    Disulfiram was studied for platelet and fibrinolytic activity in 12 healthy volunteers of both sexes (age 23-75 years). Placebo was given for 7 days, followed by disulfiram, 800 mg for 2 days and 400 mg for an additional 12 days. Finally, there was another placebo period of 14 days. With the exception of an initial platelet activation on day 2, no significant effects were found on the platelet variables studied: platelet aggregation with collagen, ADP and adrenaline, beta-thromboglobulin and platelet factor 4. Treatment for 14 days with disulfiram resulted in a decreased euglobulin clot lysis time: from 421 +/- 82 to 246 +/- 41 min (p less than 0.01). After an initial increase, plasminogen activator inhibitor activity was slightly decreased on disulfiram, from 8.4 +/- 1.6 on placebo to 6.0 +/- 1.2 U/ml (p less than 0.05) after 14 days of treatment. Plasminogen, fibrinogen and alpha 2-antiplasmin were unchanged. It is concluded that disulfiram can increase fibrinolytic activity in healthy subjects.

  5. Detection and identification of platelet antibodies and antigens in the clinical laboratory.

    PubMed

    Curtis, B R; McFarland, J G

    2009-01-01

    As a result of the unique functional properties of platelets, more-robust methods were required for detection of antibodies raised against them. Immunofluorescence detection by flow cytometry, solid-phase red cell adherence, and antigen capture ELISAs are some of the current tests that have been developed to meet the challenges of platelet antibody detection and identification and antigen phenotyping. Recently developed protein liquid bead arrays are becoming the next-generation platelet antibody tests. Fueled by development of PCR and determination of the molecular basis of the PlA1 human platelet antigen (HPA), serologic platelet typing has now been replaced by genotyping of DNA. Allele-specific PCR, melting curve analysis, and 5'-nuclease assays are now evolving into more high-throughput molecular tests. Laboratory testing for the diagnosis of immune platelet disorders has advanced considerably from its humble beginnings.

  6. Platelets and the immune continuum.

    PubMed

    Semple, John W; Italiano, Joseph E; Freedman, John

    2011-04-01

    Platelets are anucleate cells that are crucial mediators of haemostasis. Most immunologists probably don't think about platelets every day, and may even consider these cells to be 'nuisances' in certain in vitro studies. However, it is becoming increasingly clear that platelets have inflammatory functions and can influence both innate and adaptive immune responses. Here, we discuss the mechanisms by which platelets contribute to immunity: these small cells are more immunologically savvy than we once thought.

  7. Effects of acute and chronic psychological stress on platelet aggregation in mice.

    PubMed

    Matsuhisa, Fumikazu; Kitamura, Nobuo; Satoh, Eiki

    2014-03-01

    Although psychological stress has long been known to alter cardiovascular function, there have been few studies on the effect of psychological stress on platelets, which play a pivotal role in cardiovascular disease. In the present study, we investigated the effects of acute and chronic psychological stress on the aggregation of platelets and platelet cytosolic free calcium concentration ([Ca(2+)]i). Mice were subjected to both transportation stress (exposure to novel environment, psychological stress) and restraint stress (psychological stress) for 2 h (acute stress) or 3 weeks (2 h/day) (chronic stress). In addition, adrenalectomized mice were subjected to similar chronic stress (both transportation and restraint stress for 3 weeks). The aggregation of platelets from mice and [Ca(2+)]i was determined by light transmission assay and fura-2 fluorescence assay, respectively. Although acute stress had no effect on agonist-induced platelet aggregation, chronic stress enhanced the ability of the platelet agonists thrombin and ADP to stimulate platelet aggregation. However, chronic stress failed to enhance agonist-induced increase in [Ca(2+)]i. Adrenalectomy blocked chronic stress-induced enhancement of platelet aggregation. These results suggest that chronic, but not acute, psychological stress enhances agonist-stimulated platelet aggregation independently of [Ca(2+)]i increase, and the enhancement may be mediated by stress hormones secreted from the adrenal glands.

  8. Head to Head Comparison of Two Point-of-care Platelet Function Tests Used for Assessment of On-clopidogrel Platelet Reactivity in Chinese Acute Myocardial Infarction Patients Undergoing Percutaneous Coronary Intervention

    PubMed Central

    Yao, Yi; Zhang, Jia-Hui; Tang, Xiao-Fang; He, Chen; Ma, Yuan-Liang; Xu, Jing-Jing; Song, Ying; Liu, Ru; Meng, Xian-Min; Song, Lei; Wang, Miao; Gao, Run-Lin; Yuan, Jin-Qing

    2016-01-01

    Background: Platelet function tests are widely used in clinical practice to guide personalized antiplatelet therapy. In China, the thromboelastography (TEG) test has been well accepted in clinics, whereas VerifyNow, mainly used for scientific research, has not been used in routine clinical practice. The aim of the current study was to compare these two point-of-care platelet function tests and to analyze the consistency between the two tests for evaluating on-clopidogrel platelet reactivity in Chinese acute myocardial infarction patients undergoing percutaneous coronary intervention (PCI). Methods: A total of 184 patients admitted to Fuwai Hospital between August 2014 and May 2015 were enrolled in the study. On-clopidogrel platelet reactivity was assessed 3 days after PCI by TEG and VerifyNow using adenosine diphosphate as an agonist. Based on the previous reports, an inhibition of platelet aggregation (IPA) <30% for TEG or a P2Y12 reaction unit (PRU) >230 for VerifyNow was defined as high on-clopidogrel platelet reactivity (HPR). An IPA >70% or a PRU <178 was defined as low on-clopidogrel platelet reactivity (LPR). Correlation and agreement between the two methods were analyzed using the Spearman correlation coefficient (r) and kappa value (κ), respectively. Results: Our results showed that VerifyNow and TEG had a moderate but significant correlation in evaluating platelet reactivity (r = −0.511). A significant although poor agreement (κ = 0.225) in identifying HPR and a significantly moderate agreement in identifying LPR (κ = 0.412) were observed between TEG and VerifyNow. By using TEG as the reference for comparison, the cutoff values of VerifyNow for the Chinese patients in this study were identified as PRU >205 for HPR and PRU <169 for LPR. Conclusions: By comparing VerifyNow to TEG which has been widely used in clinics, VerifyNow could be an attractive alternative to TEG for monitoring on-clopidogrel platelet reactivity in Chinese patients. PMID:27647183

  9. Metabolic, membrane, and functional responses of human polymorphonuclear leukocytes to platelet-activating factor.

    PubMed

    Ingraham, L M; Coates, T D; Allen, J M; Higgins, C P; Baehner, R L; Boxer, L A

    1982-06-01

    The phospholipid mediator of anaphylaxis, platelet-activating factor (PAF) is chemotactic for polymorphonuclear leukocytes (PMN). We have examined this agent's effects on several other PMN functions. Human PMN were prepared from heparinized venous blood by Ficoll gradient. Metabolic burst was examined by measurement of O2 use and O2.- production in the presence or absence of PAF (10(-6)--10(-9) M). Unless cells were treated with cytochalasin-B (5 micrograms/ml), no significant respiratory burst was demonstrated. However, pretreatment with PAF (10(-7) M) enhanced approximately threefold the O2 utilization found when cells were subsequently stimulated with 10(-7) M FMLP. PAF also stimulated arachidonic acid metabolism in 14C-arachidonic acid-labeled PMN. Thin-layer chromatography analysis of chloroform-methanol extracts showed substances that comigrated with authentic 5-hydroxyeicosatetraenoic acid had a marked increase in radioactivity following PAF stimulation at 10(-7) M. PAF failed to stimulate release of granule enzymes, B-glucuronidase, lysozyme, or myeloperoxidase unless cytochalasin-B were added. PAF from 10(-6) M to 10(-10) M affected PMN surface responses. PMN labeled with the fluorescent dye, chlorotetracycline, showed decreased fluorescence upon addition of PAF, suggesting translocation of membrane-bound cations. Further, the rate of migration of PMN in an electric field was decreased following PAF exposure, a change consistent with reduced cell surface charge. PMN self-aggregation and adherence to endothelial cells were both influenced by PAF (10(-6) M--10(-9) M). Aggregation was markedly stimulated by the compound, and the percent PMN adhering to endothelial cell monolayers increased almost twofold in the presence of 10(-8) M PAF. Thus, PAF promotes a variety of PMN responses: enhances respiratory burst, stimulates arachidonic acid turnover, alters cell membrane cation content and surface charge, and promotes PMN self-aggregation as well as adherence to

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

    SciTech Connect

    Rudmann, S.V.

    1986-01-01

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

  11. Comparison of cytotoxic and anti-platelet activities of polyphenolic extracts from Arnica montana flowers and Juglans regia husks.

    PubMed

    Rywaniak, Joanna; Luzak, Boguslawa; Podsedek, Anna; Dudzinska, Dominika; Rozalski, Marcin; Watala, Cezary

    2015-01-01

    Polyphenolic compounds of plant origin are well known to be beneficial to human health: they exert protective effects on haemostasis and have a particular influence on blood platelets. However, the anti-platelet properties of polyphenolic compounds observed so far have not been weighed against their potential cytotoxic action against platelets. The aim of this study was to demonstrate that anti-platelet and cytotoxic effects on blood platelets may interfere and therefore, may often lead to confusion when evaluating the properties of plant extracts or other agents towards blood platelets. The anti-platelet and cytotoxic in vitro effects of plant extracts obtained from the husks of walnuts (J. regia) and flowers of arnica (A. montana) on platelet reactivity and viability were examined. Platelet function was assessed using standard methods (flow cytometry: P-selectin expression, activation of GPIIbIIIa complex, vasodilator-stimulated phosphoprotein, VASP index; turbidimetric and impedance aggregometry) and newly set assays (flow cytometric monitoring of platelet cytotoxicity). The results reveal that none of the studied plant extracts demonstrated cytotoxicity towards blood platelets. The phenolic acid-rich extract of A. montana (7.5 and 15 µg/ml) significantly reduced the ADP-induced aggregation in both whole blood and PRP, and decreased the platelet reactivity index (PRI; VASP phosphorylation) in whole blood, while showing excellent antioxidant capacity. The extract of J. regia husks significantly reduced ADP-induced platelet aggregation in whole blood when applied at 7.5 µg/ml, and only slightly decreased the PRI at 15 µg/ml. Both examined extracts suppressed platelet hyper-reactivity, and such influence did not interfere with cytotoxic effects of the extracts. Thus, its high polyphenol content, excellent antioxidant capacity and distinct anti-platelet properties, in combination with its lack of toxicity, make the extract of A. montana flowers a possible

  12. Assessment of omega-fatty-acid-supplemented human platelets for potential improvement in long-term storage.

    PubMed

    Krishnamurti, Chitra; Stewart, Michael W; Cutting, Mary A; Rothwell, Stephen W

    2002-01-15

    Uptake of omega (omega)-3 fatty acids can influence membrane stability and cell mobility. We investigated the effects of omega-3 and -6 fatty acids on the hemostatic efficacy of human platelets using an in vivo rabbit bleeding model. In vitro assays such as platelet aggregation, vWF bead-mediated ATP release and platelet adhesion to beads (measured by the residual platelet count [RPC] [free platelet count after reacting with the beads]/[baseline platelet count]x 100=%RPC; a high %RPC indicates reduced platelet function) were conducted on platelets treated with 1% fish oil (omega-3); 2% fish oil emulsion or 1% soy oil (omega-6). Oil treatment of platelets reduced the vWF bead-induced ATP release insignificantly. Addition of omega-3 agents reduced physical reactivity (%RPC) with the vWF beads by a factor of 1.2 (oil) and 1.9 (emulsion). The omega-6 oil enhanced reactivity by a factor of 1.7. After washing to remove excess reagent, platelet resuspension was most efficient with the omega-3 emulsion. Platelet function was higher with the omega-3-treated platelets (%RPC=52.3%, omega-3 oil; 63.3%, omega-3 emulsion vs. 85%, omega-6 oil; 82% untreated platelets). Ethyl-palmitate-treated thrombocytopenic rabbits were infused with human platelets. Survival times of the treated platelets, as monitored by flow cytometry (6.2-8.2 h) were comparable to untreated platelets (8.6 h). In the rabbit kidney injury model, blood loss after infusion of the treated platelets was similar to that of saline-infused rabbits (75.3+/-3.4 g). However, platelets washed prior to infusion reduced blood loss to a value comparable to that of fresh platelets (48.3+/-5 g). Furthermore, the presence of the infused platelets at the injury site was clearly visualized using FITC-tagged anti CD42a antibody. Thus, the omega-3-based agents protect the platelets from damage during the washing procedure as demonstrated in vitro by improved platelet resuspension, low %RPC, high stimulus-responsive ATP secretion

  13. Gasotransmitters and platelets.

    PubMed

    Truss, Nicola J; Warner, Timothy D

    2011-11-01

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

  14. Platelet antibodies.

    PubMed

    Pulkrabek, S M

    1996-12-01

    The proper diagnosis of patients with immune-mediated thrombocytopenias can be accomplished by using the advances made in the field of platelet serology. These techniques range from solid phase red cell adherence to sequencing platelet antigen amino acids by polymerase chain reaction. This article describes platelet antigens, the clinical tests available to detect platelet antigens and antibodies, and the value of these tests in supporting clinical diagnoses.

  15. The effects of aspirin on platelet function and lysophosphatidic acids depend on plasma concentrations of EPA and DHA

    PubMed Central

    Block, Robert C; Abdolahi, Amir; Tu, Xin; Georas, Steve N; Brenna, J. Thomas; Phipps, Richard P; Lawrence, Peter; Mousa, Shaker A

    2015-01-01

    Aspirin’s prevention of cardiovascular disease (CVD) events in individuals with type 2 diabetes mellitus is controversial. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and aspirin all affect the cyclooxygenase enzyme. The relationship between plasma EPA and DHA and aspirin’s effects has not been determined. Thirty adults with type 2 diabetes mellitus ingested aspirin (81 mg/day) for 7 days, then EPA+DHA (2.6 g/day) for 28 days, then both for another 7 days. Lysophosphatidic acid (LPA) species and more classic platelet function outcomes were determined. Plasma concentrations of total EPA+DHA were associated with 7-day aspirin reduction effects on these outcomes in a “V”-shaped manner for all 11 LPA species and ADP-induced platelet aggregation. This EPA+DHA concentration was quite consistent for each of the LPA species and ADP. These results support aspirin effects on lysolipid metabolism and platelet aggregation depending on plasma EPA+DHA concentrations in individuals with a disturbed lipid milieu. PMID:25555354

  16. Colorimetric detection of platelet-derived growth factors through competitive interactions between proteins and functional gold nanoparticles.

    PubMed

    Lin, Tzu-En; Chen, Wei-His; Shiang, Yen-Chun; Huang, Chih-Ching; Chang, Huan-Tsung

    2011-11-15

    We have developed a colorimetric assay-using aptamer modified 13-nm gold nanoparticles (Apt-Au NPs) and fibrinogen adsorbed Au NPs (Fib-Au NPs, 56nm)-for the highly selective and sensitive detection of platelet-derived growth factors (PDGF). Apt-Au NPs and Fib-Au NPs act as recognition and reporting units, respectively. PDGF-binding-aptamer (Apt(PDGF)) and 29-base-long thrombin-binding-aptamer (Apt(thr29)) are conjugated with Au NPs to prepare functional Apt-Au NPs (Apt(PDGF)/Apt(thr29)-Au NPs) for specific interaction with PDGF and thrombin, respectively. Thrombin interacts with Fib-Au NPs in solutions to catalyze the formation of insoluble fibrillar fibrin-Au NPs agglutinates through the polymerization of the unconjugated and conjugated fibrinogen. The activity of thrombin is suppressed once it interacts with the Apt(PDGF)/Apt(thr29)-Au NPs. The suppression decreases due to steric effects through the specific interaction of PDGF with Apt(PDGF), occurring on the surfaces of Apt(PDGF)/Apt(thr29)-Au NPs. Under optimal conditions [Apt(PDGF)/Apt(thr29)-Au NPs (25pM), thrombin (400pM) and Fib-Au NPs (30pM)], the Apt(PDGF)/Apt(thr29)-Au NPs/Fib-Au NPs probe responds linearly to PDGF over the concentration range of 0.5-20nM with a correlation coefficient of 0.96. The limit of detection (LOD, signal-to-noise ratio=3) for each of the three PDGF isoforms is 0.3nM in the presence of bovine serum albumin at 100μM. When using the Apt(PDGF)/Apt(thr29)-Au NPs as selectors for the enrichment of PDGF and for the removal of interferences from cell media, the LOD for PDGF provided by this probe is 35pM. The present probe reveals that the concentration of PDGF in the three cell media is 230 (±20)pM, showing its advantages of simplicity, sensitivity, and specificity.

  17. Anti-platelet therapy and aspirin resistance - clinically and chemically relevant?

    PubMed

    Rafferty, M; Walters, M R; Dawson, J

    2010-01-01

    Platelets play a central role in the pathogenesis of the atherothrombosis which ultimately causes myocardial infarction, stroke and peripheral vascular disease. Commonly used oral anti-platelet drugs include aspirin (an irreversible inhibitor of cyclo-oxygenase), clopidogrel (an ADP receptor antagonist), other thienopyridines such as ticlopidine and prasgruel, and dipyridamole (an inhibitor of adenosine reuptake and platelet phosphodiesterase). Newer agents are in development and one, ticagrelor, a reversible ADP receptor antagonist has shown promise. Despite their proven benefit, recurrent vascular events still occur in those taking anti-platelet drugs. This has led to the concept of anti-platelet resistance, most commonly aspirin resistance as this drug is the cornerstone of most regimens. The causes of aspirin resistance are numerous but potential mechanisms include lack of patient adherence, non COX-1 mediated thromboxane A2 synthesis, increased activity of alternate platelet activation pathways, interference of aspirin action by other drugs and probably pharmacogenetic factors. Measurement of platelet response to aspirin is made possible using a number of in-vitro laboratory assays of platelet function which include measurement of thromboxane A2 metabolites as well as newer point-of-care assays of platelet aggregation. The phenomenon of aspirin resistance is important as it raises the possibility of developing strategies to identify those who respond best to a particular anti-platelet regimen, or to development of newer anti-platelet therapies to which more patients respond. This review discusses important aspects of aspirin resistance both in terms of clinical medicine, alternative anti-platelet strategies, and the potential to overcome its various causes. PMID:21062249

  18. Platelet Interaction with Innate Immune Cells

    PubMed Central

    Kral, Julia Barbara; Schrottmaier, Waltraud Cornelia; Salzmann, Manuel; Assinger, Alice

    2016-01-01

    Summary Beyond their traditional role in haemostasis and thrombosis, platelets are increasingly recognised as immune modulatory cells. Activated platelets and platelet-derived microparticles can bind to leukocytes, which stimulates mutual activation and results in rapid, local release of platelet-derived cytokines. Thereby platelets modulate leukocyte effector functions and contribute to inflammatory and immune responses to injury or infection. Platelets enhance leukocyte extravasation, differentiation and cytokine release. Platelet-neutrophil interactions boost oxidative burst, neutrophil extracellular trap formation and phagocytosis and play an important role in host defence. Platelet interactions with monocytes propagate their differentiation into macrophages, modulate cytokine release and attenuate macrophage functions. Depending on the underlying pathology, platelets can enhance or diminish leukocyte cytokine production, indicating that platelet-leukocyte interactions represent a fine balanced system to restrict excessive inflammation during infection. In atherosclerosis, platelet interaction with neutrophils, monocytes and dendritic cells accelerates key steps of atherogenesis by promoting leukocyte extravasation and foam cell formation. Platelet-leukocyte interactions at sites of atherosclerotic lesions destabilise atherosclerotic plaques and promote plaque rupture. Leukocytes in turn also modulate platelet function and production, which either results in enhanced platelet destruction or increased platelet production. This review aims to summarise the key effects of platelet-leukocyte interactions in inflammation, infection and atherosclerosis. PMID:27226790

  19. Sample collection and platelet function testing: influence of vacuum or aspiration principle on PFA-100 test results.

    PubMed

    Lippi, Giuseppe; Ippolito, Luigi; Zobbi, Valentina; Sandei, Franca; Favaloro, Emmanuel J

    2013-09-01

    As for other tests of hemostasis, the investigation of platelet function is highly vulnerable to a broad series of preanalytical variables, which span from patient preparation to the final analysis of the specimen and issuance of test results. In particular, there remains much controversy about the influence of manual or vacuum aspiration of blood into primary collection tubes on platelet function testing. Accordingly, we investigated this for the PFA-100. In 12 healthy volunteers, a sample labeled as 'BD-V' was drawn into a 2.7 ml BD Vacutainer tube, whereas two additional samples were collected from the opposite arm into 5.0 ml Sarstedt S-Monovette tubes by vacuum (SD-V) or manual aspiration (SD-A). All sample were tested on PFA-100 with collagen and ADP (CADP) or collagen and epinephrine (CEPI). The values of both CEPI and CADP obtained in SD-A samples were significantly lower than those obtained in SD-V and BD-V tubes, whereas those of the two evacuated tubes did not significantly differ. On average, CEPI values were prolonged by 11% in SD-V and 13% in BD-V, whereas those of CADP were prolonged by 14% in SD-V and 10% in BD-V, respectively. These findings suggests that the lower shear stress generated by the manual aspiration of blood into the primary collection tube would prevent spurious hyper-activation of platelets, thus, preserving the integrity of their function for subsequent testing on PFA-100. This study underscores the need to define or validate local reference ranges for the PFA-100 based on the collection tube used. Different reference ranges of both CEPI and CADP may also be advisable when venous blood samples are collected with manual aspiration or vacuum principle.

  20. A Functional Assay for GPR55: Envision Protocol.

    PubMed

    Anavi-Goffer, Sharon; Ross, Ruth A

    2016-01-01

    AlphaScreen(®) SureFire(®) assay is a novel technology that combines luminescent oxygen channeling technology, nano-beads, and monocloncal antibodies to detect the level of a selected protein in a volume lower than 5 μl. This method is more sensitive compared with the traditional enzyme-linked immunosorbent assays (ELISA), and can detect an increasing number of new targets. Here, we described a method for AlphaScreen(®) SureFire(®) assay that targets ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys activation of GPR55 by L-α-lysophosphatidylinositol (LPI) and certain cannabinoids. PMID:27245893

  1. A new fabrication route for PVA/graphene platelets composites with enhanced functionalities

    NASA Astrophysics Data System (ADS)

    Lavecchia, Teresa; Tamburri, Emanuela; Angjellari, Mariglen; Savi, Damiano; Terranova, Maria Letizia

    2016-05-01

    This work deals with the synthesis and characterization of composites made of poly(vinyl alcohol) (PVA) and oxidized graphene platelets obtained from an ad hoc treatment of graphite. The composite is produced by a modified solution mixing procedure in which the in situ crosslinking of PVA with maleic anhydride has been carried out in the presence of the carbon filler. A complete characterization of the material is presented carried out by SEM, DTGA, Raman spectroscopy and I-V characteristics analysis.

  2. Platelet Activation in Human Immunodeficiency Virus Type-1 Patients Is Not Altered with Cocaine Abuse

    PubMed Central

    Kiebala, Michelle; Singh, Meera V.; Piepenbrink, Michael S.; Qiu, Xing; Kobie, James J.; Maggirwar, Sanjay B.

    2015-01-01

    Recent work has indicated that platelets, which are anucleate blood cells, significantly contribute to inflammatory disorders. Importantly, platelets also likely contribute to various inflammatory secondary disorders that are increasingly associated with Human Immunodeficiency Virus Type-1 (HIV) infection including neurological impairments and cardiovascular complications. Indeed, HIV infection is often associated with increased levels of platelet activators. Additionally, cocaine, a drug commonly abused by HIV-infected individuals, leads to increased platelet activation in humans. Considering that orchestrated signaling mechanisms are essential for platelet activation, and that nuclear factor-kappa B (NF-κB) inhibitors can alter platelet function, the role of NF-κB signaling in platelet activation during HIV infection warrants further investigation. Here we tested the hypothesis that inhibitory kappa B kinase complex (IKK) activation would be central for platelet activation induced by HIV and cocaine. Whole blood from HIV-positive and HIV-negative individuals, with or without cocaine abuse was used to assess platelet activation via flow cytometry whereas IKK activation was analyzed by performing immunoblotting and in vitro kinase assays. We demonstrate that increased platelet activation in HIV patients, as measured by CD62P expression, is not altered with reported cocaine use. Furthermore, cocaine and HIV do not activate platelets in whole blood when treated ex vivo. Finally, HIV-induced platelet activation does not involve the NF-κB signaling intermediate, IKKβ. Platelet activation in HIV patients is not altered with cocaine abuse. These results support the notion that non-IKK targeting approaches will be better suited for the treatment of HIV-associated inflammatory disorders. PMID:26076359

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

    PubMed

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

    2013-10-01

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

  4. Studies of activated GPIIb/IIIa receptors on the luminal surface of adherent platelets. Paradoxical loss of luminal receptors when platelets adhere to high density fibrinogen.

    PubMed Central

    Coller, B S; Kutok, J L; Scudder, L E; Galanakis, D K; West, S M; Rudomen, G S; Springer, K T

    1993-01-01

    The accessibility of activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to damaged blood vessels or atherosclerotic plaques is likely to play a crucial role in subsequent platelet recruitment. To define better the factors involved in this process, we developed a functional assay to assess the presence of activated, luminal GPIIb/IIIa receptors, based on their ability to bind erythrocytes containing a high density of covalently coupled RGD-containing peptides (thromboerythrocytes). Platelets readily adhered to wells coated with purified type I rat skin collagen and the adherent platelets bound a dense lawn of thromboerythrocytes. With fibrinogen-coated wells, platelet adhesion increased as the fibrinogen-coating concentration increased, reaching a plateau at about 11 micrograms/ml. Thromboerythrocyte binding to the platelets adherent to fibrinogen showed a paradoxical response, increasing at fibrinogen coating concentrations up to approximately 4-6 micrograms/ml and then dramatically decreasing at higher fibrinogen-coating concentrations. Scanning electron microscopy demonstrated that the morphology of platelets adherent to collagen was similar to that of platelets adherent to low density fibrinogen, with extensive filopodia formation and ruffling. In contrast, platelets adherent to high density fibrinogen showed a bland, flattened appearance. Immunogold staining of GPIIb/IIIa receptors demonstrated concentration of the receptors on the filopodia, and depletion of receptors on the flattened portion of the platelets. Thus, there is a paradoxical loss of accessible, activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to high density fibrinogen. Two factors may contribute to this result: engagement of GPIIb/IIIa receptors with fibrinogen on the abluminal surface leading to the loss of luminal receptors, and loss of luminal filopodia that interact with thromboerythrocytes. These data provide insight into the differences

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

    PubMed

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

    2014-07-01

    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.

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

    PubMed

    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.

  7. Functional expression of the high affinity receptor for IgE (FcepsilonRI) in human platelets and its' intracellular expression in human megakaryocytes.

    PubMed

    Hasegawa, S; Pawankar, R; Suzuki, K; Nakahata, T; Furukawa, S; Okumura, K; Ra, C

    1999-04-15

    The high affinity IgE receptor (FcepsilonRI) expressed on the cell surface of mast cells and basophils is the key molecule in triggering the IgE-mediated allergic reaction. Recently, it was elucidated that the FcepsilonRI is expressed on a variety of other cells like Langerhans cells, monocytes, and eosinophils, and the functional importance of the FcepsilonRI expression in Langerhans cells was also shown. Some studies suggest that human platelets may play important roles in allergic inflammation through the cell-surface expression of the FcepsilonRII and FcgammaRII. Here, we report that human platelets and megakaryocytes constitutively express the messenger RNA and protein for the FcepsilonRI. Although the FcepsilonRI is expressed on the cell surface of human platelets, it is only detected in the cytoplasm of human megakaryocytes. We also confirmed that human platelets express the genes for the alpha, beta, and gamma chains of the FcepsilonRI without any defined mutations. Furthermore, stimulation of human platelets via the FcepsilonRI induced the release of serotonin and RANTES (Regulated on Activation, Normal T Expressed, and presumably Secreted). Taken together, these results suggest a novel and important role for human platelets in perpetuating allergic inflammation through the expression of and activation via the FcepsilonRI.

  8. Evaluation of CK2 inhibitor (E)-3-(2,3,4,5-tetrabromophenyl)acrylic acid (TBCA) in regulation of platelet function.

    PubMed

    Ryu, Si-Yun; Kim, Soochong

    2013-11-15

    Casein Kinase II (CK2) is a serine/threonine kinase which is expressed in platelets. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a substrate of CK2 and antagonizes PI 3-kinase-mediated pathways by dephosphorylating phosphatidylinositol 3,4,5-triphosphate (PIP3). Since the role of CK2 and its signaling mechanism in platelet activation is not understood, we have examined whether CK2 plays an important role in agonist-induced platelet functional responses through the regulation of PI 3-kinase pathways by using a new class of highly selective CK2 inhibitor TBCA [(E)-3-(2,3,4,5-tetrabromophenyl)acrylic acid]. TBCA dose-dependently inhibited platelet aggregation and secretion induced by various agonists including 2-MeSADP, AYPGKF, SFLLRN, and CRP. Extent of platelet response inhibited by TBCA was similar to the extent of inhibition induced by PI 3-kinase inhibitors. CK2 regulated phosphorylation of PTEN as the inhibition of CK2 resulted in the inhibition of AYPGKF-induced PTEN phosphorylation. Agonist-induced thromboxane A2 (TxA2) generation and ERK phosphorylation were significantly inhibited by TBCA. TBCA also inhibited phosphorylation of PDK1, Akt, and GSK3β induced by AYPGKF. However, CK2 inhibition had no effect on AYPGKF-induced phosphorylation of PKC substrate plekstrin, demonstrating the selective action of TBCA through Gi-mediated PI 3-kinase pathways. Finally, platelet spreading on immobilized fibrinogen surface and clot retraction mediated by integrin αIIbβ3 signaling were significantly inhibited in the presence of TBCA. We conclude that CK2 plays a key role in platelet aggregation, secretion, TxA2 generation, and Akt and ERK phosphorylation, through the regulation of PI 3-kinase pathways. Moreover, CK2 is involved in αIIbβ3-mediated outside-in signaling in platelets. PMID:24140231

  9. A discrepancy between platelet alpha 2-receptor density and functional circulatory changes in hypertensives

    SciTech Connect

    Mores, N.; Martire, M.; Pistritto, G.; Cardillo, C.; Folli, G. )

    1990-09-01

    To investigate whether differences exist in peripheral alpha 2-adrenoceptors between normotensive and hypertensive subjects, we determined platelet alpha 2-adrenoceptor density in 10 (7 males) untreated essential hypertensives (mean age of 51.1 years, range of 44-59 years) and in 10 age- and sex-matched normotensive controls. Moreover, in hypertensive patients, we examined the relationship between receptor density and cardiovascular reactivity to mental arithmetic, static handgrip, and bicycle exercise, to verify the hypothesis that alpha 2-adrenoceptors might play a role in modulation of hemodynamic response to sympathetic stimuli. alpha 2-Adrenoceptor density, as calculated by binding of (3H)yohimbine to platelets, was significantly higher in essential hypertensives (314.8 +/- 38.7 fmol/mg) than in normotensive subjects (213.6 +/- 34.7 fmol/mg) (p less than 0.05), whereas receptor affinity was similar in both groups (4.0 +/- 0.5 nM hypertensives, 4.3 +/- 0.5 nM normotensives; p greater than 0.05). Mental arithmetic increased mean arterial pressure (MAP) by 21.5% from basal values and heart rate (HR) by 13.2%. During isometric exercise, MAP increased by 38.1% and HR by 24.7%, while during bicycle ergometry, mean increases in MAP and HR from baseline were of 27.2 and 54.3%, respectively. No correlation was found between platelet alpha 2-adrenoceptor density and percent changes in MAP induced by all tests, or between adrenoceptors and absolute basal and peak MAP values. Our findings suggest that in hypertensive patients, peripheral alpha 2-adrenoceptors are increased with respect to matched normotensives, but these receptors seem not to be involved in the modulation of cardiovascular adaptation to enhanced sympathetic activity.

  10. Receptor-mediated Modulation of Human Monocyte, Neutrophil, Lymphocyte, and Platelet Function by Phorbol Diesters

    PubMed Central

    Goodwin, Bonnie J.; Weinberg, J. Brice

    1982-01-01

    The tumor promoting phorbol diesters elicit a variety of responses from normal and leukemic blood cells in vitro by apparently interacting with cellular receptors. The biologically active ligand [20-3H] phorbol 12,13-dibutyrate ([3H]PDBu) bound specifically to intact human lymphocytes, monocytes, polymorphonuclear leukocytes (PMN), and platelets, but not to erythrocytes. Binding, which was comparable for all four blood cell types, occurred rapidly at 23° and 37°C, reaching a maximum by 20-30 min usually followed by a 30-40% decrease in cell associated radioactivity over the next 30-60 min. The time course for binding was temperature dependent with equilibrium binding occurring after 120-150 min at 4°C, with no subsequent loss of cell-associated radioactivity at this temperature. Bound [3H]PDBu could be eluted by addition of unlabeled PDBu. Scatchard analysis of data from 4°C binding studies revealed linear plots with high affinity receptors in these cell types with dissociation constants and receptors per cell of 60 nM and 7.8 × 105/cell for lymphocytes, 51 nM and 15.5 × 105/cell for monocytes, 38 nM and 4.0 × 105/cell for PMN, and 19 nM and 2.9 × 104/cell for platelets. Structure-activity studies using unlabeled phorbol-related compounds demonstrated a close correlation between their abilities to inhibit binding of [3H]PDBu to cells and their abilities to induce cellular responses (monocyte and PMN H2O2 secretion, lymphocyte 3HTdR incorporation, and platelet tritiated serotonin release); phorbol and 4-alpha phorbol were inactive while phorbol 12-myristate 13-acetate (PMA), PDBu, mezerein, and phorbol 12,13-diacetate (in decreasing order of potency) inhibited [3H]PDBu binding and elicited the various responses. Thus, these high affinity, specific receptors for the phorbol diesters, present on monocytes, lymphocytes, PMN, and platelets, mediate the pleiotypic effects induced by these ligands. PMID:6956584

  11. Differences in the influence of the interaction between acetylsalicylic acid and salicylic acid on platelet function in whole blood and isolated platelets: influence of neutrophils.

    PubMed

    González-Correa, J A; Muñoz-Marín, J; López-Villodres, J A; Navas, M D; Guerrero, A; Torres, J A; De La Cruz, J P

    2007-08-01

    The aim of this study was to characterize the influence of the interaction between acetylsalicylic acid (ASA) and salicylic acid (SA) on the inhibition by ASA of platelet aggregation in platelets isolated from whole blood, and to determine whether leukocytes influence this pharmacological interaction. This in vitro study was done in human blood from which we prepared samples of whole blood, platelet-rich plasma (PRP), PRP plus mononuclear leukocytes, and PRP plus neutrophils. The variables recorded were maximum platelet aggregation intensity, thromboxane B2 (TxB2) production, and nitric oxide (NO) production (N=10 different samples in each type of experiment). Different concentrations of ASA and SA were incubated with all samples. In PRP, the concentration of ASA that inhibited maximum aggregation by 50% (IC50) (281+/-16microM) increased with increasing SA concentration to a maximum of more than 2mM when 500microM SA was used. In whole blood, the IC50 for ASA (24.9+/-1.2microM) decreased with decreasing SA concentrations to 7.9+/-0.8microM with 50microM SA and 15.6+/-0.9microM with 125microM SA, and increased to 46.2+/-2.6microM with 250microM SA and 96.3+/-7.2microM with 500microM SA. In experiments with PRP+neutrophils the IC50 of ASA increased in the presence of all concentrations of SA. The antagonistic interactions were also reflected in the changes in TxB2 production in all samples. In samples of neutrophils incubated with ASA, the curve for NO production was shifted to the right, a finding that paralleled the changes in platelet aggregation. In conclusion, the influence of the interaction between ASA and its metabolite SA on platelet aggregation difference depending on the type of sample, and was antagonistic in PRP but partially agonistic in whole blood. Nitric oxide synthesis showed an additive effect of the two compounds.

  12. Platelet Lipidomic Profiling: Novel Insight into Cytosolic Phospholipase A2α Activity and Its Role in Human Platelet Activation.

    PubMed

    Duvernay, Matthew T; Matafonov, Anton; Lindsley, Craig W; Hamm, Heidi E

    2015-09-15

    With a newer, more selective and efficacious cytosolic phospholipase A2α (cPLA2α) inhibitor available, we revisited the role of cPLA2α activity in platelet activation and discovered that a component of platelet signaling, even larger than previously appreciated, relies on this enzyme. In a whole blood shear-based flow chamber assay, giripladib, a cPLA2α inhibitor, reduced platelet adhesion and accumulation on collagen. Moreover, giripladib differentially affected P-selectin expression and GPIIbIIIa activation depending on the agonist employed. While protease-activated receptor 1 (PAR1)-mediated platelet activation was unaffected by giripladib, the levels of PAR4- and GPVI-mediated platelet activation were significantly reduced. Meanwhile, the thromboxane A2 receptor antagonist SQ29548 had no effect on PAR-, GPVI-, or puriniergic receptor-mediated platelet activation, suggesting that another eicosanoid produced downstream of arachidonic acid liberation by cPLA2α was responsible for this large component of PAR4- and GPVI-mediated platelet activation. In parallel, we profiled PAR-mediated changes in glycerophospholipid (GPL) mass with and without giripladib to better understand cPLA2α-mediated lipid metabolism. Phosphatidylcholine and phosphatidylethanolamine (PE) demonstrated the largest consumption of mass during thrombin stimulation. Additionally, we confirm phosphatidylinositol as a major substrate of cPLA2α. A comparison of PAR1- and PAR4-induced metabolism revealed the consumption of more putative arachidonyl-PE species downstream of PAR1 activation. Instead of enhanced cPLA2α activity and therefore more arachidonic acid liberation downstream of PAR4, these results indicate the major role that cPLA2α activity plays in platelet function and suggest that a novel eicosanoid is produced in response to platelet activation that represents a large component of PAR4- and GPVI-mediated responses.

  13. Platelet Lipidomic Profiling: Novel Insight into Cytosolic Phospholipase A2α Activity and Its Role in Human Platelet Activation.

    PubMed

    Duvernay, Matthew T; Matafonov, Anton; Lindsley, Craig W; Hamm, Heidi E

    2015-09-15

    With a newer, more selective and efficacious cytosolic phospholipase A2α (cPLA2α) inhibitor available, we revisited the role of cPLA2α activity in platelet activation and discovered that a component of platelet signaling, even larger than previously appreciated, relies on this enzyme. In a whole blood shear-based flow chamber assay, giripladib, a cPLA2α inhibitor, reduced platelet adhesion and accumulation on collagen. Moreover, giripladib differentially affected P-selectin expression and GPIIbIIIa activation depending on the agonist employed. While protease-activated receptor 1 (PAR1)-mediated platelet activation was unaffected by giripladib, the levels of PAR4- and GPVI-mediated platelet activation were significantly reduced. Meanwhile, the thromboxane A2 receptor antagonist SQ29548 had no effect on PAR-, GPVI-, or puriniergic receptor-mediated platelet activation, suggesting that another eicosanoid produced downstream of arachidonic acid liberation by cPLA2α was responsible for this large component of PAR4- and GPVI-mediated platelet activation. In parallel, we profiled PAR-mediated changes in glycerophospholipid (GPL) mass with and without giripladib to better understand cPLA2α-mediated lipid metabolism. Phosphatidylcholine and phosphatidylethanolamine (PE) demonstrated the largest consumption of mass during thrombin stimulation. Additionally, we confirm phosphatidylinositol as a major substrate of cPLA2α. A comparison of PAR1- and PAR4-induced metabolism revealed the consumption of more putative arachidonyl-PE species downstream of PAR1 activation. Instead of enhanced cPLA2α activity and therefore more arachidonic acid liberation downstream of PAR4, these results indicate the major role that cPLA2α activity plays in platelet function and suggest that a novel eicosanoid is produced in response to platelet activation that represents a large component of PAR4- and GPVI-mediated responses. PMID:26295742

  14. Measurement of platelet function in dogs using a novel impedance aggregometer.

    PubMed

    Kalbantner, K; Baumgarten, A; Mischke, R

    2010-08-01

    The aim of this study was to optimise the technique and establish reference values for whole blood aggregometry in dogs using a novel multiplate analyser. Measurements were performed on the hirudin-anticoagulated blood of healthy dogs using a wide range of agonists. Optimal agonist concentrations were 10 micromol/L of adenosine diphosphate, 5 microg/mL of collagen and 1 mmol/L of arachidonic acid. Ristocetin (at 0.2 and 1 mg/mL) and thrombin receptor activating peptide (TRAP-6 at 32 and 160 micromol/L) did not consistently induce platelet aggregation. Coefficients of variance for within-run imprecision (n=10 repetitions) varied from 5% to 18%. Measurement signals were significantly higher when analyses were performed on standard samples (hirudin-anticoagulated blood) compared to citrated blood or blood samples anticoagulated with citrate buffer, regardless of whether or not re-calcification was performed (P<0.05). The findings indicate that the analyser is suitable for the investigation of platelet aggregation in dogs and analysis should be performed on hirudin-anticoagulated blood using optimised agonist concentrations. PMID:19879171

  15. Platelets and Infection – An Emerging Role of Platelets in Viral Infection

    PubMed Central

    Assinger, Alice

    2014-01-01

    Platelets are anucleate blood cells that play a crucial role in the maintenance of hemostasis. While platelet activation and elevated platelet counts (thrombocytosis) are associated with increased risk of thrombotic complications, low platelet counts (thrombocytopenia) and several platelet function disorders increase the risk of bleeding. Over the last years, more and more evidence has emerged that platelets and their activation state can also modulate innate and adaptive immune responses and low platelet counts have been identified as a surrogate marker for poor prognosis in septic patients. Viral infections often coincide with platelet activation. Host inflammatory responses result in the release of platelet activating mediators and a pro-oxidative and pro-coagulant environment, which favors platelet activation. However, viruses can also directly interact with platelets and megakaryocytes and modulate their function. Furthermore, platelets can be activated by viral antigen–antibody complexes and in response to some viruses B-lymphocytes also generate anti-platelet antibodies. All these processes contributing to platelet activation result in increased platelet consumption and removal and often lead to thrombocytopenia, which is frequently observed during viral infection. However, virus-induced platelet activation does not only modulate platelet count but also shape immune responses. Platelets and their released products have been reported to directly and indirectly suppress infection and to support virus persistence in response to certain viruses, making platelets a double-edged sword during viral infections. This review aims to summarize the current knowledge on platelet interaction with different types of viruses, the viral impact on platelet activation, and platelet-mediated modulations of innate and adaptive immune responses. PMID:25566260

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

    PubMed

    Assinger, Alice

    2014-01-01

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

  17. Arf6 controls platelet spreading and clot retraction via integrin αIIbβ3 trafficking

    PubMed Central

    Huang, Yunjie; Joshi, Smita; Xiang, Binggang; Kanaho, Yasunori; Li, Zhenyu; Bouchard, Beth A.; Moncman, Carole L.

    2016-01-01

    Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate–ribosylation factor 6 (Arf6) is a small guanosine triphosphate–binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects αIIbβ3-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)–labeled Fg. Platelets from the injected Arf6 KO mice showed lower accumulation of tagged Fg, suggesting an uptake defect. Ex vivo, Arf6 KO platelets were also defective in FITC-Fg uptake and storage. Immunofluorescence analysis showed initial trafficking of FITC-Fg to a Rab4-positive compartment followed by colocalization with Rab11-positive structures, suggesting that platelets contain and use both early and recycling endosomes. Resting and activated αIIbβ3 levels, as measured by flow cytometry, were unchanged; yet, Arf6 KO platelets exhibited enhanced spreading on Fg and faster clot retraction. This was not the result of alterations in αIIbβ3 signaling, because myosin light-chain phosphorylation and Rac1/RhoA activation were unaffected. Consistent with the enhanced clot retraction and spreading, Arf6 KO mice showed no deficits in tail bleeding or FeCl3-induced carotid injury assays. Our studies present the first mouse model for defining the functions of platelet endocytosis and suggest that altered integrin trafficking may affect the efficacy of platelet function. PMID:26738539

  18. miR-326 targets antiapoptotic Bcl-xL and mediates apoptosis in human platelets.

    PubMed

    Yu, Shifang; Huang, Huicong; Deng, Gang; Xie, Zuoting; Ye, Yincai; Guo, Ruide; Cai, Xuejiao; Hong, Junying; Qian, Dingliang; Zhou, Xiangjing; Tao, Zhihua; Chen, Bile; Li, Qiang

    2015-01-01

    Platelets play crucial roles in hemostasis, thrombosis, wound healing, inflammation, angiogenesis, and tumor metastases. Because they are anucleated blood cells, platelets lack nuclear DNA, but they do contain mitochondrial DNA, which plays a key role in regulating apoptosis. Recent evidence has suggested that miRNAs are also involved in regulating gene expression and apoptosis in platelets. Our previous study showed that the expression of miR-326 increased visibly when apheresis platelets were stored in vitro. The antiapoptotic Bcl-2 family regulator Bcl-xL has been identified as a putative target of miR-326. In the present study, dual reporter luciferase assays were used to characterize the function of miR-326 in the regulation of the apoptosis of platelet cells. These assays demonstrated that miR-326 bound to the 3'-translated region of Bcl-xL. To directly assess the functional effects of miR-326 expression, levels of Bcl-xL and the apoptotic status of stored apheresis platelets were measured after transfection of miR-326 mimic or inhibitor. Results indicated that miR-326 inhibited Bcl-xL expression and induced apoptosis in stored platelets. Additionally, miR-326 inhibited Bcl-2 protein expression and enhanced Bak expression, possibly through an indirect mechanism, though there was no effect on the expression of Bax. The effect of miR-326 appeared to be limited to apoptosis, with no significant effect on platelet activation. These results provide new insight into the molecular mechanisms affecting differential platelet gene regulation, which may increase understanding of the role of platelet apoptosis in multiple diseases.

  19. Platelet Immobilization on Supported Phospholipid Bilayers for Single Platelet Studies.

    PubMed

    Uhl, Eva; Donati, Alessia; Reviakine, Ilya

    2016-08-23

    The worldwide cardiovascular disease (CVD) epidemic is of grave concern. A major role in the etiology of CVDs is played by the platelets (thrombocytes). Platelets are anuclear cell fragments circulating in the blood. Their primary function is to catalyze clot formation, limiting traumatic blood loss in the case of injury. The same process leads to thrombosis in the case of CVDs, which are commonly managed with antiplatelet therapy. Platelets also have other, nonhemostatic functions in wound healing, inflammation, and tissue regeneration. They play a role in the early stages of atherosclerosis and the spread of cancer through metastases. Much remains to be learned about the regulation of these diverse platelet functions under physiological and pathological conditions. Breakthroughs in this regard are expected to come from single platelet studies and systems approaches. The immobilization of platelets at surfaces is advantageous for developing such approaches, but platelets are activated when they come in contact with foreign surfaces. In this work, we develop and validate a protocol for immobilizing platelets on supported lipid bilayers without activation due to immobilization. Our protocol can therefore be used for studying platelets with a wide variety of surface-sensitive techniques. PMID:27438059

  20. A novel role of h2-calponin in regulating whole blood thrombosis and platelet adhesion during physiologic flow.

    PubMed

    Hines, Patrick C; Gao, Xiufeng; White, Jennell C; D'Agostino, Ashley; Jin, Jian-Ping

    2014-12-01

    Calponin is an actin filament-associated protein reported in platelets, although the specific isoform expressed and functional role were not identified. The h2-calponin isoform is expressed in myeloid-derived peripheral blood monocytes, where it regulates adhesion. Our objective was to characterize the presence and function of the h2 isoform of calponin in platelets. H2-calponin was detected in human and mouse platelets via Western blotting. Immunofluorescent staining demonstrated h2-calponin and actin colocalized in both human and wild-type mouse platelets at rest and following collagen activation. The kinetics of platelet adhesion and whole blood thrombosis during physiologic flow was evaluated in a microfluidic flow-based thrombosis assay. The time to initiation of rapid platelet/thrombus accumulation (lag time) was significantly longer in h2-calponin knockout versus wild-type mouse blood (130.02 ± 3.74 sec and 72.95 ± 16.23 sec, respectively, P < 0.05). There was no significant difference in the rate of platelet/thrombus accumulation during the rapid phase or the maximum platelet/thrombus accumulation. H2-calponin knockout mice also had prolonged bleeding time and blood loss. H2-calponin in platelets facilitates early interactions between platelets and collagen during physiologic flow, but does not significantly affect the rate or magnitude of platelet/thrombus accumulation. H2-calponin knockout mice take 2.3 times longer to achieve hemostasis compared to wild-type controls in a tail bleeding model. The ability to delay platelet accumulation without inhibiting downstream thrombotic potential would be of significant therapeutic value, thus h2-calponin may be a novel target for therapeutic platelet inhibition. PMID:25472609

  1. A novel role of h2‐calponin in regulating whole blood thrombosis and platelet adhesion during physiologic flow

    PubMed Central

    Hines, Patrick C.; Gao, Xiufeng; White, Jennell C.; D'Agostino, Ashley; Jin, Jian‐Ping

    2014-01-01

    Abstract Calponin is an actin filament‐associated protein reported in platelets, although the specific isoform expressed and functional role were not identified. The h2‐calponin isoform is expressed in myeloid‐derived peripheral blood monocytes, where it regulates adhesion. Our objective was to characterize the presence and function of the h2 isoform of calponin in platelets. H2‐calponin was detected in human and mouse platelets via Western blotting. Immunofluorescent staining demonstrated h2‐calponin and actin colocalized in both human and wild‐type mouse platelets at rest and following collagen activation. The kinetics of platelet adhesion and whole blood thrombosis during physiologic flow was evaluated in a microfluidic flow‐based thrombosis assay. The time to initiation of rapid platelet/thrombus accumulation (lag time) was significantly longer in h2‐calponin knockout versus wild‐type mouse blood (130.02 ± 3.74 sec and 72.95 ± 16.23 sec, respectively, P < 0.05). There was no significant difference in the rate of platelet/thrombus accumulation during the rapid phase or the maximum platelet/thrombus accumulation. H2‐calponin knockout mice also had prolonged bleeding time and blood loss. H2‐calponin in platelets facilitates early interactions between platelets and collagen during physiologic flow, but does not significantly affect the rate or magnitude of platelet/thrombus accumulation. H2‐calponin knockout mice take 2.3 times longer to achieve hemostasis compared to wild‐type controls in a tail bleeding model. The ability to delay platelet accumulation without inhibiting downstream thrombotic potential would be of significant therapeutic value, thus h2‐calponin may be a novel target for therapeutic platelet inhibition. PMID:25472609

  2. The effect of the perfluorocarbon emulsion Oxycyte on platelet count and function in the treatment of decompression sickness in a swine model.

    PubMed

    Cronin, William A; Senese, Angela L; Arnaud, Francoise G; Regis, David P; Auker, Charles R; Mahon, Richard T

    2016-09-01

    Decompression from elevated ambient pressure is associated with platelet activation and decreased platelet counts. Standard treatment for decompression sickness (DCS) is hyperbaric oxygen therapy. Intravenous perfluorocarbon (PFC) emulsion is a nonrecompressive therapy being examined that improves mortality in animal models of DCS. However, PFC emulsions are associated with a decreased platelet count. We used a swine model of DCS to study the effect of PFC therapy on platelet count, function, and hemostasis. Castrated male swine (n = 50) were fitted with a vascular port, recovered, randomized, and compressed to 180 feet of sea water (fsw) for 31 min followed by decompression at 30 fsw/min. Animals were observed for DCS, administered 100% oxygen, and treated with either emulsified PFC Oxycyte (DCS-PFC) or isotonic saline (DCS-NS). Controls underwent the same procedures, but were not compressed (Sham-PFC and Sham-NS). Measurements of platelet count, thromboelastometry, and coagulation were obtained 1 h before compression and 1, 24, 48, 96, 168 and 192 h after treatment. No significant changes in normalized platelet counts were observed. Prothrombin time was elevated in DCS-PFC from 48 to 192 h compared with DCS-NS, and from 96 to 192 h compared with Sham-PFC. Normalized activated partial thromboplastin time was also elevated in DCS-PFC from 168 to 192 h compared with Sham-PFC. No bleeding events were noted. DCS treated with PFC (Oxycyte) does not impact platelet numbers, whole blood clotting by thromboelastometry, or clinical bleeding. Late changes in prothrombin time and activated partial thromboplastin time associated with PFC use in both DCS therapy and controls warrant further investigation.

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

    PubMed

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

    2011-08-18

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

  4. [Covalent chloramine inhibitors of blood platelet functions: computational indices for their reactivity and antiplatelet activity].

    PubMed

    Roshchupkin, D I; Murina, M A; Sergienko, V I

    2011-01-01

    The quantum mechanics computation of the reactivities of chloramine derivatives of amino acids and taurine has been accomplished. A pair of computational indices that reflect a predisposition of alpha amino acid chloramines to chemical decay have been revealed. One of the indices was the dihedral angle for the chain of four atoms: carbons at beta- and alpha-positions, carbon of the carboxyl group, and carbonyl oxygen. The second index was the sum of partial charges for three or two carbon atoms in the chain. The amino acid chloramines with high values of the indices showed enhanced stability. Partial charges for active chlorine in known chloramines having different structures have been computed. The charges correlate with the rate constants of the reaction between chloramines and the thiol group of reduced glutathione. New derivatives of taurine chloramines have been constructed via the introduction of different substituents into the chloramine part. Among them, the amidoderivatives had the greatest charges of active chlorine (0.19-0.23). It was found in the study of the reactions of N-acetyl-N-chlorotaurine and N-propyonyl-N-chlorotaurine with amino acids and peptides possessing the thiol, thioester, or disulphide groups that the amidoderivatives manifested the thiol chemoselectivity. N-Acetyl-N-chlorotaurine and N-propionyl-N-chlorotaurine suppress the aggregation activity of blood platelets under their activation by the agonists ADP and collagen. It is not excluded that the amidoderivatives studied prevent platelet aggregation by a modification of the critical thiol group in the purine receptor P2Y12. PMID:22117450

  5. Radioimmunoassay of factor V in human plasma and platelets

    SciTech Connect

    Tracy, P.B.; Eide, L.L.; Bowie, E.J.W.; Mann, K.G.

    1982-07-01

    Homogeneous, single-chain human factor V was used to develop a double antibody competition radioimmunoassay to measure factor V concentrations in plasma and platelets. Standard curves were constructed that allow for the detection of as little as 20 ng factor V/ml of plasma. Normal factor V concentrations range from 4 to 14 ..mu..g/ml of plasma with an average value of 7.0 +/- 2.0 ..mu..g/ml (n = 64). No correlation was observed between antigen levels and age or sex. The radioimmunoassay data are consistent with factor V clotting assays, providing freshly drawn plasma is used in the bioassay. Radioimmunoassay of washed platelets indicate that 0.63-1.93 ..mu..g of factor V is present per 2.5 X 10/sup 8/ platelets (6412-14128 molecules of factor V per platelet). When normalized to individual hematocrits and platelet count, the data indicated that platelets contribute approximately 18%-25% of the factor V found in whole blood. In addition, two individuals with functionally deficient factor V were examined and found to be deficient in both antigen and activity.

  6. Platelet dysfunction-eosinophilia syndrome in parasitized Venezuelan children.

    PubMed

    Ruiz-Sáez, Arlette; Sifontes, Luz Núñez; Feijoo, Rosa; Certad, Gabriela; Arenas-Pinto, Alejandro; Pocaterra, Leonor; Ferrara, Guiseppe; Giménez, Rita; Torres, Obdulita; Goldstein, Carlos; Bosch, Norma

    2005-08-01

    Platelet dysfunction was detected in six children with purpura and eosinophilia. We conducted clinical evaluations, hematologic and platelet function tests, clotting studies (bleeding time, prothrombin time, partial thromboplastin time, thrombin time, factor XIII, factor VIII, and von Willebrand factor), assays for IgG and IgM antibodies to platelets, and a search for stool parasites. Mild bleeding phenomena (ecchymoses, petechiae, epistaxis, and gingival) were transient. All children showed intestinal parasites and marked eosinophilia (mean count = 2,615.2 cells/muL, 95% confidence interval = 1,259.6-5,429.8). Main abnormalities included prolonged bleeding times (50%) and defective aggregation with collagen (100%) adrenaline (66%), or ADP (66%). Antibodies to platelets were not detected. Anti-parasite therapy reversed the hemorrhagic manifestations and normalized eosinophil counts and platelet alterations. No relationship could be established between excess eosinophils, intensity of bleeding, or type and degree of platelet abnormalities. Thrombocytopathic features mimicked the intrinsic defect of storage pool disease. The possible pathogenic roles of eosinophilia and parasitism are reviewed. This is the first report of this pathologic combination in Latin American children.

  7. Metalloproteolytic receptor shedding…platelets "acting their age".

    PubMed

    Andrews, Robert K; Gardiner, Elizabeth E

    2016-09-01

    Whilst significant effort has been focused on development of tools and approaches to clinically modulate activation processes that consume platelets, the platelet receptors that initiate activation processes remain untargeted. The modulation of receptor levels is also linked to underlying platelet aging processes which influence normal platelet lifespan and also the functionality and survival of stored platelets that are used in transfusion. In this review, we will focus on platelet adhesion receptors initiating thrombus formation, and discuss how regulation of levels of these receptors impact platelet function and platelet survival. PMID:27459696

  8. Platelet aggregability and in vivo platelet deposition in patients with ischemic cerebrovascular disease--evaluation by indium-111-platelet scintigraphy

    SciTech Connect

    Isaka, Y.; Kimura, K.; Uehara, A.; Hashikawa, K.; Mieno, M.; Matsumoto, M.; Handa, N.; Nakabayashi, S.; Imaizumi, M.; Kamada, T. )

    1989-12-15

    In ischemic cerebrovascular disease, it is not clear whether platelet function in vitro actually reflects the situation in vivo. Using indium-111 platelet scintigraphy as a method for detecting platelet activation in vivo, we tried to elucidate this problem. Twenty eight patients with chronic stage of ischemic cerebrovascular disease (CVD) and 17 control subjects were examined. Platelet scintigrams were positive in 9 of 28 patients in CVD, while all were negative in control. A comparison of the results obtained from qualitative platelet imaging and platelet aggregability was performed to evaluate whether threshold aggregation concentration (TAC) grade differed across the three groups (control, CVD patients without platelet deposition and CVD patients with platelet deposition). CVD patients with platelet deposition showed a higher TAC than those patients who did not show platelet deposition (P less than 0.05) or control subjects without platelet deposition (P less than 0.05). These results suggest that some patients in chronic stages of CVD may have active platelet deposition on carotid atheromatous lesions, and presence of platelet deposition in vivo could contribute to reduce platelet reactivity in peripheral blood.

  9. Combined Quantification of the Global Proteome, Phosphoproteome, and Proteolytic Cleavage to Characterize Altered Platelet Functions in the Human Scott Syndrome*

    PubMed Central

    Solari, Fiorella A.; Mattheij, Nadine J.A.; Burkhart, Julia M.; Swieringa, Frauke; Collins, Peter W.; Cosemans, Judith M.E.M.; Sickmann, Albert; Heemskerk, Johan W.M.; Zahedi, René P.

    2016-01-01

    The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca2+-dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca2+-dependent changes that are normally associated with phosphatidylserine exposure. PMID:27535140

  10. Platelet Count

    MedlinePlus

    ... rash Small purplish spots on the skin called purpura, caused by bleeding under the skin Testing may ... Idiopathic thrombocytopenia (ITP), also known as immune thrombocytopenic purpura, is the result of antibody production against platelets. ...

  11. An update on pediatric bleeding disorders: bleeding scores, benign joint hypermobility, and platelet function testing in the evaluation of the child with bleeding symptoms.

    PubMed

    O'Brien, Sarah H

    2012-05-01

    Evaluating a child with symptoms of easy bruising and/or bleeding remains a challenge in pediatric hematology, and there is no "one size fits all" approach. This review focuses on recent research in three elements of the evaluation of a child with a suspected bleeding disorder. We will first discuss the development of the standardized Pediatric Bleeding Questionnaire, and its applications in research and clinical settings. We will then discuss the relationship between benign hypermobility syndromes and hemostasis, and the importance of including a Beighton Score in the physical examination of any child presenting with unusual bruising or bleeding. While prolonged bleeding times and abnormal platelet aggregation are common findings in children with benign hypermobility, normal coagulation studies do not exclude the presence of a connective tissue disorder in a child presenting with easy bleeding and joint hypermobility on examination. Finally, we will discuss the current state of knowledge regarding the laboratory evaluation of platelet function in children. Platelet function disorders are among the most common inherited bleeding disorders. However, testing for such disorders is time-consuming and requires a step-wise approach. We will review the indications for and limitations of the most commonly utilized platelet function laboratory studies.

  12. Repurposing CRISPR/Cas9 for in situ functional assays.

    PubMed

    Malina, Abba; Mills, John R; Cencic, Regina; Yan, Yifei; Fraser, James; Schippers, Laura M; Paquet, Marilène; Dostie, Josée; Pelletier, Jerry

    2013-12-01

    RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel "all-in-one" lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an "all-in-one" system to track disrupted Trp53 in chemoresistant lymphomas in the Eμ-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens.

  13. The antidepressant 5-HT2A receptor antagonists pizotifen and cyproheptadine inhibit serotonin-enhanced platelet function.

    PubMed

    Lin, Olivia A; Karim, Zubair A; Vemana, Hari Priya; Espinosa, Enma V P; Khasawneh, Fadi T

    2014-01-01

    There is considerable interest in defining new agents or targets for antithrombotic purposes. The 5-HT2A receptor is a G-protein coupled receptor (GPCR) expressed on many cell types, and a known therapeutic target for many disease states. This serotonin receptor is also known to regulate platelet function. Thus, in our FDA-approved drug repurposing efforts, we investigated the antiplatelet activity of cyproheptadine and pizotifen, two antidepressant 5-HT2A Receptor antagonists. Our results revealed that cyproheptadine and pizotifen reversed serotonin-enhanced ADP-induced platelet aggregation in vitro and ex vivo. And the inhibitory effects of these two agents were found to be similar to that of EMD 281014, a 5-HT2A Receptor antagonist under development. In separate experiments, our studies revealed that these 5-HT2A receptor antagonists have the capacity to reduce serotonin-enhanced ADP-induced elevation in intracellular calcium levels and tyrosine phosphorylation. Using flow cytometry, we also observed that cyproheptadine, pizotifen, and EMD 281014 inhibited serotonin-enhanced ADP-induced phosphatidylserine (PS) exposure, P-selectin expression, and glycoprotein IIb-IIIa activation. Furthermore, using a carotid artery thrombosis model, these agents prolonged the time for thrombotic occlusion in mice in vivo. Finally, the tail-bleeding time was investigated to assess the effect of cyproheptadine and pizotifen on hemostasis. Our findings indicated prolonged bleeding time in both cyproheptadine- and pizotifen-treated mice. Notably, the increases in occlusion and bleeding times associated with these two agents were comparable to that of EMD 281014, and to clopidogrel, a commonly used antiplatelet drug, again, in a fashion comparable to clopidogrel and EMD 281014. Collectively, our data indicate that the antidepressant 5-HT2A antagonists, cyproheptadine and pizotifen do exert antiplatelet and thromboprotective effects, but similar to clopidogrel and EMD 281014, their

  14. [Effect of lovastatin on adhesive and aggregation function of platelets in patients with arterial hypertension and dislipidemia].

    PubMed

    Medvedev, I N; Skoriatina, I A

    2010-01-01

    The aim of the study was to evaluate efficiency of correction of lipid profile disturbances and platelet dysfunction by lovastatin in patients with arterial hypertension and dyslipidemia. Lovastatin was given to 29 patients for 4 months. The main parameters measured included dynamics of blood lipid profile, lipid peroxidation in plasma and platelets, antioxidative protection of blood fluid and platelets, platelet activity. t-Students test was used to assess statistical significance of the results. It was shown that lovastatin has beneficial effect on dyslipoproteidemia and peroxidation syndrome. Moreover, it normalizes intraplatelet regulatory mechanisms and inhibits enhanced platelet activity. Effects of lovastatin in patients with arterial hypertension and dyslipidemia persist under conditions of long-term therapy.

  15. A point mutation in the EGF-4 domain of β3 integrin is responsible for the formation of the Seca platelet alloantigen and affects receptor function

    PubMed Central

    Sachs, Ulrich J.; Bakchoul, Tamam; Eva, Olga; Giptner, Astrid; Bein, Gregor; Aster, Richard H.; Gitter, Maria; Peterson, Julie; Santoso, Sentot

    2013-01-01

    Summary Neonatal alloimmune thrombocytopenia (NAIT) is caused by fetomaternal platelet incompatibility with maternal antibodies crossing the placenta and destroying fetal platelets. Antibodies against human platelet antigen-1a (HPA-1a) and HPA-5b are responsible for the majority of NAIT cases. We observed a suspected NAIT in a newborn with a platelet count of 25 G/l and petechial haemorrhages. Serological analysis of maternal serum revealed an immunisation against αIIbβ3 on paternal platelets only, indicating the presence of an antibody against a new rare alloantigen (Seca) residing on αIIbβ3. The location of Seca on αIIbβ3 was confirmed by immunoprecipitation. Nucleotide sequence analysis of paternal β3 revealed a single nucleotide exchange (G1818T) in exon 11 of the β3 gene (ITGB3), changing Lys580 (wild-type) to Asn580 (Seca). Two additional members of the family Sec were typed Seca positive, but none of 300 blood donors. Chinese hamster ovary cells expressing Asn580, but not Lys580 αIIbβ3, bound anti-Seca, which was corroborated by immunoprecipitation. Adhesion of transfected cells onto immobilised fibrinogen showed reduced binding of the Asn580 variant compared to wild-type αIIbβ3. Analysis of transfected cells with anti-LIBS and PAC-1 antibody showed reduced binding when compared to the wild-type. No such effects were observed with Seca positive platelets, which, however, are heterozygous for the Lys580Asn mutation. In this study, we describe a NAIT case caused by maternal alloimmunisation against a new antigen on αIIbβ3. Analysis with mutant transfected cells showed that the Lys580Asn mutation responsible for the formation of the Seca antigenic determinant affects αIIbβ3 receptor function. PMID:22116617

  16. The effects of cytochalasin D and abciximab on hemostasis in canine whole blood assessed by thromboelastography and the PFA-100® platelet function analyzer system.

    PubMed

    Brainard, Benjamin M; Abed, Janan M; Koenig, Amie

    2011-07-01

    The selective inhibition of platelet function in whole blood coagulation testing may allow insights into the nature of hypercoagulability in dogs with critical illness. To determine the effects of cytochalasin D and abciximab on hemostatic parameters in canine citrated whole blood, an in-vitro study was designed using thromboelastography (TEG) and a platelet function analyzer (PFA-100®). 8 clinically healthy mixed breed dogs donated blood that was anticoagulated with 3.2% sodium citrate in a 9:1 blood-to-citrate ratio. Addition of cytochalasin D to citrated whole blood from 6 dogs at concentrations ranging from 0 µg/ml to 10 µg/ml caused a maximal reduction of TEG maximum amplitude (MA) at a concentration of 7.5 µg/ml (52.7 ± 4.3 to 14.3 ± 7.8 mm). Addition of abciximab to canine citrated whole blood at concentrations of either 20 µg/ml or 40 µg/ml did not affect the TEG tracing; however, addition of abciximab to citrated canine whole blood at concentrations of 10 µg/ml and 20 µg/ml significantly prolonged PFA-100 closure times (72.5 ± 15 to 149.2 ± 91 sec and 275.6 ± 54 sec, respectively, P < 0.04). Inhibition of canine platelet function by cytochalasin D is demonstrated by TEG, but abciximab did not change TEG tracings. Abciximab does, however, inhibit platelet aggregation under shear stress as measured by the PFA-100. Inhibition of canine platelet function with cytochalasin D may allow further TEG studies in dogs with clinical disease.

  17. Nanosecond pulsed platelet-rich plasma (nsPRP) improves mechanical and electrical cardiac function following myocardial reperfusion injury.

    PubMed

    Hargrave, Barbara; Varghese, Frency; Barabutis, Nektarios; Catravas, John; Zemlin, Christian

    2016-02-01

    Ischemia and reperfusion (I/R) of the heart is associated with biochemical and ionic changes that result in cardiac contractile and electrical dysfunction. In rabbits, platelet-rich plasma activated using nanosecond pulsed electric fields (nsPRP) has been shown to improve left ventricular pumping. Here, we demonstrate that nsPRP causes a similar improvement in mouse left ventricular function. We also show that nsPRP injection recovers electrical activity even before reperfusion begins. To uncover the mechanism of nsPRP action, we studied whether the enhanced left ventricular function in nsPRP rabbit and mouse hearts was associated with increased expression of heat-shock proteins and altered mitochondrial function under conditions of oxidative stress. Mouse hearts underwent 30 min of global ischemia and 1 h of reperfusion in situ. Rabbit hearts underwent 30 min of ischemia in vivo and were reperfused for 14 days. Hearts treated with nsPRP expressed significantly higher levels of Hsp27 and Hsp70 compared to hearts treated with vehicle. Also, pretreatment of cultured H9c2 cells with nsPRP significantly enhanced the "spare respiratory capacity (SRC)" also referred to as "respiratory reserve capacity" and ATP production in response to the uncoupler FCCP. These results suggest a cardioprotective effect of nsPRP on the ischemic heart during reperfusion. PMID:26908713

  18. Alterations in plasma membrane promote overexpression and increase of sodium influx through epithelial sodium channel in hypertensive platelets.

    PubMed

    Cerecedo, D; Martínez-Vieyra, Ivette; Sosa-Peinado, Alejandro; Cornejo-Garrido, Jorge; Ordaz-Pichardo, Cynthia; Benítez-Cardoza, Claudia

    2016-08-01

    Platelets are small, anucleated cell fragments that activate in response to a wide variety of stimuli, triggering a complex series of intracellular pathways leading to a hemostatic thrombus formation at vascular injury sites. However, in essential hypertension, platelet activation contributes to causing myocardial infarction and ischemic stroke. Reported abnormalities in platelet functions, such as platelet hyperactivity and hyperaggregability to several agonists, contribute to the pathogenesis and complications of thrombotic events associated with hypertension. Platelet membrane lipid composition and fluidity are determining for protein site accessibility, structural arrangement of platelet surface, and response to appropriate stimuli. The present study aimed to demonstrate whether structural and biochemical abnormalities in lipid membrane composition and fluidity characteristic of platelets from hypertensive patients influence the expression of the Epithelial Sodium Channel (ENaC), fundamental for sodium influx during collagen activation. Wb, cytometry and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assays demonstrated ENaC overexpression in platelets from hypertensive subjects and in relation to control subjects. Additionally, our results strongly suggest a key role of β-dystroglycan as a scaffold for the organization of ENaC and associated proteins. Understanding of the mechanisms of platelet alterations in hypertension should provide valuable information for the pathophysiology of hypertension. PMID:27137675

  19. Alterations in plasma membrane promote overexpression and increase of sodium influx through epithelial sodium channel in hypertensive platelets.

    PubMed

    Cerecedo, D; Martínez-Vieyra, Ivette; Sosa-Peinado, Alejandro; Cornejo-Garrido, Jorge; Ordaz-Pichardo, Cynthia; Benítez-Cardoza, Claudia

    2016-08-01

    Platelets are small, anucleated cell fragments that activate in response to a wide variety of stimuli, triggering a complex series of intracellular pathways leading to a hemostatic thrombus formation at vascular injury sites. However, in essential hypertension, platelet activation contributes to causing myocardial infarction and ischemic stroke. Reported abnormalities in platelet functions, such as platelet hyperactivity and hyperaggregability to several agonists, contribute to the pathogenesis and complications of thrombotic events associated with hypertension. Platelet membrane lipid composition and fluidity are determining for protein site accessibility, structural arrangement of platelet surface, and response to appropriate stimuli. The present study aimed to demonstrate whether structural and biochemical abnormalities in lipid membrane composition and fluidity characteristic of platelets from hypertensive patients influence the expression of the Epithelial Sodium Channel (ENaC), fundamental for sodium influx during collagen activation. Wb, cytometry and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assays demonstrated ENaC overexpression in platelets from hypertensive subjects and in relation to control subjects. Additionally, our results strongly suggest a key role of β-dystroglycan as a scaffold for the organization of ENaC and associated proteins. Understanding of the mechanisms of platelet alterations in hypertension should provide valuable information for the pathophysiology of hypertension.

  20. Evaluation of effects of various drugs on platelet functions using phorbol 12-myristate 13-acetate-induced megakaryocytic human erythroid leukemia cells

    PubMed Central

    Tada, Tomoki; Aki, Kensaku; Oboshi, Wataru; Kawazoe, Kazuyoshi; Yasui, Toshiyuki; Hosoi, Eiji

    2016-01-01

    Background The hyperfunction and activation of platelets have been strongly implicated in the development and recurrence of arterial occlusive disease, and various antiplatelet drugs are used to treat and prevent such diseases. New antiplatelet drugs and many other drugs have been developed, but some drugs may have adverse effects on platelet functions. Objective The aim of this study was to establish an evaluation method for evaluating the effect and adverse effect of various drugs on platelet functions. Materials and methods Human erythroid leukemia (HEL) cells were used after megakaryocytic differentiation with phorbol 12-myristate 13-acetate as an alternative to platelets. Drugs were evaluated by changes in intracellular Ca2+ concentration ([Ca2+]i) mobilization in Fura2-loaded phorbol 12-myristate 13-acetate-induced HEL cells. Aspirin and cilostazol were selected as antiplatelet drugs and ibuprofen and sodium valproate as other drugs. Results There was a positive correlation between [Ca2+]i and platelet aggregation induced by thrombin. Aspirin (5.6–560 µM) and cilostazol (5–10 µM) significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. On the other hand, ibuprofen (8–200 µM) and sodium valproate (50–1,000 µg/mL) also significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. Furthermore, the interaction effects of the simultaneous combined use of aspirin and ibuprofen or sodium valproate were evaluated. When the inhibitory effect of aspirin was higher than that of ibuprofen, the effect of aspirin was reduced, whereas when the inhibitory effect of aspirin was lower than that of ibuprofen, the effect of ibuprofen was reduced. The combination of aspirin and sodium valproate synergistically inhibited thrombin-induced [Ca2+]i. Conclusion It is possible to induce HEL cells to differentiate into megakaryocytes, which are a useful model for the study of platelet functions

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

    SciTech Connect

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

    2014-07-04

    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.

  2. Platelets, inflammation and tissue regeneration.

    PubMed

    Nurden, Alan T

    2011-05-01

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

  3. Utrophins compensate for Dp71 absence in mdx3cv in adhered platelets.

    PubMed

    Cerecedo, Doris; Mondragón, Ricardo; Candelario, Aurora; García-Sierra, Francisco; Mornet, Dominique; Rendón, Alvaro; Martínez-Rojas, Dalila

    2008-01-01

    Platelet adhesion is a critical step due to its hemostatic role in stopping bleeding after vascular damage. Short dystrophins are the most abundant dmd gene products in nonmuscle tissues, and in association with cytoskeleton proteins contribute to their intrinsic function; while utrophins are dystrophin-homologous related family proteins with structural and functional similarities. We previously demonstrated the presence of Dp71 isoforms, utrophins, and various dystrophin-associated proteins and their participation in cytoskeleton re-organization, filopodia and lamellipodia extension, and in centralizing cytoplasmic granules during the adhesion process of human platelets. To evaluate the morphologic changes and actin-based structures of mdx(3cv) platelets during the adhesion process, we compared the topographic distribution of Dp71d/Dp71Delta110(m) and dystrophin-associated protein in adhered platelets from dystrophic mdx(3cv) mouse. By confocal microscopy, we showed that absence of Dp71 isoforms in platelets from this animal model disrupted dystrophin-associated protein expression and distribution without modifying the platelet morphology displayed during the glass-adhesion process. By immunoprecipitation assays, we proved that up-regulated utrophins were associated with dystrophin-associated proteins to conform the dystrophin-associated protein complex corresponding to utrophins, which might compensate for Dp71 absence in mdx(3cv) platelets.

  4. pH-responsive drug delivery system based on AIE luminogen functionalized layered zirconium phosphate nano-platelets

    SciTech Connect

    Li, Dongdong Zhang, Yuping; Zhou, Bingbing

    2015-05-15

    Aggregation-induced emission (AIE) luminogen, quaternary tetraphenylethene cation (TPEN), was successfully incorporated into layered α-zirconium phosphate (α-ZrP) by using co-precipitation method to form inorganic–organic hybrid materials. The obtained materials show the characteristic hexagonal platelet shape with the interlayer distance did not reveal significant difference compared with pure α-ZrP. In addition, the obtained hybrid materials emit strong blue emission centered at 476 nm in aqueous media due to the electrostatic interactions of TPEN with the anionic framework of α-ZrP, which largely restrict their intramolecular rotation. More importantly, the materials provide a pH dependent release of doxorubicin (DOX), suggesting that AIE luminogen functionalized α-ZrP may be used as an imaging guided and pH-responsive delivery system for targeting therapy. - Graphical abstract: AIE luminogen was successfully incorporated into layered α-zirconium phosphate by a co-precipitation method to form inorganic–organic hybrid materials, showing a pH dependent release of DOX. - Highlights: • AIE luminogen cation was incorporated into layered α-ZrP by co-precipitation method. • The obtained material emits strong blue emission upon UV irradiation. • The material exhibits pH dependent release of DOX. • The AIE functionalized α-ZrP has potential applications in imaging guided therapy.

  5. Polymorphisms of PAI-1 and platelet GP Ia may associate with impairment of renal function and thrombocytopenia in Puumala hantavirus infection

    PubMed Central

    Laine, Outi; Joutsi-Korhonen, Lotta; Mäkelä, Satu; Mikkelsson, Jussi; Pessi, Tanja; Tuomisto, Sari; Huhtala, Heini; Libraty, Daniel; Vaheri, Antti; Karhunen, Pekka; Mustonen, Jukka

    2013-01-01

    Introduction Puumala virus (PUUV) infection is a viral hemorrhagic fever with renal syndrome (HFRS) characterized by thrombocytopenia and acute impairment of renal function. We aimed to assess whether genetic polymorphisms of platelet antigens together with those of von Willebrand factor (VWF) and plasminogen activator inhibitor (PAI-1) correlate with disease severity. Patients and methods 172 consecutive hospital-treated patients with serologically confirmed acute PUUV infection were included. Platelet glycoprotein (GP) IIIa T>C (rs5918), GP Ia T>C (rs1126643), GP Ib C>T (rs6065), GP VI T>C (rs1613662), VWF A>G (rs1063856) and PAI-1 A>G (rs2227631) were genotyped. The associations of the rarer alleles with variables reflecting the severity of the disease were analyzed. Results PAI-1 G-carriers had higher maximum creatinine level compared with the non-carriers (median 213 μmol/l, range 60–1499 μmol/l vs. median 122 μmol/l, range 51–1156 μmol/l, p=0.01). The GG-genotypes had higher creatinine levels than GA- and AA-genotypes (medians 249 μmol/l, 204 μmol/l and 122 μmol/l, respectively, p=0.03). Polymorphisms of GP VI and VWF associated with lower creatinine levels during PUUV infection. The minor C-allele of GP Ia associated with lower platelet counts (median 44×109/l, range 20–90×109/l vs median 64×109/l, range 3–238×109/l; p=0.02). Conclusions Polymorphism of PAI-1, a major regulator of fibrinolysis, has an adverse impact on the outcome of kidney function in PUUV-HFRS. Platelet collagen receptor GP Ia polymorphism associates with lower platelet count. PMID:22133274

  6. Altered bioavailability of platelet-derived factor VIII during thrombocytosis reverses phenotypic efficacy in haemophilic mice.

    PubMed

    Damon, Andrea L; Scudder, Lesley E; Gnatenko, Dmitri V; Sitaraman, Varsha; Hearing, Patrick; Jesty, Jolyon; Bahou, Wadie F

    2008-12-01

    Ectopic delivery of factor VIII (FVIII) to megakaryocytes (Mk) represents a viable approach for localized tenase generation by concentrating the FVIIIa/FIXa enzyme-cofactor complex onto activated platelet membranes. We utilized a core rat platelet factor 4 (PF4) promoter for Mk/platelet-restricted expression of human B-domain-deleted (hBDD) FVIII within the background of a haemophilia A mouse (rPF4/hBDD/FVIII-/-). Platelets from rPF4/hBDD/FVIII(-/-) mice contained approximately 122 mU FVIII:C/1 x 10(9) platelets/ml with no detectable plasmatic FVIII:C, and with no effect on alpha-granule-derived platelet factor V/Va function. Paired tenase assays (+/- thrombin) confirmed that platelet (pt) FVIII (unlike platelet FV) required thrombin cleavage for complete activation. rPF4/hBDD/FVIII(-/-) mice exposed to a thrombocytotic stimulus (thrombopoietin, TPO) demonstrated a statistically-significant 66% reduction in molar ptFVIII activity with a non-significant reduction in total ptFVIII biomass. Decreased molar ptFVIII concentration correlated with loss of phenotypic correction as evaluated using a haemostatic tail-snip assay. Comparative studies using a transgenic mouse expressing human amyloid-beta-precursor protein (hAbetaPP) from the rPF4 promoter confirmed diminished hAbetaPP expression without affecting endogenous alpha-granule PF4, establishing generalizability of these observations. While Mk/platelet-released ptFVIII (unlike pFV) is proteolytically inactive, we also conclude that thrombocytotic stimuli negatively affect ptFVIII bioavailability and phenotypic efficacy, results which correlate best with molar ptFVIII concentration, and not systemically available ptFVIII.

  7. Lack of influence of the COX inhibitors metamizol and diclofenac on platelet GPIIb/IIIa and P-selectin expression in vitro

    PubMed Central

    Scheinichen, Dirk; Elsner, Holger-Andreas; Osorio, Rodin; Jüttner, Björn; Gröschel, Werner; Jaeger, Karsten; Piepenbrock, Siegfried

    2004-01-01

    Background The effect of non-steroidal anti-inflammatory drugs (NSAIDs) for reduced platelet aggregation and thromboxane A2 synthesis has been well documented. However, the influence on platelet function is not fully explained. Aim of this study was to examine the influence of the COX-1 inhibiting NSAIDs, diclofenac and metamizol on platelet activation and leukocyte-platelet complexes, in vitro. Surface expression of GPIIb/IIIa and P-selectin on platelets, and the percentage of platelet-leukocyte complexes were investigated. Methods Whole blood was incubated with three different concentrations of diclofenac and metamizol for 5 and 30 minutes, followed by activation with TRAP-6 and ADP. Rates of GPIIb/IIIa and P-selectin expression, and the percentage of platelet-leukocyte complexes were analyzed by a flow-cytometric assay. Results There were no significant differences in the expression of GPIIb/IIIa and P-selectin, and in the formation of platelet-leukocyte complexes after activation with ADP and TRAP-6, regarding both the time of incubation and the concentrations of diclofenac and metamizol. Conclusions Accordingly, the inhibitory effect of diclofenac and metamizol on platelet aggregation is not related to a reduced surface expression of P-selectin and GPIIb/IIIa on platelets. PMID:15107131

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

    PubMed

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

    2016-01-01

    Platelets are the anucleated blood cells, wich together with the fibrin stop bleeding (hemostasis). Cellular microvesicles are membrane-surrounded microparticles released into extracellular space upon activation and/or apoptosis of various cells. Platelet-derived macrovesicles from the major population of circulating blood microparticles that play an important role in hemostasis and thrombosis. Despite numerous studies on the pathophysiology of platelet-derived macrovesicles, mechanisms of their formation and structural details remain poorly understood. Here we investigated the ultrastructure of parental platelets and platelet-derived microvesicles formed in vitro by quiescent cells as well as by cells stimulated with one of the following activators: arachidonic acid, ADP, thrombin, calcium ionophore A23187. Using transmission electron microscopy of human platelets and isolated microvesicles, we analyzed the intracellular origin, steps of formation, structural diversity, and size distributions of the subcellular particles. We have revealed that thrombin, unlike other stimuli, not only induced vesiculation of the plasma membrane but also caused break-up of the cells followed by formation of microparticles that are comparable with microvesicles by size. A fraction of these microparticles contained cellular organelles surrounded by a thin membrane. The size of platelet-derived macrovesicles varied from 30 nm to 500 nm, however, the size distributions depended on the nature of a cell-activating stimulus. The results obtained provide new information about the formation of platelet-derived macrovesicles and their structural diversity, wich is important to understand their multiple functions in normal and disease states. PMID:27228656

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

    PubMed

    Clemetson, Kenneth J

    2015-01-01

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

  10. Rupture Forces among Human Blood Platelets at different Degrees of Activation.

    PubMed

    Nguyen, Thi-Huong; Palankar, Raghavendra; Bui, Van-Chien; Medvedev, Nikolay; Greinacher, Andreas; Delcea, Mihaela

    2016-01-01

    Little is known about mechanics underlying the interaction among platelets during activation and aggregation. Although the strength of a blood thrombus has likely major biological importance, no previous study has measured directly the adhesion forces of single platelet-platelet interaction at different activation states. Here, we filled this void first, by minimizing surface mediated platelet-activation and second, by generating a strong adhesion force between a single platelet and an AFM cantilever, preventing early platelet detachment. We applied our setup to measure rupture forces between two platelets using different platelet activation states, and blockade of platelet receptors. The rupture force was found to increase proportionally to the degree of platelet activation, but reduced with blockade of specific platelet receptors. Quantification of single platelet-platelet interaction provides major perspectives for testing and improving biocompatibility of new materials; quantifying the effect of drugs on platelet function; and assessing the mechanical characteristics of acquired/inherited platelet defects.

  11. Rupture Forces among Human Blood Platelets at different Degrees of Activation

    PubMed Central

    Nguyen, Thi-Huong; Palankar, Raghavendra; Bui, Van-Chien; Medvedev, Nikolay; Greinacher, Andreas; Delcea, Mihaela

    2016-01-01

    Little is known about mechanics underlying the interaction among platelets during activation and aggregation. Although the strength of a blood thrombus has likely major biological importance, no previous study has measured directly the adhesion forces of single platelet-platelet interaction at different activation states. Here, we filled this void first, by minimizing surface mediated platelet-activation and second, by generating a strong adhesion force between a single platelet and an AFM cantilever, preventing early platelet detachment. We applied our setup to measure rupture forces between two platelets using different platelet activation states, and blockade of platelet receptors. The rupture force was found to increase proportionally to the degree of platelet activation, but reduced with blockade of specific platelet receptors. Quantification of single platelet-platelet interaction provides major perspectives for testing and improving biocompatibility of new materials; quantifying the effect of drugs on platelet function; and assessing the mechanical characteristics of acquired/inherited platelet defects. PMID:27146004

  12. Adipose-derived stem cells and platelet-rich plasma: the keys to functional periodontal tissue engineering.

    PubMed

    Tobita, Morikuni; Mizuno, Hiroshi

    2013-09-01

    Numerous different types of periodontal tissue regeneration therapies have been developed clinically with variable outcomes and serious limitations. A key goal of periodontal therapy is to regenerate the destroyed periodontal tissues including alveolar bone, cementum and periodontal ligament. The critical factors in attaining successful periodontal tissue regeneration are the correct recruitment of cells to the site and the production of a suitable extra cellular matrix consistent with the periodontal tissues. Adipose tissue, from which mesenchymal stem cells can be harvested easily and safely, is an especially attractive stem cell source, because adipose-derived stem cells have a strong potential for cell differentiation and growth factor secretion. Meanwhile, the usefulness of platelet-rich plasma in the field of dental surgery has attracted attention. Therapeutic effects of platelet-rich plasma are believed to occur through the provision of concentrated levels of platelet-derived growth factors. Further, recent reports suggested the effect of platelet-rich plasma on mesenchymal stem cell proliferation, differentiation and survival rate. Therefore, the admixture of mesenchymal stem cells and platelet-rich plasma may indicate the great potential for tissue regenerations including periodontal tissue regeneration. In this review, the potential of adipose-derived stem cells and platelet-rich plasma is introduced. Of particular interest, the usefulness in periodontal tissue regeneration and future perspective is discussed.

  13. Functional assays to define agonists and antagonists of the sigma-2 receptor

    PubMed Central

    Zeng, Chenbo; Rothfuss, Justin M.; Zhang, Jun; Vangveravong, Suwanna; Chu, Wenhua; Li, Shihong; Tu, Zhude; Xu, Jinbin; Mach, Robert H.

    2014-01-01

    The sigma-2 receptor has been identified as a biomarker in proliferating tumors. Up to date there is no well-established functional assay for defining sigma-2 agonists and antagonists. Many sigma-2 ligands with diverse structures have been shown to induce cell death in a variety of cancer cells by triggering caspase-dependent and independent apoptosis. Therefore, in the current study, we used the cell viability assay and the caspase-3 activity assay to determine sigma-2 agonists and antagonists. Three classes of sigma-2 ligands developed in our laboratory were evaluated for their potency to induce cell death in two tumor cell lines, mouse breast cancer cell line EMT-6 and human melanoma cell line MDA-MB-435. The data showed that the EC50 values of the sigma-2 ligands using the cell viability assay ranged from 11.4 μM to >200 μM, which were comparable with the EC50 values obtained using the caspase-3 assay. Based on the cytotoxicity of a sigma-2 ligand relative to that of siramesine, a commonly accepted sigma-2 agonist, we have categorized our sigma-2 ligands into agonists, partial agonists, and antagonists. The establishment of functional assays for defining sigma-2 agonists and antagonists will facilitate functional characterization of sigma-2 receptor ligands and sigma-2 receptors. PMID:24333652

  14. The role of point-of-care platelet function testing in predicting postoperative bleeding following cardiac surgery: a systematic review and meta-analysis.

    PubMed

    Corredor, C; Wasowicz, M; Karkouti, K; Sharma, V

    2015-06-01

    This systematic review and meta-analysis appraises the utility of point-of-care platelet function tests for predicting blood loss and transfusion requirements in cardiac surgical patients, and analyses whether their use within a transfusion management algorithm is associated with improved patient outcomes. We included 30 observational studies incorporating 3044 patients in the qualitative assessment, and nine randomised controlled trials including 1057 patients in the meta-analysis. Platelet function tests demonstrated significant variability in their ability to predict blood loss and transfusion requirements. Their use within a blood transfusion algorithm demonstrated a reduction in blood loss at longest follow-up (mean difference -102.9 ml (95% CI -149.9 to -56.1 ml), p < 0.001), and transfusion of packed red cells (RR 0.86 (95% CI 0.78-0.94), p = 0.001) and fresh frozen plasma (RR 0.42 (95% CI 0.30-0.59), p < 0.001). Viscoelastic methods used in combination with other platelet function tests achieved greater reduction in blood loss (mean difference -111.8 ml (95% CI -174.9 to -49.1 ml), p = 0.0005) compared with their use alone (mean difference -90.6 ml (95% CI 166.1-15.0 ml), p = 0.02). We conclude that incorporation of point-of-care platelet function tests into transfusion management algorithms is associated with a reduction in blood loss and transfusion requirements in cardiac surgery patients. PMID:25916344

  15. Platelets: crossroads of immunity and hemostasis.

    PubMed

    Jenne, Craig N

    2014-07-31

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

  16. Platelet adhesion to decorin but not collagen I correlates with the integrin α2 dimorphism E534K, the basis of the human platelet alloantigen (HPA)-5 system

    PubMed Central

    Kunicki, Thomas J.; Williams, Shirley A.; Diaz, Daniel; Farndale, Richard W.; Nugent, Diane J.

    2012-01-01

    A single nucleotide polymorphism in the integrin α2 gene ITGA2 (rs1801106; G1600A) creates the non-conservative amino acid substitution E534K, the basis of the human platelet alloantigen system HPA-5. Yet HPA-5 alleles do not influence binding of α2β1 to its primary ligand collagen I, and the effect of HPA-5 on platelet function has not been determined. We used a direct platelet adhesion assay to evaluate whether differential inheritance of HPA-5 alleles influences platelet adhesion to collagen I or an alternative ligand, decorin. Platelets from donors bearing one or more minor allele HPA-5b showed attenuated adhesion to purified decorin but not collagen I. Adhesion to decorin was significantly inhibited by human alloantibodies specific for HPA-5a but not by the collagen I sequence GFOGER or α2-specific inhibitory monoclonal antibodies. The minor allele 534K attenuates platelet adhesion to decorin but not collagen I, providing the first evidence of a functional effect of HPA-5 alleles. PMID:22133774

  17. Humanizing the Protease-Activated Receptor (PAR) Expression Profile in Mouse Platelets by Knocking PAR1 into the Par3 Locus Reveals PAR1 Expression Is Not Tolerated in Mouse Platelets

    PubMed Central

    French, Shauna L.; Paramitha, Antonia C.; Moon, Mitchell J.; Dickins, Ross A.; Hamilton, Justin R.

    2016-01-01

    Anti-platelet drugs are the mainstay of pharmacotherapy for heart attack and stroke prevention, yet improvements are continually sought. Thrombin is the most potent activator of platelets and targeting platelet thrombin receptors (protease-activated receptors; PARs) is an emerging anti-thrombotic approach. Humans express two PARs on their platelets–PAR1 and PAR4. The first PAR1 antagonist was recently approved for clinical use and PAR4 antagonists are in early clinical development. However, pre-clinical studies examining platelet PAR function are challenging because the platelets of non-primates do not accurately reflect the PAR expression profile of human platelets. Mice, for example, express Par3 and Par4. To address this limitation, we aimed to develop a genetically modified mouse that would express the same repertoire of platelet PARs as humans. Here, human PAR1 preceded by a lox-stop-lox was knocked into the mouse Par3 locus, and then expressed in a platelet-specific manner (hPAR1-KI mice). Despite correct targeting and the predicted loss of Par3 expression and function in platelets from hPAR1-KI mice, no PAR1 expression or function was detected. Specifically, PAR1 was not detected on the platelet surface nor internally by flow cytometry nor in whole cell lysates by Western blot, while a PAR1-activating peptide failed to induce platelet activation assessed by either aggregation or surface P-selectin expression. Platelets from hPAR1-KI mice did display significantly diminished responsiveness to thrombin stimulation in both assays, consistent with a Par3-/- phenotype. In contrast to the observations in hPAR1-KI mouse platelets, the PAR1 construct used here was successfully expressed in HEK293T cells. Together, these data suggest ectopic PAR1 expression is not tolerated in mouse platelets and indicate a different approach is required to develop a small animal model for the purpose of any future preclinical testing of PAR antagonists as anti-platelet drugs. PMID

  18. Extracts from Tribulus species may modulate platelet adhesion by interfering with arachidonic acid metabolism.

    PubMed

    Olas, Beata; Hamed, Arafa I; Oleszek, Wieslaw; Stochmal, Anna

    2015-01-01

    The present work was designed to study the effects of crude extracts from Tribulus pterocarpus, T. pentandrus and T. parvispinus on selected biological functions of human blood platelets in vitro. Platelet suspensions were pre-incubated with extracts from aerial parts of T. pterocarpus, T. pentandrus and T. parvispinus, at the final concentrations of 0.5, 5 and 50 µg/ml. Then, for platelet activation thrombin, was used. The effects of crude extracts from T. pterocarpus, T. pentandrus and T. parvispinus on adhesion of blood platelets to collagen were determined by method according to Tuszynski and Murphy. Arachidonic acid metabolism was measured by the level of thiobarbituric acid reactive substances (TBARS). In these studies we also compared the action of tested crude plant extracts with the effects of the polyphenolic fraction isolated from aerial parts of T. pterocarpus, which has antiplatelet and antioxidative properties. The performed assays demonstrated that the tested crude extract from T. pterocarpus and the phenolic fraction from T. pterocarpus might influence the platelet functions in vitro. The inhibitory, concentration-dependent effects of this tested extract and its phenolic fraction on adhesion of resting platelets and thrombin - stimulated platelets to collagen was found. We also observed that the crude extract from T. pterocarpus, like the polyphenolic fraction from T. pterocarpus reduced TBARS production in blood platelets. In the comparative studies, the tested crude extract from T. pterocarpus was not found to be more effective antiplatelet factor, than the polyphenolic fraction from this plant. The results obtained suggest that T. pterocarpus may be a promising source of natural compounds, valuable in the prevention of the enhanced activity of blood platelets in numerous cardiovascular diseases.

  19. Dissecting functions of the conserved oligomeric Golgi tethering complex using a cell-free assay.

    PubMed

    Cottam, Nathanael P; Wilson, Katherine M; Ng, Bobby G; Körner, Christian; Freeze, Hudson H; Ungar, Daniel

    2014-01-01

    Vesicle transport sorts proteins between compartments and is thereby responsible for generating the non-uniform protein distribution along the eukaryotic secretory and endocytic pathways. The mechanistic details of specific vesicle targeting are not yet well characterized at the molecular level. We have developed a cell-free assay that reconstitutes vesicle targeting utilizing the recycling of resident enzymes within the Golgi apparatus. The assay has physiological properties, and could be used to show that the two lobes of the conserved oligomeric Golgi tethering complex play antagonistic roles in trans-Golgi vesicle targeting. Moreover, we can show that the assay is sensitive to several different congenital defects that disrupt Golgi function and therefore cause glycosylation disorders. Consequently, this assay will allow mechanistic insight into the targeting step of vesicle transport at the Golgi, and could also be useful for characterizing some novel cases of congenital glycosylation disorders.

  20. Platelets and plasma stimulate sheep rotator cuff tendon tenocytes when cultured in an extracellular matrix scaffold.

    PubMed

    Kelly, Brian A; Proffen, Benedikt L; Haslauer, Carla M; Murray, Martha M

    2016-04-01

    The addition of platelet-rich plasma (PRP) to rotator cuff repair has not translated into improved outcomes after surgery. However, recent work stimulating ligament healing has demonstrated improved outcomes when PRP or whole blood is combined with an extracellular matrix carrier. The objective of this study was to evaluate the effect of three components of blood (plasma, platelets, and macrophages) on the in vitro activity of ovine rotator cuff cells cultured in an extracellular matrix environment. Tenocytes were obtained from six ovine infraspinatus tendons and cultured over 14 days in an extracellular matrix scaffold with the following additives: (1) plasma (PPP), (2) plasma and platelets (PAP), (3) plasma and macrophages (PPPM), (4) plasma, platelets and macrophages (PAPM), (5) phosphate buffered saline (PBS), and (6) PBS with macrophages (PBSM). Assays measuring cellular metabolism (AlamarBlue), proliferation (Quantitative DNA assay), synthesis of collagen and cytokines (SIRCOL, TNF-α and IL-10 ELISA, and MMP assay), and collagen gene expression (qPCR) were performed over the duration of the experiment, as well as histology at the conclusion. Plasma was found to stimulate cell attachment and spreading on the scaffold, as well as cellular proliferation. Platelets also stimulated cell proliferation, cellular metabolism, transition of cells to a myofibroblast phenotype, and contraction of the scaffolds. The addition of macrophages did not have any significant effect on the sheep rotator cuff cells in vitro. In vivo studies are needed to determine whether these changes in cellular function will translate into improved tendon healing.

  1. Phosphorylation of platelet actin-binding protein during platelet activation

    SciTech Connect

    Carroll, R.C.; Gerrard, J.M.

    1982-03-01

    In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide-sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2 to 3-fold labeling increases in actin-binding protein 30 to 60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P-labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

  2. Increases in ambient particulate matter air pollution, acute changes in platelet function, and effect modification by aspirin and omega-3 fatty acids: A panel study.

    PubMed

    Becerra, Adan Z; Georas, Steve; Brenna, J Thomas; Hopke, Philip K; Kane, Cathleen; Chalupa, David; Frampton, Mark W; Block, Robert; Rich, David Q

    2016-01-01

    Increased particulate matter (PM) air pollutant concentrations have been associated with platelet activation. It was postulated that elevated air pollutant concentrations would be associated with increases in measures of platelet function and that responses would be blunted when taking aspirin and/or fish oil. Data from a sequential therapy trial (30 subjects with type 2 diabetes mellitus), with 4 clinic visits (first: no supplements, second: aspirin, third: omega-3 fatty acid supplements, fourth: aspirin and omega-3 fatty acids) per subject, were utilized. Using linear mixed models, adjusted for relative humidity, temperature, visit number, and season, changes in three platelet function measures including (1) aggregation induced by adenosine diphosphate (ADP), (2) aggregation induced by collagen, and (3) thromboxane B2 production were associated with interquartile range (IQR) increases in mean concentrations of ambient PM2.5, black carbon, ultrafine particles (UFP; 10-100 nm), and accumulation mode particles (AMP; 100-500 nm) in the previous 1-96 h. IQR increases in mean UFP and AMP concentrations were associated with significant decreases in platelet response, with the largest being a -0.43 log(pg/ml) decrease in log(thromboxane B2) (95% CI = -0.8, -0.1) associated with each 582-particles/cm(3) increase in AMP, and a -1.7 ohm reduction in collagen-induced aggregation (95% CI = -3.1, -0.3) associated with each 2097-particles/cm(3) increase in UFP in the previous 72 h. This UFP effect on thromboxane B2 was significantly muted in diabetic subjects taking aspirin (-0.01 log[pg/ml]; 95% CI = -0.4, 0.3). The reason for this finding remains unknown, and needs to be investigated in future studies. PMID:27029326

  3. Increases in ambient particulate matter air pollution, acute changes in platelet function, and effect modification by aspirin and omega-3 fatty acids: A panel study.

    PubMed

    Becerra, Adan Z; Georas, Steve; Brenna, J Thomas; Hopke, Philip K; Kane, Cathleen; Chalupa, David; Frampton, Mark W; Block, Robert; Rich, David Q

    2016-01-01

    Increased particulate matter (PM) air pollutant concentrations have been associated with platelet activation. It was postulated that elevated air pollutant concentrations would be associated with increases in measures of platelet function and that responses would be blunted when taking aspirin and/or fish oil. Data from a sequential therapy trial (30 subjects with type 2 diabetes mellitus), with 4 clinic visits (first: no supplements, second: aspirin, third: omega-3 fatty acid supplements, fourth: aspirin and omega-3 fatty acids) per subject, were utilized. Using linear mixed models, adjusted for relative humidity, temperature, visit number, and season, changes in three platelet function measures including (1) aggregation induced by adenosine diphosphate (ADP), (2) aggregation induced by collagen, and (3) thromboxane B2 production were associated with interquartile range (IQR) increases in mean concentrations of ambient PM2.5, black carbon, ultrafine particles (UFP; 10-100 nm), and accumulation mode particles (AMP; 100-500 nm) in the previous 1-96 h. IQR increases in mean UFP and AMP concentrations were associated with significant decreases in platelet response, with the largest being a -0.43 log(pg/ml) decrease in log(thromboxane B2) (95% CI = -0.8, -0.1) associated with each 582-particles/cm(3) increase in AMP, and a -1.7 ohm reduction in collagen-induced aggregation (95% CI = -3.1, -0.3) associated with each 2097-particles/cm(3) increase in UFP in the previous 72 h. This UFP effect on thromboxane B2 was significantly muted in diabetic subjects taking aspirin (-0.01 log[pg/ml]; 95% CI = -0.4, 0.3). The reason for this finding remains unknown, and needs to be investigated in future studies.

  4. Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34(+) cells are functionally active in an ex vivo flow model of thrombosis.

    PubMed

    Salles, Isabelle I; Thijs, Tim; Brunaud, Christine; De Meyer, Simon F; Thys, Johan; Vanhoorelbeke, Karen; Deckmyn, Hans

    2009-12-01

    Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. In this study, we demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient mice after transplantation of unexpanded cord-blood CD34(+) cells was detected within 10 days after transplantation, with the number of circulating human platelets peaking at 2 weeks (up to 87 x 10(3)/microL). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks after transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional, as assessed by increased CD62P expression and PAC1 binding in response to collagen-related peptide and thrombin receptor-activating peptide activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human glycoprotein Ibalpha (GPIbalpha) and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets, but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo.

  5. Fast Functional Germline and Epigenetic Assays in the Nematode Caenorhabditis elegans.

    PubMed

    Lundby, Zachary; Camacho, Jessica; Allard, Patrick

    2016-01-01

    Germ cells are unique in their ability to transfer traits and genetic information from one generation to the next. The proper development and integrity of their genome are therefore of utmost importance for the health of organisms and survival of species. Many features of mammalian germ cells, including their long development span and difficulty of access, present challenges for their study in the context of toxicity assays. In light of these barriers, the model system Caenorhabditis elegans shows great potential given its ease of manipulation and genetic tractability which can be easily adapted for high-throughput analysis. In this chapter, we discuss the advantages of examining germ cell processes in C. elegans, and describe three functional germline assays for the examination of chemical impact on germline maintenance and function including assays probing germ cell differentiation, germline apoptosis, and germline epigenetic regulation. PMID:27518628

  6. The Use of Functional Nucleic Acids in Solid-Phase Fluorimetric Assays

    NASA Astrophysics Data System (ADS)

    Rupcich, Nicholas; Nutiu, Razvan; Shen, Yutu; Li, Yingfu; Brennan, John D.

    The past 15 years have seen a revolution in the area of functional nucleic acid (FNA) research since the demonstration that single-stranded RNA and DNA species can be used for both ligand binding and catalysis. An emerging area of application for such species is in the development of solid-phase fluorimetric assays for biosensing, proteomics, and drug screening purposes. In this chapter, the methods for immobilization of functional nucleic acids are briefly reviewed, with emphasis on emerging technologies such as sol-gel encapsulation. Methods for generating fluorescence signals from aptamers and nucleic acid enzymes are then described, and the use of such species in solid-phase fluorimetric assays is then discussed. Unique features of sol-gel based materials for the development of solid-phase assays are highlighted, and some emerging applications of immobilized FNA species are discussed.

  7. A rapid flow cytometric technique for the detection of platelet-monocyte complexes, activated platelets and platelet-derived microparticles.

    PubMed

    Pearson, Laura; Thom, Jim; Adams, Murray; Oostryck, Robert; Krueger, Rom; Yong, Gerald; Baker, Ross

    2009-08-01

    Platelet activation occurs in a variety of clinical situations in which it directly contributes to the pathology. This study reports a simple flow cytometric assay for platelet activation which measures platelet-derived microparticles, activated platelets and platelet-monocyte complexes. Pre- and post analytical conditions were investigated and optimized and a normal range established on 20 healthy controls. Twenty patients pre- and post percutaneous coronary intervention (PCI) were tested with the technique. Soluble activation markers sCD40 ligand and sP-selectin and plasma phospholipid levels were measured in both groups. There was a significant increase in activated platelets and platelet-monocyte complexes between normal and pre-PCI (P = 0.005 and 0.0275, respectively) suggesting an activated state. There was a significant fall in activated platelets post-PCI (P = 0.0027) which was mirrored by a fall in soluble CD40 ligand, soluble P-selectin and plasma phospholipid levels (P = 0.0066, <0.0001 and 0.0032, respectively) consistent with antiplatelet therapy administered during the process. This is a reliable and rapid method for the assessment of ex vivo platelet activation which may be an aid in diagnosis and help guide therapy for patients with thrombotic disease.

  8. Cyclosporine A enhances platelet aggregation.

    PubMed

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

    1987-12-01

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

  9. Platelets in inflammation and infection.

    PubMed

    Jenne, Craig N; Kubes, Paul

    2015-01-01

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

  10. Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays

    PubMed Central

    Urbani, Francesca; Proietti, Enrico

    2013-01-01

    The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometry- (FCM-) based assays combining different phenotypic and functional markers have been developed in the past decade for informative and longitudinal analysis of polyfunctional T-cells. These technologies were designed to address the complexity and functional heterogeneity of cancer biology and cellular immunity and to define biomarkers predicting clinical response to anticancer treatment. So far, there is still a lack of standardization of some of these immunological tests. The aim of this review is to overview the latest technologies for immune monitoring and to highlight critical steps involved in some of the FCM-based cellular immune assays. In particular, our laboratory is focused on melanoma vaccine research and thus our main goal was the validation of a functional multiparameter test (FMT) combining different functional and lineage markers to be applied in clinical trials involving patients with melanoma. PMID:24195078

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

    PubMed

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

    2013-06-01

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

  12. Acetal phosphatidic acids: novel platelet aggregating agents.

    PubMed

    Brammer, J P; Maguire, M H; Walaszek, E J; Wiley, R A

    1983-05-01

    induce platelet aggregation per se by direct action at the platelet membrane, and that the acetal function is of primary importance in their potent platelet-stimulating activity. Moreover, as the acetal phosphatidic acids are the major components of the smooth muscle-contracting acidic phospholipid tissue extract ;Darmstoff' (Vogt, 1949), their potent platelet-aggregating properties may be of physiological or pathological significance.

  13. Platelet antibody: review of detection methods

    SciTech Connect

    Schwartz, K.A.

    1988-10-01

    The driving force behind development of in vitro methods for platelet antibodies is identification of plasma factors causing platelet destruction. Early methods relied on measurement of platelet activation. Current methods are more specific and use a purified antibody against immunoglobulin or complement, which is usually labeled with /sup 125/I or tagged with an enzyme or fluorescein. Comparisons of quantitation of platelet-associated IgG show wide variability between different methods. The disparate results can be related both to differences in binding of secondary antibodies to immunoglobulin in solution compared to immunoglobulins attached to platelets and to the improper assumption that the binding ratio between the secondary detecting and primary antiplatelet antibody is one. Most assays can 1) identify neonatal isoimmune thrombocytopenia and posttransfusion purpura, 2) help to differentiate between immune and nonimmune thrombocytopenias, 3) help to sort out the offending drug when drug-induced thrombocytopenia is suspected, and 4) identify platelet alloantibodies and potential platelet donors via a cross match assay for refractory patients. However, the advantages of quantitative assays over qualitative methods with respect to predictions of patients clinical course and response to different treatments remain to be investigated. 61 references.

  14. Platelet depletion and severity of streptococcal endocarditis

    PubMed Central

    Dall, Lawrence; Miller, Todd; Herndon, Betty; Diez, Ireneo; Dew, Michelle

    1998-01-01

    OBJECTIVE: To evaluate the importance of thrombocytopenia in streptococcal endocarditis using an animal model. DESIGN: A model of human septic endocarditis was established in rats (polyethylene catheters across the aortic valve and administration of Streptococcus sanguis, 5×107 colony forming units [cfu] intravenous). Thrombocytopenia at four levels was produced by antiplatelet serum. Secondary methods of producing thrombocytopenia were also evaluated. At sacrifice (96 h after platelet depletion and 72 h after infection), vegetations were removed, weighed, diluted, plated and counted. Potential mechanisms of the dose-response relationship between vegetation density and platelet count were evaluated. SETTING: Controlled research laboratory experiments. POPULATION STUDIED: Animal models of streptococcal endocarditis. MAIN RESULTS: The bacterial density of the aortic valve vegetations significantly increased as the platelet count decreased (P=0.0007). In severely thrombocytopenic animals (two-dose antiplatelet serum), data suggest increased vegetation embolism. Platelet depletion, which was minimal with chemical methods, was produced most effectively by antithrombocyte serum. Platelet surfaces in endocarditis were found to express elevated CD62p proteins (72.7% endocarditis, 34.7% control). Platelet protein fractions were evaluated in vitro by both streptocidal (P=0.19) and phagocytosis-stimulating assays. Platelet presence in mature aortic valve vegetations averaged only about 2%. CONCLUSIONS: In platelet depletion experiments using a rat model, a dose-response relationship of peripheral circulating platelet depletion to aortic valve vegetation density was found. The mechanism relating thrombocytopenia to endocarditis severity remains unresolved. PMID:22346555

  15. An extended set of yeast-based functional assays accurately identifies human disease mutations

    PubMed Central

    Sun, Song; Yang, Fan; Tan, Guihong; Costanzo, Michael; Oughtred, Rose; Hirschman, Jodi; Theesfeld, Chandra L.; Bansal, Pritpal; Sahni, Nidhi; Yi, Song; Yu, Analyn; Tyagi, Tanya; Tie, Cathy; Hill, David E.; Vidal, Marc; Andrews, Brenda J.; Boone, Charles; Dolinski, Kara; Roth, Frederick P.

    2016-01-01

    We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (101 disease- and 78 non-disease-associated variants) from 22 human disease genes. Using the resulting reference standard, we show that experimental functional assays in a 1-billion-year diverged model organism can identify pathogenic alleles with significantly higher precision and specificity than current computational methods. PMID:26975778

  16. An extended set of yeast-based functional assays accurately identifies human disease mutations.

    PubMed

    Sun, Song; Yang, Fan; Tan, Guihong; Costanzo, Michael; Oughtred, Rose; Hirschman, Jodi; Theesfeld, Chandra L; Bansal, Pritpal; Sahni, Nidhi; Yi, Song; Yu, Analyn; Tyagi, Tanya; Tie, Cathy; Hill, David E; Vidal, Marc; Andrews, Brenda J; Boone, Charles; Dolinski, Kara; Roth, Frederick P

    2016-05-01

    We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (101 disease- and 78 non-disease-associated variants) from 22 human disease genes. Using the resulting reference standard, we show that experimental functional assays in a 1-billion-year diverged model organism can identify pathogenic alleles with significantly higher precision and specificity than current computational methods. PMID:26975778

  17. An extended set of yeast-based functional assays accurately identifies human disease mutations.

    PubMed

    Sun, Song; Yang, Fan; Tan, Guihong; Costanzo, Michael; Oughtred, Rose; Hirschman, Jodi; Theesfeld, Chandra L; Bansal, Pritpal; Sahni, Nidhi; Yi, Song; Yu, Analyn; Tyagi, Tanya; Tie, Cathy; Hill, David E; Vidal, Marc; Andrews, Brenda J; Boone, Charles; Dolinski, Kara; Roth, Frederick P

    2016-05-01

    We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (101 disease- and 78 non-disease-associated variants) from 22 human disease genes. Using the resulting reference standard, we show that experimental functional assays in a 1-billion-year diverged model organism can identify pathogenic alleles with significantly higher precision and specificity than current computational methods.

  18. Platelets kill intraerythrocytic malarial parasites and mediate survival to infection.

    PubMed

    McMorran, Brendan J; Marshall, Vikki M; de Graaf, Carolyn; Drysdale, Karen E; Shabbar, Meriam; Smyth, Gordon K; Corbin, Jason E; Alexander, Warren S; Foote, Simon J

    2009-02-01

    Platelets play a critical role in the pathogenesis of malarial infections by encouraging the sequestration of infected red blood cells within the cerebral vasculature. But platelets also have well-established roles in innate protection against microbial infections. We found that purified human platelets killed Plasmodium falciparum parasites cultured in red blood cells. Inhibition of platelet function by aspirin and other platelet inhibitors abrogated the lethal effect human platelets exert on P. falciparum parasites. Likewise, platelet-deficient and aspirin-treated mice were more susceptible to death during erythrocytic infection with Plasmodium chabaudi. Both mouse and human platelets bind malarial-infected red cells and kill the parasite within. These results indicate a protective function for platelets in the early stages of erythrocytic infection distinct from their role in cerebral malaria.

  19. Bulk fluid phase behaviour of colloidal platelet-sphere and platelet-polymer mixtures.

    PubMed

    de las Heras, Daniel; Schmidt, Matthias

    2013-04-13

    Using a geometry-based fundamental measure density functional theory, we calculate bulk fluid phase diagrams of colloidal mixtures of vanishingly thin hard circular platelets and hard spheres. We find isotropic-nematic phase separation, with strong broadening of the biphasic region, upon increasing the pressure. In mixtures with large size ratio of platelet and sphere diameters, there is also demixing between two nematic phases with differing platelet concentrations. We formulate a fundamental measure density functional for mixtures of colloidal platelets and freely overlapping spheres, which represent ideal polymers, and use it to obtain phase diagrams. We find that, for low platelet-polymer size ratio, in addition to isotropic-nematic and nematic-nematic phase coexistence, platelet-polymer mixtures also display isotropic-isotropic demixing. By contrast, we do not find isotropic-isotropic demixing in hard-core platelet-sphere mixtures for the size ratios considered.

  20. Functional C1-inhibitor diagnostics in hereditary angioedema: assay evaluation and recommendations.

    PubMed

    Wagenaar-Bos, Ineke G A; Drouet, Christian; Aygören-Pursun, Emel; Bork, Konrad; Bucher, Christoph; Bygum, Anette; Farkas, Henriette; Fust, George; Gregorek, Hanna; Hack, C Erik; Hickey, Alaco; Joller-Jemelka, Helen I; Kapusta, Maria; Kreuz, Wolfhart; Longhurst, Hilary; Lopez-Trascasa, Margarita; Madalinski, Kazimierz; Naskalski, Jerzy; Nieuwenhuys, Ed; Ponard, Denise; Truedsson, Lennart; Varga, Lilian; Nielsen, Erik Waage; Wagner, Eric; Zingale, Lorenza; Cicardi, Marco; van Ham, S Marieke

    2008-09-30

    Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent episodes of potentially life-threatening angioedema. The most widespread underlying genetic deficiency is a heterozygous deficiency of the serine protease inhibitor C1 esterase inhibitor (C1-Inh). In addition to low C4 levels, the most important laboratory parameter for correct diagnosis of HAE or angioedema due to acquired C1-Inh deficiency is reduced C1-Inh function (fC1-Inh). No direct recommendations about the assays for fC1-Inh or sample handling conditions are available, although this would prove especially useful when a laboratory first starts to offer assays on fC1-Inh for HAE diagnosis. In the present study we evaluated the performance of fC1-Inh assays in the 15 different laboratories that are specialised in HAE diagnostics and assessed inter-laboratory variation with each laboratory using their own assays and standards. A double-blind survey was conducted using plasma/serum samples from healthy donors and HAE patients and the uniformity of HAE diagnosis was evaluated. It can be concluded that the diagnosis of fC1-Inh deficiency was made correctly in most cases in this survey. We can recommend the chromogenic assay for the determination of fC1-Inh, while the complex ELISA needs further investigation.

  1. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    PubMed Central

    Israeli, Ma’ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Cohen, Ofer; Chitlaru, Theodor

    2016-01-01

    Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules. PMID:27548219

  2. Nouvelle cuisine: platelets served with inflammation.

    PubMed

    Kapur, Rick; Zufferey, Anne; Boilard, Eric; Semple, John W

    2015-06-15

    Platelets are small cellular fragments with the primary physiological role of maintaining hemostasis. In addition to this well-described classical function, it is becoming increasingly clear that platelets have an intimate connection with infection and inflammation. This stems from several platelet characteristics, including their ability to bind infectious agents and secrete many immunomodulatory cytokines and chemokines, as well as their expression of receptors for various immune effector and regulatory functions, such as TLRs, which allow them to sense pathogen-associated molecular patterns. Furthermore, platelets contain RNA that can be nascently translated under different environmental stresses, and they are able to release membrane microparticles that can transport inflammatory cargo to inflammatory cells. Interestingly, acute infections can also result in platelet breakdown and thrombocytopenia. This report highlights these relatively new aspects of platelets and, thus, their nonhemostatic nature in an inflammatory setting.

  3. Interaction of polypeptide antibiotic gramicidin S with platelets.

    PubMed

    Hackl, Ellen V; Berest, Vladimir P; Gatash, Sergey V

    2012-12-01

    Gramicidin S (GS) is a cyclic decapeptide antibiotic active against both Gram-positive and Gram-negative bacteria as well as against several pathogenic fungi. However, clinical application of GS is limited because of GS hemolytic activity. The large number of GS analogues with potentially attenuated hemolytic activity has been developed over the last two decades. For all new GS derivatives, the antimicrobial test is accompanied with the hemolytic activity assay. At the same time, neither GS nor its analogues were tested against other blood cells. In the present work, the effects of GS on platelets and platelet aggregates have been studied. GS interaction with platelets is concentration dependent and leads either to platelet swelling or platelet shape change. Effect of GS on platelets is independent of platelet aggregation mechanism. GS induces disaggregation of platelet aggregates formed in the presence of aggregation agonists. The rate of the GS interaction with platelet membranes depends on membrane lipid mobility and significantly increases with temperature. The interaction of GS with the platelet membranes depends strongly on the state of the membrane lipids. Factors affecting the membrane lipids (temperature, lipid peroxidation and ionising irradiation) modify GS interaction with platelets. Our results show that GS is active not only against erythrocytes but also against other blood cells (platelets). The estimated numbers of GS molecules per 1 µm2 of a blood cell required to induce erythrocyte hemolysis and disaggregation of platelet aggregates are comparable. This must be considered when developing new antimicrobial GS analogues with improved hemolytic properties.

  4. Development of a method to quantify platelet adhesion and aggregation under static conditions

    PubMed Central

    Baker-Groberg, Sandra M.; Cianchetti, Flor A.; Phillips, Kevin G.; McCarty, Owen J.T.

    2014-01-01

    Platelets are important players in hemostasis and thrombosis. Thus, accurate assessment of platelet function is crucial for identifying platelet function disorders and measuring the efficacy of antiplatelet therapies. We have developed a novel platelet aggregation technique that utilizes the physical parameter of platelet concentration in conjunction with volume and mass measurements to evaluate platelet adhesion and aggregation. Platelet aggregates were formed by incubating purified platelets on fibrinogen- or fibrillar collagen-coated surfaces at platelet concentrations ranging from 20,000 to 500,000 platelets/ L. Platelets formed aggregates under static conditions in a platelet concentration-dependent manner, with significantly greater mean volume and mass at higher platelet concentrations ( 400,000 platelets/ L). We show that a platelet glycoprotein IIb/IIIa inhibitor abrogated platelet-platelet aggregation, which significantly reduced the volume and mass of the platelets on the collagen surface. This static platelet aggregation technique is amenable to standardization and represents a useful tool to investigate the mechanism of platelet activation and aggregation under static conditions. PMID:24883127

  5. Platelet utilization: a Canadian Blood Services research and development symposium.

    PubMed

    Webert, Kathryn E; Alam, Asim Q; Chargé, Sophie B; Sheffield, William P

    2014-04-01

    Considerable progress has been made in recent years in understanding platelet biology and in strengthening the clinical evidence base around platelet transfusion thresholds and appropriate platelet dosing. Platelet alloimmunization rates have also declined. Nevertheless, controversies and uncertainties remain that are relevant to how these products can best be used for the benefit of platelet transfusion recipients. Platelets are unique among the blood products directly derived from whole blood or apheresis donations in requiring storage, with shaking, at ambient temperature. Storage is accordingly constrained between the need to limit the growth of any microbes in the product and the need to minimize losses in platelet function associated with storage. Proteomic and genomic approaches are being applied to the platelet storage lesion. Platelet inventory management is made challenging by these constraints. Although bacterial screening has enhanced the safety of platelet transfusions, pathogen reduction technology may offer further benefits. Continuing clinical investigations are warranted to understand the value of transfusing platelets prophylactically or only in response to bleeding in different patient groups and how best to manage the most grievously injured trauma patients. Patients refractory to platelet transfusions also require expert clinical management. The engineering of platelet substitute products is an active area of research, but considerable hurdles remain before any clinical uses may be contemplated. Roles for platelets in biological areas distinct from hemostasis are also emerging. Platelet utilization is variably affected by all of the above factors, by demographic changes, by new medications, and by new patient care approaches. PMID:24629305

  6. Platelet utilization: a Canadian Blood Services research and development symposium.

    PubMed

    Webert, Kathryn E; Alam, Asim Q; Chargé, Sophie B; Sheffield, William P

    2014-04-01

    Considerable progress has been made in recent years in understanding platelet biology and in strengthening the clinical evidence base around platelet transfusion thresholds and appropriate platelet dosing. Platelet alloimmunization rates have also declined. Nevertheless, controversies and uncertainties remain that are relevant to how these products can best be used for the benefit of platelet transfusion recipients. Platelets are unique among the blood products directly derived from whole blood or apheresis donations in requiring storage, with shaking, at ambient temperature. Storage is accordingly constrained between the need to limit the growth of any microbes in the product and the need to minimize losses in platelet function associated with storage. Proteomic and genomic approaches are being applied to the platelet storage lesion. Platelet inventory management is made challenging by these constraints. Although bacterial screening has enhanced the safety of platelet transfusions, pathogen reduction technology may offer further benefits. Continuing clinical investigations are warranted to understand the value of transfusing platelets prophylactically or only in response to bleeding in different patient groups and how best to manage the most grievously injured trauma patients. Patients refractory to platelet transfusions also require expert clinical management. The engineering of platelet substitute products is an active area of research, but considerable hurdles remain before any clinical uses may be contemplated. Roles for platelets in biological areas distinct from hemostasis are also emerging. Platelet utilization is variably affected by all of the above factors, by demographic changes, by new medications, and by new patient care approaches.

  7. Mutation in the gene encoding the. alpha. chain of platelet glycoprotein Ib in platelet-type von Willebrand disease

    SciTech Connect

    Miller, J.L.; Cunningham, D.; Lyle, V.A.; Finch, C.N. )

    1991-06-01

    Platelet-type von Willebrand disease (PT-vWD) is an autosomal dominant bleeding disorder characterized by abnormally enhanced binding of von Willebrand factor (vWF) by patient platelets. Although the platelet glycoprotein (GP) Ib/IX complex is known to constitute the platelet's ristocetin-dependent receptor for vWF, a unique structural abnormality within this complex has not previously been identified in PT-vWD. Using the poly merase chain reaction to amplify genomic DNA coding for the {alpha} chain of GP Ib (GP IB{alpha}) and then sequencing the amplified DNA following cloning into M13mp18 and M13mp19 phage vectors, the authors have found a single point mutation in the GP Ib{alpha} coding region of PT-vWD DNA resulting in the substitution of valine for glycine at residue 233. This substitution within the vWF-binding region of GP Ib{alpha} is likely to exert a significant influence on the conformation of the resulting protein. Competitive oligonucleotide primer assay for this mutation showed a homozygous wild-type pattern in genomic DNA from the 161 normal volunteers studied and from 6 phenotypically normal members of a PT-vWD family. All 7 affected members of this family studied were heterozygous for the mutant allele. Platelet GP Ib{alpha} mRNA reverse-transcribed and studied by competitive oligonucleotide primer assay showed similar expression of the mutant and wild-type alleles in the affected PT-vWD patients. Absence in the normal population, tight linkage with phenotypic expression of disease, and absence of any additional abnormality of GP Ib{alpha} in these patients identify the glycine-to-valine substitution as a point mutation underlying functional abnormality of the vWF receptor in PT-vWD.

  8. A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

    PubMed

    Polireddy, Kishore; Khan, Mohiuddin Md Taimur; Chavan, Hemantkumar; Young, Susan; Ma, Xiaochao; Waller, Anna; Garcia, Matthew; Perez, Dominique; Chavez, Stephanie; Strouse, Jacob J; Haynes, Mark K; Bologa, Cristian G; Oprea, Tudor I; Tegos, George P; Sklar, Larry A; Krishnamurthy, Partha

    2012-01-01

    ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity. PMID:22808084

  9. Tethered Function Assays as Tools to Elucidate the Molecular Roles of RNA-Binding Proteins.

    PubMed

    Bos, Tomas J; Nussbacher, Julia K; Aigner, Stefan; Yeo, Gene W

    2016-01-01

    Dynamic regulation of RNA molecules is critical to the survival and development of cells. Messenger RNAs are transcribed in the nucleus as intron-containing pre-mRNAs and bound by RNA-binding proteins, which control their fate by regulating RNA stability, splicing, polyadenylation, translation, and cellular localization. Most RBPs have distinct mRNA-binding and functional domains; thus, the function of an RBP can be studied independently of RNA-binding by artificially recruiting the RBP to a reporter RNA and then measuring the effect of RBP recruitment on reporter splicing, stability, translational efficiency, or intracellular trafficking. These tethered function assays therefore do not require prior knowledge of the RBP's endogenous RNA targets or its binding sites within these RNAs. Here, we provide an overview of the experimental strategy and the strengths and limitations of common tethering systems. We illustrate specific examples of the application of the assay in elucidating the function of various classes of RBPs. We also discuss how classic tethering assay approaches and insights gained from them have been empowered by more recent technological advances, including efficient genome editing and high-throughput RNA-sequencing. PMID:27256382

  10. Tethered Function Assays as Tools to Elucidate the Molecular Roles of RNA-Binding Proteins.

    PubMed

    Bos, Tomas J; Nussbacher, Julia K; Aigner, Stefan; Yeo, Gene W

    2016-01-01

    Dynamic regulation of RNA molecules is critical to the survival and development of cells. Messenger RNAs are transcribed in the nucleus as intron-containing pre-mRNAs and bound by RNA-binding proteins, which control their fate by regulating RNA stability, splicing, polyadenylation, translation, and cellular localization. Most RBPs have distinct mRNA-binding and functional domains; thus, the function of an RBP can be studied independently of RNA-binding by artificially recruiting the RBP to a reporter RNA and then measuring the effect of RBP recruitment on reporter splicing, stability, translational efficiency, or intracellular trafficking. These tethered function assays therefore do not require prior knowledge of the RBP's endogenous RNA targets or its binding sites within these RNAs. Here, we provide an overview of the experimental strategy and the strengths and limitations of common tethering systems. We illustrate specific examples of the application of the assay in elucidating the function of various classes of RBPs. We also discuss how classic tethering assay approaches and insights gained from them have been empowered by more recent technological advances, including efficient genome editing and high-throughput RNA-sequencing.

  11. (Dicer)phering roles of microRNA in platelets.

    PubMed

    Boilard, Eric; Belleannée, Clémence

    2016-04-01

    In this issue of Blood, Rowley et al report that noncoding RNAs precisely regulate the messenger RNA (mRNA) profile in platelets. Interfering in this process using genetically engineered mice affects hemostatic and thrombotic functions of platelets. PMID:27056990

  12. Calreticulin-independent regulation of the platelet integrin alphaIIbbeta3 by the KVGFFKR alphaIIb-cytoplasmic motif.

    PubMed

    Reilly, Dermot; Larkin, Deirdre; Devocelle, Marc; Fitzgerald, Desmond J; Moran, Niamh

    2004-02-01

    The platelet integrin alphaIIbbeta3 alters conformation in response to platelet activation and ligand binding, although the molecular mechanisms involved are not known. We previously showed that a lipid modified peptide, corresponding to the membrane proximal 989KVGFFKR995 portion of the alphaIIb cytoplasmic tail, independently activates platelet alphaIIbbeta3. Calreticulin (CRT) is a potential integrin regulatory protein based on its interaction with the highly conserved alpha-integrin sequence KxGFFKR. We therefore examined the possible interaction of calreticulin and alphaIIbbeta3 in human platelets. We demonstrate that calreticulin in platelets is localised to the granulomere. In contrast, the known integrin-binding protein talin accumulates at the periphery of spreading platelets and colocalises with alphaIIbbeta3 during the process of adhesion. An interaction between calreticulin and alphaIIbbeta3 could not be demonstrated using co-immunoprecipitation techniques under various platelet activation states, even in the presence of covalent chemical crosslinkers. Thus, calreticulin does not functionally interact with the major integrin in human platelets. In order to identify proteins that interact with the integrin KVGFFKR motif we then used a peptide 'pull-down' assay from platelet lysates with biotinylated peptides and demonstrate that only the alphaIIb and beta3 subunits selectively and individually interact with this sequence. This interaction is divalent cation-dependent, has high-affinity, and occurs both with purified alphaIIbbeta3 complex and with electroeluted alpha and beta subunits. Thus, our data show that the conserved integrin KVGFFKR domain interacts primarily with the alpha and beta cytoplasmic tails and not with CRT in human platelets. PMID:14985176

  13. Toll-Like Receptor Signalling Is Not Involved in Platelet Response to Streptococcus pneumoniae In Vitro or In Vivo

    PubMed Central

    Schaap, Marianne C. L.; Hou, Baidong; van der Poll, Tom; Nieuwland, Rienk; van ‘t Veer, Cornelis

    2016-01-01

    Streptococcus (S.) pneumoniae strains vary considerably in their ability to cause invasive disease in humans, which is at least in part determined by the capsular serotype. Platelets have been implicated as sentinel cells in the circulation for host defence. One of their utensils for this function is the expression of Toll-like receptors (TLRs). We here aimed to investigate platelet response to S. pneumoniae and a role for TLRs herein. Platelets were stimulated using four serotypes of S. pneumonia including an unencapsulated mutant strain. In vitro aggregation and flow cytometry assays were performed using blood of healthy volunteers, or blood of TLR knock out and WT mice. For in vivo pneumonia experiments, platelet specific Myd88 knockout (Plt-Myd88-/-) mice were used. We found that platelet aggregation was induced by unencapsulated S. pneumoniae only. Whole blood incubation with all S. pneumoniae serotypes tested resulted in platelet degranulation and platelet-leukocyte complex formation. Platelet activation was TLR independent, as responses were not inhibited by TLR blocking antibodies, not induced by TLR agonists and were equally induced in wild-type and Tlr2-/-, Tlr4-/-, Tlr2/4-/-, Tlr9-/- and Myd88-/- blood. Plt-Myd88-/- and control mice displayed no differences in bacterial clearance or immune response to pneumonia by unencapsulated S. pneumoniae. In conclusion, S. pneumoniae activates platelets through a TLR-independent mechanism that is impeded by the bacterial capsule. Additionally, platelet MyD88-dependent TLR signalling is not involved in host defence to unencapsulated S. pneumoniae in vivo. PMID:27253707

  14. A new mutation in the KINDLIN-3 gene ablates integrin-dependent leukocyte, platelet, and osteoclast function in a patient with leukocyte adhesion deficiency-III.

    PubMed

    Crazzolara, Roman; Maurer, Kathrin; Schulze, Harald; Zieger, Barbara; Zustin, Jozef; Schulz, Ansgar S

    2015-09-01

    Disabling mutations in integrin-mediated cell signaling have been a major focus of interest over the last decade for patients affected with leukocyte adhesion deficiency-III (LAD-III). In this study, we identified a new C>T point mutation in exon 13 in the FERMT3 gene in an infant diagnosed with LAD-III and showed that KINDLIN-3 expression is required for platelet aggregation and leukocyte function, but also osteoclast-mediated bone resorption. After allogeneic bone marrow transplant, all overt symptoms disappeared. This newly identified mutation along with its novel role in dysregulation of bone homeostasis extends our understanding of KINDLIN-3 in humans.

  15. Impact of experimental haemodilution on platelet function, thrombin generation and clot firmness: effects of different coagulation factor concentrates

    PubMed Central

    Caballo, Carolina; Escolar, Gines; Diaz-Ricart, Maribel; Lopez-Vílchez, Irene; Lozano, Miguel; Cid, Joan; Pino, Marcos; Beltrán, Joan; Basora, Misericordia; Pereira, Arturo; Galan, Ana M.

    2013-01-01

    Background Haemodilution during resuscitation after massive haemorrhage may worsen the coagulopathy and perpetuate bleeding. Materials and methods Blood samples from healthy donors were diluted (30 and-60%) using crystalloids (saline, Ringer’s lactate, PlasmalyteTM) or colloids (6% hydroxyethylstarch [HES130/0.4], 5% human albumin, and gelatin). The effects of haemodilution on platelet adhesion (Impact R), thrombin generation (TG), and thromboelastometry (TEM) parameters were analysed as were the effects of fibrinogen, prothrombin complex concentrates (PCC), activated recombinant factor VII (FVIIa), and cryoprecipates on haemodilution. Results Platelet interactions was already significantly reduced at 30% haemodilution. Platelet reactivity was not improved by addition of any of the concentrates tested. A decrease in TG and marked alterations of TEM parameters were noted at 60% haemodilution. HES130/0.4 was the expander with the most deleterious action. TG was significantly enhanced by PCC whereas rFVIIa only caused a mild acceleration of TG initiation. Fibrinogen restored the alterations of TEM parameters caused by haemodilution including those caused by HES 130/0.4. Cryoprecipitates significantly improved the alterations caused by haemodilution on TG and TEM parameters; the effects on TG disappeared after ultracentrifugation of the cryoprecipitates. Discussion The haemostatic alterations caused by haemodilution are multifactorial and affect both blood cells and coagulation. In our in vitro approach, HES 130/0.4 had the most deleterious effect on haemostasis parameters. Coagulation factor concentrates did not improve platelet interactions in the Impact R, but did have favourable effects on coagulation parameters measured by TG and TEM. Fibrinogen notably improved TEM parameters without increasing thrombin generation, suggesting that this concentrate may help to preserve blood clotting abilities during haemodilution without enhancing the prothrombotic risk. PMID

  16. Reduced platelet-mediated and enhanced leukocyte-mediated fibrinolysis in experimentally induced diabetes in rats

    SciTech Connect

    Winocour, P.D.; Colwell, J.A.

    1985-05-01

    Studies of fibrinolytic activity in diabetes mellitus have produced conflicting results. This may be a result of methodologic insensitivity or of variable contributions of the different blood components to whole blood fibrinolysis. To explore these two possibilities, the authors used a sensitive solid-phase radiometric assay to examine the fibrinolytic activity of whole blood, platelet-rich plasma, leukocytes, and platelet- and leukocyte-poor plasma prepared from control rats and rats with streptozocin-induced diabetes at various times after induction of diabetes. Fibrinolytic activity of whole blood from diabetic rats after 7 days was significantly reduced, and remained reduced after longer durations of diabetes up to 28 days. Platelet-rich plasma from diabetic rats had decreased fibrinolytic activity, which followed the same time course of changes as in whole blood. The platelet contribution to whole blood fibrinolysis was further reduced in vivo after 14 days of diabetes by a reduced whole blood platelet count. In contrast, fibrinolytic activity of leukocytes from diabetic rats became enhanced after 7 days of diabetes. After 49 days of diabetes, the whole blood leukocyte count was reduced, and in vivo would offset the enhanced activity. Plasma fibrinolytic activity was small compared with that of whole blood and was unaltered in diabetic rats. The authors conclude that altered platelet function contributes to decreased fibrinolytic activity of whole blood in diabetic rats, and that this may be partially offset by enhanced leukocyte-mediated fibrinolysis.

  17. beta-Dystroglycan modulates the interplay between actin and microtubules in human-adhered platelets.

    PubMed

    Cerecedo, Doris; Cisneros, Bulmaro; Suárez-Sánchez, Rocío; Hernández-González, Enrique; Galván, Iván

    2008-05-01

    To maintain the continuity of an injured blood vessel, platelets change shape, secrete granule contents, adhere, aggregate, and retract in a haemostatic plug. Ordered arrays of microtubules, microfilaments, and associated proteins are responsible for these platelet responses. In full-spread platelets, microfilament bundles in association with other cytoskeleton proteins are anchored in focal contacts. Recent studies in migrating cells suggest that co-ordination and direct physical interaction of microtubules and actin network modulate adhesion development. In platelets, we have proposed a feasible association between these two cytoskeletal systems, as well as the participation of the dystrophin-associated protein complex, as part of the focal adhesion complex. The present study analysed the participation of microtubules and actin during the platelet adhesion process. Confocal microscopy, fluorescence resonance transfer energy and immunoprecipitation assays were used to provide evidence of a cross-talk between these two cytoskeletal systems. Interestingly, beta-dystroglycan was found to act as an interplay protein between actin and microtubules and an additional communication between these two cytoskeleton networks was maintained through proteins of focal adhesion complex. Altogether our data are indicative of a dynamic co-participation of actin filaments and microtubules in modulating focal contacts to achieve platelet function.

  18. Detection of microbial contamination in platelets

    NASA Astrophysics Data System (ADS)

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

    2005-03-01

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

  19. Platelet MicroRNAs: An Overview.

    PubMed

    Dahiya, Neetu; Sarachana, Tewarit; Vu, Long; Becker, Kevin G; Wood, William H; Zhang, Yongqing; Atreya, Chintamani D

    2015-10-01

    MicroRNAs (miRNAs) are short ~22-nucleotide noncoding RNA that have been found to influence the expression of many genes and cellular processes by either repressing translation or degrading messenger RNA transcripts. Platelet miRNA expression has been shown to be perturbed during ex vivo storage of platelets and in platelet-associated disorders. Although bioinformatics-based miRNA target predictions have been established, direct experimental validation of the role of miRNAs in platelet biology has been rather slow. Target prediction studies are, nonetheless, valuable in directing the design of appropriate experiments to test specific miRNA:messenger RNA interactions relevant to the underlying mechanisms of platelet function in general and in disease as well as in ex vivo storage-associated "storage lesions," a collective term used to include physiologic, biochemical, and morphologic changes that occur in stored platelets. This brief review will focus on emerging human platelet miRNA studies to emphasize their potential role relevant to transfusion medicine field in terms of regulating platelet signaling pathways, markers of platelet associated disorders, and remote impactors of gene expression (intercellular biomodulators) as well as potential platelet quality markers of storage and pathogen reduction treatments.

  20. Pavlovian fear conditioning as a behavioral assay for hippocampus and amygdala function: cautions and caveats.

    PubMed

    Maren, Stephen

    2008-10-01

    Pavlovian fear conditioning has become an important model for investigating the neural substrates of learning and memory in rats, mice and humans. The hippocampus and amygdala are widely believed to be essential for fear conditioning to contexts and discrete cues, respectively. Indeed, this parsing of function within the fear circuit has been used to leverage fear conditioning as a behavioral assay of hippocampal and amygdala function, particularly in transgenic mouse models. Recent work, however, blurs the anatomical segregation of cue and context conditioning and challenges the necessity for the hippocampus and amygdala in fear learning. Moreover, nonassociative factors may influence the performance of fear responses under a variety of conditions. Caution must therefore be exercised when using fear conditioning as a behavioral assay for hippocampal- and amygdala-dependent learning.

  1. Pavlovian fear conditioning as a behavioral assay for hippocampus and amygdala function: cautions and caveats.

    PubMed

    Maren, Stephen

    2008-10-01

    Pavlovian fear conditioning has become an important model for investigating the neural substrates of learning and memory in rats, mice and humans. The hippocampus and amygdala are widely believed to be essential for fear conditioning to contexts and discrete cues, respectively. Indeed, this parsing of function within the fear circuit has been used to leverage fear conditioning as a behavioral assay of hippocampal and amygdala function, particularly in transgenic mouse models. Recent work, however, blurs the anatomical segregation of cue and context conditioning and challenges the necessity for the hippocampus and amygdala in fear learning. Moreover, nonassociative factors may influence the performance of fear responses under a variety of conditions. Caution must therefore be exercised when using fear conditioning as a behavioral assay for hippocampal- and amygdala-dependent learning. PMID:18973583

  2. Stability of lyophilized human platelets loaded with small molecule carbohydrates.

    PubMed

    Wang, J X; Yang, C; Wan, W; Liu, M X; Ren, S P; Quan, G B; Han, Y

    2011-01-01

    Long-term preservation of platelets is a great challenge for blood transfusion centers, due to the required narrow storage temperature arange (22 ± 2 degree C). Short shelf life and potential bacterial growth often lead to the shortage of high-quality platelets. Freeze-dried preservation is thus believed to be a potential solution for long-term platelet storage without losing the hemostasis function. Here we report a new platelet preservation method, which uses small molecule carbohydrates to extend storage time and to maintain platelet function. The activities of lyophilized platelets that were stabilized with small molecule carbohydrate (e.g., cell viability, mean platelet volume, activation characteristics, and aggregation kinetics) were maintained after storage of 30, 60, and 90 days at room temperature, 4 degree C, and -20 degree C. The recovery of freeze-dried platelets was 87 percent in comparison to fresh platelets. The mean platelet volume of rehydrated platelets increased (from 6.8 fl to 8.0 fl). About 40 percent of rehydrated platelets was in the early-activated stage (PCA-1 positive) and 30 percent was in the terminal-activated stage (CD62P positive). The cell viability was about 60 percent as measured with CMFDA vital probes. The aggregation rate of rehydrated platelets after 90-day storage was similar to fresh platelets stored at 22 degree C ± 2 degree C.

  3. Mean platelet size related to glycoprotein-specific autoantibodies and platelet-associated IgG.

    PubMed

    Javela, K; Kekomäki, R

    2007-12-01

    Recent evidence suggests that platelet-associated glycoprotein-specific (GP) antibodies represent true positive autoantibodies and can therefore be taken as the gold standard. Earlier tests, which aimed at detecting platelet-associated IgG (PA-IgG), might have been hampered, e.g. by the variation of platelet size in thrombocytopenic patients. In this study, 206 samples with increased PA-IgG from consecutive thrombocytopenic patients were tested further for GP-specific antibodies with a monoclonal antibody immobilized platelet antigen test (MAIPA) using a combination of a GP IIbIIIa-specific and a GP IbIX-specific antibody for immobilization or, in a separate assay, GP V-specific antibody. Mean platelet size was recorded as forward scatter (FSC) of platelets in flow cytometric analysis of PA-IgG. GP-specific antibodies were detected in 49 (24%) of the 206 patient samples. Their presence correlated well with increased PA-IgG (R = 0.769). The mean platelet size and mean fluorescence intensity (MFI) of PA-IgG were both significantly increased in patients compared with healthy controls (n = 112; P < 0.0001). Notably, PA-IgG was associated with platelet size within the platelet population of both healthy controls and patients (R = 0.999). Further, the probability of GP IIbIIIa and/or IbIX and GP V-specific PA-IgG tended to increase with the mean platelet size of the patients (P = 0.045). In conclusion, large platelets bound more IgG than platelets of normal size, which may explain at least in part the reported low specificity of total PA-IgG measurement. As the PA-IgG displays low specificity compared with the gold standard, its use as such may be abandoned and replaced by tests for platelet-associated GP-specific autoantibodies. PMID:17988298

  4. Mean platelet size related to glycoprotein-specific autoantibodies and platelet-associated IgG

    PubMed Central

    JAVELA, K; KEKOMÄKI, R

    2007-01-01

    Recent evidence suggests that platelet-associated glycoprotein-specific (GP) antibodies represent true positive autoantibodies and can therefore be taken as the gold standard. Earlier tests, which aimed at detecting platelet-associated IgG (PA-IgG), might have been hampered, e.g. by the variation of platelet size in thrombocytopenic patients. In this study, 206 samples with increased PA-IgG from consecutive thrombocytopenic patients were tested further for GP-specific antibodies with a monoclonal antibody immobilized platelet antigen test (MAIPA) using a combination of a GP IIbIIIa-specific and a GP IbIX-specific antibody for immobilization or, in a separate assay, GP V-specific antibody. Mean platelet size was recorded as forward scatter (FSC) of platelets in flow cytometric analysis of PA-IgG. GP-specific antibodies were detected in 49 (24%) of the 206 patient samples. Their presence correlated well with increased PA-IgG (R = 0.769). The mean platelet size and mean fluorescence intensity (MFI) of PA-IgG were both significantly increased in patients compared with healthy controls (n = 112; P < 0.0001). Notably, PA-IgG was associated with platelet size within the platelet population of both healthy controls and patients (R = 0.999). Further, the probability of GP IIbIIIa and/or IbIX and GP V-specific PA-IgG tended to increase with the mean platelet size of the patients (P = 0.045). In conclusion, large platelets bound more IgG than platelets of normal size, which may explain at least in part the reported low specificity of total PA-IgG measurement. As the PA-IgG displays low specificity compared with the gold standard, its use as such may be abandoned and replaced by tests for platelet-associated GP-specific autoantibodies. PMID:17988298

  5. Utility of preoperative in vitro platelet function tests for predicting bleeding risk in patients undergoing functional endoscopic sinus surgery

    PubMed Central

    Lee, A-Jin; Kim, Sang-Gyung

    2016-01-01

    Background It is necessary to predict the bleeding risk in patients undergoing functional endoscopic sinus surgery (FESS). To evaluate the adequacy of primary hemostasis, preoperative hemostatic screening tests are used. In the present study, we determined whether there is a positive correlation between prolonged closure time (CT) with collagen/epinephrine (CT-epi), prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT) and bleeding during FESS. Patients and methods We reviewed the medical records of 90 patients without bleeding histories who had undergone FESS from March 2013 to June 2014. More than 200 mL of blood loss was defined as moderate bleeding during surgery. With respect to bleeding during surgery, we determined the sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of CT-epi, PT, INR and aPTT. Results Of the 90 patients, 17 (18.9%) patients had preoperative prolonged CT values and three (17.6%) patients had bleeding. In comparison, five (6.8%) of the 73 (81.1%) patients who had undergone FESS with preoperative normal PFA values experienced bleeding (P=0.171). On the other hand, patients with prolonged PT values (2, 2.2%), prolonged INR values (3, 3.3%) or prolonged PTT values (1, 1.1%) had no bleeding episode. Preoperative CT had low sensitivity (44.4%) and PPV (23.5%). Conclusion During preoperative period, the hemostatic screening may not be helpful to detect the bleeding tendency in adult patients undergoing FESS. Routine measurement of CT-epi, PT, INR and aPTT for preoperative screening may not be recommended for FESS patients. PMID:27799837

  6. Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

    PubMed Central

    Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J.; Smithgall, Thomas E.

    2015-01-01

    The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

  7. Mouse ES-cell-based functional assay to evaluate mutations in BRCA2

    PubMed Central

    Kuznetsov, Sergey G.; Liu, Pentao; Sharan, Shyam K.

    2009-01-01

    Individuals with mutations in breast cancer susceptibility genes, BRCA1 and BRCA2 have up to 80% risk of developing breast cancer by the age of 70. Sequencing based genetic tests are now available to identify mutation carriers in effort to reduce mortality through prevention and early diagnosis. However, lack of a suitable functional assay hinders risk assessment of more than 1900 BRCA1 and BRCA2 variants in the Breast Cancer Information Core database that do not clearly disrupt the gene product. We have established a simple, versatile and reliable assay to test for functional significance of mutations in BRCA2 using mouse embryonic stem cells (ES-cells) and bacterial artificial chromosomes (BACs) and have used it to classify 17 sequence variants. The assay is based on the ability of human BRCA2 to complement the loss of endogenous Brca2 in mouse ES-cells. This technique may also serve as a paradigm for functional analysis of mutations found in other human disease genes. PMID:18607349

  8. Association of membrane/lipid rafts with the platelet cytoskeleton and the caveolin PY14: participation in the adhesion process.

    PubMed

    Cerecedo, Doris; Martínez-Vieyra, Ivette; Maldonado-García, Deneb; Hernández-González, Enrique; Winder, Steve J

    2015-11-01

    Platelets are the most prominent elements of blood tissue involved in hemostasis at sites of blood vessel injury. Platelet cytoskeleton is responsible for their shape modifications observed during activation and adhesion to the substratum; therefore the interactions between cytoskeleton and plasma membrane are critical to modulate blood platelet functions. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to membrane/lipid rafts (MLR) and regulate lateral diffusion of membrane proteins and lipids. Resting, thrombin-activated, and adherent human platelets were processed for biochemical studies including western-blot and immunprecipitation assays and confocal analysis were performed to characterize the interaction of MLR with the main cytoskeleton elements and β-dystroglycan as well as with the association of caveolin-1 PY14 with focal adhesion proteins. We transfected a megakaryoblast cell line (Meg-01) to deplete β-dystroglycan, subsequent to their differentiation to the platelet progenitors. Our data showed a direct interaction of the MLR with cytoskeleton to regulate platelet shape, while an association of caveolin-1 PY14 with vinculin is needed to establish focal adhesions, which are modulated for β-dystroglycan. In conclusion, caveolin-1 PY14 in association with platelet cytoskeleton participate in focal adhesions dynamics.

  9. Rapid platelet turnover in WASP(−) mice correlates with increased ex vivo phagocytosis of opsonized WASP(−) platelets

    PubMed Central

    Prislovsky, Amanda; Marathe, Bindumadhav; Hosni, Amira; Bolen, Alyssa L.; Nimmerjahn, Falk; Jackson, Carl W.; Weiman, Darryl; Strom, Ted S.

    2008-01-01

    Objective Our objective was to determine a mechanism for the thrombocytopenia of murine Wiskott-Aldrich syndrome (WAS). Materials and Methods Consumption rates of WAS protein (WASP)( −) and wild-type (WT) platelets were measured by injection of 5-chloromethylfluorescein diacetate (CMFDA)-labeled platelets into WT or WASP(−) recipients, and by in vivo biotinylation. Platelet and reticulated platelet counts were performed using quantitative flow cytometry. Bone marrow megakaryocyte number and ploidy was assessed by flow cytometry. Phagocytosis of CMFDA-labeled, opsonized platelets was assessed using bone marrow–derived macrophages. Serum antiplatelet antibodies were assayed via their binding to WT platelets. Results CMFDA-labeled WASP(−) platelets are consumed more rapidly than WT platelets in either WT or WASP(−) recipients. In vivo biotinylation studies corroborate these findings and show a normal consumption rate for WASP(−) reticulated platelets. The number of reticulated platelets is reduced in WASP(−) mice, but a significant number of the mice show an increased proportion of reticulated platelets and more severe thrombocytopenia. Sera from some of the latter group contain antiplatelet antibodies. Compared to WT platelets, WASP(−) platelets opsonized with anti-CD61 or 6A6 antibody are taken up more rapidly by bone marrow–derived macrophages. In vivo consumption rates of WASP(−) platelets are more accelerated by opsonization than are those of WT platelets. Conclusion Both rapid clearance and impaired production contribute to the thrombocytopenia of murine WAS. Increased susceptibility of opsonized WASP(−) platelets to phagocytosis leads to increased in vivo clearance. This correlates with a higher incidence of individuals with an elevated fraction of reticulated platelets, a more severe thrombocytopenia, and antiplatelet antibodies. PMID:18346836

  10. [Prophylactic platelet transfusions].

    PubMed

    Ilmakunnas, Minna; Remes, Kari; Hiippala, Seppo; Mäkisalo, Heikki; Åberg, Fredrik

    2016-01-01

    The consumption of platelet products in Finland is exceptionally high. For the most part, platelets are transfused pre-operatively to thrombocytopenic patients in order to prevent hemorrhage. Most of the minor procedures could, however, be conducted even if the patients'platelet levels would be lower than usual. In cardiac surgery, platelets are used because of the hemorrhagic diathesis associated with platelet inhibitors. Platelet inhibitors will, however, also bind to transfused platelets, whereby instead of prophylactic platelet transfusions it would be more sensible to leave the thorax open and not carry out ineffective platelet transfusions until the effect of the inhibitors has run out. We outline the prophylactic use of platelets based on recent international clinical practice guidelines. PMID:27400590

  11. Platelets contribute to growth and metastasis in hepatocellular carcinoma.

    PubMed

    Bihari, Chhagan; Rastogi, Archana; Shasthry, Saggere Muralikrishna; Bajpai, Meenu; Bhadoria, Ajeet Singh; Rajesh, S; Mukund, Amar; Kumar, Anupam; Sarin, Shiv K

    2016-09-01

    To determine the association of platelets with hepatocellular carcinoma (HCC) growth and its metastasis. We examined platelets, laboratory, and radiological data of consecutive 420 HCC and 1008 cirrhosis cases. Follow-up information of platelet count in cirrhosis to HCC, pre- to post-therapy, and post-therapy to HCC outcome was analyzed. Cytokine profiling was performed in HCC and cirrhosis (n = 10 each). On the basis of imaging, HCC was divided into six subgroups. Cytosmears of HCC were assessed for platelet clustering around tumor cells. An in vitro Matrigel invasion assay was performed on human HCC cell lines using graded concentration of platelets. Baseline platelet numbers and platelet/lymphocyte ratios (PLRs) were significantly higher (p < 0.001) in HCC than cirrhosis. IL-1, IL-6, FGF, G-CSF, thrombopoietin, and VEGF were higher in HCC than cirrhosis. Platelet counts were increased after HCC conversion of cirrhosis (p < 0.001) and decreased (p < 0.001) after therapy. Platelets and PLR in recurrence cases were higher than in responders at baseline. AFP, PIVKAII, platelets, and PLR increase (p < 0.001 each) with advancement in HCC growth. Multivariate analysis showed platelets (p = 0.002), PLR (p = 0.004), and AFP (p < 0.001) associated with distant metastasis. Platelet clustering seen in 75.7% of HCC group 3, 45% in group 2, and 12.5% in group 1 cases (p < 0.001). Invaded cells in Matrigel assay positively correlated with platelet concentration. Platelets can contribute to the development, growth, invasion, and metastasis of HCC. Rising platelet count after HCC therapy is indicative of incomplete response or recurrence. PMID:27457354

  12. Migraine: the platelet hypothesis after 10 years.

    PubMed

    Hanington, E

    1989-01-01

    The proposal that migraine is a blood disorder and caused by a primary abnormality of platelet behaviour was first put forward in 1978. This paper outlines the basis on which the proposal was made and the way in which the platelet hypothesis can account for the many facets of the disorder. It also reports further studies of platelet composition and function which have been undertaken by a large number of independent workers during the past ten years. The results of their investigations provide strong additional support for the platelet hypothesis in migraine.

  13. Platelet cytoskeleton and its hemostatic role.

    PubMed

    Cerecedo, Doris

    2013-12-01

    Upon vascular injury, platelets adhere to the exposed extracellular matrix, which triggers the platelet activation and aggregation to form a hemostatic plug to seal the wound. All of these events involve dramatic changes in shape because of the cytoskeleton reorganization. The versatility of the cytoskeleton's main elements depends on the biochemical nature of the elements, as well as on the associated proteins that confer multiple functions within the cell. The list of these associated proteins grows actively, increasing our knowledge concerning the complexity of platelet cytoskeleton machinery. The present review evidences the recently described platelet proteins that promote characteristic modifications in their cytoskeleton organization, with special focus on the dystrophin-glycoprotein complex.

  14. Purification of Protein Chaperones and Their Functional Assays with Intermediate Filaments.

    PubMed

    Perng, Ming-Der; Huang, Yu-Shan; Quinlan, Roy A

    2016-01-01

    Intermediate filament (IF) scaffolds facilitate small heat shock protein (sHSP) function, while IF function is sHSP dependent. sHSPs interact with IFs and the importance of this interaction is to maintain the individuality of the IFs and to modulate interfilament interactions both in networks and in assembly intermediates. Mutations in both sHSPs and their interacting IF proteins phenocopy each other in the human diseases they cause. This establishes a key functional relationship between these two very distinct protein families, and it also evidences the role of this cytoskeleton-chaperone complex in the cellular stress response. In this chapter, we describe the detailed experimental protocols for the preparation of purified IF proteins and sHSPs to facilitate the study in vitro of their functional interactions. In addition, we describe the detailed biochemical procedures to assess the effect of sHSP on the assembly of IFs, the binding to IFs, and the prevention of noncovalent filament-filament interactions using in vitro cosedimentation, electron microscopy, and viscosity assays. These assays are valuable research tools to study and manipulate sHSP-IF complexes in vitro and therefore to determine the structure-function detail of this complex, and how it contributes to cellular, tissue, and organismal homeostasis and the in vivo stress response.

  15. Decreased Pasteur effect in platelets of aged individuals.

    PubMed

    D'Aurelio, M; Merlo Pich, M; Catani, L; Sgarbi, G L; Bovina, C; Formiggini, G; Parenti Castelli, G; Baum, H; Tura, S; Lenaz, G

    2001-06-01

    We have investigated the mitochondrial energy state in human platelets of young (19-30 years old) and aged individuals (65-87 years old) exploiting the Pasteur effect, i.e. stimulation of lactate production by incubation of the purified platelets with the mitochondrial respiratory chain inhibitor, antimycin A. This assay allows the determination of mitochondrial function with respect to glycolysis, and the ratio of mitochondrial adenosine triphosphate (ATP) to glycolytic ATP. A significant increase of basal, non-stimulated lactate production and decrease of the stimulation by antimycin A were observed in the older individuals, suggesting that the impairment of oxidative phosphorylation detectable in post-mitotic tissues of aged individuals can be observed also in easily collectable blood cells.

  16. Recombinant human factor VIIa (rFVIIa) can activate factor FIX on activated platelets.

    PubMed

    Gabriel, D A; Li, X; Monroe, D M; Roberts, H R

    2004-10-01

    The studies reported here show that factor (F)VIIa can activate factor (F)IX on activated platelets in the absence of tissue factor. Both FIX and FIXa bind to the activated platelet surface with a K(d) of 8 nM and 2 nM, respectively. With factor (F)VIIIa, FIXa binds more tightly to platelets (K(d) 0.6 nM). At rFVIIa concentrations < 100 nm, no direct binding to the activated platelet surface can be detected with electrophoretic light scattering. However, in the presence of FIX, rFVIIa binding to platelets at concentrations as low as 10 nm rFVIIa can be detected. This is reflected by a decrease in the FIX K(d) from 8 to 1.6 nM. When rFVIIa is added to activated platelets in the presence of both FIX and FVIIIa, the K(d) for FIX decreases to 0.6, suggesting that rFVIIa activates FIX on the surface of activated platelets in the absence of tissue factor. The activation of FIX by FVIIa on activated platelets can also be demonstrated by a functional assay for FIXa. These data show that pharmacological doses of rFVIIa result in the direct activation of FIX by rFVIIa to form additional tenase complexes ultimately resulting in improved thrombin generation. These results may explain, at least in part, the mechanism of action of rFVIIa in hemorrhagic conditions seen in otherwise normal patients who develop an acquired coagulopathy due to trauma, surgery or a variety of other events in which rFVIIa has been found to be effective.

  17. In vitro pharmacological characterization of vorapaxar, a novel platelet thrombin receptor antagonist.

    PubMed

    Hawes, Brian E; Zhai, Ying; Hesk, David; Wirth, Mark; Wei, Huijun; Chintala, Madhu; Seiffert, Dietmar

    2015-09-01

    Vorapaxar is a novel protease-activated receptor-1 (PAR1) antagonist recently approved for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. The present study provides a comprehensive in vitro pharmacological characterization of vorapaxar interaction with the PAR1 receptor on human platelets. Similar studies were performed with a metabolite of vorapaxar (M20). Vorapaxar and M20 were competitive PAR1 antagonists that demonstrated concentration-dependent, saturable, specific, and slowly reversible binding to the receptor present on intact human platelets. The affinities of vorapaxar and M20 for the PAR1 receptor were in the low nanomolar range, as determined by saturation-, kinetic- and competitive binding studies. The calculated Kd and Ki values for vorapaxar increased in the presence of plasma, indicating a decrease in the free fraction available for binding to the PAR1 receptor on human platelets. Vorapaxar was also evaluated in functional assays using thrombin or a PAR1 agonist peptide (SFLLRN). Vorapaxar and M20 completely blocked thrombin-stimulated PAR1/β-arrestin association in recombinant cells and abolished thrombin-stimulated calcium influx in washed human platelets and vascular smooth muscle cells. Moreover, vorapaxar and M20 inhibited PAR1 agonist peptide-mediated platelet aggregation in human platelet rich plasma with a steep concentration response relationship. Vorapaxar exhibited high selectivity for inhibition of PAR1 over other platelet GPCRs. In conclusion, vorapaxar is a potent PAR1 antagonist exhibiting saturable, reversible, selective binding with slow off-rate kinetics and effectively inhibits thrombin's PAR1-mediated actions on human platelets. PMID:26022529

  18. A TR-FRET-based functional assay for screening activators of CARM1.

    PubMed

    Zeng, Hao; Wu, Jiacai; Bedford, Mark T; Sbardella, Gianluca; Hoffmann, F Michael; Bi, Kun; Xu, Wei

    2013-05-10

    Epigenetics is an emerging field that demands selective cell-permeable chemical probes to perturb, especially in vivo, the activity of specific enzymes involved in modulating the epigenetic codes. Coactivator-associated arginine methyltransferase 1 (CARM1) is a coactivator of estrogen receptor α (ERα), the main target in human breast cancer. We previously showed that twofold overexpression of CARM1 in MCF7 breast cancer cells increased the expression of ERα-target genes involved in differentiation and reduced cell proliferation, thus leading to the hypothesis that activating CARM1 by chemical activators might be therapeutically effective in breast cancer. Selective, potent, cell-permeable CARM1 activators will be essential to test this hypothesis. Here we report the development of a cell-based, time-resolved (TR) FRET assay that uses poly(A) binding protein 1 (PABP1) methylation to monitor cellular activity of CARM1. The LanthaScreen TR-FRET assay uses MCF7 cells expressing GFP-PABP1 fusion protein through BacMam gene delivery system, methyl-PABP1 specific antibody, and terbium-labeled secondary antibody. This assay has been validated as reflecting the expression and/or activity of CARM1 and optimized for high throughput screening to identify CARM1 allosteric activators. This TR-FRET platform serves as a generic tool for functional screening of cell-permeable, chemical modulators of CARM1 for elucidation of its in vivo functions. PMID:23585185

  19. A simple behavioral assay for testing visual function in Xenopus laevis.

    PubMed

    Viczian, Andrea S; Zuber, Michael E

    2014-01-01

    Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once. PMID:24962702

  20. The acute extracellular flux (XF) assay to assess compound effects on mitochondrial function.

    PubMed

    Wang, Ruolan; Novick, Steven J; Mangum, James B; Queen, Kennedy; Ferrick, David A; Rogers, George W; Stimmel, Julie B

    2015-03-01

    Numerous investigations have linked mitochondrial dysfunction to adverse health outcomes and drug-induced toxicity. The pharmaceutical industry is challenged with identifying mitochondrial liabilities earlier in drug development and thereby reducing late-stage attrition. Consequently, there is a demand for reliable, higher-throughput screening methods for assessing the impact of drug candidates on mitochondrial function. The extracellular flux (XF) assay described here is a plate-based method in which galactose-conditioned HepG2 cells were acutely exposed to test compounds, then real-time changes in the oxygen consumption rate and extracellular acidification rate were simultaneously measured using a Seahorse Bioscience XF-96 analyzer. The acute XF assay was validated using marketed drugs known to modulate mitochondrial function, and data analysis was automated using a spline curve fitting model developed at GlaxoSmithKline. We demonstrate that the acute XF assay is a robust, sensitive screening platform for evaluating drug-induced effects on mitochondrial activity in whole cells.

  1. Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response

    PubMed Central

    Changsom, Don; Lerdsamran, Hatairat; Wiriyarat, Witthawat; Chakritbudsabong, Warunya; Siridechadilok, Bunpote; Prasertsopon, Jarunee; Noisumdaeng, Pirom; Masamae, Wanibtisam; Puthavathana, Pilaipan

    2016-01-01

    Influenza neuraminidase (NA) proteins expressed in TK− cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity. Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross

  2. Gain-of-function assays in the axolotl (Ambystoma mexicanum) to identify signaling pathways that induce and regulate limb regeneration.

    PubMed

    Lee, Jangwoo; Aguilar, Cristian; Gardiner, David

    2013-01-01

    The adult salamander has been studied as a model for regeneration of complex tissues for many decades. Only recently with the development of gain-of-function assays for regeneration, has it been possible to screen for and assay the function of the multitude of signaling factors that have been identified in studies of embryonic development and tumorigenesis. Given the conservation of function of these regulatory pathways controlling growth and pattern formation, it is now possible to use the functional assays in the salamander to test the ability of endogenous as well as small-molecule signaling factors to induce a regenerative response.

  3. Gain-of-function assays in the axolotl (Ambystoma mexicanum) to identify signaling pathways that induce and regulate limb regeneration.

    PubMed

    Lee, Jangwoo; Aguilar, Cristian; Gardiner, David

    2013-01-01

    The adult salamander has been studied as a model for regeneration of complex tissues for many decades. Only recently with the development of gain-of-function assays for regeneration, has it been possible to screen for and assay the function of the multitude of signaling factors that have been identified in studies of embryonic development and tumorigenesis. Given the conservation of function of these regulatory pathways controlling growth and pattern formation, it is now possible to use the functional assays in the salamander to test the ability of endogenous as well as small-molecule signaling factors to induce a regenerative response. PMID:24029949

  4. A Nonradioactive Uptake Assay for Rapid Analysis of Thyroid Hormone Transporter Function.

    PubMed

    Jayarama-Naidu, Roopa; Johannes, Jörg; Meyer, Franziska; Wirth, Eva Katrin; Schomburg, Lutz; Köhrle, Josef; Renko, Kostja

    2015-07-01

    Thyroid hormones (TH) are actively taken up into target cells via TH-transmembrane transporters (THTT). Their activity and expression patterns define a layer of endocrine regulation that is poorly understood. Therefore, THTT are potential targets for interfering agents (endocrine disruptors) as well as for pharmacological interventions. Inactivating mutations have been identified as the underlying cause of heritable diseases (monocarboxylate transporter 8-associated Allan-Herndon-Dudley syndrome) and might also define a class of subclinical TH insensitivity. As a basic tool to solve questions regarding THTT substrate specificity, activation or inactivation by compounds and functional changes from mutations, uptake assays with radiolabeled tracers are standard. Due to the need for radioactive isotopes, this technique is limited to screening of labelled substrates and disadvantageous regarding handling, setup, and regulatory issues. To overcome these hurdles, we developed an uptake assay protocol using nonradioactive ligands. In brief, uptake of nonradioactive iodine-containing substrate molecules was monitored via Sandell-Kolthoff reaction. The novel assay was designed to the common microtiter plate layout. As a prove-of-principle, we measured TH uptake by monocarboxylate transporter 8-transfected MDCK1 cells. Titrations with bromosulphthalein as an example for inhibitor screening setups and a side-by-side comparison with the radioactive method prove this assay to be reliable, sensitive, and convenient. Furthermore, the method was applicable on primary murine astrocytes, which enables high-throughput screening studies on in vitro model systems with physiological transporter regulation. Due to its design, it is applicable for high-throughput screening of modulatory compounds, but it is also a safe, inexpensive and an easily accessible method for functional testing of THTT in basic science. PMID:25910050

  5. Comparative analysis of human ex vivo–generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale

    PubMed Central

    Wang, Yuhuan; Hayes, Vincent; Jarocha, Danuta; Sim, Xiuli; Harper, Dawn C.; Fuentes, Rudy; Sullivan, Spencer K.; Gadue, Paul; Chou, Stella T.; Torok-Storb, Beverly J.; Marks, Michael S.; French, Deborah L.

    2015-01-01

    Thrombopoiesis is the process by which megakaryocytes release platelets that circulate as uniform small, disc-shaped anucleate cytoplasmic fragments with critical roles in hemostasis and related biology. The exact mechanism of thrombopoiesis and the maturation pathways of platelets released into the circulation remain incompletely understood. We showed that ex vivo–generated murine megakaryocytes infused into mice release platelets within the pulmonary vasculature. Here we now show that infused human megakaryocytes also release platelets within the lungs of recipient mice. In addition, we observed a population of platelet-like particles (PLPs) in the infusate, which include platelets released during ex vivo growth conditions. By comparing these 2 platelet populations to human donor platelets, we found marked differences: platelets derived from infused megakaryocytes closely resembled infused donor platelets in morphology, size, and function. On the other hand, the PLP was a mixture of nonplatelet cellular fragments and nonuniform-sized, preactivated platelets mostly lacking surface CD42b that were rapidly cleared by macrophages. These data raise a cautionary note for the clinical use of human platelets released under standard ex vivo conditions. In contrast, human platelets released by intrapulmonary-entrapped megakaryocytes appear more physiologic in nature and nearly comparable to donor platelets for clinical application. PMID:25852052

  6. Platelets and infections - complex interactions with bacteria.

    PubMed

    Hamzeh-Cognasse, Hind; Damien, Pauline; Chabert, Adrien; Pozzetto, Bruno; Cognasse, Fabrice; Garraud, Olivier

    2015-01-01

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

  7. Platelets: Covert Regulators of Lymphatic Development

    PubMed Central

    Bertozzi, Cara C.; Hess, Paul R.; Kahn, Mark L.

    2010-01-01

    The field of platelet biology has rapidly expanded beyond the classical role of platelets in preventing blood loss and orchestrating clot formation. Despite the lack of transcriptional ability of these anuclear cell fragments, platelet function is now thought to encompass such diverse contexts as tissue repair, immune activation, primary tumor formation, and metastasis. Recent studies from multiple groups have turned the spotlight on an exciting new role for platelets in the formation of lymphatic vessels during embryonic development. Genetic experiments demonstrate that Podoplanin, a transmembrane protein expressed on lymphatic endothelial cells, engages the platelet CLEC-2 receptor when exposed to blood, leading to SYK-SLP-76-dependent platelet activation. When components of this pathway are disrupted, aberrant vascular connections form, resulting in blood-lymphatic mixing. Furthermore, platelet-null embryos manifest identical blood-lymphatic mixing. The identification of platelets as the critical cell type mediating blood-lymphatic vascular separation raises new questions in our understanding of lymphatic development and platelet biology. PMID:21071706

  8. An industrial perspective on utilizing functional ion channel assays for high throughput screening.

    PubMed

    Worley, Jennings F; Main, Martin J

    2002-01-01

    The ability to apply large-scale screening formats to measures of ion channel function offers immense opportunities for drug discovery and academic research. Technologies have been developed over the last several years that now provide the ability to screen large numbers of compounds and natural products on ion channel function to find novel drugs. Application of these technologies has vastly improved the capabilities of ion channel drug discovery and provides an avenue to accelerate discoveries of ion channel biology. These advances have largely arisen from the development and application of instruments and reporters of membrane potential and ion movements in cells used to measure functional activity of ion channels. This article endeavors to describe the practical applications of these technologies in developing, validating, and implementing high throughput screening assay formats to different types of ion channels.

  9. Current and emerging approaches for evaluating platelet disorders.

    PubMed

    Podda, G; Femia, E A; Cattaneo, M

    2016-05-01

    Platelets play a central role in physiological hemostasis and also in pathological thrombosis. It is well established that congenital or acquired abnormalities of platelet function are associated with a heightened risk of bleeding of variable severity and excessive hemorrhage after surgery or trauma. Several kinds of different platelet function tests have been developed over the years to identify or diagnose platelet function disorders. The use of these tests for the assessment of thrombotic risk or for monitoring the effects of drugs inhibiting platelet function is not well established. Light transmission aggregometry (LTA) is the gold standard for the study of patients with defects of platelet function. Its results are affected by several pre-analytical and analytical variables. The Subcommittee on Platelet Physiology of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis published official guidelines for the standardization of the variables affecting LTA, which should be followed to harmonize the procedures across different laboratories worldwide. The lumi-aggregometer, a modification of LTA that measures platelet secretion in parallel with aggregation, is preferable to LTA for diagnosing inherited defects of platelet function, because it is more sensitive to the most common disorders, which are characterized by abnormalities of platelet secretion. LTA (or lumi-aggregometry) is useful as a first screening test of patients with the clinical suspicion of defects of platelet function, because it helps to provide an interim diagnostic hypothesis, which can then be confirmed or discounted using appropriate and specific tests. PMID:27426860

  10. Platelet generation in vivo and in vitro.

    PubMed

    Wang, Biao; Zheng, Jiansheng

    2016-01-01

    Platelet (PLT) transfusion, which is the primary cell therapy for thrombocytopenia, has been a source of concern in recent years due to its limitations of donor-dependent supply and soaring costs. In vitro platelet generation on an industrial scale is a possible solution requiring exploration. The technology of platelet generation ex vivo has been widely studied across the world, though the mechanisms of physiological thrombopoiesis and platelet biology function in vivo still remain elusive today. Various culture systems have been studied, most of which proved quite inefficient in generating functional platelets ex vivo, so there is still a long way to reach our ultimate goal of generating a fully functional platelet in vitro on an industrial scale. This review integrates the latest research into physiological platelet biogenesis and ex vivo-platelet/megakaryocyte (MK) generation protocols with a focus on the ability to generate PLT/MK in large quantities, summarizes current culture systems based on induced human pluripotent stem cells and adipose-derived stem cells, and discusses significant challenges that must be overcome for these approaches to be perfected. PMID:27390629

  11. Assaying effector function in planta using double-barreled particle bombardment.

    PubMed

    Kale, Shiv D; Tyler, Brett M

    2011-01-01

    The biolistic transient gene expression assay is a beneficial tool for studying gene function in vivo. However, biolistic transient assay systems have inherent pitfalls that often cause experimental inaccuracies such as poor transformation efficiency, which can be confused with biological phenomena. The double-barreled gene gun device is an inexpensive and highly effective attachment that enables statistically significant data to be obtained with one-tenth the number of experimental replicates compared to conventional biolistic assays. The principle behind the attachment is to perform two simultaneous bombardments with control and test DNA preparations onto the same leaf. The control bombardment measures the efficiency of the transformation while the ratio of the test bombardment to the control bombardment measures the activity of the gene of interest. With care, the ratio between the pair of bombardments can be highly reproducible from bombardment to bombardment. The double-barreled attachment has been used to study plant resistance (R) gene-mediated responses to effectors, induction and suppression of cell death by a wide variety of pathogen and host molecules, and the role of oömycete effector RXLR motifs in cell reentry.

  12. Unscheduled DNA Synthesis: The Clinical and Functional Assay for Global Genomic DNA Nucleotide Excision Repair

    PubMed Central

    Latimer, Jean J.; Kelly, Crystal M.

    2016-01-01

    The unscheduled DNA synthesis (UDS) assay measures the ability of a cell to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique by creating 6-4 photoproducts and pyrimidine dimers using UV-C irradiation. This procedure is designed specifically for quantification of the 6-4 photoproducts. Repair is quantified by the amount of radioactive thymidine incorporated during repair synthesis after this insult, and radioactivity is evaluated by grain counting after autoradiography. The results are used to clinically diagnose human DNA repair deficiency disorders and provide a basis for investigation of repair deficiency in human tissues or tumors. No other functional assay is available that directly measures the capacity to perform NER on the entire genome without the use of specific antibodies. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation (HCR), as discussed in Chapter 37, is not an equivalent technique, as it measures only transcription-coupled repair (TCR) at active genes, a small subset of total NER. PMID:24623250

  13. Aspirin sensitivity of platelet aggregation in diabetes mellitus.

    PubMed

    Albert, Stewart G; Hasnain, Bibi I; Ritter, Detlef G; Joist, J Heinrich; Mooradian, Arshag D

    2005-11-01

    Although aspirin is cardioprotective in high-risk populations, many with diabetes mellitus (DM) are unresponsive to these benefits. We questioned whether cardiovascular unresponsiveness might be demonstrated by lack of aspirin sensitivity to in vitro platelet functions especially in subjects with poorly controlled diabetes. Six women and 4 men (48+/-8 years [mean+/-S.D.]), selected for poor control (glycohemoglobin 11.9+/-2.2%) and 10 sex-age (+/-5 years) matched controls received 81 mg aspirin daily. There was a 2-week washout from aspirin and related drugs. After the aspirin dose on day-7, blood for platelet aggregation assays, and 24-h urine for 2,3 dinor thromboxane B2 (TxB2) and 2,3 dinor 6-keto (PGF1alpha) were obtained. Aspirin sensitivity was defined as inhibition (i.e., lower than expected) platelet aggregation after exposure to an agonist. Those with diabetes and controls were sensitive to aspirin inhibition of platelet aggregation induced by 1.6 mM arachidonic acid (9.5+/-3.9% versus 9.1+/-3.1%, normal range 40-100%) and by 0.83 microg/mL collagen (17.4+/-13.9% versus 13.2+/-9.3%, normal range 60-93%), respectively. Aspirin sensitivity to 2 microM ADP was present in five with diabetes and five controls. Urinary prostaglandin metabolites were suppressed below reference ranges, without differences between those with DM or controls for TxB2 (350+/-149 pg/mg versus 348+/-93 pg/mg creatinine) and PGF1alpha (255+/-104 pg/mg versus 222+/-88 pg/mg creatinine). In conclusion, in poorly controlled diabetes, there was no differential lack of aspirin sensitivity to platelet aggregation, or lack of aspirin suppression of urinary TxB2 or PGF1alpha, compared with controls on aspirin. Despite suppression of urinary prostaglandin metabolites, aspirin resistance was most apparent to ADP-mediated platelet aggregation. It is not known what level of inhibition of in vitro tests is necessary for the cardioprotective benefits of aspirin in diabetes mellitus. Thus, the lack of

  14. Isolation, Culture, Functional Assays, and Immunofluorescence of Myofiber-Associated Satellite Cells.

    PubMed

    Vogler, Thomas O; Gadek, Katherine E; Cadwallader, Adam B; Elston, Tiffany L; Olwin, Bradley B

    2016-01-01

    Adult skeletal muscle stem cells, termed satellite cells, regenerate and repair the functional contractile cells in adult skeletal muscle called myofibers. Satellite cells reside in a niche between the basal lamina and sarcolemma of myofibers. Isolating single myofibers and their associated satellite cells provides a culture system that partially mimics the in vivo environment. We describe methods for isolating and culturing intact individual myofibers and their associated satellite cells from the mouse extensor digitorum longus muscle. Following dissection and isolation of individual myofibers we provide protocols for myofiber transplantation, satellite cell transfection, immune detection of satellite cell antigens, and assays to examine satellite cell self-renewal and proliferation.

  15. Isolation of Mouse Hair Follicle Bulge Stem Cells and Their Functional Analysis in a Reconstitution Assay.

    PubMed

    Zheng, Ying; Hsieh, Jen-Chih; Escandon, Julia; Cotsarelis, George

    2016-01-01

    The hair follicle (HF) is a dynamic structure readily accessible within the skin, and contains various pools of stem cells that have a broad regenerative potential during normal homeostasis and in response to injury. Recent discoveries demonstrating the multipotent capabilities of hair follicle stem cells and the easy access to skin tissue make the HF an attractive source for isolating stem cells and their subsequent application in tissue engineering and regenerative medicine. Here, we describe the isolation and purification of hair follicle bulge stem cells from mouse skin, and hair reconstitution assays that allows the functional analysis of multipotent stem cells. PMID:27431247

  16. Isolation, Culture, Functional Assays, and Immunofluorescence of Myofiber-Associated Satellite Cells.

    PubMed

    Vogler, Thomas O; Gadek, Katherine E; Cadwallader, Adam B; Elston, Tiffany L; Olwin, Bradley B

    2016-01-01

    Adult skeletal muscle stem cells, termed satellite cells, regenerate and repair the functional contractile cells in adult skeletal muscle called myofibers. Satellite cells reside in a niche between the basal lamina and sarcolemma of myofibers. Isolating single myofibers and their associated satellite cells provides a culture system that partially mimics the in vivo environment. We describe methods for isolating and culturing intact individual myofibers and their associated satellite cells from the mouse extensor digitorum longus muscle. Following dissection and isolation of individual myofibers we provide protocols for myofiber transplantation, satellite cell transfection, immune detection of satellite cell antigens, and assays to examine satellite cell self-renewal and proliferation. PMID:27492171

  17. Binding assays with streptavidin-functionalized superparamagnetic nanoparticles and biotinylated analytes using fluxgate magnetorelaxometry

    NASA Astrophysics Data System (ADS)

    Heim, Erik; Ludwig, Frank; Schilling, Meinhard

    2009-05-01

    Binding assays based on the magnetorelaxation of superparamagnetic nanoparticles as markers are presented utilizing a differential fluxgate system. As ligand and receptor, streptavidin and biotin, respectively, are used. Superparamagnetic nanoparticles are functionalized with streptavidin and bound to two types of biotinylated analytes: agarose beads and bovine serum (BSA) proteins. The size difference of the two analytes causes a different progress of the reaction. As a consequence, the analysis of the relaxation signal is carried out dissimilarly for the two analytes. In addition, we studied the reaction kinetics of the two kinds of analytes with the fluxgate system.

  18. Functional Analysis of Protein Tyrosine Phosphatases in Thrombosis and Hemostasis.

    PubMed

    Rahmouni, Souad; Hego, Alexandre; Delierneux, Céline; Wéra, Odile; Musumeci, Lucia; Tautz, Lutz; Oury, Cécile

    2016-01-01

    Platelets are small blood cells derived from cytoplasmic fragments of megakaryocytes and play an essential role in thrombosis and hemostasis. Platelet activation depends on the rapid phosphorylation and dephosphorylation of key signaling molecules, and a number of kinases and phosphatases have been identified as major regulators of platelet function. However, the investigation of novel signaling proteins has suffered from technical limitations due to the anucleate nature of platelets and their very limited levels of mRNA and de novo protein synthesis. In the past, experimental methods were restricted to the generation of genetically modified mice and the development of specific antibodies. More recently, novel (phospho)proteomic technologies and pharmacological approaches using specific small-molecule inhibitors have added additional capabilities to investigate specific platelet proteins.In this chapter, we report methods for using genetic and pharmacological approaches to investigate the function of platelet signaling proteins. While the described experiments focus on the role of the dual-specificity phosphatase 3 (DUSP3) in platelet signaling, the presented methods are applicable to any signaling enzyme. Specifically, we describe a testing strategy that includes (1) aggregation and secretion experiments with mouse and human platelets, (2) immunoprecipitation and immunoblot assays to study platelet signaling events, (3) detailed protocols to use selected animal models in order to investigate thrombosis and hemostasis in vivo, and (4) strategies for utilizing pharmacological inhibitors on human platelets. PMID:27514813

  19. Autologous Platelet-Rich Plasma Preparations

    PubMed Central

    Schippinger, Gert; Prüller, Florian; Divjak, Manuela; Mahla, Elisabeth; Fankhauser, Florian; Rackemann, Steve; Raggam, Reinhard Bernd

    2015-01-01

    Background Autologous platelet-rich plasma (PRP) has been widely used for the treatment of sports injuries. It has been associated with improved healing and regeneration of soft tissues in elite athletes. Athletes are commonly receiving nonsteroidal anti-inflammatory drugs (NSAIDs). As yet, the effect of these drugs on platelet function in PRP formulations has not been taken into consideration. Hypothesis The function of platelets in PRP produced under the influence of NSAIDs is inhibited and may lessen a possible healing effect on the site of injury. Study Design Controlled laboratory study. Methods PRP was collected from patients receiving NSAIDs after elective orthopaedic surgery, and platelet function was evaluated using light transmission aggregometry (LTA). Results were compared with those obtained from healthy volunteers without a history of NSAID intake during the previous 2 weeks. Two different systems for blood collection and PRP production (Arthrex ACP double-syringe system and standard 4.5-mL sodium citrate blood collection tubes) were used and compared regarding the quality of PRP that was produced. Results For both groups, the baseline platelet counts of whole blood and the platelet counts of PRP formulations were found to be in the normal range. Both collection systems for PRP produced comparable results without significant differences between the groups. Platelet function testing with LTA revealed significantly impaired platelet aggregation in both PRP preparations, obtained from patients taking NSAIDs, irrespective of the type of NSAID (P < .001). All subjects from the control group showed normal platelet aggregation patterns when tested with LTA. Conclusion Autologous PRP produced from subjects after NSAID medication shows significantly impaired platelet function and may result in lower quality regarding the content of bioactive compounds. Clinical Relevance If required, the administration of NSAIDs should be performed after blood collection for

  20. Platelets as immune cells in infectious diseases.

    PubMed

    Speth, Cornelia; Löffler, Jürgen; Krappmann, Sven; Lass-Flörl, Cornelia; Rambach, Günter

    2013-11-01

    Platelets have been shown to cover a broad range of functions. Besides their role in hemostasis, they have immunological functions and thus participate in the interaction between pathogens and host defense. Platelets have a broad repertoire of receptor molecules that enable them to sense invading pathogens and infection-induced inflammation. Consequently, platelets exert antimicrobial effector mechanisms, but also initiate an intense crosstalk with other arms of the innate and adaptive immunity, including neutrophils, monocytes/macrophages, dendritic cells, B cells and T cells. There is a fragile balance between beneficial antimicrobial effects and detrimental reactions that contribute to the pathogenesis, and many pathogens have developed mechanisms to influence these two outcomes. This review aims to highlight aspects of the interaction strategies between platelets and pathogenic bacteria, viruses, fungi and parasites, in addition to the subsequent networking between platelets and other immune cells, and the relevance of these processes for the pathogenesis of infections.

  1. The role of adenosine triphosphate citrate lyase in the metabolism of acetyl coenzyme a and function of blood platelets in diabetes mellitus.

    PubMed

    Michno, Anna; Skibowska, Anna; Raszeja-Specht, Anna; Cwikowska, Justyna; Szutowicz, Andrzej

    2004-01-01

    Diabetes is known to increase blood platelet activity. Activities of pyruvate dehydrogenase (PDH), adenosine triphosphate (ATP)-citrate lyase (ATPCL), acetyl-coenzyme A (acetyl-CoA) content, malonyl dialdehyde (MDA), synthesis, and platelet aggregation in resting conditions and after activation with thrombin were measured in diabetic subjects and in age- and sex-matched healthy subjects. Activities of ATPCL and PDH, acetyl-CoA content, and thrombin-evoked MDA synthesis as well as platelet aggregation in diabetes were 31%, 51%, 62%, 35%, and 21%, respectively, higher than in healthy subjects. In addition, activation of diabetic platelets caused 2 times greater release of acetyl-CoA from their mitochondria than in controls. Both 1.0 mmol/L (-)hydroxycitrate and 0.1 mmol/L SB-204490 decreased acetyl-CoA content in platelet cytoplasm along with suppression of MDA synthesis and platelet aggregation. These inhibitory effects were about 2 times greater in diabetic than in control platelets. The data presented indicate that the ATPCL pathway is operative in human platelets and may be responsible for provision of about 50% of acetyl units from their mitochondrial to cytoplasmic compartment. Increased acetyl-CoA synthesis in diabetic platelets may be the cause of their excessive activity in the course of the disease. ATPCL may be a target for its specific inhibitors as factors decreasing platelet activity. PMID:14681844

  2. Interactions of human blood-platelets with vaccinia

    SciTech Connect

    Vernon, C.E.B.

    1989-01-01

    These investigations were conducted to determine whether vaccinia (strain WR) adsorbs to the human platelet and alters specific platelet activities, namely, the uptake of {sup 14}C-serotonin, the release of {sup 14}C-serotonin and also the release of {sup 14}C-serotonin stimulated by thrombin. Vaccinia did not alter the platelet uptake of {sup 14}C-serotonin. To determine if vaccinia induces a release of {sup 14}C-serotonin from platelets, vaccinia was added to washed or unwashed {sup 14}C-serotonin labeled platelets, and the release of {sup 14}C-Serotonin into the supernatant was measured. Less than 8% of the {sup 14}C-Serotonin was released. The action of vaccinia to alter the platelet release of {sup 14}C-serotonin induced by thrombin was monitored by measuring the radioactivity released from thrombin stimulated {sup 14}C-serotonin labeled platelets incubated with or without vaccinia. Vaccinia inhibited the thrombin induced release of {sup 14}C-serotonin from platelets at a virus to platelet ratios of 5 through 80 plaque forming units (p.f.u.)/platelet. The inhibition was dose dependent. The binding of virus to platelets was determined by a plaque assay of a washed mixture of vaccinia virus and platelets. After inoculation of mixture onto a monolayer of BSC40 cells at a virus to platelet ratio of 0.1 p.f.u./platelet, 50 cell-bound-virus per 130,000-150,000 platelets were enumerated. Vaccinia was observed to inhibit the thrombin induced clot formation of plasma by a thrombin clotting time test. Scanning electron micrographs of the clot formed in the presence of vaccinia revealed a close packed fibrous structure lacking the cross-linked mesh-like pattern seen in a normal clot. Transmission electron micrographs showed an increase in the length and a close packing of the fibrin threads.

  3. Micro-Arrayed Human Embryonic Stem Cells-Derived Cardiomyocytes for In Vitro Functional Assay

    PubMed Central

    Serena, Elena; Cimetta, Elisa; Zatti, Susi; Zaglia, Tania; Zagallo, Monica; Keller, Gordon; Elvassore, Nicola

    2012-01-01

    Introduction The heart is one of the least regenerative organs in the body and any major insult can result in a significant loss of heart cells. The development of an in vitro-based cardiac tissue could be of paramount importance for many aspects of the cardiology research. In this context, we developed an in vitro assay based on human cardiomyocytes (hCMs) and ad hoc micro-technologies, suitable for several applications: from pharmacological analysis to physio-phatological studies on transplantable hCMs. We focused on the development of an assay able to analyze not only hCMs viability, but also their functionality. Methods hCMs were cultured onto a poly-acrylamide hydrogel with tunable tissue-like mechanical properties and organized through micropatterning in a 20×20 array. Arrayed hCMs were characterized by immunofluorescence, GAP-FRAP analyses and live and dead assay. Their functionality was evaluated monitoring the excitation-contraction coupling. Results Micropatterned hCMs maintained the expression of the major cardiac markers (cTnT, cTnI, Cx43, Nkx2.5, α-actinin) and functional properties. The spontaneous contraction frequency was (0.83±0.2) Hz, while exogenous electrical stimulation lead to an increase up to 2 Hz. As proof of concept that our device can be used for screening the effects of pathological conditions, hCMs were exposed to increasing levels of H2O2. Remarkably, hCMs viability was not compromised with exposure to 0.1 mM H2O2, but hCMs contractility was dramatically suppressed. As proof of concept, we also developed a microfluidic platform to selectively treat areas of the cell array, in the perspective of performing multi-parametric assay. Conclusions Such system could be a useful tool for testing the effects of multiple conditions on an in vitro cell model representative of human heart physiology, thus potentially helping the processes of therapy and drug development. PMID:23152776

  4. Platelet antibody in idiopathic thrombocytopenic purpura and other thrombocytopenias

    SciTech Connect

    Sugiura, K.; Steiner, M.; Baldini, M.G.

    1980-10-01

    Platelet-associated immunoglobulin was measured by the use of fluorescent anti-1gG antibody. The method is simple, rapid, and sensitive and provides a precise quantitive assay of bound (direct) and free (indirect) 1gG with platelet specificity. We have evaluated this test in 30 normal volunteers and in 50 patients with immune and nonimmune, treated and untreated thrombocytopenias. All patients with immune thrombocytopenias (acute and chronic idiopathic thrombocytopenic purpura and systemic lupus erythematosus) having platelet counts < 100,000/..mu..l had elevated levels of platelet-bound 1gG and 86% had also positive results in the indirect assay. All patients with nonimmunological thrombocytopenias showed normal results in the direct and indirect assay of platelet-associated immunoglobulin. In patients studied repeatedly during the course of their illness, an inverse relation was found between platelet count and level of platelet-bound 1gG. Patients with systemic lupus erythematosus presented clear exceptions to this rule. Investigations of the absorbability of platelet autoantibodies and alloantibodies showed that this assay can readily differentiate between these two antibody species and can also identify specificities of alloantibodies.

  5. Aminopropyltriethoxysilane-mediated surface functionalization of hydroxyapatite nanoparticles: synthesis, characterization, and in vitro toxicity assay

    PubMed Central

    Wang, Shige; Wen, Shihui; Shen, Mingwu; Guo, Rui; Cao, Xueyan; Wang, Jianhua; Shi, Xiangyang

    2011-01-01

    Background We report on aminopropyltriethoxysilane (APTS)-mediated surface modification of nanohydroxyapatite with different surface functional groups for potential biomedical applications. In this study, nanohydroxyapatite covalently linked with APTS (n-HA-APTS) was reacted with acetic anhydride or succinic anhydride to produce neutralized (n-HA-APTS. Ac) or negatively charged (n-HA-APTS.SAH) nanohydroxyapatite, respectively. Nanohydroxyapatite formed with amine, acetyl, and carboxyl groups was extensively characterized using Fourier transform infrared spectroscopy, transmission electron microscopy, 1H nuclear magnetic resonance spectroscopy, X-ray diffraction, inductively coupled plasma-atomic emission spectroscopy, and zeta potential measurements. Results In vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay revealed that the slight toxicity of the amine-functionalized n-HA-APTS could be eliminated by post-functionalization of APTS amines to form acetyl and carboxyl groups. Blood compatibility assessment demonstrated that the negligible hemolytic activity of the pristine nanohydroxyapatite particles did not appreciably change after APTS-mediated surface functionalization. Conclusion APTS-mediated functionalization of nanohydroxyapatite with different surface groups may be useful for further functionalization of nanohydroxyapatite with biologically active materials, thereby providing possibilities for a broad range of biomedical applications. PMID:22267929

  6. The prowess of platelets in immunity and inflammation.

    PubMed

    Koenen, Rory R

    2016-09-27

    Platelets not only serve as essential haemostatic cells, they also have important roles in immune defence and inflammation. Despite not having a nucleus, platelets contain physiologically relevant amounts of RNA, which can be spliced and translated into functional proteins. In addition, platelets have the ability to bind to numerous other cells, such as leukocytes and vascular cells. During those interactions, platelets can modulate cellular responses, resulting in e. g. inflammatory activation or apoptosis. Recent studies have demonstrated that platelets can influence the outcomes of bacterial and viral infection, as well as the extent of tissue injury after ischaemia. Platelets also carry considerable amounts of cytokines and growth factors in their secretory granules, preformed for rapid secretion. Those properties in combination with the sheer amount of platelets circulating in the blood stream make them an important force in the immune response during health and disease. In this overview, recent findings concerning those interesting properties of platelets beyond haemostasis are discussed.

  7. An advanced application of protein microarrays: cell-based assays for functional genomics.

    PubMed

    Carbone, Roberta

    2009-01-01

    Microarrays have become common tools for approaching different experimental questions: DNA, protein and peptide arrays offer the power of multiplexing the assay and by means of miniaturization technology, the possibility to reduce cost and amount of samples and reagents. Recently, a novel technology for functional assays has been proposed. Sabatini and co-workers have shown a cell-based microarrays method (1) that relies on the deposition and immobilization of an array of cDNA plasmids on a slide where cells are subsequently plated; the cDNA is then internalized by "reverse transfection" and cells overexpress or downregulate in each single spot the genes of interest. This approach allows the screening of different phenotypes in living cells of many genes in parallel on a single slide. To overcome some relevant limitations of this approach, we have implemented the technology by means of viral immobilization (2) on a novel surface of cluster-assembled nanostructured TiO2 (3) previously functionalized with an array of a docking protein. In this work, we present the detailed development of the "reverse infection cell-microarray based technology" in U2OS cells on a novel coated slide that represents an advanced application of protein arrays.

  8. Surveys, simulation and single-cell assays relate function and phylogeny in a lake ecosystem.

    PubMed

    Preheim, Sarah P; Olesen, Scott W; Spencer, Sarah J; Materna, Arne; Varadharajan, Charuleka; Blackburn, Matthew; Friedman, Jonathan; Rodríguez, Jorge; Hemond, Harold; Alm, Eric J

    2016-01-01

    Much remains unknown about what drives microbial community structure and diversity. Highly structured environments might offer clues. For example, it may be possible to identify metabolically similar species as groups of organisms that correlate spatially with the geochemical processes they carry out. Here, we use a 16S ribosomal RNA gene survey in a lake that has chemical gradients across its depth to identify groups of spatially correlated but phylogenetically diverse organisms. Some groups had distributions across depth that aligned with the distributions of metabolic processes predicted by a biogeochemical model, suggesting that these groups performed biogeochemical functions. A single-cell genetic assay showed, however, that the groups associated with one biogeochemical process, sulfate reduction, contained only a few organisms that have the genes required to reduce sulfate. These results raise the possibility that some of these spatially correlated groups are consortia of phylogenetically diverse and metabolically different microbes that cooperate to carry out geochemical functions. PMID:27562262

  9. Surveys, simulation and single-cell assays relate function and phylogeny in a lake ecosystem.

    PubMed

    Preheim, Sarah P; Olesen, Scott W; Spencer, Sarah J; Materna, Arne; Varadharajan, Charuleka; Blackburn, Matthew; Friedman, Jonathan; Rodríguez, Jorge; Hemond, Harold; Alm, Eric J

    2016-08-15

    Much remains unknown about what drives microbial community structure and diversity. Highly structured environments might offer clues. For example, it may be possible to identify metabolically similar species as groups of organisms that correlate spatially with the geochemical processes they carry out. Here, we use a 16S ribosomal RNA gene survey in a lake that has chemical gradients across its depth to identify groups of spatially correlated but phylogenetically diverse organisms. Some groups had distributions across depth that aligned with the distributions of metabolic processes predicted by a biogeochemical model, suggesting that these groups performed biogeochemical functions. A single-cell genetic assay showed, however, that the groups associated with one biogeochemical process, sulfate reduction, contained only a few organisms that have the genes required to reduce sulfate. These results raise the possibility that some of these spatially correlated groups are consortia of phylogenetically diverse and metabolically different microbes that cooperate to carry out geochemical functions.

  10. Cross-match-compatible platelets improve corrected count increments in patients who are refractory to randomly selected platelets

    PubMed Central

    Elhence, Priti; Chaudhary, Rajendra K.; Nityanand, Soniya

    2014-01-01

    Background Cross-match-compatible platelets are used for the management of thrombocytopenic patients who are refractory to transfusions of randomly selected platelets. Data supporting the effectiveness of platelets that are compatible according to cross-matching with a modified antigen capture enzyme-linked immunosorbent assay (MAC-ELISA or MACE) are limited. This study aimed to determine the effectiveness of cross-match-compatible platelets in an unselected group of refractory patients. Materials and methods One hundred ABO compatible single donor platelet transfusions given to 31 refractory patients were studied. Patients were defined to be refractory if their 24-hour corrected count increment (CCI) was <5×109/L following two consecutive platelet transfusions. Platelets were cross-matched by MACE and the CCI was determined to monitor the effectiveness of platelet transfusions. Results The clinical sensitivity, specificity, positive predictive value and negative predictive value of the MACE-cross-matched platelets for post-transfusion CCI were 88%, 54.6%, 39.3% and 93.2%, respectively. The difference between adequate and inadequate post-transfusion 24-hour CCI for MACE cross-matched-compatible vs incompatible single donor platelet transfusions was statistically significant (p=0.000). The 24-hour CCI (mean±SD) was significantly higher for cross-match-compatible platelets (9,250±026.6) than for incompatible ones (6,757.94±2,656.5) (p<0.0001). Most of the incompatible cross-matches (73.2%) were due to anti-HLA antibodies, alone (55.3% of cases) or together with anti-platelet glycoprotein antibodies (17.9%). Discussion The clinical sensitivity and negative predictive value of platelet cross-matching by MACE were high in this study and such tests may, therefore, be used to select compatible platelets for refractory patients. A high negative predictive value demonstrates the greater chance of an adequate response with cross-matched-compatible platelets. PMID

  11. Relative performance of a TGS for the assay of drummed waste as function of collimator opening

    SciTech Connect

    Kane, S.C.; Croft, S.; McClay, P.; Venkataraman, R.; Villani, M.F.

    2007-07-01

    , one may wish to widen the repertoire but there is currently little guidance as to the trade-off involved. In this work, we address this weakness by studying the performance of a typical TGS arrangement as a function of collimator opening, scan pattern and scan time for a representative selection of simulated waste forms. Our focus is on assessing the impact on the precision and accuracy of the quantitative assay result but we also report the utility of the imaging information in confirming acceptable knowledge about the packages. (authors)

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

    PubMed

    Pietersz, R N I

    2009-10-01

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

  13. Platelet peripheral benzodiazepine receptors are decreased in Parkinson's disease

    SciTech Connect

    Bonuccelli, U.; Nuti, A.; Del Dotto, P.; Piccini, P.; Martini, C.; Giannacccini, G.; Lucacchini, A.; Muratorio, A. )

    1991-01-01

    Peripheral benzodiazepine (BDZ) receptors are located in a variety of tissues, including platelets, in the nuclear and/or mitochondrial membranes. The authors studied the density of peripheral BDZ receptors in platelets of 10 de novo Parkinson's disease (PD) patients, 18 PD patients treated with a levodopa/carbidopa combination, and in 15 healthy subjects matched for sex and age. The binding assay was conducted using ({sup 3}H)PK 11195, a specific ligand for peripheral BDZ receptors. A significant decrease in the density of ({sup 3}H)PK 11195 binding sites has been observed in PD patients with respect to controls but not between de novo and treated PD patients. No correlation has been found between the decrease in density of ({sup 3}H)PK 11195 binding sites in platelets and either the duration or severity of PD. Peripheral BDZ receptors are implicated in the regulation of mitochondrial respiratory function. Thus, their decrease in PD might parallel the abnormalities in mitochondrial function recently found in this neurologic disease.

  14. Suitability of measurement of swirling as a marker of platelet shape change in concentrates stored for transfusion.

    PubMed

    Mathai, Jaisy; Resmi, K R; Sulochana, P V; Sathyabhama, S; Baby Saritha, G; Krishnan, Lissy K

    2006-09-01

    Platelet discoid shape is known to correlate with in vivo viability after transfusion. Measurement of shape change requires invasive sampling and laborious assays, which is difficult to perform in a blood transfusion center as a routine test for quality control of stored platelets. The objective of this study was to establish suitability of swirling assessment in stored platelet suspension as a routine test for quality check, by comparing platelet shape change measurement carried out in parallel. The study was done in two types of bags obtained from different manufactures (Groups 1 and 2). Platelet concentrates (PC) were stored for 120 h and samples drawn at 24-h intervals, optical analysis at 540 nm was carried out to quantify shape change in response to an agonist adenosine diphosphate (ADP). The same bags were subjected to swirling assessment, by two blood bank personnel independently and graded as positive (+, ++, +++) or as negative, based on the silky appearance of the suspension. Swirling negative platelets were prepared by storing platelets at 4 degrees C for 24 h and were compared with swirling positive platelets. Other parameters studied in the samples drawn at 24-h intervals were platelet count, mean platelet volume, and blood gases. Swirling assessment results correlated well with shape change measurement at each study period with a P value significant at 0.02 and 0.04 for group 1 and 2 bags, respectively. In the negative swirling controls, extent of shape change was lower than the extent in test bags, showing reduced capacity to respond to ADP at 4 degrees C. The results of the study indicate that by simple swirling measurements, stored PC can be monitored for loss of discoid shape before they are transfused. Gas tension and pH were with in permissible limits. Therefore, inspection of swirling can be a reliable method of quality control as it correlates with platelet function. The platelets that retain the discoid shape in vitro at the time of

  15. A reporter assay in lamprey embryos reveals both functional conservation and elaboration of vertebrate enhancers.

    PubMed

    Parker, Hugo J; Sauka-Spengler, Tatjana; Bronner, Marianne; Elgar, Greg

    2014-01-01

    The sea lamprey is an important model organism for investigating the evolutionary origins of vertebrates. As more vertebrate genome sequences are obtained, evolutionary developmental biologists are becoming increasingly able to identify putative gene regulatory elements across the breadth of the vertebrate taxa. The identification of these regions makes it possible to address how changes at the genomic level have led to changes in developmental gene regulatory networks and ultimately to the evolution of morphological diversity. Comparative genomics approaches using sea lamprey have already predicted a number of such regulatory elements in the lamprey genome. Functional characterisation of these sequences and other similar elements requires efficient reporter assays in lamprey. In this report, we describe the development of a transient transgenesis method for lamprey embryos. Focusing on conserved non-coding elements (CNEs), we use this method to investigate their functional conservation across the vertebrate subphylum. We find instances of both functional conservation and lineage-specific functional evolution of CNEs across vertebrates, emphasising the utility of functionally testing homologous CNEs in their host species.

  16. A Functional Assay to Assess Connexin 43-Mediated Cell-to-Cell Communication of Second Messengers in Cultured Bone Cells.

    PubMed

    Stains, Joseph P; Civitelli, Roberto

    2016-01-01

    Cell-to-cell transfer of small molecules is a fundamental way by which multicellular organisms coordinate function. Recent work has highlighted the complexity of biologic responses downstream of gap junctions. As the connexin-regulated effectors are coming into focus, there is a need to develop functional assays that allow specific testing of biologically relevant second messengers. Here, we describe a modification of the classic gap junction parachute assay to assess biologically relevant molecules passed through gap junctions. PMID:27207296

  17. Glutamate Receptor Interacting Protein 1 Mediates Platelet Adhesion and Thrombus Formation.

    PubMed

    Modjeski, Kristina L; Ture, Sara K; Field, David J; Cameron, Scott J; Morrell, Craig N

    2016-01-01

    Thrombosis-associated pathologies, such as myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. Because platelets are necessary for hemostasis and thrombosis, platelet directed therapies must balance inhibiting platelet function with bleeding risk. Glutamate receptor interacting protein 1 (GRIP1) is a large scaffolding protein that localizes and organizes interacting proteins in other cells, such as neurons. We have investigated the role of GRIP1 in platelet function to determine its role as a molecular scaffold in thrombus formation. Platelet-specific GRIP1-/- mice were used to determine the role of GRIP1 in platelets. GRIP1-/- mice had normal platelet counts, but a prolonged bleeding time and delayed thrombus formation in a FeCl3-induced vessel injury model. In vitro stimulation of WT and GRIP1-/- platelets with multiple agonists showed no difference in platelet activation. However, in vivo platelet rolling velocity after endothelial stimulation was significantly greater in GRIP1-/- platelets compared to WT platelets, indicating a potential platelet adhesion defect. Mass spectrometry analysis of GRIP1 platelet immunoprecipitation revealed enrichment of GRIP1 binding to GPIb-IX complex proteins. Western blots confirmed the mass spectrometry findings that GRIP1 interacts with GPIbα, GPIbβ, and 14-3-3. Additionally, in resting GRIP1-/- platelets, GPIbα and 14-3-3 have increased interaction compared to WT platelets. GRIP1 interactions with the GPIb-IX binding complex are necessary for normal platelet adhesion to a stimulated endothelium. PMID:27631377

  18. Glutamate Receptor Interacting Protein 1 Mediates Platelet Adhesion and Thrombus Formation

    PubMed Central

    Modjeski, Kristina L.; Ture, Sara K.; Field, David J.; Cameron, Scott J.; Morrell, Craig N.

    2016-01-01

    Thrombosis-associated pathologies, such as myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. Because platelets are necessary for hemostasis and thrombosis, platelet directed therapies must balance inhibiting platelet function with bleeding risk. Glutamate receptor interacting protein 1 (GRIP1) is a large scaffolding protein that localizes and organizes interacting proteins in other cells, such as neurons. We have investigated the role of GRIP1 in platelet function to determine its role as a molecular scaffold in thrombus formation. Platelet-specific GRIP1-/- mice were used to determine the role of GRIP1 in platelets. GRIP1-/- mice had normal platelet counts, but a prolonged bleeding time and delayed thrombus formation in a FeCl3-induced vessel injury model. In vitro stimulation of WT and GRIP1-/- platelets with multiple agonists showed no difference in platelet activation. However, in vivo platelet rolling velocity after endothelial stimulation was significantly greater in GRIP1-/- platelets compared to WT platelets, indicating a potential platelet adhesion defect. Mass spectrometry analysis of GRIP1 platelet immunoprecipitation revealed enrichment of GRIP1 binding to GPIb-IX complex proteins. Western blots confirmed the mass spectrometry findings that GRIP1 interacts with GPIbα, GPIbβ, and 14-3-3. Additionally, in resting GRIP1-/- platelets, GPIbα and 14-3-3 have increased interaction compared to WT platelets. GRIP1 interactions with the GPIb-IX binding complex are necessary for normal platelet adhesion to a stimulated endothelium. PMID:27631377

  19. Nanoparticle biointerfacing by platelet membrane cloaking.

    PubMed

    Hu, Che-Ming J; Fang, Ronnie H; Wang, Kuei-Chun; Luk, Brian T; Thamphiwatana, Soracha; Dehaini, Diana; Nguyen, Phu; Angsantikul, Pavimol; Wen, Cindy H; Kroll, Ashley V; Carpenter, Cody; Ramesh, Manikantan; Qu, Vivian; Patel, Sherrina H; Zhu, Jie; Shi, William; Hofman, Florence M; Chen, Thomas C; Gao, Weiwei; Zhang, Kang; Chien, Shu; Zhang, Liangfang

    2015-10-01

    Development of functional nanoparticles can be encumbered by unanticipated material properties and biological events, which can affect nanoparticle effectiveness in complex, physiologically relevant systems. Despite the advances in bottom-up nanoengineering and surface chemistry, reductionist functionalization approaches remain inadequate in replicating the complex interfaces present in nature and cannot avoid exposure of foreign materials. Here we report on the preparation of polymeric nanoparticles enclosed in the plasma membrane of human platelets, which are a unique population of cellular fragments that adhere to a variety of disease-relevant substrates. The resulting nanoparticles possess a right-side-out unilamellar membrane coating functionalized with immunomodulatory and adhesion antigens associated with platelets. Compared to uncoated particles, the platelet membrane-cloaked nanoparticles have reduced cellular uptake by macrophage-like cells and lack particle-induced complement activation in autologous human plasma. The cloaked nanoparticles also display platelet-mimicking properties such as selective adhesion to damaged human and rodent vasculatures as well as enhanced binding to platelet-adhering pathogens. In an experimental rat model of coronary restenosis and a mouse model of systemic bacterial infection, docetaxel and vancomycin, respectively, show enhanced therapeutic efficacy when delivered by the platelet-mimetic nanoparticles. The multifaceted biointerfacing enabled by the platelet membrane cloaking method provides a new approach in developing functional nanoparticles for disease-targeted delivery.

  20. Functional recombinant MHC class II molecules and high-throughput peptide-binding assays

    PubMed Central

    Justesen, Sune; Harndahl, Mikkel; Lamberth, Kasper; Nielsen, Lise-Lotte B; Buus, Søren

    2009-01-01

    Background Molecules of the class II major histocompability complex (MHC-II) specifically bind and present exogenously derived peptide epitopes to CD4+ T helper cells. The extreme polymorphism of the MHC-II hampers the complete analysis of peptide binding. It is also a significant hurdle in the generation of MHC-II molecules as reagents to study and manipulate specific T helper cell responses. Methods to generate functional MHC-II molecules recombinantly, and measure their interaction with peptides, would be highly desirable; however, no consensus methodology has yet emerged. Results We generated α and β MHC-II chain constructs, where the membrane-spanning regions were replaced by dimerization motifs, and the C-terminal of the β chains was fused to a biotinylation signal peptide (BSP) allowing for in vivo biotinylation. These chains were produced separately as inclusion bodies in E. coli , extracted into urea, and purified under denaturing and non-reducing conditions using conventional column chromatography. Subsequently, diluting the two chains into a folding reaction with appropriate peptide resulted in efficient peptide-MHC-II complex formation. Several different formats of peptide-binding assay were developed including a homogeneous, non-radioactive, high-throughput (HTS) binding assay. Binding isotherms were generated allowing the affinities of interaction to be determined. The affinities of the best binders were found to be in the low nanomolar range. Recombinant MHC-II molecules and accompanying HTS peptide-binding assay were successfully developed for nine different MHC-II molecules including the DPA1*0103/DPB1*0401 (DP401) and DQA1*0501/DQB1*0201, where both α and β chains are polymorphic, illustrating the advantages of producing the two chains separately. Conclusion We have successfully developed versatile MHC-II resources, which may assist in the generation of MHC class II -wide reagents, data, and tools. PMID:19416502

  1. Arf6 plays an early role in platelet activation by collagen and convulxin.

    PubMed

    Choi, Wangsun; Karim, Zubair A; Whiteheart, Sidney W

    2006-04-15

    Small GTPases play critical roles in hemostasis, though the roster of such molecules in platelets is not complete. In this study, we report the presence of Ras-related GTPases of the ADP-ribosylation factor (Arf) family. Platelets contain Arf1 or 3 and Arf6, with the latter being predominantly membrane associated. Using effector domain pull-down assays, we show, counter to other GTPases, that Arf6-GTP is present in resting platelets and decreases rapidly upon activation with collagen or convulxin. This decrease does not completely rely on secondary agonists (ADP and thromboxane A2) or require integrin signaling. The decrease in free Arf6-GTP temporally precedes activation of Rho family GTPases (RhoA, Cdc42, and Rac1). Using a membrane-permeant, myristoylated peptide, which mimics the N-terminus of Arf6, we show that the Arf6-GTP decrease is essential for collagen- and convulxin-induced aggregation, platelet adherence, and spreading on collagen-coated glass. Treatment with this peptide also affects the activation of Rho family GTPases, but has little effect on RalA and Rap1 or on agonist-induced calcium mobilization. These data show that Arf6 is a key element in activation through GPVI, and is required for activation of the Rho family GTPases and the subsequent cytoskeletal rearrangements needed for full platelet function. PMID:16352809

  2. Platelet hyperaggregability in obesity: is there a role for nitric oxide impairment and oxidative stress?

    PubMed

    Leite, Natália Rodrigues Pereira; Siqueira de Medeiros, Mariana; Mury, Wanda Vianna; Matsuura, Cristiane; Perszel, Monique Bandeira Moss; Noronha Filho, Gerson; Brunini, Tatiana Mc; Mendes-Ribeiro, Antônio Claúdio

    2016-08-01

    Epidemiological evidence has shown that platelet activation markers are consistently elevated in obesity, contributing to its prothrombotic state. In order to improve the understanding of the regulation of platelet function in obesity, the aim of this study was to investigate the l-arginine-nitric oxide (NO) pathway in obese adults without other cardiovascular risk factor. Seventeen obese (body mass index [BMI] 35.9±1.0 kg/m(2) ) and eighteen age-matched normal weight subjects (BMI 22.0±0.6 kg/m(2) ) were included in this study. l-arginine influx was measured with incubation of l-[(3) H]-arginine. NO synthase (NOS) and arginase activities were determined by the citrulline assay and the conversion of l-[(14) C]-arginine to [(14) C]-urea, respectively. Cyclic guanosine monophosphate (cGMP) content was evaluated by enzyme-linked immunosorbent assay. In addition, the study analyzed: platelet aggregation; intraplatelet antioxidant enzymes, via superoxide dismutase (SOD) and catalase activities; and systemic levels of l-arginine, fibrinogen, and C-reactive protein (CRP). Obese patients presented a significant decrease of platelet l-arginine influx, NOS activity, and cGMP levels, along with platelet hyperaggregability. On the presence of NO donor, platelet aggregation was similar between the groups. The fibrinogen and CRP systemic levels were significantly higher and SOD activity was reduced in obesity. No significant differences were observed in plasma levels of l-arginine and intraplatelet arginase and catalase activities between groups. The diminished NO bioavailability associated with inflammatory status and impaired enzymatic antioxidant defence may contribute to future cardiovascular complications in obesity. PMID:27145241

  3. Outcomes of an automated procedure for the selection of effective platelets for patients refractory to random donors based on cross-matching locally available platelet products.

    PubMed

    Rebulla, Paolo; Morelati, Fernanda; Revelli, Nicoletta; Villa, Maria Antonietta; Paccapelo, Cinzia; Nocco, Angela; Greppi, Noemi; Marconi, Maurizio; Cortelezzi, Agostino; Fracchiolla, Nicola; Martinelli, Giovanni; Deliliers, Giorgio Lambertenghi

    2004-04-01

    In 1999, we implemented an automated platelet cross-matching (XM) programme to select compatible platelets from the local inventory for patients refractory to random donor platelets. In this study, we evaluated platelet count increments in 40 consecutive refractory patients (8.3% of 480 consecutive platelet recipients) given 569 cross-match-negative platelets between April 1999 and December 2001. XM was performed automatically with a commercially available immunoadherence assay. Pre-, 1- and 24-h post-transfusion platelet counts (mean +/- SD) for the 569 XM-negative platelet transfusions containing 302 +/- 71 x 109 platelets were 7.7 +/- 5.5, 32.0 +/- 21.0 and 16.8 +/- 15.5 x 109/l respectively. Increments were significantly higher (P < 0.05, t-test) than those observed in the same patients given 303 random platelet pools (dose = 318 +/- 52 x 109 platelets) during the month before refractoriness was detected, when pre-, 1- and 24-h post-transfusion counts were 7.0 +/- 8.6, 15.9 +/- 16.1 and 9.6 +/- 12.8 x 109/l respectively. The cost of the platelet XM disposable kit per transfusion to produce 1-h post-transfusion platelet count increments >10 x 109/l was euro 447. This programme enabled the rapid selection of effective platelets for refractory patients, from the local inventory. PMID:15015974

  4. Outcomes of an automated procedure for the selection of effective platelets for patients refractory to random donors based on cross-matching locally available platelet products.

    PubMed

    Rebulla, Paolo; Morelati, Fernanda; Revelli, Nicoletta; Villa, Maria Antonietta; Paccapelo, Cinzia; Nocco, Angela; Greppi, Noemi; Marconi, Maurizio; Cortelezzi, Agostino; Fracchiolla, Nicola; Martinelli, Giovanni; Deliliers, Giorgio Lambertenghi

    2004-04-01

    In 1999, we implemented an automated platelet cross-matching (XM) programme to select compatible platelets from the local inventory for patients refractory to random donor platelets. In this study, we evaluated platelet count increments in 40 consecutive refractory patients (8.3% of 480 consecutive platelet recipients) given 569 cross-match-negative platelets between April 1999 and December 2001. XM was performed automatically with a commercially available immunoadherence assay. Pre-, 1- and 24-h post-transfusion platelet counts (mean +/- SD) for the 569 XM-negative platelet transfusions containing 302 +/- 71 x 109 platelets were 7.7 +/- 5.5, 32.0 +/- 21.0 and 16.8 +/- 15.5 x 109/l respectively. Increments were significantly higher (P < 0.05, t-test) than those observed in the same patients given 303 random platelet pools (dose = 318 +/- 52 x 109 platelets) during the month before refractoriness was detected, when pre-, 1- and 24-h post-transfusion counts were 7.0 +/- 8.6, 15.9 +/- 16.1 and 9.6 +/- 12.8 x 109/l respectively. The cost of the platelet XM disposable kit per transfusion to produce 1-h post-transfusion platelet count increments >10 x 109/l was euro 447. This programme enabled the rapid selection of effective platelets for refractory patients, from the local inventory.

  5. The effect of dabigatran and rivaroxaban on platelet reactivity and inflammatory markers.

    PubMed

    Zemer-Wassercug, Noa; Haim, Moti; Leshem-Lev, Dorit; Orvin, Katia L; Vaduganathan, Muthiah; Gutstein, Ariel; Kadmon, Ehud; Mager, Aviv; Kornowski, Ran; Lev, Eli I; Lev, Eli L

    2015-10-01

    The new oral anticoagulants (NOACs) reduce stroke and systemic embolism in patients with non-valvular atrial fibrillation (AF), but dabigatran may increase risk of coronary ischemic events for unclear reasons. Thus, this study assessed the effects of dabigatran and rivaroxaban on platelet reactivity and inflammatory markers in patients with non-valvular AF. Patients with non-valvular AF planned to begin treatment with NOACs were included. Seventeen patients were prescribed dabigatran and ten rivaroxaban. Platelet function (as assessed by multiple-electrode aggregometry, Impact-R shear-induced platelet deposition, P-selectin expression and plasma RANTES levels) and high-sensitivity C-reactive protein (hs-CRP) were measured at enrollment (prior to initiation of NOAC treatment) and at least 7 days into treatment with either dabigratran or rivaroxaban. Seventeen patients treated with dabigatran (mean age 69 ± 7 years, 35 % women, mean CHADS2 score 2.6 ± 1.2), and ten patients treated with rivaroxaban (mean age 73 ± 9 years, 20 % women, mean CHADS2 score 2.7 ± 1.6) completed the study. In both groups, there were no significant differences in platelet reactivity between the baseline and on-anticoagulant treatment time-points, as measured by each of the platelet-specific assays. There was a trend towards increased platelet reactivity in response to arachidonic acid from baseline to on-treatment in both groups, probably as a result of aspirin discontinuation in 33 % of patients. No significant differences were noted between baseline and on-treatment in hs-CRP in both anticoagulant groups. Treatment with dabigatran and rivaroxaban does not appear to be associated with changes in markers of platelet reactivity or systemic inflammation.

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

    SciTech Connect

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

    2010-01-22

    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.

  7. Actin filaments and microtubule dual-granule transport in human adhered platelets: the role of alpha-dystrobrevins.

    PubMed

    Cerecedo, Doris; Cisneros, Bulmaro; Mondragón, Ricardo; González, Sirenia; Galván, Iván J

    2010-04-01

    Upon activation with physiological stimuli, human platelets undergo morphological changes, centralizing their organelles and secreting effector molecules at the site of vascular injury. Previous studies have indicated that the actin filaments and microtubules of suspension-activated platelets play a critical role in granule movement and exocytosis; however, the participation of these cytoskeleton elements in adhered platelets remains unexplored. alpha- and beta-dystrobrevin members of the dystrophin-associated protein complex in muscle and non-muscle cells have been described as motor protein receptors that might participate in the transport of cellular components in neurons. Recently, we characterized the expression of dystrobrevins in platelets; however, their functional diversity within this cellular model had not been elucidated. The present study examined the contribution of actin filaments and microtubules in granule trafficking during the platelet adhesion process using cytoskeleton-disrupting drugs, quantification of soluble P-selectin, fluorescence resonance transfer energy analysis and immunoprecipitation assays. Likewise, we assessed the interaction of alpha-dystrobrevins with the ubiquitous kinesin heavy chain. Our results strongly suggest that microtubules and actin filaments participate in the transport of alpha and dense granules in the platelet adhesion process, during which alpha-dystrobrevins play the role of regulatory and adaptor proteins that govern trafficking events.

  8. Identification of APC mutations and evaluation of their expression level using a functional screening assay

    SciTech Connect

    Varesco, L.; Gismondi, V.; Bafico, A.

    1994-09-01

    A functional screen for chain-terminating mutations in the APC gene recently has been developed. It is based on the PCR and cloning of a segment of the gene in-frame with a colorimetric marker gene (lacz) followed by screening for the level of activity of the marker polypeptide (beta-galactosidase). This method scores colony number with different blue colors that are produced by bacteria containing normal and mutant APC segments. In the present work this method was used to screen the entire APC coding region by using eight primer pairs. DNA segments with known APC mutations at different positions in the gene were used as controls and were clearly identifiable with this assay. In addition, the entire APC coding region has been examined in 21 APC patients in whom PCR-SSCP did not identify an APC mutation. Novel mutations (n=14) were identified by the blue/white assay and were all confirmed by sequence analysis. This method also was used to quantitate the expression of paternal and maternal APC alleles taking advantage of an RsaI site polymorphism at position 1458 in a small number of informative individuals. Differential expression of some known mutant APC mRNAs was observed.

  9. [Determination of tolerance ability of platelet to the change of solution osmotic pressure and its significance].

    PubMed

    Ouyang, Xi-Lin; Liu, Jing-Han; Gao, Dayong

    2003-02-01

    In order to determine the tolerance ability of platelet to change of osmotic pressure in solution, the isotonic fresh platelets were exposed to a series of crystal salt solutions with osmotic pressure range from 47 to 611 mOsm for 15 minutes. Then the platelets were returned to isotonic condition and kept for 15 minutes. The expressions of phosphatidylserine and CD62p were assayed in platelets. The results showed that the phosphatidylserine and CD62p expressions were increased when the osmotic pressure of solution was below 238 mOsm, but no significant rise was detected when the platelets were exposed to 611 mOsm solution. No increases of positive rate of CD62p and phosphatidylserine were detected in platelets returned to isotonic condition. It is concluded that platelets are sensitive to hypoosmotic solution and tolerated to hyperosmotic solution. Exceeding the platelet safe volume limitation may lead to injure of platelet osmosis in crystal salt solution. PMID:12667298

  10. Gut Microbial Metabolite TMAO Enhances Platelet Hyperreactivity and Thrombosis Risk.

    PubMed

    Zhu, Weifei; Gregory, Jill C; Org, Elin; Buffa, Jennifer A; Gupta, Nilaksh; Wang, Zeneng; Li, Lin; Fu, Xiaoming; Wu, Yuping; Mehrabian, Margarete; Sartor, R Balfour; McIntyre, Thomas M; Silverstein, Roy L; Tang, W H Wilson; DiDonato, Joseph A; Brown, J Mark; Lusis, Aldons J; Hazen, Stanley L

    2016-03-24

    Normal platelet function is critical to blood hemostasis and maintenance of a closed circulatory system. Heightened platelet reactivity, however, is associated with cardiometabolic diseases and enhanced potential for thrombotic events. We now show gut microbes, through generation of trimethylamine N-oxide (TMAO), directly contribute to platelet hyperreactivity and enhanced thrombosis potential. Plasma TMAO levels in subjects (n > 4,000) independently predicted incident (3 years) thrombosis (heart attack, stroke) risk. Direct exposure of platelets to TMAO enhanced sub-maximal stimulus-dependent platelet activation from multiple agonists through augmented Ca(2+) release from intracellular stores. Animal model studies employing dietary choline or TMAO, germ-free mice, and microbial transplantation collectively confirm a role for gut microbiota and TMAO in modulating platelet hyperresponsiveness and thrombosis potential and identify microbial taxa associated with plasma TMAO and thrombosis potential. Collectively, the present results reveal a previously unrecognized mechanistic link between specific dietary nutrients, gut microbes, platelet function, and thrombosis risk.

  11. Gut Microbial Metabolite TMAO Enhances Platelet Hyperreactivity and Thrombosis Risk.

    PubMed

    Zhu, Weifei; Gregory, Jill C; Org, Elin; Buffa, Jennifer A; Gupta, Nilaksh; Wang, Zeneng; Li, Lin; Fu, Xiaoming; Wu, Yuping; Mehrabian, Margarete; Sartor, R Balfour; McIntyre, Thomas M; Silverstein, Roy L; Tang, W H Wilson; DiDonato, Joseph A; Brown, J Mark; Lusis, Aldons J; Hazen, Stanley L

    2016-03-24

    Normal platelet function is critical to blood hemostasis and maintenance of a closed circulatory system. Heightened platelet reactivity, however, is associated with cardiometabolic diseases and enhanced potential for thrombotic events. We now show gut microbes, through generation of trimethylamine N-oxide (TMAO), directly contribute to platelet hyperreactivity and enhanced thrombosis potential. Plasma TMAO levels in subjects (n > 4,000) independently predicted incident (3 years) thrombosis (heart attack, stroke) risk. Direct exposure of platelets to TMAO enhanced sub-maximal stimulus-dependent platelet activation from multiple agonists through augmented Ca(2+) release from intracellular stores. Animal model studies employing dietary choline or TMAO, germ-free mice, and microbial transplantation collectively confirm a role for gut microbiota and TMAO in modulating platelet hyperresponsiveness and thrombosis potential and identify microbial taxa associated with plasma TMAO and thrombosis potential. Collectively, the present results reveal a previously unrecognized mechanistic link between specific dietary nutrients, gut microbes, platelet function, and thrombosis risk. PMID:26972052

  12. Characterization of canine platelet adhesion to extracellular matrix proteins.

    PubMed

    Pelagalli, Alessandra; Pero, Maria Elena; Mastellone, Vincenzo; Cestaro, Anna; Signoriello, Simona; Lombardi, Pietro; Avallone, Luigi

    2011-07-01

    Canine platelets have been extensively studied but little is known about specific aspects such as adhesion. Platelet adhesion is a critical step during haemostasis and thrombosis as well as during inflammatory and immunopathogenic responses. The aim of this study was to evaluate the adhesive properties of canine platelets using fibrinogen and collagen as substrates immobilized on plates. Adhesion was monitored for 120 min and the effect of adenosine 5'-diphosphate (ADP) was assayed. The results showed that canine platelets displayed good adhesion activity that was significantly time-dependent. Moreover, ADP was able to enhance platelet adhesion in a dose-dependent manner. The findings aid knowledge of the adhesion process and suggest a specific role of surface platelet receptors in mediating the interaction with extracellular matrix proteins.

  13. Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay

    PubMed Central

    Sharma, Vasundhara; Jordan, Jennifer J.; Ciribilli, Yari; Resnick, Michael A.; Bisio, Alessandra; Inga, Alberto

    2015-01-01

    The NF-κB transcription factor family plays a central role in innate immunity and inflammation processes and is frequently dysregulated in cancer. We developed an NF-κB functional assay in yeast to investigate the following issues: transactivation specificity of NF-κB proteins acting as homodimers or heterodimers; correlation between transactivation capacity and in vitro DNA binding measurements; impact of co-expressed interacting proteins or of small molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 cDNAs were cloned into centromeric expression vectors under inducible GAL1 promoter in order to vary their expression levels. Since p50 lacks a transactivation domain (TAD), a chimeric construct containing the TAD derived from p65 was also generated (p50TAD) to address its binding and transactivation potential. The p50TAD and p65 had distinct transactivation specificities towards seventeen different κB response elements (κB-REs) where single nucleotide changes could greatly impact transactivation. For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα. Transactivation results in yeast correlated only partially with in vitro measured DNA binding affinities, suggesting that features other than strength of interaction with naked DNA affect transactivation, although factors such as chromatin context are kept constant in our isogenic yeast assay. The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators. PMID:26147604

  14. What Is Vinculin Needed for in Platelets?

    PubMed Central

    Mitsios, John V.; Prévost, Nicolas; Kasirer-Friede, Ana; Gutierrez, Edgar; Groisman, Alex; Abrams, Charles S.; Wang, Yanfeng; Litvinov, Rustem I.; Zemljic-Harpf, Alice; Ross, Robert S.; Shattil, Sanford J.

    2010-01-01

    Summary Background Vinculin links integrins to the cell cytoskeleton by virtue of its binding to proteins such as talin and F-actin. It has been implicated in the transmission of mechanical forces from the extracellular matrix to the cytoskeleton of migrating cells. Vinculin’s function in platelets is unknown. Objective To determine whether vinculin is required for the functions of platelets and their major integrin, αIIbβ3. Methods The murine vinculin gene (Vcl) was deleted in the megakaryocyte/platelet lineage by breeding Vcl fl/fl mice with Pf4-Cre mice. Platelet and integrin functions were studied in vivo and ex vivo. Results Vinculin was undetectable in platelets from Vcl fl/fl Cre+ mice, as determined by immunoblotting and fluorescence microscopy. Vinculin-deficient megakaryocytes exhibited increased membrane tethers in response to mechanical pulling on αIIbβ3 with laser tweezers, suggesting that vinculin helps to maintain membrane cytoskeleton integrity. Surprisingly, vinculin-deficient platelets displayed normal agonist-induced fibrinogen binding to αIIbβ3, aggregation, spreading, actin polymerization/organization, clot retraction and the ability to form a procoagulant surface. Furthermore, vinculin-deficient platelets adhered to immobilized fibrinogen or collagen normally, both under static and flow conditions. Tail bleeding times were prolonged in 59% of vinculin-deficient mice. However, these mice exhibited no spontaneous bleeding and they formed occlusive platelet thrombi comparable to wild-type littermates in response to carotid artery injury with FeCl3. Conclusion Despite promoting membrane cytoskeleton integrity when mechanical force is applied to αIIbβ3, vinculin is not required for the traditional functions of αIIbβ3 or the platelet actin cytoskeleton. PMID:20670372

  15. Functional gold nanoparticles coupled with microporous membranes: a flow controlled assay for colorimetric visualization of proteins.

    PubMed

    Chen, Yu-Yuan; Unnikrishnan, Binesh; Li, Yu-Jia; Huang, Chih-Ching

    2014-11-21

    We report a rapid and simple assay for colorimetric visualization of thrombin at nanomolar levels using functional gold nanoparticles (FAuNPs) coupled with microporous membranes. We used a 29-mer thiolated-thrombin-binding-aptamer (TBA29) to prepare TBA29 functionalized AuNPs (TBA29-AuNPs) for the selective detection of human thrombin. The sensing mechanism is based on the principle of TBA29-AuNPs flowing down through the nitrocellulose membrane (NCM) pores at different flow rates after binding to thrombin. Compared with free TBA29-AuNPs, when thrombin-TBA29-AuNPs were dropped on the NCM, the particles flowed down more easily through the NCM pores along with the buffer solution due to the increase in the gravity of particles. Therefore, color intensities of TBA29-AuNPs on the NCM depended on the concentration of thrombin; the color intensity was lighter when the concentration of thrombin was higher. Thrombin can be detected at the nanomolar level with the naked eye using this colorime